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1
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Zhao W, Zhu X, Huang G, Gu H, Bi Y, Tang D, Ren H. Application of Multiple Base-Editing Mediated by Polycistronic tRNA-gRNA-Processing System in Pig Cells. Biotechnol Bioeng 2025; 122:779-791. [PMID: 39844444 DOI: 10.1002/bit.28931] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2024] [Revised: 01/04/2025] [Accepted: 01/10/2025] [Indexed: 01/24/2025]
Abstract
Gene edited pigs have extensive and important application value in the fields of agriculture and biomedicine. With the increasing demand in medical research and agricultural markets, more and more application scenarios require gene edited pigs to possess two or even more advantageous phenotypes simultaneously. The current production of multi gene edited pigs is inefficient, time-consuming, and costly, and there is an urgent need to develop efficient and accurate multi gene editing application technologies. The polycistronic tRNA-gRNA-processing system (PTG), developed based on endogenous tRNA self-processing systems, has been shown to exhibit efficient multi gene editing in plants. This study aims to combine a PTG strategy with multiple gRNA production functions with an adenine base editor (ABE) to test its feasibility for efficient and precise multi gene base editing in pig cells. The results indicate that the PTG based integrated ABE plasmid can perform efficient base editing at multiple gene loci in pig cells. And while the gene editing efficiency was significantly improved, no indel and sgRNA dependent off target effects caused by DSB were detected. This work permit will provide a solid foundation for the production of multi gene edited pigs with agricultural and medical applications.
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Affiliation(s)
- Wudi Zhao
- Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, China
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, Gene Editing Technology Center of Guangdong Province, Foshan University, Foshan, China
| | - Xiangxing Zhu
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, Gene Editing Technology Center of Guangdong Province, Foshan University, Foshan, China
| | - Guobin Huang
- Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, China
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, Gene Editing Technology Center of Guangdong Province, Foshan University, Foshan, China
| | - Hao Gu
- Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, China
| | - Yanzhen Bi
- Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, China
| | - Dongsheng Tang
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, Gene Editing Technology Center of Guangdong Province, Foshan University, Foshan, China
| | - Hongyan Ren
- Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, China
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2
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Chen P, Wu Y, Wang H, Liu H, Zhou J, Chen J, Lei J, Sun Z, Paek C, Yin L. Highly parallel profiling of the activities and specificities of Cas12a variants in human cells. Nat Commun 2025; 16:3022. [PMID: 40155371 PMCID: PMC11953374 DOI: 10.1038/s41467-025-57150-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2024] [Accepted: 02/11/2025] [Indexed: 04/01/2025] Open
Abstract
Several Cas12a variants have been developed to broaden its targeting range, improve the gene editing specificity or the efficiency. However, selecting the appropriate Cas12a among the many orthologs for a given target sequence remains difficult. Here, we perform high-throughput analyses to evaluate the activity and compatibility with specific PAMs of 24 Cas12a variants and develop deep learning models for these Cas12a variants to predict gene editing activities at target sequences of interest. Furthermore, we reveal and enhance the truncation in the integrated tag sequence that may hinder off-targeting detection for Cas12a by GUIDE-seq. This enhanced system, which we term enGUIDE-seq, is used to evaluate and compare the off-targeting and translocations of these Cas12a variants.
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Affiliation(s)
- Peng Chen
- Department of Pediatric Research Institute; Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children's Hospital of Chongqing Medical University, School of Basic Medical Sciences, Chongqing Medical University, Chongqing, China
- State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China
| | - Yankang Wu
- State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China
| | - Hongjian Wang
- State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China
| | - Huan Liu
- State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China
| | - Jin Zhou
- Department of Pediatric Research Institute; Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children's Hospital of Chongqing Medical University, School of Basic Medical Sciences, Chongqing Medical University, Chongqing, China
- State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China
- Wuhan Biorun Biosciences Co., Ltd., Wuhan, China
| | - Jingli Chen
- School of Medicine, Wuhan University of Science and Technology, Wuhan, China
| | - Jun Lei
- Department of Pediatric Research Institute; Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children's Hospital of Chongqing Medical University, School of Basic Medical Sciences, Chongqing Medical University, Chongqing, China
- State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China
| | - Zaiqiao Sun
- State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China
| | - Chonil Paek
- State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China
| | - Lei Yin
- Department of Pediatric Research Institute; Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children's Hospital of Chongqing Medical University, School of Basic Medical Sciences, Chongqing Medical University, Chongqing, China.
- State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China.
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Zheng X, Huang CH, Yan S, Rong MD. Advances and applications of genome-edited animal models for severe combined immunodeficiency. Zool Res 2025; 46:249-260. [PMID: 39846200 DOI: 10.24272/j.issn.2095-8137.2024.195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2025] Open
Abstract
Severe combined immunodeficiency disease (SCID), characterized by profound immune system dysfunction, can lead to life-threatening infections and death. Animal models play a pivotal role in elucidating biological processes and advancing therapeutic strategies. Recent advances in gene-editing technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, and base editing, have significantly enhanced the generation of SCID models. These models have not only deepened our understanding of disease pathophysiology but have also driven progress in cancer therapy, stem cell transplantation, organ transplantation, and infectious disease management. This review provides a comprehensive overview of current SCID models generated using novel gene-editing approaches, highlighting their potential applications in translational medicine and their role in advancing biomedical research.
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Affiliation(s)
- Xiao Zheng
- Stomatological Hospital, School of Stomatology, Southern Medical University, Guangzhou, Guangdong 510280, China
| | - Chun-Hui Huang
- Guangdong Key Laboratory of Non-Human Primate Models, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong 510632, China
- School of medicine, Jinan University, Guangzhou, Guangdong 510632, China
| | - Sen Yan
- Guangdong Key Laboratory of Non-Human Primate Models, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong 510632, China
- School of medicine, Jinan University, Guangzhou, Guangdong 510632, China
- Department of Neurology, Guangzhou Red Cross Hospital, Faculty of Medical Science, Jinan University, Guangzhou, Guangdong 510220, China. E-mail:
| | - Ming-Deng Rong
- Stomatological Hospital, School of Stomatology, Southern Medical University, Guangzhou, Guangdong 510280, China. E-mail:
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Shi W, Jin E, Fang L, Sun Y, Fan Z, Zhu J, Liang C, Zhang YP, Zhang YQ, Wang GD, Zhao W. VDGE: a data repository of variation database for gene-edited animals across multiple species. Nucleic Acids Res 2025; 53:D1250-D1260. [PMID: 39470732 PMCID: PMC11701559 DOI: 10.1093/nar/gkae956] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2024] [Revised: 10/05/2024] [Accepted: 10/10/2024] [Indexed: 11/01/2024] Open
Abstract
Gene-edited animals are crucial for addressing fundamental questions in biology and medicine and hold promise for practical applications. In light of the rapid advancement of gene editing technologies over the past decade, a dramatically increased number of gene-edited animals have been generated. Genome editing at off-target sites can, however, introduce genomic variations, potentially leading to unintended functional consequences in these animals. So, there is an urgent need to systematically collect and collate these variations in gene-edited animals to aid data mining and integrative in-depth analyses. However, existing databases are currently insufficient to meet this need. Here, we present the Variation Database of Gene-Edited animals (VDGE, https://ngdc.cncb.ac.cn/vdge), the first open-access repository to present genomic variations and annotations in gene-edited animals, with a particular focus on larger animals such as monkeys. At present, VDGE houses 151 on-target mutations from 210 samples, and 115,710 variations identified from 107 gene-edited and wild-type animal trios through unified and standardized analysis and concurrently provides comprehensive annotation details for each variation, thus facilitating the assessment of their functional consequences and promoting mechanistic studies and practical applications for gene-edited animals.
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Affiliation(s)
- Wenwen Shi
- State Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
| | - Enhui Jin
- National Genomics Data Center, China National Center for Bioinformation, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
- Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
- University of Chinese Academy of Sciences, No.1 Yanqihu East Rd, Huairou District, Beijing 101408, China
| | - Lu Fang
- Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
| | - Yanling Sun
- National Genomics Data Center, China National Center for Bioinformation, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
- Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
- Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Cambridge Street, Houston, TX 77030, USA
- Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Cambridge Street, Houston, TX 77030, USA
| | - Zhuojing Fan
- National Genomics Data Center, China National Center for Bioinformation, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
- Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
| | - Junwei Zhu
- National Genomics Data Center, China National Center for Bioinformation, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
- Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
| | - Chengzhi Liang
- University of Chinese Academy of Sciences, No.1 Yanqihu East Rd, Huairou District, Beijing 101408, China
- Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
| | - Ya-Ping Zhang
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, No.17 Longxin Road, Panlong District, Kunming 650201, China
| | - Yong Q Zhang
- State Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
- University of Chinese Academy of Sciences, No.1 Yanqihu East Rd, Huairou District, Beijing 101408, China
- School of Life Sciences, Hubei University, 368 Youyi Avenue, Wuchang District, Wuhan 430062, China
| | - Guo-Dong Wang
- University of Chinese Academy of Sciences, No.1 Yanqihu East Rd, Huairou District, Beijing 101408, China
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, No.17 Longxin Road, Panlong District, Kunming 650201, China
| | - Wenming Zhao
- National Genomics Data Center, China National Center for Bioinformation, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
- Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China
- University of Chinese Academy of Sciences, No.1 Yanqihu East Rd, Huairou District, Beijing 101408, China
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Pi W, Feng G, Liu M, Nie C, Chen C, Wang J, Wang L, Wan P, Liu C, Liu Y, Zhou P. Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes. Int J Mol Sci 2024; 25:9145. [PMID: 39273092 PMCID: PMC11395511 DOI: 10.3390/ijms25179145] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2024] [Revised: 08/18/2024] [Accepted: 08/20/2024] [Indexed: 09/15/2024] Open
Abstract
The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.
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Affiliation(s)
- Wenhui Pi
- College of Animal Science and Technology, Shihezi University, Shihezi 832003, China
| | - Guangyu Feng
- College of Animal Science and Technology, Shihezi University, Shihezi 832003, China
| | - Minghui Liu
- College of Animal Science and Technology, Shihezi University, Shihezi 832003, China
| | - Cunxi Nie
- College of Animal Science and Technology, Shihezi University, Shihezi 832003, China
| | - Cheng Chen
- College of Animal Science and Technology, Shihezi University, Shihezi 832003, China
| | - Jingjing Wang
- State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China
| | - Limin Wang
- State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China
| | - Pengcheng Wan
- State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China
| | - Changbin Liu
- State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China
| | - Yi Liu
- State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China
| | - Ping Zhou
- State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China
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6
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Yao YG, Lu L, Ni RJ, Bi R, Chen C, Chen JQ, Fuchs E, Gorbatyuk M, Lei H, Li H, Liu C, Lv LB, Tsukiyama-Kohara K, Kohara M, Perez-Cruz C, Rainer G, Shan BC, Shen F, Tang AZ, Wang J, Xia W, Xia X, Xu L, Yu D, Zhang F, Zheng P, Zheng YT, Zhou J, Zhou JN. Study of tree shrew biology and models: A booming and prosperous field for biomedical research. Zool Res 2024; 45:877-909. [PMID: 39004865 PMCID: PMC11298672 DOI: 10.24272/j.issn.2095-8137.2024.199] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2024] [Accepted: 07/03/2024] [Indexed: 07/16/2024] Open
Abstract
The tree shrew ( Tupaia belangeri) has long been proposed as a suitable alternative to non-human primates (NHPs) in biomedical and laboratory research due to its close evolutionary relationship with primates. In recent years, significant advances have facilitated tree shrew studies, including the determination of the tree shrew genome, genetic manipulation using spermatogonial stem cells, viral vector-mediated gene delivery, and mapping of the tree shrew brain atlas. However, the limited availability of tree shrews globally remains a substantial challenge in the field. Additionally, determining the key questions best answered using tree shrews constitutes another difficulty. Tree shrew models have historically been used to study hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, myopia, and psychosocial stress-induced depression, with more recent studies focusing on developing animal models for infectious and neurodegenerative diseases. Despite these efforts, the impact of tree shrew models has not yet matched that of rodent or NHP models in biomedical research. This review summarizes the prominent advancements in tree shrew research and reflects on the key biological questions addressed using this model. We emphasize that intensive dedication and robust international collaboration are essential for achieving breakthroughs in tree shrew studies. The use of tree shrews as a unique resource is expected to gain considerable attention with the application of advanced techniques and the development of viable animal models, meeting the increasing demands of life science and biomedical research.
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Affiliation(s)
- Yong-Gang Yao
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
- KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China. E-mail:
| | - Li Lu
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
- KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China
| | - Rong-Jun Ni
- Mental Health Center and Psychiatric Laboratory, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
- Sichuan Clinical Medical Research Center for Mental Disorders, Chengdu, Sichuan 610044, China
| | - Rui Bi
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
- KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China
| | - Ceshi Chen
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
| | - Jia-Qi Chen
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
| | - Eberhard Fuchs
- German Primate Center, Leibniz Institute of Primate Research, Göttingen 37077, Germany
| | - Marina Gorbatyuk
- Department of Optometry and Vision Science, School of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Hao Lei
- State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China
- Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China
| | - Hongli Li
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
- KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
| | - Chunyu Liu
- Soong Ching Ling Institute of Maternity and Child Health, International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China
| | - Long-Bao Lv
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
| | - Kyoko Tsukiyama-Kohara
- Transboundary Animal Diseases Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima-city, Kagoshima 890-8580, Japan
| | - Michinori Kohara
- Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan
| | | | - Gregor Rainer
- Department of Medicine, University of Fribourg, Fribourg CH-1700, Switzerland
| | - Bao-Ci Shan
- Beijing Engineering Research Center of Radiographic Techniques and Equipment, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China
- School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Fang Shen
- CAS Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing 100101, China
- Department of Psychology, University of Chinese Academy of Sciences, Beijing 100101, China
| | - An-Zhou Tang
- Department of Otorhinolaryngology Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530000, China
- Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor (Guangxi Medical University), Ministry of Education, Nanning, Guangxi 530000, China
| | - Jing Wang
- Department of Neurobiology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, Jiangsu 211166, China
| | - Wei Xia
- Department of Otorhinolaryngology Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530000, China
- Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor (Guangxi Medical University), Ministry of Education, Nanning, Guangxi 530000, China
| | - Xueshan Xia
- School of Public Health, Kunming Medical University, Kunming, Yunnan 650500, China
| | - Ling Xu
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
- KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China
| | - Dandan Yu
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
- KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China
| | - Feng Zhang
- Soong Ching Ling Institute of Maternity and Child Health, International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China
| | - Ping Zheng
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
- KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China
| | - Yong-Tang Zheng
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), and National Resource Center for Non-Human Primates, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650107, China
- KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
- Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China
| | - Jumin Zhou
- Key Laboratory of Genetic Evolution and Animal Models, Yunnan Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650204, China
| | - Jiang-Ning Zhou
- CAS Key Laboratory of Brain Function and Diseases, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China
- Institute of Brain Science, First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China
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7
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Mishra S, Nayak S, Tuteja N, Poosapati S, Swain DM, Sahoo RK. CRISPR/Cas-Mediated Genome Engineering in Plants: Application and Prospectives. PLANTS (BASEL, SWITZERLAND) 2024; 13:1884. [PMID: 39065411 PMCID: PMC11279650 DOI: 10.3390/plants13141884] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/22/2024] [Revised: 06/21/2024] [Accepted: 06/28/2024] [Indexed: 07/28/2024]
Abstract
Genetic engineering has become an essential element in developing climate-resilient crops and environmentally sustainable solutions to respond to the increasing need for global food security. Genome editing using CRISPR/Cas [Clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas)] technology is being applied to a variety of organisms, including plants. This technique has become popular because of its high specificity, effectiveness, and low production cost. Therefore, this technology has the potential to revolutionize agriculture and contribute to global food security. Over the past few years, increasing efforts have been seen in its application in developing higher-yielding, nutrition-rich, disease-resistant, and stress-tolerant "crops", fruits, and vegetables. Cas proteins such as Cas9, Cas12, Cas13, and Cas14, among others, have distinct architectures and have been used to create new genetic tools that improve features that are important for agriculture. The versatility of Cas has accelerated genomic analysis and facilitated the use of CRISPR/Cas to manipulate and alter nucleic acid sequences in cells of different organisms. This review provides the evolution of CRISPR technology exploring its mechanisms and contrasting it with traditional breeding and transgenic approaches to improve different aspects of stress tolerance. We have also discussed the CRISPR/Cas system and explored three Cas proteins that are currently known to exist: Cas12, Cas13, and Cas14 and their potential to generate foreign-DNA-free or non-transgenic crops that could be easily regulated for commercialization in most countries.
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Affiliation(s)
- Swetaleena Mishra
- Department of Biotechnology, Centurion University of Technology and Management, Bhubaneswar 752050, India;
| | - Subhendu Nayak
- Vidya USA Corporation, Otis Stone Hunter Road, Bunnell, FL 32100, USA;
| | - Narendra Tuteja
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi 110067, India;
| | - Sowmya Poosapati
- Plant Biology Laboratory, Salk Institute for Biological Studies, San Diego, CA 92037, USA
| | - Durga Madhab Swain
- MU Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA
| | - Ranjan Kumar Sahoo
- Department of Biotechnology, Centurion University of Technology and Management, Bhubaneswar 752050, India;
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Mishu MA, Nath SK, Sohidullah M, Hossain MT. Advancement of animal and poultry nutrition: Harnessing the power of CRISPR-Cas genome editing technology. J Adv Vet Anim Res 2024; 11:483-493. [PMID: 39101073 PMCID: PMC11296187 DOI: 10.5455/javar.2024.k798] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2024] [Revised: 05/31/2024] [Accepted: 06/02/2024] [Indexed: 08/06/2024] Open
Abstract
CRISPR-associated proteins and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) technology has emerged as a groundbreaking advancement in animal and poultry nutrition to improve feed conversion efficiency, enhance disease resistance, and improve the nutritional quality of animal products. Despite significant advancements, there is a research gap in the systematic understanding and comprehensive use of the CRISPR-Cas method in animal and poultry nutrition. The purpose of this study is to elucidate the latest advancements in animal and poultry nutrition through CRISPR-Cas genome editing technology, focusing on gene manipulation in metabolism, immunity, and growth. Following preferred reporting items in meta-analysis and systematic reviews guidelines, we conducted a systematic search using several databases, including Scopus, PubMed, and Web of Science, until May 2024, and finally, we included a total of 108 articles in this study. This article explores the use of the CRISPR-Cas system in the advancement of feed additives like probiotics and enzymes, which could reduce the use of antibiotics in animal production. Furthermore, the article discusses ethical and regulatory issues related to gene editing in animal and poultry nutrition, including concerns about animal welfare, food safety, and environmental impacts. Overall, the CRISPR-Cas system holds substantial promise to overcome the challenges in modern animal agriculture. By enriching the nutritional quality of animal products, increasing disease resistance, and improving feed efficiency, it offers sustainable and cost-effective solutions that can revolutionize animal and poultry nutrition.
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Affiliation(s)
- Mahbuba Akther Mishu
- Department of Agricultural Finance, Co-operatives and Banking, Khulna Agricultural University, Khulna, Bangladesh
| | - Sabuj Kanti Nath
- Department of Animal Nutrition, Khulna Agricultural University, Khulna, Bangladesh
| | - M. Sohidullah
- Department of Microbiology and Public Health, Khulna Agricultural University, Khulna, Bangladesh
| | - Md. Taslim Hossain
- Department of Animal Nutrition, Khulna Agricultural University, Khulna, Bangladesh
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9
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Dolezel M, Lang A, Greiter A, Miklau M, Eckerstorfer M, Heissenberger A, Willée E, Züghart W. Challenges for the Post-Market Environmental Monitoring in the European Union Imposed by Novel Applications of Genetically Modified and Genome-Edited Organisms. BIOTECH 2024; 13:14. [PMID: 38804296 PMCID: PMC11130885 DOI: 10.3390/biotech13020014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Revised: 05/03/2024] [Accepted: 05/08/2024] [Indexed: 05/29/2024] Open
Abstract
Information on the state of the environment is important to achieve the objectives of the European Green Deal, including the EU's Biodiversity Strategy for 2030. The existing regulatory provisions for genetically modified organisms (GMOs) foresee an obligatory post-market environmental monitoring (PMEM) of potential adverse effects upon release into the environment. So far, GMO monitoring activities have focused on genetically modified crops. With the advent of new genomic techniques (NGT), novel GMO applications are being developed and may be released into a range of different, non-agricultural environments with potential implications for ecosystems and biodiversity. This challenges the current monitoring concepts and requires adaptation of existing monitoring programs to meet monitoring requirements. While the incorporation of existing biodiversity monitoring programs into GMO monitoring at the national level is important, additional monitoring activities will also be required. Using case examples, we highlight that monitoring requirements for novel GMO applications differ from those of GM crop plants previously authorized for commercial use in the European Union.
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Affiliation(s)
- Marion Dolezel
- Land Use & Biosafety Unit, Umweltbundesamt–Environment Agency Austria (EAA), Spittelauer Laende 5, 1090 Vienna, Austria; (A.G.); (M.M.); (M.E.); (A.H.)
| | - Andreas Lang
- Büro Lang, Hoernlehof, Gresgen 108, 79669 Zell im Wiesental, Germany;
- Research Group Environmental Geosciences, Department of Environmental Sciences, University of Basel, Bernoullistr. 30, 4056 Basel, Switzerland
| | - Anita Greiter
- Land Use & Biosafety Unit, Umweltbundesamt–Environment Agency Austria (EAA), Spittelauer Laende 5, 1090 Vienna, Austria; (A.G.); (M.M.); (M.E.); (A.H.)
| | - Marianne Miklau
- Land Use & Biosafety Unit, Umweltbundesamt–Environment Agency Austria (EAA), Spittelauer Laende 5, 1090 Vienna, Austria; (A.G.); (M.M.); (M.E.); (A.H.)
| | - Michael Eckerstorfer
- Land Use & Biosafety Unit, Umweltbundesamt–Environment Agency Austria (EAA), Spittelauer Laende 5, 1090 Vienna, Austria; (A.G.); (M.M.); (M.E.); (A.H.)
| | - Andreas Heissenberger
- Land Use & Biosafety Unit, Umweltbundesamt–Environment Agency Austria (EAA), Spittelauer Laende 5, 1090 Vienna, Austria; (A.G.); (M.M.); (M.E.); (A.H.)
| | - Eva Willée
- Division of Terrestrial Monitoring, Federal Agency for Nature Conservation (BfN), Konstantinstr. 110, 53179 Bonn, Germany (W.Z.)
| | - Wiebke Züghart
- Division of Terrestrial Monitoring, Federal Agency for Nature Conservation (BfN), Konstantinstr. 110, 53179 Bonn, Germany (W.Z.)
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10
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Yuan YG, Liu SZ, Farhab M, Lv MY, Zhang T, Cao SX. Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock. Funct Integr Genomics 2024; 24:81. [PMID: 38709433 DOI: 10.1007/s10142-024-01364-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2024] [Revised: 04/29/2024] [Accepted: 05/01/2024] [Indexed: 05/07/2024]
Abstract
One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.
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Affiliation(s)
- Yu-Guo Yuan
- College of Veterinary Medicine/Key Laboratory of Animal Genetic Engineering, Yangzhou University, Yangzhou, 225009, Jiangsu, China.
- Jiangsu Co-Innovation Center of Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China.
| | - Song-Zi Liu
- College of Veterinary Medicine/Key Laboratory of Animal Genetic Engineering, Yangzhou University, Yangzhou, 225009, Jiangsu, China
- Jiangsu Co-Innovation Center of Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Muhammad Farhab
- College of Veterinary Medicine/Key Laboratory of Animal Genetic Engineering, Yangzhou University, Yangzhou, 225009, Jiangsu, China
- Jiangsu Co-Innovation Center of Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Mei-Yun Lv
- College of Veterinary Medicine/Key Laboratory of Animal Genetic Engineering, Yangzhou University, Yangzhou, 225009, Jiangsu, China
- Jiangsu Co-Innovation Center of Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Ting Zhang
- School of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry, Jurong, 212499, China
| | - Shao-Xiao Cao
- Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China
- Jiangsu Provincial Engineering Research Center for Precision animal Breeding, Nanjing, 210014, China
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11
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Wang X, Liu TX, Zhang Y, Xu LW, Yuan SL, Cui AL, Guo WW, Wang YF, Yang SM, Zhao JG. Genetically modified pigs: Emerging animal models for hereditary hearing loss. Zool Res 2024; 45:284-291. [PMID: 38485498 PMCID: PMC11017082 DOI: 10.24272/j.issn.2095-8137.2023.231] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Accepted: 12/05/2023] [Indexed: 03/19/2024] Open
Abstract
Hereditary hearing loss (HHL), a genetic disorder that impairs auditory function, significantly affects quality of life and incurs substantial economic losses for society. To investigate the underlying causes of HHL and evaluate therapeutic outcomes, appropriate animal models are necessary. Pigs have been extensively used as valuable large animal models in biomedical research. In this review, we highlight the advantages of pig models in terms of ear anatomy, inner ear morphology, and electrophysiological characteristics, as well as recent advancements in the development of distinct genetically modified porcine models of hearing loss. Additionally, we discuss the prospects, challenges, and recommendations regarding the use pig models in HHL research. Overall, this review provides insights and perspectives for future studies on HHL using porcine models.
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Affiliation(s)
- Xiao Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
- State Key Laboratory of Animal Biotech Breeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Tian-Xia Liu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Ying Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Liang-Wei Xu
- Department of Otolaryngology-Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Beijing 100853, China
| | - Shuo-Long Yuan
- Department of Otolaryngology-Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Beijing 100853, China
| | - A-Long Cui
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230022, China
| | - Wei-Wei Guo
- Department of Otolaryngology-Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Beijing 100853, China
| | - Yan-Fang Wang
- State Key Laboratory of Animal Biotech Breeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Shi-Ming Yang
- Department of Otolaryngology-Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Beijing 100853, China. E-mail:
| | - Jian-Guo Zhao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China. E-mail:
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12
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Han Y, Zhou J, Zhang R, Liang Y, Lai L, Li Z. Genome-edited rabbits: Unleashing the potential of a promising experimental animal model across diverse diseases. Zool Res 2024; 45:253-262. [PMID: 38287906 PMCID: PMC11017087 DOI: 10.24272/j.issn.2095-8137.2023.201] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2023] [Accepted: 12/05/2023] [Indexed: 01/31/2024] Open
Abstract
Animal models are extensively used in all aspects of biomedical research, with substantial contributions to our understanding of diseases, the development of pharmaceuticals, and the exploration of gene functions. The field of genome modification in rabbits has progressed slowly. However, recent advancements, particularly in CRISPR/Cas9-related technologies, have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases, including cardiovascular disorders, immunodeficiencies, aging-related ailments, neurological diseases, and ophthalmic pathologies. These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice. This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine, underscoring their impact and future potential in translational medicine.
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Affiliation(s)
- Yang Han
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun, Jilin 130062, China
| | - Jiale Zhou
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun, Jilin 130062, China
| | - Renquan Zhang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun, Jilin 130062, China
| | - Yuru Liang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun, Jilin 130062, China
| | - Liangxue Lai
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun, Jilin 130062, China
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China
- Guangzhou Regenerative Medicine and Health Guang Dong Laboratory (GRMH-GDL), Guangzhou, Guangdong 510005, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China. E-mail:
| | - Zhanjun Li
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun, Jilin 130062, China. E-mail:
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13
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Wu J, Kang Y, Luo X, Dai S, Shi Y, Li Z, Tang Z, Chen Z, Zhu R, Yang P, Li Z, Wang H, Chen X, Zhao Z, Ji W, Niu Y. Long-term in vivo chimeric cells tracking in non-human primate. Protein Cell 2024; 15:207-222. [PMID: 37758041 PMCID: PMC10903985 DOI: 10.1093/procel/pwad049] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2023] [Accepted: 08/31/2023] [Indexed: 10/03/2023] Open
Abstract
Non-human primates (NHPs) are increasingly used in preclinical trials to test the safety and efficacy of biotechnology therapies. Nonetheless, given the ethical issues and costs associated with this model, it would be highly advantageous to use NHP cellular models in clinical studies. However, developing and maintaining the naïve state of primate pluripotent stem cells (PSCs) remains difficult as does in vivo detection of PSCs, thus limiting biotechnology application in the cynomolgus monkey. Here, we report a chemically defined, xeno-free culture system for culturing and deriving monkey PSCs in vitro. The cells display global gene expression and genome-wide hypomethylation patterns distinct from monkey-primed cells. We also found expression of signaling pathways components that may increase the potential for chimera formation. Crucially for biomedical applications, we were also able to integrate bioluminescent reporter genes into monkey PSCs and track them in chimeric embryos in vivo and in vitro. The engineered cells retained embryonic and extra-embryonic developmental potential. Meanwhile, we generated a chimeric monkey carrying bioluminescent cells, which were able to track chimeric cells for more than 2 years in living animals. Our study could have broad utility in primate stem cell engineering and in utilizing chimeric monkey models for clinical studies.
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Affiliation(s)
- Junmo Wu
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Yu Kang
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Xiang Luo
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Shaoxing Dai
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Yuxi Shi
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Zhuoyao Li
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Zengli Tang
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Zhenzhen Chen
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Ran Zhu
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Pengpeng Yang
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Zifan Li
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Hong Wang
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Xinglong Chen
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Ziyi Zhao
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Weizhi Ji
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
| | - Yuyu Niu
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming 650500, China
- Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China
- Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
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14
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Pacesa M, Pelea O, Jinek M. Past, present, and future of CRISPR genome editing technologies. Cell 2024; 187:1076-1100. [PMID: 38428389 DOI: 10.1016/j.cell.2024.01.042] [Citation(s) in RCA: 81] [Impact Index Per Article: 81.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2023] [Revised: 01/23/2024] [Accepted: 01/26/2024] [Indexed: 03/03/2024]
Abstract
Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes and treat the underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency and easy programmability, stand at the forefront of this revolution. In this Review, we discuss the current state of CRISPR gene editing technologies in both research and therapy, highlighting limitations that constrain them and the technological innovations that have been developed in recent years to address them. Additionally, we examine and summarize the current landscape of gene editing applications in the context of human health and therapeutics. Finally, we outline potential future developments that could shape gene editing technologies and their applications in the coming years.
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Affiliation(s)
- Martin Pacesa
- Laboratory of Protein Design and Immunoengineering, École Polytechnique Fédérale de Lausanne and Swiss Institute of Bioinformatics, Station 19, CH-1015 Lausanne, Switzerland
| | - Oana Pelea
- Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Martin Jinek
- Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
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15
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Duo T, Liu X, Mo D, Bian Y, Cai S, Wang M, Li R, Zhu Q, Tong X, Liang Z, Jiang W, Chen S, Chen Y, He Z. Single-base editing in IGF2 improves meat production and intramuscular fat deposition in Liang Guang Small Spotted pigs. J Anim Sci Biotechnol 2023; 14:141. [PMID: 37919760 PMCID: PMC10621156 DOI: 10.1186/s40104-023-00930-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2023] [Accepted: 08/06/2023] [Indexed: 11/04/2023] Open
Abstract
BACKGROUND Chinese indigenous pigs are popular with consumers for their juiciness, flavour and meat quality, but they have lower meat production. Insulin-like growth factor 2 (IGF2) is a maternally imprinted growth factor that promotes skeletal muscle growth by regulating cell proliferation and differentiation. A single nucleotide polymorphism (SNP) within intron 3 of porcine IGF2 disrupts a binding site for the repressor, zinc finger BED-type containing 6 (ZBED6), leading to up-regulation of IGF2 and causing major effects on muscle growth, heart size, and backfat thickness. This favorable mutation is common in Western commercial pig populations, but absent in most Chinese indigenous pig breeds. To improve meat production of Chinese indigenous pigs, we used cytosine base editor 3 (CBE3) to introduce IGF2-intron3-C3071T mutation into porcine embryonic fibroblasts (PEFs) isolated from a male Liang Guang Small Spotted pig (LGSS), and single-cell clones harboring the desired mutation were selected for somatic cell nuclear transfer (SCNT) to generate the founder line of IGF2T/T pigs. RESULTS We found the heterozygous progeny IGF2C/T pigs exhibited enhanced expression of IGF2, increased lean meat by 18%-36%, enlarged loin muscle area by 3%-17%, improved intramuscular fat (IMF) content by 18%-39%, marbling score by 0.75-1, meat color score by 0.53-1.25, and reduced backfat thickness by 5%-16%. The enhanced accumulation of intramuscular fat in IGF2C/T pigs was identified to be regulated by the PI3K-AKT/AMPK pathway, which activated SREBP1 to promote adipogenesis. CONCLUSIONS We demonstrated the introduction of IGF2-intron3-C3071T in Chinese LGSS can improve both meat production and quality, and first identified the regulation of IMF deposition by IGF2 through SREBP1 via the PI3K-AKT/AMPK signaling pathways. Our study provides a further understanding of the biological functions of IGF2 and an example for improving porcine economic traits through precise base editing.
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Affiliation(s)
- Tianqi Duo
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Xiaohong Liu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Delin Mo
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Yu Bian
- Department of Pharmacology (the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, 150081, China
| | - Shufang Cai
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
- Hunan Provincial Key Laboratory for Genetic Improvement of Domestic Animal, College of Animal Science and Technology, Hunan Agricultural University, Changsha, 410128, China
| | - Min Wang
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Ruiqiang Li
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Qi Zhu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Xian Tong
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Ziyun Liang
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Weilun Jiang
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Shiyi Chen
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China
| | - Yaosheng Chen
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China.
| | - Zuyong He
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, Guangdong, China.
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Wang JH, Wu SJ, Li Y, Zhao Y, Liu ZM, Deng SL, Lian ZX. Improving the Efficiency of Precise Genome Editing with CRISPR/Cas9 to Generate Goats Overexpressing Human Butyrylcholinesterase. Cells 2023; 12:1818. [PMID: 37508483 PMCID: PMC10378061 DOI: 10.3390/cells12141818] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Revised: 07/02/2023] [Accepted: 07/04/2023] [Indexed: 07/30/2023] Open
Abstract
The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It is the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated genome-edited primary goat fetal fibroblast cells (GFFs). Improving the double-strand break (DSB) efficiency of Cas9 in primary cells would improve the homologous repair (HR) efficiency. The low efficiency of HR remains a major hurdle in CRISPR/Cas9-mediated precise genome editing, increasing the work required to screen the genome-edited primary cell clones. In this study, we modified several essential parameters that affect the efficiency of the CRISPR/Cas9-mediated knock-in GFF cloning system, including establishing a high-efficiency transfection system for primary cells via nucleofection and optimizing homology arm (HA) length during HR. Here, we specifically inserted a recombinant human butyrylcholinesterase gene (rhBChE) into the goat fibroblast growth factor (FGF)-5 locus through the CRISPR/Cas9 system, thereby achieving simultaneous rhBChE insertion and FGF5 knock-out. First, this study introduced the Cas9, FGF5 knock-out small guide RNA, and rhBChE knock-in donors into GFFs by electroporation and obtained positive cell clones without off-target effects. Then, we demonstrated the expression of rhBChE in GFF clones and verified its function. Finally, we obtained a CRISPR/Cas9-mediated rhBChE-overexpression goat.
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Affiliation(s)
- Jia-Hao Wang
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
- Department of Biomedical Engineering, College of Future Technology, Peking University, Beijing 100871, China
| | - Su-Jun Wu
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
| | - Yan Li
- Laboratory Animal Center of the Academy of Military Medical Sciences, Beijing 100071, China;
| | - Yue Zhao
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
| | - Zhi-Mei Liu
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
| | - Shou-Long Deng
- NHC Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing 100021, China
| | - Zheng-Xing Lian
- Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
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17
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Yang SP, Zhu XX, Qu ZX, Chen CY, Wu YB, Wu Y, Luo ZD, Wang XY, He CY, Fang JW, Wang LQ, Hong GL, Zheng ST, Zeng JM, Yan AF, Feng J, Liu L, Zhang XL, Zhang LG, Miao K, Tang DS. Production of MSTN knockout porcine cells using adenine base-editing-mediated exon skipping. In Vitro Cell Dev Biol Anim 2023:10.1007/s11626-023-00763-5. [PMID: 37099179 DOI: 10.1007/s11626-023-00763-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2023] [Accepted: 03/24/2023] [Indexed: 04/27/2023]
Abstract
Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9 and cytosine base editing (CBE) technologies, adenine base editing (ABE) shows better safety and accuracy in gene modification. However, because of the characteristics of gene sequences, the ABE system cannot be widely used in gene knockout. Alternative splicing of mRNA is an important biological mechanism in eukaryotes for the formation of proteins with different functional activities. The splicing apparatus recognizes conserved sequences of the 5' end splice donor and 3' end splice acceptor motifs of introns in pre-mRNA that can trigger exon skipping, leading to the production of new functional proteins, or causing gene inactivation through frameshift mutations. This study aimed to construct a MSTN knockout pig by inducing exon skipping with the aid of the ABE system to expand the application of the ABE system for the preparation of knockout pigs. In this study, first, we constructed ABEmaxAW and ABE8eV106W plasmid vectors and found that their editing efficiencies at the targets were at least sixfold and even 260-fold higher than that of ABEmaxAW by contrasting the editing efficiencies at the gene targets of endogenous CD163, IGF2, and MSTN in pigs. Subsequently, we used the ABE8eV106W system to realize adenine base (the base of the antisense strand is thymine) editing of the conserved splice donor sequence (5'-GT) of intron 2 of the porcine MSTN gene. A porcine single-cell clone carrying a homozygous mutation (5'-GC) in the conserved sequence (5'-GT) of the intron 2 splice donor of the MSTN gene was successfully generated after drug selection. Unfortunately, the MSTN gene was not expressed and, therefore, could not be characterized at this level. No detectable genomic off-target edits were identified by Sanger sequencing. In this study, we verified that the ABE8eV106W vector had higher editing efficiency and could expand the editing scope of ABE. Additionally, we successfully achieved the precise modification of the alternative splice acceptor of intron 2 of the porcine MSTN gene, which may provide a new strategy for gene knockout in pigs.
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Affiliation(s)
- Shuai-Peng Yang
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Sciences and Engineering, Foshan University, Foshan, 528225, China
| | - Xiang-Xing Zhu
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Sciences and Engineering, Foshan University, Foshan, 528225, China.
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China.
| | - Zi-Xiao Qu
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Sciences and Engineering, Foshan University, Foshan, 528225, China
| | - Cai-Yue Chen
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Yao-Bing Wu
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Yue Wu
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Zi-Dan Luo
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Xin-Yi Wang
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Chu-Yu He
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Jia-Wen Fang
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Ling-Qi Wang
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Guang-Long Hong
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Shu-Tao Zheng
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Jie-Mei Zeng
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Ai-Fen Yan
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Juan Feng
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Lian Liu
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Xiao-Li Zhang
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Li-Gang Zhang
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China
| | - Kai Miao
- Centre for Precision Medicine Research and Training, Faculty of Health Sciences, University of Macau, Macau SAR, China.
| | - Dong-Sheng Tang
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Sciences and Engineering, Foshan University, Foshan, 528225, China.
- Gene Editing Technology Center of Guangdong Province, School of Medicine, Foshan University, Foshan, 528225, China.
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18
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Bhokisham N, Laudermilch E, Traeger LL, Bonilla TD, Ruiz-Estevez M, Becker JR. CRISPR-Cas System: The Current and Emerging Translational Landscape. Cells 2023; 12:cells12081103. [PMID: 37190012 DOI: 10.3390/cells12081103] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2023] [Revised: 04/03/2023] [Accepted: 04/04/2023] [Indexed: 05/17/2023] Open
Abstract
CRISPR-Cas technology has rapidly changed life science research and human medicine. The ability to add, remove, or edit human DNA sequences has transformative potential for treating congenital and acquired human diseases. The timely maturation of the cell and gene therapy ecosystem and its seamless integration with CRISPR-Cas technologies has enabled the development of therapies that could potentially cure not only monogenic diseases such as sickle cell anemia and muscular dystrophy, but also complex heterogenous diseases such as cancer and diabetes. Here, we review the current landscape of clinical trials involving the use of various CRISPR-Cas systems as therapeutics for human diseases, discuss challenges, and explore new CRISPR-Cas-based tools such as base editing, prime editing, CRISPR-based transcriptional regulation, CRISPR-based epigenome editing, and RNA editing, each promising new functionality and broadening therapeutic potential. Finally, we discuss how the CRISPR-Cas system is being used to understand the biology of human diseases through the generation of large animal disease models used for preclinical testing of emerging therapeutics.
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Affiliation(s)
| | - Ethan Laudermilch
- Corporate Research Material Labs, 3M Center, 3M Company, Maplewood, MN 55144, USA
| | - Lindsay L Traeger
- Corporate Research Material Labs, 3M Center, 3M Company, Maplewood, MN 55144, USA
| | - Tonya D Bonilla
- Corporate Research Material Labs, 3M Center, 3M Company, Maplewood, MN 55144, USA
| | | | - Jordan R Becker
- Corporate Research Material Labs, 3M Center, 3M Company, Maplewood, MN 55144, USA
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19
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Tao M, Mao J, Bao Y, Liu F, Mai Y, Guan S, Luo S, Huang Y, Li Z, Zhong Y, Wei B, Pan J, Wang Q, Zheng L, Situ B. A Blood-Responsive AIE Bioprobe for the Ultrasensitive Detection and Assessment of Subarachnoid Hemorrhage. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2205435. [PMID: 36683187 PMCID: PMC10015902 DOI: 10.1002/advs.202205435] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/19/2022] [Revised: 12/19/2022] [Indexed: 06/17/2023]
Abstract
Subarachnoid hemorrhage (SAH) is a severe subtype of stroke caused by the rupturing of blood vessels in the brain. The ability to accurately assess the degree of bleeding in an SAH model is crucial for understanding the brain-damage mechanisms and developing therapeutic strategies. However, current methods are unable to monitor microbleeding owing to their limited sensitivities. Herein, a new bleeding assessment system using a bioprobe TTVP with aggregation-induced emission (AIE) characteristics is demonstrated. TTVP is a water-soluble, small-molecule probe that specifically interacts with blood. Taking advantage of its AIE characteristics, cell membranes affinity, and albumin-targeting ability, TTVP fluoresces in bleeding areas and detects the presence of blood with a high signal-to-noise (S/N) ratio. The degree of SAH bleeding in an endovascular perforation model is clearly evaluated based on the intensity of the fluorescence observed in the brain, which enables the ultrasensitive detection of mirco-bleeding in the SAH model in a manner that outperforms the current imaging strategies. This method serves as a promising tool for the sensitive analysis of the degree of bleeding in SAHs and other hemorrhagic diseases.
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Affiliation(s)
- Maliang Tao
- Department of Laboratory MedicineNanfang HospitalSouthern Medical UniversityGuangzhou510515China
- Guangdong Engineering and Technology Research Center for Rapid Diagnostic BiosensorsNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Jian Mao
- Department of NeurosurgeryNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Yun Bao
- Department of NeurosurgeryNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Fan Liu
- Department of NeurosurgeryNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Yiying Mai
- The Second Clinical CollegeSouthern Medical UniversityGuangzhou510515China
| | - Shujuan Guan
- Department of Laboratory MedicineNanfang HospitalSouthern Medical UniversityGuangzhou510515China
- Guangdong Engineering and Technology Research Center for Rapid Diagnostic BiosensorsNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Shihua Luo
- Center for Clinical Laboratory Diagnosis and Researchthe Affiliated Hospital of Youjiang Medical University for NationalitiesBaise533000China
| | - Yifang Huang
- Department of Clinical Laboratorythe First Affiliated Hospital of Guangxi Medical UniversityNanning530021China
| | - Zixiong Li
- Department of Laboratory MedicineNanfang HospitalSouthern Medical UniversityGuangzhou510515China
- Guangdong Engineering and Technology Research Center for Rapid Diagnostic BiosensorsNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Yuan Zhong
- Department of Laboratory MedicineNanfang HospitalSouthern Medical UniversityGuangzhou510515China
- Guangdong Engineering and Technology Research Center for Rapid Diagnostic BiosensorsNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Binbin Wei
- Department of Laboratory MedicineNanfang HospitalSouthern Medical UniversityGuangzhou510515China
- Guangdong Engineering and Technology Research Center for Rapid Diagnostic BiosensorsNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Jun Pan
- Department of NeurosurgeryNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Qian Wang
- Department of Laboratory MedicineNanfang HospitalSouthern Medical UniversityGuangzhou510515China
- Guangdong Engineering and Technology Research Center for Rapid Diagnostic BiosensorsNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Lei Zheng
- Department of Laboratory MedicineNanfang HospitalSouthern Medical UniversityGuangzhou510515China
- Guangdong Engineering and Technology Research Center for Rapid Diagnostic BiosensorsNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Bo Situ
- Department of Laboratory MedicineNanfang HospitalSouthern Medical UniversityGuangzhou510515China
- Guangdong Engineering and Technology Research Center for Rapid Diagnostic BiosensorsNanfang HospitalSouthern Medical UniversityGuangzhou510515China
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20
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Cas9-mediated replacement of expanded CAG repeats in a pig model of Huntington's disease. Nat Biomed Eng 2023; 7:629-646. [PMID: 36797418 DOI: 10.1038/s41551-023-01007-3] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Accepted: 01/20/2023] [Indexed: 02/18/2023]
Abstract
The monogenic nature of Huntington's disease (HD) and other neurodegenerative diseases caused by the expansion of glutamine-encoding CAG repeats makes them particularly amenable to gene therapy. Here we show the feasibility of replacing expanded CAG repeats in the mutant HTT allele with a normal CAG repeat in genetically engineered pigs mimicking the selective neurodegeneration seen in patients with HD. A single intracranial or intravenous injection of adeno-associated virus encoding for Cas9, a single-guide RNA targeting the HTT gene, and donor DNA containing the normal CAG repeat led to the depletion of mutant HTT in the animals and to substantial reductions in the dysregulated expression and neurotoxicity of mutant HTT and in neurological symptoms. Our findings support the further translational development of virally delivered Cas9-based gene therapies for the treatment of genetic neurodegenerative diseases.
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21
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Lee HJ, Kim HJ, Lee SJ. Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3'-Truncated sgRNA. CRISPR J 2023; 6:52-61. [PMID: 36576897 PMCID: PMC9942177 DOI: 10.1089/crispr.2022.0071] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The CRISPR-Cas system has been used as a convenient tool for genome editing because the nuclease that cuts the target DNA and the guide RNA that recognizes the target are separated into modules. Cas12f1, which has a smaller size than that of other Cas nucleases, is easily loaded into vectors and is emerging as a new genome editing tool. In this study, AsCas12f1 was used to negatively select only Escherichia coli cells obtained by oligonucleotide-directed genome editing. Although double-, triple-, and quadruple-base substitutions were accurately and efficiently performed in the genome, the performance of single-base editing was poor. To resolve this limitation, we serially truncated the 3'-end of sgRNAs and determined the maximal truncation required to maintain the target DNA cleavage activity of Cas12f1. Negative selection of single-nucleotide-edited cells was efficiently performed with the maximally 3'-truncated sgRNA-Cas12f1 complex in vivo. Moreover, Sanger sequencing showed that the accuracy of single-nucleotide substitution, insertion, and deletion in the microbial genome was improved. These results demonstrated that a truncated sgRNA approach could be widely used for accurate CRISPR-mediated genome editing.
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Affiliation(s)
- Ho Joung Lee
- Department of Systems Biotechnology, Institute of Microbiomics, Chung-Ang University, Anseong, Republic of Korea
| | - Hyun Ju Kim
- Department of Systems Biotechnology, Institute of Microbiomics, Chung-Ang University, Anseong, Republic of Korea
| | - Sang Jun Lee
- Department of Systems Biotechnology, Institute of Microbiomics, Chung-Ang University, Anseong, Republic of Korea.,Address correspondence to: Sang Jun Lee, Department of Systems Biotechnology, Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea.
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22
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Haughan J, Ortved KF, Robinson MA. Administration and detection of gene therapy in horses: A systematic review. Drug Test Anal 2023; 15:143-162. [PMID: 36269665 DOI: 10.1002/dta.3394] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2022] [Revised: 09/30/2022] [Accepted: 10/19/2022] [Indexed: 11/09/2022]
Abstract
Gene therapy uses genetic modification of cells to produce a therapeutic effect. Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression. Gene therapy is currently prohibited in both human and equine athletes because of the potential to induce production of performance-enhancing proteins in the athlete's body, also referred to as "gene doping." Detection of gene doping is challenging and necessitates development of creative, novel analytical methods for doping control. Methods for detection of gene doping must be specific to and will vary depending on the type of gene therapy. The purpose of this paper is to present the results of a systematic review of gene editing, gene therapy, and detection of gene doping in horses. Based on the published literature, gene therapy has been administered to horses in a large number of experimental studies and a smaller number of clinical cases. Detection of gene therapy is possible using a combination of PCR and sequencing technologies. This summary can provide a basis for discussion of appropriate and inappropriate uses for gene therapy in horses by the veterinary community and guide expansion of methods to detect inappropriate uses by the regulatory community.
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Affiliation(s)
- Joanne Haughan
- Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania, USA
| | - Kyla F Ortved
- Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania, USA
| | - Mary A Robinson
- Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania, USA.,Pennsylvania Equine Toxicology & Research Center, West Chester University, West Chester, Pennsylvania, USA
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23
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Sheikh Beig Goharrizi MA, Ghodsi S, Memarjafari MR. Implications of CRISPR-Cas9 Genome Editing Methods in Atherosclerotic Cardiovascular Diseases. Curr Probl Cardiol 2023; 48:101603. [PMID: 36682390 DOI: 10.1016/j.cpcardiol.2023.101603] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2023] [Accepted: 01/17/2023] [Indexed: 01/21/2023]
Abstract
Today, new methods have been developed to treat or modify the natural course of cardiovascular diseases (CVDs), including atherosclerosis, by the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system. Genome-editing tools are CRISPR-related palindromic short iteration systems such as CRISPR-Cas9, a valuable technology for achieving somatic and germinal genomic manipulation in model cells and organisms for various applications, including the creation of deletion alleles. Mutations in genomic deoxyribonucleic acid and new genes' placement have emerged. Based on World Health Organization fact sheets, 17.9 million people die from CVDs each year, an estimated 32% of all deaths worldwide. 85% of all CVD deaths are due to acute coronary events and strokes. This review discusses the applications of CRISPR-Cas9 technology throughout atherosclerotic disease research and the prospects for future in vivo genome editing therapies. We also describe several limitations that must be considered to achieve the full scientific and therapeutic potential of cardiovascular genome editing in the treatment of atherosclerosis.
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Affiliation(s)
| | - Saeed Ghodsi
- Department of Cardiology, Sina Hospital, Tehran University of Medical Sciences, Tehran, Iran
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24
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Jin Q, Liu X, Zhuang Z, Huang J, Gou S, Shi H, Zhao Y, Ouyang Z, Liu Z, Li L, Mao J, Ge W, Chen F, Yu M, Guan Y, Ye Y, Tang C, Huang R, Wang K, Lai L. Doxycycline-dependent Cas9-expressing pig resources for conditional in vivo gene nullification and activation. Genome Biol 2023; 24:8. [PMID: 36650523 PMCID: PMC9843877 DOI: 10.1186/s13059-023-02851-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2022] [Accepted: 01/06/2023] [Indexed: 01/18/2023] Open
Abstract
BACKGROUND CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals. RESULTS Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53-sgRNA, LKB1-sgRNA, and mutant human KRAS gene into the adult pancreases. CONCLUSIONS Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.
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Affiliation(s)
- Qin Jin
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
| | - Xiaoyi Liu
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Zhenpeng Zhuang
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Jiayuan Huang
- Guangdong Provincial Key Laboratory of Laboratory Animals, Guangdong Laboratory Animals Monitoring Institute, Guangzhou, 510633, China
| | - Shixue Gou
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
| | - Hui Shi
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
| | - Yu Zhao
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, 529020, China
| | - Zhen Ouyang
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, 529020, China
| | - Zhaoming Liu
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, 529020, China
| | - Lei Li
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Junjie Mao
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
| | - Weikai Ge
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, 529020, China
| | - Fangbing Chen
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, 529020, China
| | - Manya Yu
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
| | - Yezhi Guan
- Guangdong Provincial Key Laboratory of Laboratory Animals, Guangdong Laboratory Animals Monitoring Institute, Guangzhou, 510633, China
| | - Yinghua Ye
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China
| | - Chengcheng Tang
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, 529020, China
| | - Ren Huang
- Guangdong Provincial Key Laboratory of Laboratory Animals, Guangdong Laboratory Animals Monitoring Institute, Guangzhou, 510633, China
| | - Kepin Wang
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China.
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China.
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, 529020, China.
| | - Liangxue Lai
- China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China.
- Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, 572000, China.
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, 529020, China.
- Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China.
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25
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Wani AK, Akhtar N, Singh R, Prakash A, Raza SHA, Cavalu S, Chopra C, Madkour M, Elolimy A, Hashem NM. Genome centric engineering using ZFNs, TALENs and CRISPR-Cas9 systems for trait improvement and disease control in Animals. Vet Res Commun 2023; 47:1-16. [PMID: 35781172 DOI: 10.1007/s11259-022-09967-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Accepted: 06/24/2022] [Indexed: 01/27/2023]
Abstract
Livestock is an essential life commodity in modern agriculture involving breeding and maintenance. The farming practices have evolved mainly over the last century for commercial outputs, animal welfare, environment friendliness, and public health. Modifying genetic makeup of livestock has been proposed as an effective tool to create farmed animals with characteristics meeting modern farming system goals. The first technique used to produce transgenic farmed animals resulted in random transgene insertion and a low gene transfection rate. Therefore, genome manipulation technologies have been developed to enable efficient gene targeting with a higher accuracy and gene stability. Genome editing (GE) with engineered nucleases-Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) regulates the targeted genetic alterations to facilitate multiple genomic modifications through protein-DNA binding. The application of genome editors indicates usefulness in reproduction, animal models, transgenic animals, and cell lines. Recently, CRISPR/Cas system, an RNA-dependent genome editing tool (GET), is considered one of the most advanced and precise GE techniques for on-target modifications in the mammalian genome by mediating knock-in (KI) and knock-out (KO) of several genes. Lately, CRISPR/Cas9 tool has become the method of choice for genome alterations in livestock species due to its efficiency and specificity. The aim of this review is to discuss the evolution of engineered nucleases and GETs as a powerful tool for genome manipulation with special emphasis on its applications in improving economic traits and conferring resistance to infectious diseases of animals used for food production, by highlighting the recent trends for maintaining sustainable livestock production.
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Affiliation(s)
- Atif Khurshid Wani
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Nahid Akhtar
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Reena Singh
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Ajit Prakash
- Department of Biochemistry and Biophysics, University of North Carolina, 120 Mason Farm Road, CB# 7260, 3093 Genetic Medicine, Chapel Hill, NC, 27599-2760, USA
| | - Sayed Haidar Abbas Raza
- College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
| | - Simona Cavalu
- Faculty of Medicine and Pharmacy, University of Oradea, P -ta 1Decembrie 10, 410073, Oradea, Romania
| | - Chirag Chopra
- School of Bioengineering and Biosciences, Lovely Professional University, Punjab, 144411, India
| | - Mahmoud Madkour
- Animal Production Department, National Research Centre, Dokki, Giza, 12622, Egypt
| | - Ahmed Elolimy
- Animal Production Department, National Research Centre, Dokki, Giza, 12622, Egypt
| | - Nesrein M Hashem
- Department of Animal and Fish Production, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, 21545, Egypt.
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26
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Functional genomic tools for emerging model species. Trends Ecol Evol 2022; 37:1104-1115. [PMID: 35914975 DOI: 10.1016/j.tree.2022.07.004] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 07/08/2022] [Accepted: 07/11/2022] [Indexed: 01/12/2023]
Abstract
Most studies in the field of ecology and evolution aiming to connect genotype to phenotype rarely validate identified loci using functional tools. Recent developments in RNA interference (RNAi) and clustered regularly interspaced palindromic repeats (CRISPR)-Cas genome editing have dramatically increased the feasibility of functional validation. However, these methods come with specific challenges when applied to emerging model organisms, including limited spatial control of gene silencing, low knock-in efficiencies, and low throughput of functional validation. Moreover, many functional studies to date do not recapitulate ecologically relevant variation, and this limits their scope for deeper insights into evolutionary processes. We therefore argue that increased use of gene editing by allelic replacement through homology-directed repair (HDR) would greatly benefit the field of ecology and evolution.
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27
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Jin Q, Yang X, Gou S, Liu X, Zhuang Z, Liang Y, Shi H, Huang J, Wu H, Zhao Y, Ouyang Z, Zhang Q, Liu Z, Chen F, Ge W, Xie J, Li N, Lai C, Zhao X, Wang J, Lian M, Li L, Quan L, Ye Y, Lai L, Wang K. Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression. SCIENCE CHINA. LIFE SCIENCES 2022; 65:2269-2286. [PMID: 35596888 DOI: 10.1007/s11427-021-2088-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/05/2021] [Accepted: 01/20/2022] [Indexed: 06/15/2023]
Abstract
Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.
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Affiliation(s)
- Qin Jin
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Xiaoyu Yang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Shixue Gou
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Xiaoyi Liu
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Zhenpeng Zhuang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Yanhui Liang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Hui Shi
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Jiayuan Huang
- Guangdong Provincial Key Laboratory of Laboratory Animals, Guangdong Laboratory Animals Monitoring Institute, Guangzhou, 510633, China
| | - Han Wu
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Yu Zhao
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China
| | - Zhen Ouyang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China
| | - Quanjun Zhang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Zhaoming Liu
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China
| | - Fangbing Chen
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China
| | - Weikai Ge
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China
| | - Jingke Xie
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China
| | - Nan Li
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China
| | - Chengdan Lai
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China
| | - Xiaozhu Zhao
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Jiaowei Wang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Meng Lian
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Lei Li
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Longquan Quan
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Yinghua Ye
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China
- Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Liangxue Lai
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China.
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China.
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China.
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China.
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China.
| | - Kepin Wang
- CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou, 510530, China.
- Sanya institute of Swine resource, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Hainan Provincial Research Center of Laboratory Animals, Sanya, 572000, China.
- Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, 510530, China.
- Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China.
- Guangdong Provincial Key Laboratory of Large Animal models for Biomedicine, Wuyi University, Jiangmen, 529020, China.
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28
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Dog models of human atherosclerotic cardiovascular diseases. Mamm Genome 2022:10.1007/s00335-022-09965-w. [PMID: 36243810 DOI: 10.1007/s00335-022-09965-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Accepted: 10/06/2022] [Indexed: 10/17/2022]
Abstract
Cardiovascular diseases (CVD) are one of the leading causes of death worldwide. Eighty-five percent of CVD-associated deaths are due to heart attacks and stroke. Atherosclerosis leads to heart attack and stroke through a slow progression of lesion formation and luminal narrowing of arteries. Dogs are similar to humans in terms of their cardiovascular physiology, size, and anatomy. Dog models have been developed to recapitulate the complex phenotype of human patients and understand the underlying mechanism of CVD. Different methods, including high-fat, high-cholesterol diet and genetic modification, have been used to generate dog models of human CVD. Remarkably, the location and severity of atherosclerotic lesions in the coronary arteries and branches of the carotid arteries of dog models closely resemble those of human CVD patients. Overt clinical manifestations such as stroke caused by plaque rupture and thrombosis were observed in dog models. Thus, dog models can help define the pathophysiological mechanisms of atherosclerosis and develop potential strategy for preventing and treating CVD. In this review, we summarize the progress in generating and characterizing canine models to investigate CVD and discuss the advantages and limitations of canine CVD models.
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29
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Liang W, He J, Mao C, Yu C, Meng Q, Xue J, Wu X, Li S, Wang Y, Yi H. Gene editing monkeys: Retrospect and outlook. Front Cell Dev Biol 2022; 10:913996. [PMID: 36158194 PMCID: PMC9493099 DOI: 10.3389/fcell.2022.913996] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2022] [Accepted: 08/15/2022] [Indexed: 11/13/2022] Open
Abstract
Animal models play a key role in life science research, especially in the study of human disease pathogenesis and drug screening. Because of the closer proximity to humans in terms of genetic evolution, physiology, immunology, biochemistry, and pathology, nonhuman primates (NHPs) have outstanding advantages in model construction for disease mechanism study and drug development. In terms of animal model construction, gene editing technology has been widely applied to this area in recent years. This review summarizes the current progress in the establishment of NHPs using gene editing technology, which mainly focuses on rhesus and cynomolgus monkeys. In addition, we discuss the limiting factors in the applications of genetically modified NHP models as well as the possible solutions and improvements. Furthermore, we highlight the prospects and challenges of the gene-edited NHP models.
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Affiliation(s)
- Weizheng Liang
- Central Laboratory, The First Affiliated Hospital of Hebei North University, Zhangjiakou, China
- *Correspondence: Weizheng Liang, ; Shanliang Li, ; Yukai Wang, ; Hongyang Yi,
| | - Junli He
- Department of Pediatrics, Shenzhen University General Hospital, Shenzhen, China
| | - Chenyu Mao
- University of Pennsylvania, Philadelphia, PA, United States
| | - Chengwei Yu
- School of Future Technology, University of Chinese Academy of Sciences, Beijing, China
| | - Qingxue Meng
- Central Laboratory, The First Affiliated Hospital of Hebei North University, Zhangjiakou, China
| | - Jun Xue
- Department of General Surgery, The First Affiliated Hospital of Hebei North University, Zhangjiakou, China
| | - Xueliang Wu
- Department of General Surgery, The First Affiliated Hospital of Hebei North University, Zhangjiakou, China
| | - Shanliang Li
- Department of Pharmacology, Guangxi University of Chinese Medicine, Nanning, Guangxi, China
- *Correspondence: Weizheng Liang, ; Shanliang Li, ; Yukai Wang, ; Hongyang Yi,
| | - Yukai Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
- National Stem Cell Resource Center, Chinese Academy of Sciences, Beijing, China
- *Correspondence: Weizheng Liang, ; Shanliang Li, ; Yukai Wang, ; Hongyang Yi,
| | - Hongyang Yi
- National Clinical Research Centre for Infectious Diseases, The Third People’s Hospital of Shenzhen and The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen, China
- *Correspondence: Weizheng Liang, ; Shanliang Li, ; Yukai Wang, ; Hongyang Yi,
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30
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CRISPR/Cas9 system: a reliable and facile genome editing tool in modern biology. Mol Biol Rep 2022; 49:12133-12150. [PMID: 36030476 PMCID: PMC9420241 DOI: 10.1007/s11033-022-07880-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Accepted: 08/17/2022] [Indexed: 11/10/2022]
Abstract
Genome engineering has always been a versatile technique in biological research and medicine, with several applications. In the last several years, the discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 technology has swept the scientific community and revolutionised the speed of modern biology, heralding a new era of disease detection and rapid biotechnology discoveries. It enables successful gene editing by producing targeted double-strand breaks in virtually any organism or cell type. So, this review presents a comprehensive knowledge about the mechanism and structure of Cas9-mediated RNA-guided DNA targeting and cleavage. In addition, genome editing via CRISPR-Cas9 technology in various animals which are being used as models in scientific research including Non-Human Primates Pigs, Dogs, Zebra, fish and Drosophila has been discussed in this review. This review also aims to understand the applications, serious concerns and future perspective of CRISPR/Cas9-mediated genome editing.
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31
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Ontogeny of cellular organization and LGR5 expression in porcine cochlea revealed using tissue clearing and 3D imaging. iScience 2022; 25:104695. [PMID: 35865132 PMCID: PMC9294204 DOI: 10.1016/j.isci.2022.104695] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2022] [Revised: 04/20/2022] [Accepted: 06/27/2022] [Indexed: 11/23/2022] Open
Abstract
Over 11% of the world's population experience hearing loss. Although there are promising studies to restore hearing in rodent models, the size, ontogeny, genetics, and frequency range of hearing of most rodents' cochlea do not match that of humans. The porcine cochlea can bridge this gap as it shares many anatomical, physiological, and genetic similarities with its human counterpart. Here, we provide a detailed methodology to process and image the porcine cochlea in 3D using tissue clearing and light-sheet microscopy. The resulting 3D images can be employed to compare cochleae across different ages and conditions, investigate the ontogeny of cochlear cytoarchitecture, and produce quantitative expression maps of LGR5, a marker of cochlear progenitors in mice. These data reveal that hair cell organization, inner ear morphology, cellular cartography in the organ of Corti, and spatiotemporal expression of LGR5 are dynamic over developmental stages in a pattern not previously documented.
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32
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Kim M, Hyun SH. Neurotrophic factors in the porcine ovary: Their effects on follicular growth, oocyte maturation, and developmental competence. Front Vet Sci 2022; 9:931402. [PMID: 36032306 PMCID: PMC9399750 DOI: 10.3389/fvets.2022.931402] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2022] [Accepted: 07/20/2022] [Indexed: 12/01/2022] Open
Abstract
Pigs are cost-effective industrial animals because they produce a large number of offspring and have shorter rebreeding intervals compared with other animals, such as non-human primates. The reproductive physiology of pigs has been studied over the past several decades. However, there is not enough research on the effects of the neurotrophic factors on the ovarian physiology and development in pigs. As the ovary is a highly innervated organ, various neurotrophic factors during ovarian development can promote the growth of nerve fibers and improve the development of ovarian cells. Thus, investigating the role of neurotrophic factors on ovarian development, and the relationship between neurotrophic factors and porcine female reproduction is worth studying. In this review, we focused on the physiological roles of various neurotrophic factors in porcine ovaries and summarized the current status of the studies related to the relationship between neurotrophic factors and porcine ovarian development.
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Affiliation(s)
- Mirae Kim
- Laboratory of Veterinary Embryology and Biotechnology, Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, South Korea
- Institute of Stem Cell and Regenerative Medicine, Chungbuk National University, Cheongju, South Korea
| | - Sang-Hwan Hyun
- Laboratory of Veterinary Embryology and Biotechnology, Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, South Korea
- Institute of Stem Cell and Regenerative Medicine, Chungbuk National University, Cheongju, South Korea
- Graduate School of Veterinary Biosecurity and Protection, Chungbuk National University, Cheongju, South Korea
- *Correspondence: Sang-Hwan Hyun
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33
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Fortier AV, Meisner OC, Nair AR, Chang SWC. Prefrontal Circuits guiding Social Preference: Implications in Autism Spectrum Disorder. Neurosci Biobehav Rev 2022; 141:104803. [PMID: 35908593 PMCID: PMC10122914 DOI: 10.1016/j.neubiorev.2022.104803] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Revised: 07/10/2022] [Accepted: 07/27/2022] [Indexed: 11/30/2022]
Abstract
Although Autism Spectrum Disorder (ASD) is increasing in diagnostic prevalence, treatment options are inadequate largely due to limited understanding of ASD's underlying neural mechanisms. Contributing to difficulties in treatment development is the vast heterogeneity of ASD, from physiological causes to clinical presentations. Recent studies suggest that distinct genetic and neurological alterations may converge onto similar underlying neural circuits. Therefore, an improved understanding of neural circuit-level dysfunction in ASD may be a more productive path to developing broader treatments that are effective across a greater spectrum of ASD. Given the social preference behavioral deficits commonly seen in ASD, dysfunction in circuits mediating social preference may contribute to the atypical development of social cognition. We discuss some of the animal models used to study ASD and examine the function and effects of dysregulation of the social preference circuits, notably the medial prefrontal cortex-amygdala and the medial prefrontal cortex-nucleus accumbens circuits, in these animal models. Using the common circuits underlying similar behavioral disruptions of social preference behaviors as an example, we highlight the importance of identifying disruption in convergent circuits to improve the translational success of animal model research for ASD treatment development.
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Affiliation(s)
- Abigail V Fortier
- Department of Psychology, Yale University, New Haven, CT 06520, USA; Department of Molecular, Cellular, Developmental Biology, New Haven, CT 06520, USA
| | - Olivia C Meisner
- Department of Psychology, Yale University, New Haven, CT 06520, USA; Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA
| | - Amrita R Nair
- Department of Psychology, Yale University, New Haven, CT 06520, USA
| | - Steve W C Chang
- Department of Psychology, Yale University, New Haven, CT 06520, USA; Department of Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA; Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06510, USA; Wu Tsai Institute, Yale University, New Haven, CT 06510, USA
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34
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Abstract
Cardiovascular disease remains the leading cause of morbidity and mortality in the developed world. In recent decades, extraordinary effort has been devoted to defining the molecular and pathophysiological characteristics of the diseased heart and vasculature. Mouse models have been especially powerful in illuminating the complex signaling pathways, genetic and epigenetic regulatory circuits, and multicellular interactions that underlie cardiovascular disease. The advent of CRISPR genome editing has ushered in a new era of cardiovascular research and possibilities for genetic correction of disease. Next-generation sequencing technologies have greatly accelerated the identification of disease-causing mutations, and advances in gene editing have enabled the rapid modeling of these mutations in mice and patient-derived induced pluripotent stem cells. The ability to correct the genetic drivers of cardiovascular disease through delivery of gene editing components in vivo, while still facing challenges, represents an exciting therapeutic frontier. In this review, we provide an overview of cardiovascular disease mechanisms and the potential applications of CRISPR genome editing for disease modeling and correction. We also discuss the extent to which mice can faithfully model cardiovascular disease and the opportunities and challenges that lie ahead.
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Affiliation(s)
- Ning Liu
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas
| | - Eric N Olson
- Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas
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35
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Mutations in rhodopsin, endothelin B receptor, and CC chemokine receptor 5 in large animals: Modeling human diseases. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2022; 189:155-178. [PMID: 35595348 DOI: 10.1016/bs.pmbts.2022.02.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
G protein-coupled receptors (GPCRs) are the largest family of cell membrane receptors involved in modulating almost all physiological processes by transducing extracellular signals into the cytoplasm. Dysfunctions of GPCR-regulated signaling result in diverse human diseases, making GPCRs the most popular drug targets for human medicine. Large animals share higher similarities (in physiology and metabolism) with humans than rodents. Similar to findings in human genetics, diverse diseases caused by mutations in GPCR genes have also been discovered in large animals. Rhodopsin, endothelin B receptor, and CC chemokine receptor type 5 have been shown to be involved in human retinitis pigmentosa, Hirschsprung disease, and HIV infection/AIDS, respectively, and several mutations of these GPCRs have also been identified from large animals. The large animals with naturally occurring mutations of these GPCRs provide an opportunity to gain a better understanding of the pathogenesis of human diseases, and can be used for preclinical trials of therapies for human diseases. In this review, we aim to summarize the naturally occurring mutations of these three GPCRs in large animals and humans.
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36
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Lin Y, Li J, Li C, Tu Z, Li S, Li XJ, Yan S. Application of CRISPR/Cas9 System in Establishing Large Animal Models. Front Cell Dev Biol 2022; 10:919155. [PMID: 35656550 PMCID: PMC9152178 DOI: 10.3389/fcell.2022.919155] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2022] [Accepted: 05/02/2022] [Indexed: 11/13/2022] Open
Abstract
The foundation for investigating the mechanisms of human diseases is the establishment of animal models, which are also widely used in agricultural industry, pharmaceutical applications, and clinical research. However, small animals such as rodents, which have been extensively used to create disease models, do not often fully mimic the key pathological changes and/or important symptoms of human disease. As a result, there is an emerging need to establish suitable large animal models that can recapitulate important phenotypes of human diseases for investigating pathogenesis and developing effective therapeutics. However, traditional genetic modification technologies used in establishing small animal models are difficultly applied for generating large animal models of human diseases. This difficulty has been overcome to a great extent by the recent development of gene editing technology, especially the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). In this review, we focus on the applications of CRISPR/Cas9 system to establishment of large animal models, including nonhuman primates, pigs, sheep, goats and dogs, for investigating disease pathogenesis and treatment. We also discuss the limitations of large animal models and possible solutions according to our current knowledge. Finally, we sum up the applications of the novel genome editing tool Base Editors (BEs) and its great potential for gene editing in large animals.
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37
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Genome-wide association study reveals the genetic basis of growth trait in yellow catfish with sexual size dimorphism. Genomics 2022; 114:110380. [PMID: 35533968 DOI: 10.1016/j.ygeno.2022.110380] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 04/20/2022] [Accepted: 05/02/2022] [Indexed: 01/14/2023]
Abstract
Sexual size dimorphism has been widely observed in a large number of animals including fish species. Genome-wide association study (GWAS) is a powerful tool to dissect the genetic basis of complex traits, whereas the sex-differences in the genomics of animal complex traits have been ignored in the GWAS analysis. Yellow catfish (Pelteobagrus fulvidraco) is an important aquaculture fish in China with significant sexual size dimorphism. In this study, GWAS was conducted to identify candidate SNPs and genes related to body length (BL) and body weight (BW) in 125 female yellow catfish from a breeding population. In total, one BL-related SNP and three BW-related SNPs were identified to be significantly associated with the traits. Besides, one of these SNPs (Chr15:19195072) was shared in both the BW and BL traits in female yellow catfish, which was further validated in 185 male individuals and located on the exon of stat5b gene. Transgenic yellow catfish and zebrafish that expressed yellow catfish stat5b showed increased growth rate and reduction of sexual size dimorphism. These results not only reveal the genetic basis of growth trait and sexual size dimorphism in fish species, but also provide useful information for the marker-assisted breeding in yellow catfish.
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38
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Martin-Collado D, Byrne TJ, Crowley JJ, Kirk T, Ripoll G, Whitelaw CBA. Gene-Edited Meat: Disentangling Consumers' Attitudes and Potential Purchase Behavior. Front Nutr 2022; 9:856491. [PMID: 35449542 PMCID: PMC9017286 DOI: 10.3389/fnut.2022.856491] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Accepted: 03/14/2022] [Indexed: 11/18/2022] Open
Abstract
Novel gene-editing (GE) technologies provide promising opportunities to increase livestock productivity and to tackle several global livestock production sustainability and food security challenges. However, these technologies, as with previous genetic modification technologies in food production, are very likely to generate social controversy and opposition toward their use in the meat industry. Here, we explored public attitudes and consumption predisposition toward gene-edited meat products and their potential added benefits to livestock farming. Our results show that societal perception currently comes as a package, where the use of gene-editing technology acts as an extrinsic cue of meat products quality, and is used to make a range of inferences about all quality facets at once. Although consumers with anti-GE attitudinal positions generally were not sensitive to price discounts or added benefits, added benefits increased the consumption predisposition of most moderate and pro-GE consumers, where benefits related to animal welfare had larger effects than those relating to the environment or human health issues.
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Affiliation(s)
- Daniel Martin-Collado
- Department of Animal Science, Agrifood Research and Technology Centre of Aragon (CITA), Zaragoza, Spain.,AgriFood Institute of Aragon - IA2 (CITA-University of Zaragoza), Zaragoza, Spain
| | - Tim J Byrne
- AbacusBio International Limited, Roslin Innovation Centre, Edinburgh, United Kingdom
| | - Jonh J Crowley
- AbacusBio International Limited, Roslin Innovation Centre, Edinburgh, United Kingdom.,Department of Agriculture, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada
| | - Tom Kirk
- AbacusBio International Limited, Roslin Innovation Centre, Edinburgh, United Kingdom
| | - Guillermo Ripoll
- Department of Animal Science, Agrifood Research and Technology Centre of Aragon (CITA), Zaragoza, Spain.,AgriFood Institute of Aragon - IA2 (CITA-University of Zaragoza), Zaragoza, Spain
| | - C B A Whitelaw
- The Roslin Institute, Royal (Dick) School of Veterinary Sciences, University of Edinburgh, Edinburgh, United Kingdom
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39
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Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Cell Res 2022; 32:383-400. [PMID: 34848870 PMCID: PMC8976023 DOI: 10.1038/s41422-021-00592-9] [Citation(s) in RCA: 53] [Impact Index Per Article: 17.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2021] [Accepted: 11/04/2021] [Indexed: 12/19/2022] Open
Abstract
Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.
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40
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Maniego J, Pesko B, Habershon-Butcher J, Hincks P, Taylor P, Tozaki T, Ohnuma A, Stewart G, Proudman C, Ryder E. Use of mitochondrial sequencing to detect gene doping in horses via gene editing and somatic cell nuclear transfer. Drug Test Anal 2022; 14:1429-1437. [PMID: 35362263 DOI: 10.1002/dta.3267] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2022] [Revised: 03/24/2022] [Accepted: 03/29/2022] [Indexed: 11/09/2022]
Abstract
Gene editing and subsequent cloning techniques offer great potential not only in genetic disease correction in domestic animals, but also in livestock production by enhancement of desirable traits. The existence of the technology, however, leaves it open to potential misuse in performance-led sports such as horseracing and other equestrian events. Recent advances in equine gene editing, regarding the generation of gene-edited embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer, has highlighted the need to develop tools to detect potential prohibited use of the technology. One possible method involves the characterisation of the mitochondrial genome (which is not routinely preserved during cloning) and comparing it to the sequence of the registered dam. We present here our approach to whole-mitochondrial sequencing using tiled long-range PCR and next-generation sequencing. To determine whether the background mutation rate in the mitochondrial genome could potentially confound results, we sequenced ten sets of dam and foal duos. We found variation between duos but none within duos, indicating that this method is feasible for future screening systems. Analysis of WGS data from over one hundred Thoroughbred horses revealed wide variation in the mitochondria sequence within the breed, further displaying the utility of this approach.
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Affiliation(s)
- Jillian Maniego
- Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, UK
| | - Bogusia Pesko
- Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, UK
| | | | - Pamela Hincks
- Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, UK
| | - Polly Taylor
- Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, UK
| | - Teruaki Tozaki
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Japan
| | - Aoi Ohnuma
- Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Japan
| | - Graham Stewart
- School of Biosciences and Medicine, University of Surrey, Guildford, UK
| | - Christopher Proudman
- School of Veterinary Medicine, Daphne Jackson Road, University of Surrey, Guildford, UK
| | - Edward Ryder
- Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, UK
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41
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New pathogenic insights from large animal models of neurodegenerative diseases. Protein Cell 2022; 13:707-720. [PMID: 35334073 PMCID: PMC9233730 DOI: 10.1007/s13238-022-00912-8] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2022] [Accepted: 02/23/2022] [Indexed: 12/12/2022] Open
Abstract
Animal models are essential for investigating the pathogenesis and developing the treatment of human diseases. Identification of genetic mutations responsible for neurodegenerative diseases has enabled the creation of a large number of small animal models that mimic genetic defects found in the affected individuals. Of the current animal models, rodents with genetic modifications are the most commonly used animal models and provided important insights into pathogenesis. However, most of genetically modified rodent models lack overt neurodegeneration, imposing challenges and obstacles in utilizing them to rigorously test the therapeutic effects on neurodegeneration. Recent studies that used CRISPR/Cas9-targeted large animal (pigs and monkeys) have uncovered important pathological events that resemble neurodegeneration in the patient’s brain but could not be produced in small animal models. Here we highlight the unique nature of large animals to model neurodegenerative diseases as well as the limitations and challenges in establishing large animal models of neurodegenerative diseases, with focus on Huntington disease, Amyotrophic lateral sclerosis, and Parkinson diseases. We also discuss how to use the important pathogenic insights from large animal models to make rodent models more capable of recapitulating important pathological features of neurodegenerative diseases.
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42
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Reith MEA, Kortagere S, Wiers CE, Sun H, Kurian MA, Galli A, Volkow ND, Lin Z. The dopamine transporter gene SLC6A3: multidisease risks. Mol Psychiatry 2022; 27:1031-1046. [PMID: 34650206 PMCID: PMC9008071 DOI: 10.1038/s41380-021-01341-5] [Citation(s) in RCA: 39] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Revised: 09/28/2021] [Accepted: 10/01/2021] [Indexed: 02/02/2023]
Abstract
The human dopamine transporter gene SLC6A3 has been consistently implicated in several neuropsychiatric diseases but the disease mechanism remains elusive. In this risk synthesis, we have concluded that SLC6A3 represents an increasingly recognized risk with a growing number of familial mutants associated with neuropsychiatric and neurological disorders. At least five loci were related to common and severe diseases including alcohol use disorder (high activity variant), attention-deficit/hyperactivity disorder (low activity variant), autism (familial proteins with mutated networking) and movement disorders (both regulatory variants and familial mutations). Association signals depended on genetic markers used as well as ethnicity examined. Strong haplotype selection and gene-wide epistases support multimarker assessment of functional variations and phenotype associations. Inclusion of its promoter region's functional markers such as DNPi (rs67175440) and 5'VNTR (rs70957367) may help delineate condensate-based risk action, testing a locus-pathway-phenotype hypothesis for one gene-multidisease etiology.
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Affiliation(s)
- Maarten E A Reith
- Department of Psychiatry, New York University School of Medicine, New York City, NY, 10016, USA
| | - Sandhya Kortagere
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, 19129, USA
| | - Corinde E Wiers
- Laboratory of Neuroimaging, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD, 20817, USA
- Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Hui Sun
- Laboratory of Neuroimaging, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD, 20817, USA
| | - Manju A Kurian
- Molecular Neurosciences, Developmental Neurosciences, Zayed Centre for Research into Rare Diseases in Children, UCL Great Ormond Street Institute of Child Health, and Department of Neurology, Great Ormond Street Hospital, London, WC1N 1EH, UK
| | - Aurelio Galli
- Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, 35294, USA
| | - Nora D Volkow
- Laboratory of Neuroimaging, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD, 20817, USA
- National Institute on Drug Abuse, Bethesda, MD, 20817, USA
| | - Zhicheng Lin
- Laboratory of Psychiatric Neurogenomics, McLean Hospital, and Department of Psychiatry, Harvard Medical School, Belmont, MA, 02478, USA.
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Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells. Biotechnol Lett 2022; 44:59-76. [PMID: 34997407 DOI: 10.1007/s10529-021-03214-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2021] [Accepted: 11/28/2021] [Indexed: 12/26/2022]
Abstract
Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.
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Tu CF, Chuang CK, Yang TS. The application of new breeding technology based on gene editing in pig industry. Anim Biosci 2022; 35:791-803. [PMID: 34991204 PMCID: PMC9066036 DOI: 10.5713/ab.21.0390] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2021] [Accepted: 12/07/2021] [Indexed: 12/02/2022] Open
Abstract
Genome/gene-editing (GE) techniques, characterized by a low technological barrier, high efficiency, and broad application among organisms, are now being employed not only in medical science but also in agriculture/veterinary science. Different engineered CRISPR/Cas9s have been identified to expand the application of this technology. In pig production, GE is a precise new breeding technology (NBT), and promising outcomes in improving economic traits, such as growth, lean or healthy meat production, animal welfare, and disease resistance, have already been documented and reviewed. These promising achievements in porcine gene editing, including the Myostatin gene knockout (KO) in indigenous breeds to improve lean meat production, the uncoupling protein 1 (UCP1) gene knock-in to enhance piglet thermogenesis and survival under cold stress, the generation of GGTA1 and CMP-N-glycolylneuraminic acid hydroxylase (CMAH) gene double KO (dKO) pigs to produce healthy red meat, and the KO or deletion of exon 7 of the CD163 gene to confer resistance to porcine reproductive and respiratory syndrome virus infection, are described in the present article. Other related approaches for such purposes are also discussed. The current trend of global regulations or legislation for GE organisms is that they are exempted from classification as genetically modified organisms (GMOs) if no exogenes are integrated into the genome, according to product-based and not process-based methods. Moreover, an updated case study in the EU showed that current GMO legislation is not fit for purpose in term of NBTs, which contribute to the objectives of the EU’s Green Deal and biodiversity strategies and even meet the United Nations’ sustainable development goals for a more resilient and sustainable agri-food system. The GE pigs generated via NBT will be exempted from classification as GMOs, and their global valorization and commercialization can be foreseen.
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Affiliation(s)
- Ching-Fu Tu
- Division of Animal Technology, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu City 30093, Taiwan
| | - Chin-Kai Chuang
- Division of Animal Technology, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu City 30093, Taiwan
| | - Tien-Shuh Yang
- Division of Animal Technology, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu City 30093, Taiwan.,Department of Biotechnology and Animal Science, National Ilan University, Yilan City, 26047 Taiwan
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Yang W, Chen X, Li S, Li XJ. Genetically modified large animal models for investigating neurodegenerative diseases. Cell Biosci 2021; 11:218. [PMID: 34933675 PMCID: PMC8690884 DOI: 10.1186/s13578-021-00729-8] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Accepted: 12/07/2021] [Indexed: 12/02/2022] Open
Abstract
Neurodegenerative diseases represent a large group of neurological disorders including Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington’s disease. Although this group of diseases show heterogeneous clinical and pathological phenotypes, they share important pathological features characterized by the age-dependent and progressive degeneration of nerve cells that is caused by the accumulation of misfolded proteins. The association of genetic mutations with neurodegeneration diseases has enabled the establishment of various types of animal models that mimic genetic defects and have provided important insights into the pathogenesis. However, most of genetically modified rodent models lack the overt and selective neurodegeneration seen in the patient brains, making it difficult to use the small animal models to validate the effective treatment on neurodegeneration. Recent studies of pig and monkey models suggest that large animals can more faithfully recapitulate pathological features of neurodegenerative diseases. In this review, we discuss the important differences in animal models for modeling pathological features of neurodegenerative diseases, aiming to assist the use of animal models to better understand the pathogenesis and to develop effective therapeutic strategies.
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Affiliation(s)
- Weili Yang
- Guangdong Key Laboratory of Non-Human Primate Research, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, 510632, China.
| | - Xiusheng Chen
- Guangdong Key Laboratory of Non-Human Primate Research, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, 510632, China
| | - Shihua Li
- Guangdong Key Laboratory of Non-Human Primate Research, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, 510632, China
| | - Xiao-Jiang Li
- Guangdong Key Laboratory of Non-Human Primate Research, Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, 510632, China.
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46
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Monzani PS, Adona PR, Long SA, Wheeler MB. Cows as Bioreactors for the Production of Nutritionally and Biomedically Significant Proteins. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1354:299-314. [PMID: 34807448 DOI: 10.1007/978-3-030-85686-1_15] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
Dairy and beef cattle make a vital contribution to global nutrition, and since their domestication, they have been continuously exposed to natural and artificial selection to improve production characteristics. The technologies of transgenesis and gene editing used in cattle are responsible for generating news characteristics in bovine breeding, such as alteration of nutritional components of milk and meat enhancing human health benefits, disease resistance decreasing production costs and offering safe products for human food, as well as the recombinant protein production of biomedical significance. Different methodologies have been used to generate transgenic cattle as bioreactors. These methods include the microinjection of vectors in pronuclear, oocyte or zygote, sperm-mediate transgenesis, and somatic cell nuclear transfer. Gene editing has been applied to eliminate unwanted genes related to human and animal health, such as allergy, infection, or disease, and to insert transgenes into specific sites in the host genome. Methodologies for the generation of genetically modified cattle are laborious and not very efficient. However, in the last 30 years, transgenic animals were produced using many biotechnological tools. The result of these modifications includes (1) the change of nutritional components, including proteins, amino acids and lipids for human nutrition; (2) the removal allergic proteins milk; (3) the production of cows resistant to disease; or (4) the production of essential proteins used in biomedicine (biomedical proteins) in milk and blood plasma. The genetic modification of cattle is a powerful tool for biotechnology. It allows for the generation of new or modified products and functionality that are not currently available in this species.
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Affiliation(s)
- P S Monzani
- Instituto Chico Mendes de Conservação da Biodiversidade/Centro Nacional de Pesquisa e Conservação da Biodiversidade Aquática Continental, Pirassununga, SP, Brasil.
| | - P R Adona
- Saúde e Produção de Ruminantes, Universidade Norte do Paraná, Arapongas, PR, Brasil
| | - S A Long
- Departments of Animal Sciences and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - M B Wheeler
- Departments of Animal Sciences and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.,Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
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Entine J, Felipe MSS, Groenewald JH, Kershen DL, Lema M, McHughen A, Nepomuceno AL, Ohsawa R, Ordonio RL, Parrott WA, Quemada H, Ramage C, Slamet-Loedin I, Smyth SJ, Wray-Cahen D. Regulatory approaches for genome edited agricultural plants in select countries and jurisdictions around the world. Transgenic Res 2021; 30:551-584. [PMID: 33970411 PMCID: PMC8316157 DOI: 10.1007/s11248-021-00257-8] [Citation(s) in RCA: 63] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2020] [Accepted: 04/21/2021] [Indexed: 12/28/2022]
Abstract
Genome editing in agriculture and food is leading to new, improved crops and other products. Depending on the regulatory approach taken in each country or region, commercialization of these crops and products may or may not require approval from the respective regulatory authorities. This paper describes the regulatory landscape governing genome edited agriculture and food products in a selection of countries and regions.
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Affiliation(s)
- Jon Entine
- Genetic Literacy Project, Cincinnati, OH, USA
| | - Maria Sueli S Felipe
- Genomic Sciences and Biotechnology Program, Catholic University of Brasília, Brasília, DF, Brazil
| | | | | | - Martin Lema
- Departamento de Ciencia Y Tecnología and Maestría en Ciencia, Tecnología y Sociedad, Universidad Nacional de Quilmes, Bernal Buenos Aires, Argentina
| | - Alan McHughen
- Botany and Plant Sciences, University of California, Riverside, CA, USA.
| | | | - Ryo Ohsawa
- Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Reynante L Ordonio
- Crop Biotechnology Center, Philippine Rice Research Institute, Maligaya, Science City of Munoz, Philippines
| | - Wayne A Parrott
- Department of Crop and Soil Sciences and Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA, USA
| | - Hector Quemada
- Department of Biological Sciences, Western Michigan University, Kalamazoo, MI, USA
| | - Carl Ramage
- Office of the Deputy Vice-Chancellor (Research and Industry Engagement), Rautaki Solutions Pty Ltd, La Trobe University, Melbourne, VIC, Australia
| | - Inez Slamet-Loedin
- Fellow of The World Academy of Sciences, Cluster Lead-Trait and Genome Engineering, International Rice Research Institute, Manila, Philippines
| | - Stuart J Smyth
- Department of Agricultural and Resource Economics, University of Saskatchewan, Saskatoon, SK, Canada
| | - Diane Wray-Cahen
- United States Department of Agriculture, Foreign Agricultural Service, Washington, DC, USA
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Application of the modified cytosine base-editing in the cultured cells of bama minipig. Biotechnol Lett 2021; 43:1699-1714. [PMID: 34189671 DOI: 10.1007/s10529-021-03159-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2021] [Accepted: 06/24/2021] [Indexed: 12/26/2022]
Abstract
Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed "all-in-one"-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.
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The Moral Status of Cognitively Enhanced Monkeys and Other Novel Beings. Camb Q Healthc Ethics 2021; 30:492-503. [PMID: 34109929 DOI: 10.1017/s0963180120001048] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The discussion about the moral status of novel beings tends to focus on artificial intelligence, robots, and other man-made systems. We should, however, also consider a likelier kind of novel beings: animals that are genetically modified to develop human-like cognitive capabilities. This paper focuses on the possibility of conferring human characteristics on nonhuman primates (NHPs) in the context of neuroscientific research. It first discusses the use of NHPs for neuroscientific research and then, second, describes recent developments that promise to revolutionize the field and how that may lead to NHPs attaining human-like cognitive capabilities. Third, an account of moral status is developed to ground the central claim, that making the NHP brain more human-like is unproblematic as long as the NHPs do not become persons. In conclusion, this paper discusses the implications for the moral status of cognitively enhanced NHPs, as well as the implications for other novel beings.
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50
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Feng S, Huang H, Wang N, Wei Y, Liu Y, Qin D. Sleep Disorders in Children With Autism Spectrum Disorder: Insights From Animal Models, Especially Non-human Primate Model. Front Behav Neurosci 2021; 15:673372. [PMID: 34093147 PMCID: PMC8173056 DOI: 10.3389/fnbeh.2021.673372] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Accepted: 04/16/2021] [Indexed: 02/05/2023] Open
Abstract
Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental disorder with deficient social skills, communication deficits and repetitive behaviors. The prevalence of ASD has increased among children in recent years. Children with ASD experience more sleep problems, and sleep appears to be essential for the survival and integrity of most living organisms, especially for typical synaptic development and brain plasticity. Many methods have been used to assess sleep problems over past decades such as sleep diaries and parent-reported questionnaires, electroencephalography, actigraphy and videosomnography. A substantial number of rodent and non-human primate models of ASD have been generated. Many of these animal models exhibited sleep disorders at an early age. The aim of this review is to examine and discuss sleep disorders in children with ASD. Toward this aim, we evaluated the prevalence, clinical characteristics, phenotypic analyses, and pathophysiological brain mechanisms of ASD. We highlight the current state of animal models for ASD and explore their implications and prospects for investigating sleep disorders associated with ASD.
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Affiliation(s)
- Shufei Feng
- Department of Pediatric Rehabilitation Medicine, Kunming Children’s Hospital, Kunming, China
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, China
| | - Haoyu Huang
- Department of Pediatric Rehabilitation Medicine, Kunming Children’s Hospital, Kunming, China
| | - Na Wang
- School of Basic Medical Sciences, Yunnan University of Chinese Medicine, Kunming, China
| | - Yuanyuan Wei
- School of Basic Medical Sciences, Yunnan University of Chinese Medicine, Kunming, China
| | - Yun Liu
- Department of Pediatric Rehabilitation Medicine, Kunming Children’s Hospital, Kunming, China
| | - Dongdong Qin
- Department of Pediatric Rehabilitation Medicine, Kunming Children’s Hospital, Kunming, China
- State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, China
- School of Basic Medical Sciences, Yunnan University of Chinese Medicine, Kunming, China
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