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He S, Liu Y, Zhang Z, Cai M, Hao Y, Hu H. Gene Editing in Ganoderma lucidum: Development, Challenges, and Future Prospects. J Fungi (Basel) 2025; 11:310. [PMID: 40278130 PMCID: PMC12029067 DOI: 10.3390/jof11040310] [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: 03/10/2025] [Revised: 04/05/2025] [Accepted: 04/09/2025] [Indexed: 04/26/2025] Open
Abstract
As an emerging and innovative technology, gene-editing technology has been widely applied in crop breeding, human disease treatment, animal model research, drug and vaccine development, and microbial engineering. We mainly introduce the development of gene-editing technology, the application of clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) in Ganoderma lucidum breeding, the current challenges and optimization strategies in the use of gene-editing technology in Ganoderma breeding, as well as the current status of gene-editing technology in Ganoderma breeding. Finally, the future research directions and innovative strategies that gene editing may explore in Ganoderma breeding are prospects given the existing background, future research directions, and innovative strategies that gene editing may explore in Ganoderma breeding prospects.
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Affiliation(s)
- Shiqi He
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China; (S.H.); (Y.L.); (Z.Z.); (M.C.); (Y.H.)
| | - Yuanchao Liu
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China; (S.H.); (Y.L.); (Z.Z.); (M.C.); (Y.H.)
| | - Zhi Zhang
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China; (S.H.); (Y.L.); (Z.Z.); (M.C.); (Y.H.)
- Guangdong Yuewei Biotechnology Co., Ltd., Shaoguan 512029, China
| | - Manjun Cai
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China; (S.H.); (Y.L.); (Z.Z.); (M.C.); (Y.H.)
| | - Yufan Hao
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China; (S.H.); (Y.L.); (Z.Z.); (M.C.); (Y.H.)
| | - Huiping Hu
- National Health Commission Science and Technology Innovation Platform for Nutrition and Safety of Microbial Food, Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China; (S.H.); (Y.L.); (Z.Z.); (M.C.); (Y.H.)
- Guangdong Yuewei Biotechnology Co., Ltd., Shaoguan 512029, China
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Miura I, Hasegawa Y, Ito M, Ezaz T, Ogata M. Disruption of Sex-Linked Sox3 Causes ZW Female-to-Male Sex Reversal in the Japanese Frog Glandirana rugosa. Biomolecules 2024; 14:1566. [PMID: 39766273 PMCID: PMC11673724 DOI: 10.3390/biom14121566] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2024] [Revised: 11/30/2024] [Accepted: 12/04/2024] [Indexed: 01/11/2025] Open
Abstract
Sox3 is an ancestral homologous gene of the male-determining Sry in eutherian mammals and determines maleness in medaka fish. In the Japanese frog, Glandirana rugosa, Sox3 is located on the Z and W chromosomes. To assess the sex-determining function of Sox3 in this frog, we investigated its expression in gonads during early tadpole development and conducted genome-editing experiments. We found that the Sox3 mRNA levels in the gonads/mesonephroi were much higher in ZW females than that in ZZ males, and that the W-borne allele was dominantly expressed. A higher expression in ZW females preceded the onset of the sexually dimorphic expression of other autosomal sex differentiation genes. The Sox3 protein was detected by immunostaining in the somatic cells of early tadpole gonads around the boundary between the medulla and cortex in ZW females, whereas it was outside the gonads in ZZ males. Disrupting Sox3 using TALEN, which targets two distinct sites, generated sex-reversed ZW males and hermaphrodites, whereas no sex reversal was observed in ZZ males. These results suggest that the sex-linked Sox3 is involved in female determination in the ZZ-ZW sex-determining system of the frog, an exact opposite function to the male determination of medaka Sox3y and eutherian Sry.
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Affiliation(s)
- Ikuo Miura
- Amphibian Research Center, Hiroshima University, Higashi-Hiroshima 739-8526, Japan
- Institute for Applied Ecology, University of Canberra, Bruce, ACT 2617, Australia;
| | | | - Michihiko Ito
- School of Science, Kitasato University, Sagamihara 252-0373, Japan;
| | - Tariq Ezaz
- Institute for Applied Ecology, University of Canberra, Bruce, ACT 2617, Australia;
| | - Mitsuaki Ogata
- Preservation and Research Center, City of Yokohama, Yokohama 241-0804, Japan;
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Maeda Y, Ding J, Saeki M, Kuwayama N, Kishi Y. A simple method for gene expression in endo- and ectodermal cells in mouse embryos before neural tube closure. Dev Biol 2024; 516:114-121. [PMID: 39102935 DOI: 10.1016/j.ydbio.2024.08.001] [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: 03/18/2024] [Revised: 07/18/2024] [Accepted: 08/02/2024] [Indexed: 08/07/2024]
Abstract
The lack of a widely accessible method for expressing genes of interest in wild-type embryos is a fundamental obstacle to understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, which is a period of drastic development for many tissues. In this study, we present a simple technique for injecting vectors into the amniotic cavity and allowing them to reach the ectodermal cells and the epithelia of endodermal organs of mouse embryos at E8.0 via in utero injection, using only a widely used optical fiber with an illuminator. Using this technique, retroviruses can be introduced to facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. We also demonstrated in utero electroporation of plasmid DNA into E7.0 and E8.0 embryos. Taking advantage of this method, we reveal the association between Ldb1 and the activity of the Neurog2 transcription factor in the mouse neocortex. This technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure.
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Affiliation(s)
- Yurie Maeda
- Laboratory of Molecular Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
| | - Jingwen Ding
- Laboratory of Molecular Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
| | - Mai Saeki
- Laboratory of Molecular Neurobiology, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo 113-0032, Japan
| | - Naohiro Kuwayama
- Laboratory of Molecular Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
| | - Yusuke Kishi
- Laboratory of Molecular Biology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan; Laboratory of Molecular Neurobiology, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo 113-0032, Japan.
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4
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Huang T, Fakurazi S, Cheah PS, Ling KH. Chromosomal and cellular therapeutic approaches for Down syndrome: A research update. Biochem Biophys Res Commun 2024; 735:150664. [PMID: 39260337 DOI: 10.1016/j.bbrc.2024.150664] [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: 06/01/2024] [Revised: 08/20/2024] [Accepted: 09/03/2024] [Indexed: 09/13/2024]
Abstract
In individuals with Down syndrome (DS), an additional HSA21 chromosome copy leads to the overexpression of a myriad of HSA21 genes, disrupting the transcription of the entire genome. This dysregulation in transcription and post-transcriptional modifications contributes to abnormal phenotypes across nearly all tissues and organs in DS individuals. The array of severe clinical symptoms associated with trisomy 21 poses a considerable challenge in the quest for a cure for DS. Fortunately, a wealth of research suggests that chromosome therapy, hinging on cutting-edge genome editing technologies, can potentially eliminate the extra copy of the human chromosome 21. Genome editing tools have demonstrated their efficacy in restoring trisomy to a normal diploid state in vitro DS cell models. Furthermore, we delve into the noteworthy findings in cellular therapy for DS, with recent studies showcasing the increasing feasibility of strategies involving stem cells and CAR T-cells to address corresponding clinical phenotypes.
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Affiliation(s)
- Tan Huang
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
| | - Sharida Fakurazi
- Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
| | - Pike-See Cheah
- Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia; Malaysian Research Institute on Ageing (MyAgeing(TM)), Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
| | - King-Hwa Ling
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia; Malaysian Research Institute on Ageing (MyAgeing(TM)), Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.
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Chen X, Gao Y, Zhang Y. Allogeneic CAR-T cells for cancer immunotherapy. Immunotherapy 2024; 16:1079-1090. [PMID: 39378059 PMCID: PMC11492692 DOI: 10.1080/1750743x.2024.2408048] [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: 06/17/2024] [Accepted: 09/19/2024] [Indexed: 10/19/2024] Open
Abstract
Autologous chimeric antigen receptor (CAR)-modified T (CAR-T) cell therapy has displayed high efficacy in the treatment of hematological malignancies. Up to now, 11 autologous CAR-T cell products have been approved for the management of malignancies globally. However, the application of autologous CAR-T cell therapy has many individual limitations, long time-consuming, highly cost, and the risk of manufacturing failure. Indeed, some patients would not benefit from autologous CAR-T cell products because of rapid disease progression. Allogeneic CAR-T cells especially universal CAR-T (U-CAR-T) cell therapy are superior to these challenges of autologous CAR-T cells. In this review, we describe basic study and clinical trials of U-CAR-T cell therapeutic methods for malignancies. In addition, we summarize the problems encountered and potential solutions.
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Affiliation(s)
- Xinfeng Chen
- Biotherapy Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, China
- Cancer Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, China
| | - Yaoxin Gao
- Biotherapy Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, China
| | - Yi Zhang
- Biotherapy Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, China
- Cancer Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, China
- State Key Laboratory of Esophageal Cancer Prevention & Treatment, Zhengzhou, Henan, 450052, China
- School of Life Sciences, Zhengzhou University, Zhengzhou, Henan, 450052, China
- Tianjian Laboratory of Advanced Biomedical Sciences, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, 450052, China
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Castillo SR, Simone BW, Clark KJ, Devaux P, Ekker SC. Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs. Hum Gene Ther 2024; 35:798-813. [PMID: 39212664 PMCID: PMC11511777 DOI: 10.1089/hum.2024.073] [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: 05/16/2024] [Accepted: 08/05/2024] [Indexed: 09/04/2024] Open
Abstract
DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5' bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5'-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5' bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.
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Affiliation(s)
- Santiago R. Castillo
- Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, Minnesota, USA
- Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Brandon W. Simone
- Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA
| | - Karl J. Clark
- Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA
| | - Patricia Devaux
- Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, Minnesota, USA
- Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Stephen C. Ekker
- Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA
- Department of Pediatrics and Department of Molecular Biosciences, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA
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Joo J, Kim KJ, Lim J, Choi SY, Koh W, Lee CJ. Generation of Astrocyte-specific BEST1 Conditional Knockout Mouse with Reduced Tonic GABA Inhibition in the Brain. Exp Neurobiol 2024; 33:180-192. [PMID: 39266474 PMCID: PMC11411089 DOI: 10.5607/en24019] [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: 08/15/2024] [Revised: 08/29/2024] [Accepted: 08/31/2024] [Indexed: 09/14/2024] Open
Abstract
Bestrophin-1 (BEST1) is a Ca2+-activated anion channel known for its role in astrocytes. Best1 is permeable to gliotransmitters, including GABA, to contribute to tonic GABA inhibition and modulate synaptic transmission in neighboring neurons. Despite the crucial functions of astrocytic BEST1, there is an absence of genetically engineered cell-type specific conditional mouse models addressing these roles. In this study, we developed an astrocyte-specific BEST1 conditional knock-out (BEST1 aKO) mouse line. Using the embryonic stem cell (ES cell) targeting method, we developed Best1 floxed mice (C57BL/6JCya-Best1em1flox/Cya), which have exon 3, 4, 5, and 6 of Best1 flanked by two loxP sites. By crossing with hGFAP-CreERT2 mice, we generated Best1 floxed/hGFAP-CreERT2 mice, which allowed for the tamoxifen-inducible deletion of Best1 under the human GFAP promoter. We characterized its features across various brain regions, including the striatum, hippocampal dentate gyrus (HpDG), and Parafascicular thalamic nucleus (Pf). Compared to the Cre-negative control, we observed significantly reduced BEST1 protein expression in immunohistochemistry (IHC) and tonic GABA inhibition in patch clamp recordings. The reduction in tonic GABA inhibition was 66.7% in the striatum, 46.4% in the HpDG, and 49.6% in the Pf. Our findings demonstrate that the BEST1 channel in astrocytes significantly contributes to tonic inhibition in the local brain areas. These mice will be valuable for future studies not only on tonic GABA release but also on tonic release of gliotransmitters mediated by astrocytic BEST1.
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Affiliation(s)
- Jinhyeong Joo
- Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon 34126, Korea
- IBS School, Korea University of Science and Technology (UST), Daejeon 34113, Korea
| | - Ki Jung Kim
- Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon 34126, Korea
| | - Jiwoon Lim
- Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon 34126, Korea
- IBS School, Korea University of Science and Technology (UST), Daejeon 34113, Korea
| | - Sun Yeong Choi
- Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon 34126, Korea
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Korea
| | - Wuhyun Koh
- Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon 34126, Korea
| | - C Justin Lee
- Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon 34126, Korea
- IBS School, Korea University of Science and Technology (UST), Daejeon 34113, Korea
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8
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Castillo SR, Simone BW, Clark KJ, Devaux P, Ekker SC. Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.13.593977. [PMID: 38798498 PMCID: PMC11118498 DOI: 10.1101/2024.05.13.593977] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2024]
Abstract
DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs are often deployed as pairs, with each arm comprised of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this constraint, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated αDdCBEs that contain modified TALE proteins engineered to recognize all 5' bases. Notably, 5'-T-noncompliant, canonical DdCBEs efficiently edited mtDNA at diverse loci. However, DdCBEs were frequently outperformed by αDdCBEs, which consistently displayed significant improvements in activity and specificity, regardless of the 5'-most bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with DddA tox and its derivatives DddA6, and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes at a single target site. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.
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9
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Wang J, Zhang X, Chen H, Ren H, Zhou M, Zhao Y. Engineered stem cells by emerging biomedical stratagems. Sci Bull (Beijing) 2024; 69:248-279. [PMID: 38101962 DOI: 10.1016/j.scib.2023.12.006] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Revised: 09/24/2023] [Accepted: 11/09/2023] [Indexed: 12/17/2023]
Abstract
Stem cell therapy holds immense potential as a viable treatment for a widespread range of intractable disorders. As the safety of stem cell transplantation having been demonstrated in numerous clinical trials, various kinds of stem cells are currently utilized in medical applications. Despite the achievements, the therapeutic benefits of stem cells for diseases are limited, and the data of clinical researches are unstable. To optimize tthe effectiveness of stem cells, engineering approaches have been developed to enhance their inherent abilities and impart them with new functionalities, paving the way for the next generation of stem cell therapies. This review offers a detailed analysis of engineered stem cells, including their clinical applications and potential for future development. We begin by briefly introducing the recent advances in the production of stem cells (induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs)). Furthermore, we present the latest developments of engineered strategies in stem cells, including engineered methods in molecular biology and biomaterial fields, and their application in biomedical research. Finally, we summarize the current obstacles and suggest future prospects for engineered stem cells in clinical translations and biomedical applications.
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Affiliation(s)
- Jinglin Wang
- Department of Vascular Surgery, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China; Division of Hepatobiliary Surgery and Transplantation Surgery, Department of General Surgery, Nanjing Drum Tower Hospital, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
| | - Xiaoxuan Zhang
- Division of Hepatobiliary Surgery and Transplantation Surgery, Department of General Surgery, Nanjing Drum Tower Hospital, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
| | - Hanxu Chen
- Division of Hepatobiliary Surgery and Transplantation Surgery, Department of General Surgery, Nanjing Drum Tower Hospital, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
| | - Haozhen Ren
- Division of Hepatobiliary Surgery and Transplantation Surgery, Department of General Surgery, Nanjing Drum Tower Hospital, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
| | - Min Zhou
- Department of Vascular Surgery, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China.
| | - Yuanjin Zhao
- Department of Vascular Surgery, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China; Division of Hepatobiliary Surgery and Transplantation Surgery, Department of General Surgery, Nanjing Drum Tower Hospital, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China; Shenzhen Research Institute, Southeast University, Shenzhen 518038, China.
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Abstract
Chronic hepatitis B virus (HBV) infection is a serious disease that currently has no cure. Key forms of HBV include covalently closed circular DNA, which mediates chronic persistence, and integrated DNA, which contributes to immune evasion and carcinogenesis. These forms are not targeted by current therapies; however, gene editing technologies have emerged as promising tools for disrupting HBV DNA. Gene editor-induced double-stranded breaks at precise locations within the HBV genome can induce effects ranging from inactivation of target genes to complete degradation of the target genome. Although promising, several challenges remain in efficacy and safety that require solutions.
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Affiliation(s)
- Henrik Zhang
- Westmead Institute for Medical Research, University of Sydney School of Medicine and Health, 176 Hawkesbury Road, Westmead, NSW 2145, Australia
| | - Thomas Tu
- Westmead Institute for Medical Research, University of Sydney School of Medicine and Health, 176 Hawkesbury Road, Westmead, NSW 2145, Australia.
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11
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Ma J, Zhang P, Zheng M, Wang B, Gao P, Qu L, Zheng F. A strain of Vibrio alginolyticus isolated from Azumapecten farreri and its pathogenic mechanism using CRISPR-Cas9 technology. Biotechnol Lett 2023; 45:1279-1291. [PMID: 37505340 DOI: 10.1007/s10529-023-03394-8] [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: 09/27/2022] [Revised: 04/26/2023] [Accepted: 04/30/2023] [Indexed: 07/29/2023]
Abstract
Scallops have become an important aquaculture species in China because they contain high-quality protein, and scallops are important health food that combines multiple effects and high economic benefits. However, scallop aquaculture is perennially threatened by various pathogenic Vibrio species, leading to great economic losses. We obtained a strain of pathogenic bacteria, identified as Vibrio alginolyticus, from the diseased Azumapecten farreri in the scallop farming area of Huangdao District in 2018, and V. alginolyticus is one of the major shellfish pathogens. We showed that V. alginolyticus was isolated and identified as a pathogen in A. farreri for the first time. In this study, we evaluated its morphology and performed a phylogenetic analysis based on 16S rRNA gene sequencing. In addition, we performed a preliminary analysis of its pathogenic mechanisms. The Hfq protein in V. alginolyticus is an important RNA-binding protein in the quorum-sensing system that not only affects the sensitivity of Vibrio to environmental stress but also regulates a variety of functions, such as cell membrane formation, motility, and virulence towards the host. However, its effect on the pathogenesis of V. alginolyticus to A. farreri is unclear. To further investigate the pathogenic mechanism of the Hfq protein in V. alginolyticus to A. farreri, we used the CRISPR-Cas9 system to target and deplete the hfq gene fragment in V. alginolyticus and obtained the mutant strain V. ΔHfq-. We found that the peripheral flagellum of the mutant strain was lost, which reduced the motility of V. alginolyticus. Therefore, the deletion of target genes by the CRISPR/Cas9 genome editing system confirmed that the Hfq protein played a key role in reducing the ability of V. alginolyticus to infect A. farreri. In conclusion, our current findings provided valuable insights into the healthy culture of scallops.
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Affiliation(s)
- Jingxue Ma
- First Institute of Oceanography MNR, Qingdao, People's Republic of China
- Key Laboratory of Science of Engineering for Marine Ecology and Environment, The First Institute of Oceanography, Ministry of Natural Resources, Qingdao, People's Republic of China
- Laboratory of Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, People's Republic of China
- Qingdao University, Qingdao, People's Republic of China
| | - Peiyu Zhang
- Qingdao University, Qingdao, People's Republic of China
| | - Minggang Zheng
- First Institute of Oceanography MNR, Qingdao, People's Republic of China
- Key Laboratory of Science of Engineering for Marine Ecology and Environment, The First Institute of Oceanography, Ministry of Natural Resources, Qingdao, People's Republic of China
- Laboratory of Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, People's Republic of China
| | - Bo Wang
- First Institute of Oceanography MNR, Qingdao, People's Republic of China
- Key Laboratory of Science of Engineering for Marine Ecology and Environment, The First Institute of Oceanography, Ministry of Natural Resources, Qingdao, People's Republic of China
- Laboratory of Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, People's Republic of China
| | - Ping Gao
- First Institute of Oceanography MNR, Qingdao, People's Republic of China
- Key Laboratory of Science of Engineering for Marine Ecology and Environment, The First Institute of Oceanography, Ministry of Natural Resources, Qingdao, People's Republic of China
- Laboratory of Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, People's Republic of China
| | - Lingyun Qu
- First Institute of Oceanography MNR, Qingdao, People's Republic of China
- Key Laboratory of Science of Engineering for Marine Ecology and Environment, The First Institute of Oceanography, Ministry of Natural Resources, Qingdao, People's Republic of China
- Laboratory of Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, People's Republic of China
| | - Fengrong Zheng
- First Institute of Oceanography MNR, Qingdao, People's Republic of China.
- Key Laboratory of Science of Engineering for Marine Ecology and Environment, The First Institute of Oceanography, Ministry of Natural Resources, Qingdao, People's Republic of China.
- Laboratory of Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, People's Republic of China.
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12
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Mazloum A, Karagyaur M, Chernyshev R, van Schalkwyk A, Jun M, Qiang F, Sprygin A. Post-genomic era in agriculture and veterinary science: successful and proposed application of genetic targeting technologies. Front Vet Sci 2023; 10:1180621. [PMID: 37601766 PMCID: PMC10434572 DOI: 10.3389/fvets.2023.1180621] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Accepted: 07/20/2023] [Indexed: 08/22/2023] Open
Abstract
Gene editing tools have become an indispensable part of research into the fundamental aspects of cell biology. With a vast body of literature having been generated based on next generation sequencing technologies, keeping track of this ever-growing body of information remains challenging. This necessitates the translation of genomic data into tangible applications. In order to address this objective, the generated Next Generation Sequencing (NGS) data forms the basis for targeted genome editing strategies, employing known enzymes of various cellular machinery, in generating organisms with specifically selected phenotypes. This review focuses primarily on CRISPR/Cas9 technology in the context of its advantages over Zinc finger proteins (ZNF) and Transcription activator-like effector nucleases (TALEN) and meganucleases mutagenesis strategies, for use in agricultural and veterinary applications. This review will describe the application of CRISPR/Cas9 in creating modified organisms with custom-made properties, without the undesired non-targeted effects associated with virus vector vaccines and bioactive molecules produced in bacterial systems. Examples of the successful and unsuccessful applications of this technology to plants, animals and microorganisms are provided, as well as an in-depth look into possible future trends and applications in vaccine development, disease resistance and enhanced phenotypic traits will be discussed.
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Affiliation(s)
- Ali Mazloum
- Federal Center for Animal Health, Vladimir, Russia
| | - Maxim Karagyaur
- Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, Moscow, Russia
| | | | - Antoinette van Schalkwyk
- Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort, South Africa
- Department of Biotechnology, University of the Western Cape, Bellville, South Africa
| | - Ma Jun
- School of Life Sciences and Engineering, Foshan University, Foshan, China
| | - Fu Qiang
- School of Life Sciences and Engineering, Foshan University, Foshan, China
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13
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Zhang Z, Bao X, Lin CP. Progress and Prospects of Gene Editing in Pluripotent Stem Cells. Biomedicines 2023; 11:2168. [PMID: 37626665 PMCID: PMC10452926 DOI: 10.3390/biomedicines11082168] [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: 06/30/2023] [Revised: 07/16/2023] [Accepted: 07/18/2023] [Indexed: 08/27/2023] Open
Abstract
Applying programmable nucleases in gene editing has greatly shaped current research in basic biology and clinical translation. Gene editing in human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), is highly relevant to clinical cell therapy and thus should be examined with particular caution. First, since all mutations in PSCs will be carried to all their progenies, off-target edits of editors will be amplified. Second, due to the hypersensitivity of PSCs to DNA damage, double-strand breaks (DSBs) made by gene editing could lead to low editing efficiency and the enrichment of cell populations with defective genomic safeguards. In this regard, DSB-independent gene editing tools, such as base editors and prime editors, are favored due to their nature to avoid these consequences. With more understanding of the microbial world, new systems, such as Cas-related nucleases, transposons, and recombinases, are also expanding the toolbox for gene editing. In this review, we discuss current applications of programmable nucleases in PSCs for gene editing, the efforts researchers have made to optimize these systems, as well as new tools that can be potentially employed for differentiation modeling and therapeutic applications.
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Affiliation(s)
| | | | - Chao-Po Lin
- School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; (Z.Z.); (X.B.)
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14
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Li KL, Nakashima K, Hisata K, Satoh N. Expression and possible functions of a horizontally transferred glycosyl hydrolase gene, GH6-1, in Ciona embryogenesis. EvoDevo 2023; 14:11. [PMID: 37434168 DOI: 10.1186/s13227-023-00215-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2023] [Accepted: 07/01/2023] [Indexed: 07/13/2023] Open
Abstract
BACKGROUND The Tunicata or Urochordata is the only animal group with the ability to synthesize cellulose directly and cellulose is a component of the tunic that covers the entire tunicate body. The genome of Ciona intestinalis type A contains a cellulose synthase gene, CesA, that it acquired via an ancient, horizontal gene transfer. CesA is expressed in embryonic epidermal cells and functions in cellulose production. Ciona CesA is composed of both a glycosyltransferase domain, GT2, and a glycosyl hydrolase domain, GH6, which shows a mutation at a key position and seems functionless. Interestingly, the Ciona genome contains a glycosyl hydrolase gene, GH6-1, in which the GH6 domain seems intact. This suggests expression and possible functions of GH6-1 during Ciona embryogenesis. Is GH6-1 expressed during embryogenesis? If so, in what tissues is the gene expressed? Does GH6-1 serve a function? If so, what is it? Answers to these questions may advance our understanding of evolution of this unique animal group. RESULTS Quantitative reverse transcription PCR and in situ hybridization revealed that GH6-1 is expressed in epidermis of tailbud embryos and in early swimming larvae, a pattern similar to that of CesA. Expression is downregulated at later stages and becomes undetectable in metamorphosed juveniles. The GH6-1 expression level is higher in the anterior-trunk region and caudal-tip regions of late embryos. Single-cell RNA sequencing analysis of the late tailbud stage showed that cells of three clusters with epidermal identity express GH6-1, and that some of them co-express CesA. TALEN-mediated genome editing was used to generate GH6-1 knockout Ciona larvae. Around half of TALEN-electroporated larvae showed abnormal development of adhesive papillae and altered distribution of surface cellulose. In addition, three-fourths of TALEN-electroporated animals failed to complete larval metamorphosis. CONCLUSIONS This study showed that tunicate GH6-1, a gene that originated by horizontal gene transfer of a prokaryote gene, is recruited into the ascidian genome, and that it is expressed and functions in epidermal cells of ascidian embryos. Although further research is required, this observation demonstrates that both CesA and GH6-1 are involved in tunicate cellulose metabolism, impacting tunicate morphology and ecology.
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Affiliation(s)
- Kun-Lung Li
- Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, 904-0495, Japan.
- Institute of Cellular and Organismic Biology, Academia Sinica, Taipei City, 115, Taiwan.
| | - Keisuke Nakashima
- Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, 904-0495, Japan
| | - Kanako Hisata
- Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, 904-0495, Japan
| | - Noriyuki Satoh
- Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, 904-0495, Japan
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15
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Cuevas-Ocaña S, Yang JY, Aushev M, Schlossmacher G, Bear CE, Hannan NRF, Perkins ND, Rossant J, Wong AP, Gray MA. A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells. Int J Mol Sci 2023; 24:10266. [PMID: 37373413 PMCID: PMC10299534 DOI: 10.3390/ijms241210266] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2023] [Revised: 06/09/2023] [Accepted: 06/09/2023] [Indexed: 06/29/2023] Open
Abstract
Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3-6 weeks in order to understand genetic determinants of disease and precision medicine.
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Affiliation(s)
- Sara Cuevas-Ocaña
- Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; (G.S.); (N.D.P.); (M.A.G.)
- Biodiscovery Institute, Translational Medical Sciences, School of Medicine, University of Nottingham, Nottingham NG7 2RD, UK;
| | - Jin Ye Yang
- Programme in Developmental & Stem Cell Biology, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; (J.Y.Y.); (J.R.); (A.P.W.)
| | - Magomet Aushev
- Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Biomedicine West Wing, Centre for Life, Times Square, Newcastle upon Tyne NE1 3BZ, UK;
| | - George Schlossmacher
- Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; (G.S.); (N.D.P.); (M.A.G.)
| | - Christine E. Bear
- Programme in Molecular Medicine, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada;
| | - Nicholas R. F. Hannan
- Biodiscovery Institute, Translational Medical Sciences, School of Medicine, University of Nottingham, Nottingham NG7 2RD, UK;
| | - Neil D. Perkins
- Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; (G.S.); (N.D.P.); (M.A.G.)
| | - Janet Rossant
- Programme in Developmental & Stem Cell Biology, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; (J.Y.Y.); (J.R.); (A.P.W.)
| | - Amy P. Wong
- Programme in Developmental & Stem Cell Biology, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; (J.Y.Y.); (J.R.); (A.P.W.)
| | - Michael A. Gray
- Biosciences Institute, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; (G.S.); (N.D.P.); (M.A.G.)
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16
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Papaioannou NY, Patsali P, Naiisseh B, Papasavva PL, Koniali L, Kurita R, Nakamura Y, Christou S, Sitarou M, Mussolino C, Cathomen T, Kleanthous M, Lederer CW. High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis. Front Genome Ed 2023; 5:1141618. [PMID: 36969374 PMCID: PMC10030607 DOI: 10.3389/fgeed.2023.1141618] [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: 01/10/2023] [Accepted: 02/17/2023] [Indexed: 03/11/2023] Open
Abstract
Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34+ cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34+ cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34+ cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools. Results and Discussion: Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.
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Affiliation(s)
- Nikoletta Y. Papaioannou
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Petros Patsali
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Basma Naiisseh
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Panayiota L. Papasavva
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Lola Koniali
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Ryo Kurita
- Research and Development Department, Central Blood Institute, Blood Service Headquarters Japanese Red Cross Society, Tokyo, Japan
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Research Center, Tsukuba, Japan
| | - Soteroula Christou
- Thalassaemia Centre, State Health Services Organisation of Cyprus, Nicosia, Cyprus
| | - Maria Sitarou
- Thalassaemia Centre, State Health Services Organisation of Cyprus, Larnaca, Cyprus
| | - Claudio Mussolino
- Institute for Transfusion Medicine and Gene Therapy, Medical Center—University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center—University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Marina Kleanthous
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Carsten W. Lederer
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
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17
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Sony SK, Kaul T, Motelb KFA, Thangaraj A, Bharti J, Kaul R, Verma R, Nehra M. CRISPR/Cas9-mediated homology donor repair base editing confers glyphosate resistance to rice ( Oryza sativa L.). FRONTIERS IN PLANT SCIENCE 2023; 14:1122926. [PMID: 36959937 PMCID: PMC10027715 DOI: 10.3389/fpls.2023.1122926] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/13/2022] [Accepted: 02/07/2023] [Indexed: 06/18/2023]
Abstract
Globally, CRISPR-Cas9-based genome editing has ushered in a novel era of crop advancements. Weeds pose serious a threat to rice crop productivity. Among the numerous herbicides, glyphosate [N-(phosphonomethyl)-glycine] has been employed as a post-emergent, broad-spectrum herbicide that represses the shikimate pathway via inhibition of EPSPS (5'-enolpyruvylshikimate-3-phosphate synthase) enzyme in chloroplasts. Here, we describe the development of glyphosate-resistant rice lines by site-specific amino acid substitutions (G172A, T173I, and P177S: GATIPS-mOsEPSPS) and modification of phosphoenolpyruvate-binding site in the native OsEPSPS gene employing fragment knockout and knock-in of homology donor repair (HDR) template harboring desired mutations through CRISPR-Cas9-based genome editing. The indigenously designed two-sgRNA OsEPSPS-NICTK-1_pCRISPR-Cas9 construct harboring rice codon-optimized SpCas9 along with OsEPSPS-HDR template was transformed into rice. Stable homozygous T2 edited rice lines revealed significantly high degree of glyphosate-resistance both in vitro (4 mM/L) and field conditions (6 ml/L; Roundup Ready) in contrast to wild type (WT). Edited T2 rice lines (ER1-6) with enhanced glyphosate resistance revealed lower levels of endogenous shikimate (14.5-fold) in contrast to treated WT but quite similar to WT. ER1-6 lines exhibited increased aromatic amino acid contents (Phe, two-fold; Trp, 2.5-fold; and Tyr, two-fold) than WT. Interestingly, glyphosate-resistant Cas9-free EL1-6 rice lines displayed a significant increment in grain yield (20%-22%) in comparison to WT. Together, results highlighted that the efficacy of GATIPS mutations in OsEPSPS has tremendously contributed in glyphosate resistance (foliar spray of 6 ml/L), enhanced aromatic amino acids, and improved grain yields in rice. These results ensure a novel strategy for weed management without yield penalties, with a higher probability of commercial release.
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18
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Rhiel M, Geiger K, Andrieux G, Rositzka J, Boerries M, Cathomen T, Cornu TI. T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds. Front Genome Ed 2023; 5:1130736. [PMID: 36890979 PMCID: PMC9986454 DOI: 10.3389/fgeed.2023.1130736] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2022] [Accepted: 02/06/2023] [Indexed: 02/22/2023] Open
Abstract
Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the CCR5 and TRAC loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing.
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Affiliation(s)
- Manuel Rhiel
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, Freiburg, Germany
| | - Kerstin Geiger
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, Freiburg, Germany
- Ph.D. Program, Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Geoffroy Andrieux
- Institute of Medical Bioinformatics and Systems Medicine, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- German Cancer Consortium (DKTK), Partner Site Freiburg, and German Cancer Research Center (DKFZ), Heidelberg, Germany
- Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Julia Rositzka
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, Freiburg, Germany
| | - Melanie Boerries
- Institute of Medical Bioinformatics and Systems Medicine, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- German Cancer Consortium (DKTK), Partner Site Freiburg, and German Cancer Research Center (DKFZ), Heidelberg, Germany
- Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Comprehensive Cancer Center Freiburg (CCCF), Medical Center—University of Freiburg, Freiburg, Germany
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, Freiburg, Germany
- Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Tatjana I. Cornu
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, Freiburg, Germany
- Faculty of Medicine, University of Freiburg, Freiburg, Germany
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19
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Abstract
Transcription activator-like effector (TALE) nuclease (TALEN) is the second-generation genome editing tool consisting of TALE protein containing customizable DNA-binding repeats and nuclease domain of FokI enzyme. Each DNA-binding repeat recognizes one base of double-strand DNA, and functional TALEN can be created by a simple modular assembly of these repeats. To easily and efficiently assemble the highly repetitive DNA-binding repeat arrays, various construction systems such as Golden Gate assembly, serial ligation, and ligation-independent cloning have been reported. In this chapter, we summarize the updated situation of these systems and publicly available reagents and protocols, enabling optimal selection of best suited systems for every researcher who wants to utilize TALENs in various research fields.
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Affiliation(s)
- Tetsushi Sakuma
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
| | - Takashi Yamamoto
- Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
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20
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Isola G. Prospective Advances in Genome Editing Investigation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1396:301-313. [DOI: 10.1007/978-981-19-5642-3_19] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
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21
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Mitra S, Anand U, Ghorai M, Kant N, Kumar M, Radha, Jha NK, Swamy MK, Proćków J, de la Lastra JMP, Dey A. Genome editing technologies, mechanisms and improved production of therapeutic phytochemicals: Opportunities and prospects. Biotechnol Bioeng 2023; 120:82-94. [PMID: 36224758 PMCID: PMC10091730 DOI: 10.1002/bit.28260] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2022] [Revised: 09/10/2022] [Accepted: 10/08/2022] [Indexed: 11/09/2022]
Abstract
Plants produce a large number of secondary metabolites, known as phytometabolites that may be employed as medicines, dyes, poisons, and insecticides in the field of medicine, agriculture, and industrial use, respectively. The rise of genome management approaches has promised a factual revolution in genetic engineering. Targeted genome editing in living entities permits the understanding of the biological systems very clearly, and also sanctions to address a wide-ranging objective in the direction of improving features of plant and their yields. The last few years have introduced a number of unique genome editing systems, including transcription activator-like effector nucleases, zinc finger nucleases, and miRNA-regulated clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). Genome editing systems have helped in the transformation of metabolic engineering, allowing researchers to modify biosynthetic pathways of different secondary metabolites. Given the growing relevance of editing genomes in plant research, the exciting novel methods are briefly reviewed in this chapter. Also, this chapter highlights recent discoveries on the CRISPR-based modification of natural products in different medicinal plants.
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Affiliation(s)
- Sicon Mitra
- Department of Biotechnology, School of Engineering & TechnologySharda UniversityGreater NoidaUttar PradeshIndia
| | | | - Mimosa Ghorai
- Department of Life SciencesPresidency UniversityKolkataWest BengalIndia
| | - Nishi Kant
- Department of Chemical EngineeringIndian Institute of Technology DelhiDelhiNew DelhiIndia
| | - Manoj Kumar
- Chemical and Biochemical Processing DivisionICAR‐Central Institute for Research on Cotton TechnologyMumbaiMaharashtraIndia
| | - Radha
- School of Biological and Environmental SciencesShoolini University of Biotechnology and Management SciencesSolanHimachal PradeshIndia
| | - Niraj K. Jha
- Department of Biotechnology, School of Engineering & TechnologySharda UniversityGreater NoidaUttar PradeshIndia
- Department of Biotechnology Engineering and Food TechnologyChandigarh UniversityMohaliPunjabIndia
- Department of Biotechnology, School of Applied & Life SciencesUttaranchal UniversityDehradunUttarakhandIndia
| | - Mallappa K. Swamy
- Department of BiotechnologyEast West First Grade College of ScienceBengaluruKarnatakaIndia
| | - Jarosław Proćków
- Department of Plant Biology, Institute of Environmental BiologyWrocław University of Environmental and Life SciencesWrocławPoland
| | - José M. Pérez de la Lastra
- Biotechnology of Macromolecules Research Group, Department of Life and Earth SciencesInstituto de Productos Naturales y Agrobiología‐Consejo Superior de Investigaciones Científicas, (IPNA‐CSIC)San Cristóbal de La LagunaTenerifeSpain
| | - Abhijit Dey
- Department of Life SciencesPresidency UniversityKolkataWest BengalIndia
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22
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CRISPR-Cas9 Technology for the Creation of Biological Avatars Capable of Modeling and Treating Pathologies: From Discovery to the Latest Improvements. Cells 2022; 11:cells11223615. [PMID: 36429042 PMCID: PMC9688409 DOI: 10.3390/cells11223615] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Revised: 11/10/2022] [Accepted: 11/13/2022] [Indexed: 11/18/2022] Open
Abstract
This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein 9 (CRISPR-Cas9), which have integrated deeply into the research field within a short period due to its effectiveness. It became a standard tool utilized in a broad spectrum of biological and therapeutic applications. Furthermore, reliable disease models are required to improve the quality of healthcare. CRISPR-Cas9 has the potential to diversify our knowledge in genetics by generating cellular models, which can mimic various human diseases to better understand the disease consequences and develop new treatments. Precision in genome editing offered by CRISPR-Cas9 is now paving the way for gene therapy to expand in clinical trials to treat several genetic diseases in a wide range of species. This review article will discuss genome-editing tools: CRISPR-Cas9, Zinc Finger Nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs). It will also encompass the importance of CRISPR-Cas9 technology in generating cellular disease models for novel therapeutics, its applications in gene therapy, and challenges with novel strategies to enhance its specificity.
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Li R, Wang Q, She K, Lu F, Yang Y. CRISPR/Cas systems usher in a new era of disease treatment and diagnosis. MOLECULAR BIOMEDICINE 2022; 3:31. [PMID: 36239875 PMCID: PMC9560888 DOI: 10.1186/s43556-022-00095-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2022] [Accepted: 09/27/2022] [Indexed: 11/21/2022] Open
Abstract
The discovery and development of the CRISPR/Cas system is a milestone in precise medicine. CRISPR/Cas nucleases, base-editing (BE) and prime-editing (PE) are three genome editing technologies derived from CRISPR/Cas. In recent years, CRISPR-based genome editing technologies have created immense therapeutic potential with safe and efficient viral or non-viral delivery systems. Significant progress has been made in applying genome editing strategies to modify T cells and hematopoietic stem cells (HSCs) ex vivo and to treat a wide variety of diseases and disorders in vivo. Nevertheless, the clinical translation of this unique technology still faces many challenges, especially targeting, safety and delivery issues, which require further improvement and optimization. In addition, with the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), CRISPR-based molecular diagnosis has attracted extensive attention. Growing from the specific set of molecular biological discoveries to several active clinical trials, CRISPR/Cas systems offer the opportunity to create a cost-effective, portable and point-of-care diagnosis through nucleic acid screening of diseases. In this review, we describe the development, mechanisms and delivery systems of CRISPR-based genome editing and focus on clinical and preclinical studies of therapeutic CRISPR genome editing in disease treatment as well as its application prospects in therapeutics and molecular detection.
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Affiliation(s)
- Ruiting Li
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, 610041, Sichuan, China
| | - Qin Wang
- School of Pharmacy, Southwest Minzu University, Chengdu, 610225, Sichuan, China
| | - Kaiqin She
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, 610041, Sichuan, China
- Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Fang Lu
- Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Yang Yang
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, 610041, Sichuan, China.
- Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
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Demirel F, Germec M, Turhan I. Fermentable sugars production from wheat bran and rye bran: response surface model optimization of dilute sulfuric acid hydrolysis. ENVIRONMENTAL TECHNOLOGY 2022; 43:3779-3800. [PMID: 34029158 DOI: 10.1080/09593330.2021.1934563] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Accepted: 05/17/2021] [Indexed: 06/12/2023]
Abstract
ABSTRACTOptimization of hydrolysis conditions of lignocellulosic biomass is crucial to able to produce value-added products by fermentation. This study not only determines optimal dilute sulfuric acid (H2SO4) hydrolysis conditions of wheat bran (WB) and rye bran (RB) by using one-factor-at-a-time method and subsequently Box-Behnken design but also elucidates chemical composition of hydrolysates yielded under optimal hydrolysis conditions. Based on the results, optimal hydrolysis conditions of WB and RB were 121 and 130°C of temperature, 1/8 and 1/8 w/v of solid to liquid ratio, 2.66 and 1.58% v/v of dilute H2SO4 ratio, and 30 and 16 min of implementation time, respectively. Hydrolysates obtained from WB and RB at these conditions contained 72.7 (0.58 g sugar/g biomass) and 89.4 g/L (0.72 g sugar/g biomass) of reducing sugar concentration, respectively. Hydrolysis rates of WB and RB were 87.79 and 91.33%, respectively. Main reducing sugar in RB hydrolysate was glucose with 31.17 g/L (0.25 g glucose/g biomass) while glucose and xylose were the main monosaccharides with 20.90 (0.17 g glucose/g biomass) and 18.69 g/L (0.15 g xylose/g biomass) in WB hydrolysate, respectively. With acidic hydrolysis of WB and RB, inhibitors such as phenolics, 5-Hydroxymethylfurfural, 2-Furaldehyde (not for RB), acetic acid, and formic acid (not for WB) formed. Catalytic efficiency values of H2SO4 for WB and RB were 15.2 and 24.4 g /g, respectively, indicating that inhibitor concentration in WB hydrolysate was higher than that of RB. These results indicated that WB and RB have a high potential in production of value-added products by fermentation.
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Affiliation(s)
- Fadime Demirel
- Department of Food Engineering, Akdeniz University, Antalya, Turkey
| | - Mustafa Germec
- Department of Food Engineering, Akdeniz University, Antalya, Turkey
| | - Irfan Turhan
- Department of Food Engineering, Akdeniz University, Antalya, Turkey
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Huang C, Li Q, Li J. Site-specific genome editing in treatment of inherited diseases: possibility, progress, and perspectives. MEDICAL REVIEW (BERLIN, GERMANY) 2022; 2:471-500. [PMID: 37724161 PMCID: PMC10388762 DOI: 10.1515/mr-2022-0029] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/02/2022] [Accepted: 10/11/2022] [Indexed: 09/20/2023]
Abstract
Advancements in genome editing enable permanent changes of DNA sequences in a site-specific manner, providing promising approaches for treating human genetic disorders caused by gene mutations. Recently, genome editing has been applied and achieved significant progress in treating inherited genetic disorders that remain incurable by conventional therapy. Here, we present a review of various programmable genome editing systems with their principles, advantages, and limitations. We introduce their recent applications for treating inherited diseases in the clinic, including sickle cell disease (SCD), β-thalassemia, Leber congenital amaurosis (LCA), heterozygous familial hypercholesterolemia (HeFH), etc. We also discuss the paradigm of ex vivo and in vivo editing and highlight the promise of somatic editing and the challenge of germline editing. Finally, we propose future directions in delivery, cutting, and repairing to improve the scope of clinical applications.
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Affiliation(s)
- Chao Huang
- Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, Zhejiang, China
| | - Qing Li
- State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Jinsong Li
- Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, Zhejiang, China
- State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
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Sun W, Jiang Z, Jiang W, Yang R. Universal chimeric antigen receptor T cell therapy - The future of cell therapy: A review providing clinical evidence. Cancer Treat Res Commun 2022; 33:100638. [PMID: 36184307 DOI: 10.1016/j.ctarc.2022.100638] [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: 07/29/2022] [Revised: 09/15/2022] [Accepted: 09/21/2022] [Indexed: 12/14/2022]
Abstract
Autologous CAR-T therapy has shown promising outcomes in the treatment of tumors, particularly hematological malignancies over the past years. However, the application of autologous CAR-T therapy is limited, due to undesirable patient and/or peripheral blood characteristics, the high cost and long time period of manufacturing, and other challenges. Universal CAR-T therapy could overcome major limitations of autologous CAR-T therapy. In this review, we described the research and development status of universal CAR-T therapy for hematological malignancies. In addition, we also summarized the challenges had been encountered and the current solutions.
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Affiliation(s)
| | | | - Wen Jiang
- Gobroad Healthcare Group, Beijing, China
| | - Rui Yang
- Gobroad Healthcare Group, Beijing, China.
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Kong W, Lu C, Ding Y, Meng Y. Update of treatment for Gaucher disease. Eur J Pharmacol 2022; 926:175023. [DOI: 10.1016/j.ejphar.2022.175023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2022] [Revised: 04/18/2022] [Accepted: 05/09/2022] [Indexed: 11/03/2022]
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Houghton BC, Panchal N, Haas SA, Chmielewski KO, Hildenbeutel M, Whittaker T, Mussolino C, Cathomen T, Thrasher AJ, Booth C. Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP. Front Genome Ed 2022; 4:828489. [PMID: 35677600 PMCID: PMC9168036 DOI: 10.3389/fgeed.2022.828489] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Accepted: 04/06/2022] [Indexed: 12/27/2022] Open
Abstract
X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the SH2D1A gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of SAP cDNA at the first exon of the SH2D1A locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of SAP gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients.
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Affiliation(s)
- Benjamin C. Houghton
- Molecular and Cellular Immunology, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
| | - Neelam Panchal
- Molecular and Cellular Immunology, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
| | - Simone A. Haas
- Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Kay O. Chmielewski
- Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Faculty of Biology, University of Freiburg, Freiburg, Germany
| | - Markus Hildenbeutel
- Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Thomas Whittaker
- Molecular and Cellular Immunology, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
| | - Claudio Mussolino
- Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Adrian J Thrasher
- Molecular and Cellular Immunology, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
| | - Claire Booth
- Molecular and Cellular Immunology, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
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Yamashiro K, Ikegaya Y, Matsumoto N. In Utero Electroporation for Manipulation of Specific Neuronal Populations. MEMBRANES 2022; 12:membranes12050513. [PMID: 35629839 PMCID: PMC9147339 DOI: 10.3390/membranes12050513] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/17/2022] [Revised: 04/27/2022] [Accepted: 05/06/2022] [Indexed: 02/05/2023]
Abstract
The complexity of brain functions is supported by the heterogeneity of brain tissue and millisecond-scale information processing. Understanding how complex neural circuits control animal behavior requires the precise manipulation of specific neuronal subtypes at high spatiotemporal resolution. In utero electroporation, when combined with optogenetics, is a powerful method for precisely controlling the activity of specific neurons. Optogenetics allows for the control of cellular membrane potentials through light-sensitive ion channels artificially expressed in the plasma membrane of neurons. Here, we first review the basic mechanisms and characteristics of in utero electroporation. Then, we discuss recent applications of in utero electroporation combined with optogenetics to investigate the functions and characteristics of specific regions, layers, and cell types. These techniques will pave the way for further advances in understanding the complex neuronal and circuit mechanisms that underlie behavioral outputs.
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Affiliation(s)
- Kotaro Yamashiro
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan; (K.Y.); (Y.I.)
| | - Yuji Ikegaya
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan; (K.Y.); (Y.I.)
- Institute for AI and Beyond, The University of Tokyo, Tokyo 113-0033, Japan
- Center for Information and Neural Networks, National Institute of Information and Communications Technology, Osaka 565-0871, Japan
| | - Nobuyoshi Matsumoto
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan; (K.Y.); (Y.I.)
- Institute for AI and Beyond, The University of Tokyo, Tokyo 113-0033, Japan
- Correspondence:
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Genome Editing: A Promising Approach for Achieving Abiotic Stress Tolerance in Plants. Int J Genomics 2022; 2022:5547231. [PMID: 35465040 PMCID: PMC9033345 DOI: 10.1155/2022/5547231] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Accepted: 03/24/2022] [Indexed: 12/26/2022] Open
Abstract
The susceptibility of crop plants towards abiotic stresses is highly threatening to assure global food security as it results in almost 50% annual yield loss. To address this issue, several strategies like plant breeding and genetic engineering have been used by researchers from time to time. However, these approaches are not sufficient to ensure stress resilience due to the complexity associated with the inheritance of abiotic stress adaptive traits. Thus, researchers were prompted to develop novel techniques with high precision that can address the challenges connected to the previous strategies. Genome editing is the latest approach that is in the limelight for improving the stress tolerance of plants. It has revolutionized crop research due to its versatility and precision. The present review is an update on the different genome editing tools used for crop improvement so far and the various challenges associated with them. It also highlights the emerging potential of genome editing for developing abiotic stress-resilient crops.
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Li G, Li X, Zhuang S, Wang L, Zhu Y, Chen Y, Sun W, Wu Z, Zhou Z, Chen J, Huang X, Wang J, Li D, Li W, Wang H, Wei W. Gene editing and its applications in biomedicine. SCIENCE CHINA. LIFE SCIENCES 2022; 65:660-700. [PMID: 35235150 PMCID: PMC8889061 DOI: 10.1007/s11427-021-2057-0] [Citation(s) in RCA: 40] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 10/27/2021] [Accepted: 12/06/2021] [Indexed: 02/06/2023]
Abstract
The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats (CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.
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Affiliation(s)
- Guanglei Li
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Xiangyang Li
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
| | - Songkuan Zhuang
- Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, 518035, China
| | - Liren Wang
- Shanghai Frontiers Science Research Base of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Yifan Zhu
- Shanghai Frontiers Science Research Base of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Yangcan Chen
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
- Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China
| | - Wen Sun
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
- Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China
| | - Zeguang Wu
- Biomedical Pioneering Innovation Center, Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, 100871, China
| | - Zhuo Zhou
- Biomedical Pioneering Innovation Center, Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, 100871, China
| | - Jia Chen
- Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
| | - Xingxu Huang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.
| | - Jin Wang
- Department of Clinical Laboratory, Shenzhen Institute of Translational Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, 518035, China.
| | - Dali Li
- Shanghai Frontiers Science Research Base of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, China.
| | - Wei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
- University of Chinese Academy of Sciences, Beijing, 100049, China.
- Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China.
- Bejing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101, China.
- HIT Center for Life Sciences, Harbin Institute of Technology, Harbin, 150001, China.
| | - Haoyi Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
- University of Chinese Academy of Sciences, Beijing, 100049, China.
- Institute for Stem Cell and Regenerative Medicine, Chinese Academy of Sciences, Beijing, 100101, China.
| | - Wensheng Wei
- Biomedical Pioneering Innovation Center, Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, 100871, China.
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Ravendran S, Hernández SS, König S, Bak RO. CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome. Front Genome Ed 2022; 4:793010. [PMID: 35373187 PMCID: PMC8969908 DOI: 10.3389/fgeed.2022.793010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Accepted: 02/14/2022] [Indexed: 12/17/2022] Open
Abstract
Defects in the DOCK8 gene causes combined immunodeficiency termed DOCK8 immunodeficiency syndrome (DIDS). DIDS previously belonged to the disease category of autosomal recessive hyper IgE syndrome (AR-HIES) but is now classified as a combined immunodeficiency (CID). This genetic disorder induces early onset of susceptibility to severe recurrent viral and bacterial infections, atopic diseases and malignancy resulting in high morbidity and mortality. This pathological state arises from impairment of actin polymerization and cytoskeletal rearrangement, which induces improper immune cell migration-, survival-, and effector functions. Owing to the severity of the disease, early allogenic hematopoietic stem cell transplantation is recommended even though it is associated with risk of unintended adverse effects, the need for compatible donors, and high expenses. So far, no alternative therapies have been developed, but the monogenic recessive nature of the disease suggests that gene therapy may be applied. The advent of the CRISPR/Cas gene editing system heralds a new era of possibilities in precision gene therapy, and positive results from clinical trials have already suggested that the tool may provide definitive cures for several genetic disorders. Here, we discuss the potential application of different CRISPR/Cas-mediated genetic therapies to correct the DOCK8 gene. Our findings encourage the pursuit of CRISPR/Cas-based gene editing approaches, which may constitute more precise, affordable, and low-risk definitive treatment options for DOCK8 deficiency.
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Affiliation(s)
| | | | | | - Rasmus O. Bak
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
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33
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Forner J, Kleinschmidt D, Meyer EH, Fischer A, Morbitzer R, Lahaye T, Schöttler MA, Bock R. Targeted introduction of heritable point mutations into the plant mitochondrial genome. NATURE PLANTS 2022; 8:245-256. [PMID: 35301443 PMCID: PMC8940627 DOI: 10.1038/s41477-022-01108-y] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Accepted: 02/10/2022] [Indexed: 05/05/2023]
Abstract
The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.
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Affiliation(s)
- Joachim Forner
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany
| | - Dennis Kleinschmidt
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany
| | - Etienne H Meyer
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany
- Institut für Pflanzenphysiologie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany
| | - Axel Fischer
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany
| | - Robert Morbitzer
- ZMBP, Allgemeine Genetik, Universität Tübingen, Tübingen, Germany
| | - Thomas Lahaye
- ZMBP, Allgemeine Genetik, Universität Tübingen, Tübingen, Germany
| | - Mark A Schöttler
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany
| | - Ralph Bock
- Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany.
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Ashoti A, Limone F, van Kranenburg M, Alemany A, Baak M, Vivié J, Piccioni F, Dijkers PF, Creyghton M, Eggan K, Geijsen N. Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen. PLoS One 2022; 17:e0263262. [PMID: 35176052 PMCID: PMC8853573 DOI: 10.1371/journal.pone.0263262] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Accepted: 01/17/2022] [Indexed: 12/26/2022] Open
Abstract
Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens.
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MESH Headings
- CRISPR-Cas Systems
- Cell Survival
- Gene Expression Regulation
- Homeodomain Proteins/antagonists & inhibitors
- Homeodomain Proteins/genetics
- Homeodomain Proteins/metabolism
- Humans
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology
- Muscle Cells/metabolism
- Muscle Cells/pathology
- Muscular Dystrophy, Facioscapulohumeral/genetics
- Muscular Dystrophy, Facioscapulohumeral/metabolism
- Muscular Dystrophy, Facioscapulohumeral/pathology
- Myoblasts/metabolism
- Myoblasts/pathology
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Affiliation(s)
- Ator Ashoti
- Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
- * E-mail: (AA); (FL); (NG); (KE)
| | - Francesco Limone
- Department of Stem Cell and Regenerative Biology, Harvard University Cambridge, MA, United States of America
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, United States of America
- * E-mail: (AA); (FL); (NG); (KE)
| | - Melissa van Kranenburg
- Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
| | - Anna Alemany
- Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
| | - Mirna Baak
- Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
| | - Judith Vivié
- Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
- Single Cell Discoveries, Utrecht, The Netherlands
| | | | - Pascale F. Dijkers
- Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
| | - Menno Creyghton
- Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
| | - Kevin Eggan
- Department of Stem Cell and Regenerative Biology, Harvard University Cambridge, MA, United States of America
- Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, United States of America
- * E-mail: (AA); (FL); (NG); (KE)
| | - Niels Geijsen
- Hubrecht Institute, Developmental Biology and Stem Cell Research, Utrecht, The Netherlands
- * E-mail: (AA); (FL); (NG); (KE)
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Rezalotfi A, Fritz L, Förster R, Bošnjak B. Challenges of CRISPR-Based Gene Editing in Primary T Cells. Int J Mol Sci 2022; 23:ijms23031689. [PMID: 35163611 PMCID: PMC8835901 DOI: 10.3390/ijms23031689] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Accepted: 01/29/2022] [Indexed: 12/30/2022] Open
Abstract
Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T-cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system, allowing for the deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs that were important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches.
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Affiliation(s)
- Alaleh Rezalotfi
- Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany; (A.R.); (L.F.); (R.F.)
| | - Lea Fritz
- Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany; (A.R.); (L.F.); (R.F.)
| | - Reinhold Förster
- Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany; (A.R.); (L.F.); (R.F.)
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, 30625 Hannover, Germany
- German Centre for Infection Research (DZIF), Partner Site Hannover, 30625 Hannover, Germany
| | - Berislav Bošnjak
- Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany; (A.R.); (L.F.); (R.F.)
- Correspondence: ; Tel.: +49-511-532-9731
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Li Q, Jia E, Yan Y, Ma R, Dong J, Ma P. Using the Strategy of Inducing and Genetically Transforming Plant Suspension Cells to Produce High Value-Added Bioactive Substances. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2022; 70:699-710. [PMID: 35018771 DOI: 10.1021/acs.jafc.1c05712] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Plants can produce many functional bioactive substances. The suspension cell system of plants can be constructed based on its characteristics to realize the large-scale production of valuable products. In this review, we mainly talk about the main strategies, elicitation, and genetic transformation to improve the yield of active substances by using this system. Meanwhile, we focus on the challenges hiding in the practical application and the future prospects and provide new ideas and the theoretical basis for obtaining numerous bioactive substances from plants.
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Affiliation(s)
- Qian Li
- College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China
| | - Entong Jia
- College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China
| | - Yurong Yan
- College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China
| | - Rui Ma
- Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, Jilin 130033, People's Republic of China
| | - Juane Dong
- College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China
| | - Pengda Ma
- College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China
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An Overview of Zebrafish Modeling Methods in Drug Discovery and Development. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1387:145-169. [PMID: 34961915 DOI: 10.1007/5584_2021_684] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
Animal studies are recognized as a significant step forward in the bridging between drug discovery and clinical applications. Animal models, due to their relative genetic, molecular, physiological, and even anatomical similarities to humans, can provide a suitable platform for unraveling the mechanisms underlying human diseases and discovering new therapeutic approaches as well. Recently, zebrafish has attracted attention as a valuable experimental and pharmacological model in drug discovery and development studies due to its prominent characteristics such as the high degree of genetic similarity with humans, genetic manipulability, and prominent clinical features. Since advancing a theory to a valid and reliable observation requires the manipulation of animals, it is, therefore, essential to use efficient modeling methods appropriate to the different aspects of experimental conditions. In this context, applying several various approaches such as using chemicals, pathogens, and genetic manipulation approaches allows zebrafish development into a preferable model that mimics some human disease pathophysiology. Thus, such modeling approaches not only can provide a framework for a comprehensive understanding of the human disease mechanisms that have a counterpart in zebrafish but also can pave the way for discovering new drugs that are accompanied by higher amelioration effects on different human diseases.
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Cui Z, Liu H, Zhang H, Huang Z, Tian R, Li L, Fan W, Chen Y, Chen L, Zhang S, Das BC, Severinov K, Hitzeroth II, Debata PR, Jin Z, Liu J, Huang Z, Xie W, Xie H, Lang B, Ma J, Weng H, Tian X, Hu Z. The comparison of ZFNs, TALENs, and SpCas9 by GUIDE-seq in HPV-targeted gene therapy. MOLECULAR THERAPY. NUCLEIC ACIDS 2021; 26:1466-1478. [PMID: 34938601 PMCID: PMC8655392 DOI: 10.1016/j.omtn.2021.08.008] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Accepted: 08/10/2021] [Indexed: 12/26/2022]
Abstract
Zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR-associated Cas9 endonucleases are three major generations of genome editing tools. However, no parallel comparison about the efficiencies and off-target activity of the three nucleases has been reported, which is critical for the final clinical decision. We for the first time developed the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method in ZFNs and TALENs with novel bioinformatics algorithms to evaluate the off-targets. By targeting human papillomavirus 16 (HPV16), we compared the performance of ZFNs, TALENs, and SpCas9 in vivo. Our data showed that ZFNs with similar targets could generate distinct massive off-targets (287–1,856), and the specificity could be reversely correlated with the counts of middle “G” in zinc finger proteins (ZFPs). We also compared the TALENs with different N-terminal domains (wild-type [WT]/αN/βN) and G recognition modules (NN/NH) and found the design (αN or NN) to improve the efficiency of TALEN inevitably increased off-targets. Finally, our results showed that SpCas9 was more efficient and specific than ZFNs and TALENs. Specifically, SpCas9 had fewer off-target counts in URR (SpCas9, n = 0; TALEN, n = 1; ZFN, n = 287), E6 (SpCas9, n = 0; TALEN, n = 7), and E7 (SpCas9, n = 4; TALEN, n = 36). Taken together, we suggest that for HPV gene therapies, SpCas9 is a more efficient and safer genome editing tool. Our off-target data could be used to improve the design of ZFNs and TALENs, and the universal in vivo off-target detection pipeline for three generations of artificial nucleases provided useful tools for genome engineering-based gene therapy.
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Affiliation(s)
- Zifeng Cui
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
| | - Hui Liu
- Department of Pathology, Xi’an People’s Hospital (Xi’an Fourth Hospital), Shaanxi, China
| | - Hongfeng Zhang
- Department of Pathology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
| | - Zhaoyue Huang
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
| | - Rui Tian
- Center for Translational Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, Guangdong, China
| | - Lifang Li
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
| | - Weiwen Fan
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
| | - Yili Chen
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
| | - Lijie Chen
- Graduate School, Bengbu Medical College, Bengbu, Anhui 233000, China
| | - Sen Zhang
- Graduate School, Bengbu Medical College, Bengbu, Anhui 233000, China
| | - Bhudev C. Das
- Amity Institute of Molecular Medicine & Stem Cell Research, Amity University Uttar Pradesh, Sector 125, Noida 201313, India
| | - Konstantin Severinov
- Skolkovo Institute of Science and Technology 100 Novaya Street, Skolkovo, Moscow Region 143025, Russia
| | - Inga Isabel Hitzeroth
- Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Cape Town 7701, South Africa
| | - Priya Ranjan Debata
- Department of Zoology, North Orissa University, Takatpur, Baripada, Odisha 757003, India
| | - Zhuang Jin
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
| | - Jiashuo Liu
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
| | - Zheying Huang
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
| | - Weiling Xie
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
| | - Hongxian Xie
- Generulor Company Bio-X Lab, Guangzhou 510006, Guangdong, China
| | - Bin Lang
- School of Health Sciences and Sports, Macao Polytechnic Institute, Macao 999078, China
| | - Ji Ma
- Department of Pathology, The Central Hospital of Sui Zhou, Hubei, China
| | - Haiyan Weng
- Department of Pathology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230036, China
- Intelligent Pathology Institute, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230036, China
- Corresponding author: Haiyan Weng, Department of Pathology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230036, China.
| | - Xun Tian
- Department of Obstetrics and Gynecology, Academician Expert Workstation, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei, China
- Corresponding author: Xun Tian, Department of Obstetrics and Gynecology, Academician Expert Workstation, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, Hubei, China.
| | - Zheng Hu
- Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China
- Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
- Corresponding author: Zheng Hu, Department of Gynecological Oncology, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan 2nd Road, Yuexiu, Guangzhou 510080, Guangdong, China.
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Schneider N, Sundaresan Y, Gopalakrishnan P, Beryozkin A, Hanany M, Levanon EY, Banin E, Ben-Aroya S, Sharon D. Inherited retinal diseases: Linking genes, disease-causing variants, and relevant therapeutic modalities. Prog Retin Eye Res 2021; 89:101029. [PMID: 34839010 DOI: 10.1016/j.preteyeres.2021.101029] [Citation(s) in RCA: 100] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2021] [Revised: 11/11/2021] [Accepted: 11/16/2021] [Indexed: 12/11/2022]
Abstract
Inherited retinal diseases (IRDs) are a clinically complex and heterogenous group of visual impairment phenotypes caused by pathogenic variants in at least 277 nuclear and mitochondrial genes, affecting different retinal regions, and depleting the vision of affected individuals. Genes that cause IRDs when mutated are unique by possessing differing genotype-phenotype correlations, varying inheritance patterns, hypomorphic alleles, and modifier genes thus complicating genetic interpretation. Next-generation sequencing has greatly advanced the identification of novel IRD-related genes and pathogenic variants in the last decade. For this review, we performed an in-depth literature search which allowed for compilation of the Global Retinal Inherited Disease (GRID) dataset containing 4,798 discrete variants and 17,299 alleles published in 31 papers, showing a wide range of frequencies and complexities among the 194 genes reported in GRID, with 65% of pathogenic variants being unique to a single individual. A better understanding of IRD-related gene distribution, gene complexity, and variant types allow for improved genetic testing and therapies. Current genetic therapeutic methods are also quite diverse and rely on variant identification, and range from whole gene replacement to single nucleotide editing at the DNA or RNA levels. IRDs and their suitable therapies thus require a range of effective disease modelling in human cells, granting insight into disease mechanisms and testing of possible treatments. This review summarizes genetic and therapeutic modalities of IRDs, provides new analyses of IRD-related genes (GRID and complexity scores), and provides information to match genetic-based therapies such as gene-specific and variant-specific therapies to the appropriate individuals.
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Affiliation(s)
- Nina Schneider
- Department of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel
| | - Yogapriya Sundaresan
- Department of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel
| | - Prakadeeswari Gopalakrishnan
- Department of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel
| | - Avigail Beryozkin
- Department of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel
| | - Mor Hanany
- Department of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel
| | - Erez Y Levanon
- The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, 5290002, Israel
| | - Eyal Banin
- Department of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel
| | - Shay Ben-Aroya
- The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, 5290002, Israel
| | - Dror Sharon
- Department of Ophthalmology, Hadassah Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel.
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Büning H, Fehse B, Ivics Z, Kochanek S, Koehl U, Kupatt C, Mussolino C, Nettelbeck DM, Schambach A, Uckert W, Wagner E, Cathomen T. Gene Therapy "Made in Germany": A Historical Perspective, Analysis of the Status Quo, and Recommendations for Action by the German Society for Gene Therapy. Hum Gene Ther 2021; 32:987-996. [PMID: 34662229 DOI: 10.1089/hum.2021.29178.hbu] [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] [Indexed: 12/16/2022] Open
Abstract
Gene therapies have been successfully applied to treat severe inherited and acquired disorders. Although research and development are sufficiently well funded in Germany and while the output of scientific publications and patents is comparable with the leading nations in gene therapy, the country lags noticeably behind with regard to the number of both clinical studies and commercialized gene therapy products. In this article, we give a historical perspective on the development of gene therapy in Germany, analyze the current situation from the standpoint of the German Society for Gene Therapy (DG-GT), and define recommendations for action that would enable our country to generate biomedical and economic advantages from innovations in this sector, instead of merely importing advanced therapy medicinal products. Inter alia, we propose (1) to harmonize and simplify regulatory licensing processes to enable faster access to advanced therapies, and (2) to establish novel coordination, support and funding structures that facilitate networking of the key players. Such a center would provide the necessary infrastructure and know-how to translate cell and gene therapies to patients on the one hand, and pave the way for commercialization of these promising and innovative technologies on the other. Hence, these courses of action would not only benefit the German biotech and pharma landscape but also the society and the patients in need of new treatment options.
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Affiliation(s)
- Hildegard Büning
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Boris Fehse
- Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, Germany
| | - Zoltán Ivics
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | | | - Ulrike Koehl
- Fraunhofer Institute for Cell Therapy and Immunology (IZI) and Institute of Clinical Immunology, University of Leipzig, Leipzig, Germany.,Institute for Cellular Therapeutics, Hannover Medical School, Hannover, Germany
| | - Christian Kupatt
- Klinik und Poliklinik für Innere Medizin I, Klinikum rechts der Isar, Technical University Munich, Munich, Germany.,German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Munich, Germany
| | - Claudio Mussolino
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency (CCI), Medical Faculty, University of Freiburg, Freiburg, Germany
| | - Dirk M Nettelbeck
- Clinical Cooperation Unit Virotherapy, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Axel Schambach
- Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
| | - Wolfgang Uckert
- Department of Molecular Cell Biology and Gene Therapy, Max-Delbrück-Center for Molecular Medicine (MDC), Berlin, Germany
| | - Ernst Wagner
- Pharmaceutical Biotechnology, Center for System-based Drug Research, Center for NanoScience (CeNS), Ludwig-Maximilians-University of Munich, Munich, Germany
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany.,Center for Chronic Immunodeficiency (CCI), Medical Faculty, University of Freiburg, Freiburg, Germany
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González Castro N, Bjelic J, Malhotra G, Huang C, Alsaffar SH. Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies. Int J Mol Sci 2021; 22:10355. [PMID: 34638696 PMCID: PMC8509008 DOI: 10.3390/ijms221910355] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2021] [Revised: 08/26/2021] [Accepted: 09/24/2021] [Indexed: 12/15/2022] Open
Abstract
Recent advances in programmable nucleases including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) have propelled genome editing from explorative research to clinical and industrial settings. Each technology, however, features distinct modes of action that unevenly impact their applicability across the entire genome and are often tested under significantly different conditions. While CRISPR-Cas is currently leading the field due to its versatility, quick adoption, and high degree of support, it is not without limitations. Currently, no technology can be regarded as ideal or even applicable to every case as the context dictates the best approach for genetic modification within a target organism. In this review, we implement a four-pillar framework (context, feasibility, efficiency, and safety) to assess the main genome editing platforms, as a basis for rational decision-making by an expanding base of users, regulators, and consumers. Beyond carefully considering their specific use case with the assessment framework proposed here, we urge stakeholders interested in genome editing to independently validate the parameters of their chosen platform prior to commitment. Furthermore, safety across all applications, particularly in clinical settings, is a paramount consideration and comprehensive off-target detection strategies should be incorporated within workflows to address this. Often neglected aspects such as immunogenicity and the inadvertent selection of mutants deficient for DNA repair pathways must also be considered.
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Affiliation(s)
- Nicolás González Castro
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
| | - Jan Bjelic
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
| | - Gunya Malhotra
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
| | - Cong Huang
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
| | - Salman Hasan Alsaffar
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
- Biotechnology Department, Environment and Life Sciences Research Center, Kuwait Institute for Scientific Research, Shuwaikh 13109, Kuwait
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Chuang CK, Lin WM. Points of View on the Tools for Genome/Gene Editing. Int J Mol Sci 2021; 22:9872. [PMID: 34576035 PMCID: PMC8470269 DOI: 10.3390/ijms22189872] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2021] [Revised: 08/26/2021] [Accepted: 09/09/2021] [Indexed: 12/25/2022] Open
Abstract
Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.
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Affiliation(s)
- Chin-Kai Chuang
- Animal Technology Research Center, Division of Animal Technology, Agricultural Technology Research Institute, No. 52, Kedong 2nd Rd., Zhunan Township, Miaoli County 35053, Taiwan;
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Atkins A, Chung CH, Allen AG, Dampier W, Gurrola TE, Sariyer IK, Nonnemacher MR, Wigdahl B. Off-Target Analysis in Gene Editing and Applications for Clinical Translation of CRISPR/Cas9 in HIV-1 Therapy. Front Genome Ed 2021; 3:673022. [PMID: 34713260 PMCID: PMC8525399 DOI: 10.3389/fgeed.2021.673022] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Accepted: 06/21/2021] [Indexed: 12/26/2022] Open
Abstract
As genome-editing nucleases move toward broader clinical applications, the need to define the limits of their specificity and efficiency increases. A variety of approaches for nuclease cleavage detection have been developed, allowing a full-genome survey of the targeting landscape and the detection of a variety of repair outcomes for nuclease-induced double-strand breaks. Each approach has advantages and disadvantages relating to the means of target-site capture, target enrichment mechanism, cellular environment, false discovery, and validation of bona fide off-target cleavage sites in cells. This review examines the strengths, limitations, and origins of the different classes of off-target cleavage detection systems including anchored primer enrichment (GUIDE-seq), in situ detection (BLISS), in vitro selection libraries (CIRCLE-seq), chromatin immunoprecipitation (ChIP) (DISCOVER-Seq), translocation sequencing (LAM PCR HTGTS), and in vitro genomic DNA digestion (Digenome-seq and SITE-Seq). Emphasis is placed on the specific modifications that give rise to the enhanced performance of contemporary techniques over their predecessors and the comparative performance of techniques for different applications. The clinical relevance of these techniques is discussed in the context of assessing the safety of novel CRISPR/Cas9 HIV-1 curative strategies. With the recent success of HIV-1 and SIV-1 viral suppression in humanized mice and non-human primates, respectively, using CRISPR/Cas9, rigorous exploration of potential off-target effects is of critical importance. Such analyses would benefit from the application of the techniques discussed in this review.
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Affiliation(s)
- Andrew Atkins
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, United States
- Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Cheng-Han Chung
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, United States
- Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Alexander G. Allen
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, United States
- Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Will Dampier
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, United States
- Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Theodore E. Gurrola
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, United States
- Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Ilker K. Sariyer
- Department of Neuroscience and Center for Neurovirology, Temple University Lewis Katz School of Medicine, Philadelphia, PA, United States
| | - Michael R. Nonnemacher
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, United States
- Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Brian Wigdahl
- Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, United States
- Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
- Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, United States
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Arnesen JA, Hoof JB, Kildegaard HF, Borodina I. Genome Editing of Eukarya. Metab Eng 2021. [DOI: 10.1002/9783527823468.ch10] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
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Xu M, Weng Q, Ji J. Applications and advances of CRISPR/Cas9 in animal cancer model. Brief Funct Genomics 2021; 19:235-241. [PMID: 32124927 DOI: 10.1093/bfgp/elaa002] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2019] [Revised: 01/07/2020] [Indexed: 01/18/2023] Open
Abstract
The recent developments of clustered regularly interspaced short palindromic repeats(CRISPR)/-associate protein 9 (CRISPR/Cas9) have got scientific interests due to the straightforward, efficient and versatile talents of it. Furthermore, the CRISPR/Cas9 system has democratized access to gene editing in many biological fields, including cancer. Cancer development is a multistep process caused by innate and acquired mutations and leads to the initiation and progression of tumorigenesis. It is obvious that establishing appropriate animal cancer models which can simulate human cancers is crucial for cancer research currently. Since the emergence of CRISPR/Cas9, considerable efforts have been taken by researchers to apply this technology in generating animal cancer models. Although there is still a long way to go we are happy to see the achievements we have made and the promising future we have.
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Smith T, Singh P, Chmielewski KO, Bloom K, Cathomen T, Arbuthnot P, Ely A. Improved Specificity and Safety of Anti-Hepatitis B Virus TALENs Using Obligate Heterodimeric FokI Nuclease Domains. Viruses 2021; 13:v13071344. [PMID: 34372550 PMCID: PMC8310341 DOI: 10.3390/v13071344] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Accepted: 07/02/2021] [Indexed: 01/04/2023] Open
Abstract
Persistent hepatitis B virus (HBV) infection remains a serious medical problem worldwide, with an estimated global burden of 257 million carriers. Prophylactic and therapeutic interventions, in the form of a vaccine, immunomodulators, and nucleotide and nucleoside analogs, are available. Vaccination, however, offers no therapeutic benefit to chronic sufferers and has had a limited impact on infection rates. Although immunomodulators and nucleotide and nucleoside analogs have been licensed for treatment of chronic HBV, cure rates remain low. Transcription activator-like effector nucleases (TALENs) designed to bind and cleave viral DNA offer a novel therapeutic approach. Importantly, TALENs can target covalently closed circular DNA (cccDNA) directly with the potential of permanently disabling this important viral replicative intermediate. Potential off-target cleavage by engineered nucleases leading to toxicity presents a limitation of this technology. To address this, in the context of HBV gene therapy, existing TALENs targeting the viral core and surface open reading frames were modified with second- and third-generation FokI nuclease domains. As obligate heterodimers these TALENs prevent target cleavage as a result of FokI homodimerization. Second-generation obligate heterodimeric TALENs were as effective at silencing viral gene expression as first-generation counterparts and demonstrated an improved specificity in a mouse model of HBV replication.
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Affiliation(s)
- Tiffany Smith
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
| | - Prashika Singh
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
| | - Kay Ole Chmielewski
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg & Medical Faculty, University of Freiburg, 79106 Freiburg, Germany; (K.O.C.); (T.C.)
| | - Kristie Bloom
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg & Medical Faculty, University of Freiburg, 79106 Freiburg, Germany; (K.O.C.); (T.C.)
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
| | - Abdullah Ely
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa; (T.S.); (P.S.); (K.B.); (P.A.)
- Correspondence: ; Tel.: +27-(0)11-717-2561
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Morvan MG, Teque F, Ye L, Moreno ME, Wang J, VandenBerg S, Stoddart CA, Kan YW, Levy JA. Genetically edited CD34 + cells derived from human iPS cells in vivo but not in vitro engraft and differentiate into HIV-resistant cells. Proc Natl Acad Sci U S A 2021; 118:e2102404118. [PMID: 33975958 PMCID: PMC8158014 DOI: 10.1073/pnas.2102404118] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34+ cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34+ cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34+ cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34+ cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.
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Affiliation(s)
- Maelig G Morvan
- Department of Medicine, Division of Hematology and Oncology, University of California, San Francisco, CA 94143-1270
| | - Fernando Teque
- Department of Medicine, Division of Hematology and Oncology, University of California, San Francisco, CA 94143-1270
| | - Lin Ye
- Department of Medicine, Institute of Human Genetics, University of California, San Francisco, CA 94143
| | - Mary E Moreno
- Department of Medicine, Division of Experimental Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94110
| | - Jiaming Wang
- Department of Medicine, Institute of Human Genetics, University of California, San Francisco, CA 94143
| | - Scott VandenBerg
- Helen Diller Family Comprehensive Cancer Center, Biorepository and Tissue Biomarker Technology Core, University of California, San Francisco, CA 94143-0875
| | - Cheryl A Stoddart
- Department of Medicine, Division of Experimental Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94110
| | - Yuet Wai Kan
- Department of Medicine, Institute of Human Genetics, University of California, San Francisco, CA 94143;
| | - Jay A Levy
- Department of Medicine, Division of Hematology and Oncology, University of California, San Francisco, CA 94143-1270;
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Manzari MT, Shamay Y, Kiguchi H, Rosen N, Scaltriti M, Heller DA. Targeted drug delivery strategies for precision medicines. NATURE REVIEWS. MATERIALS 2021; 6:351-370. [PMID: 34950512 PMCID: PMC8691416 DOI: 10.1038/s41578-020-00269-6] [Citation(s) in RCA: 486] [Impact Index Per Article: 121.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 11/24/2020] [Indexed: 05/05/2023]
Abstract
Progress in the field of precision medicine has changed the landscape of cancer therapy. Precision medicine is propelled by technologies that enable molecular profiling, genomic analysis, and optimized drug design to tailor treatments for individual patients. Although precision medicines have resulted in some clinical successes, the use of many potential therapeutics has been hindered by pharmacological issues, including toxicities and drug resistance. Drug delivery materials and approaches have now advanced to a point where they can enable the modulation of a drug's pharmacological parameters without compromising the desired effect on molecular targets. Specifically, they can modulate a drug's pharmacokinetics, stability, absorption, and exposure to tumours and healthy tissues, and facilitate the administration of synergistic drug combinations. This Review highlights recent progress in precision therapeutics and drug delivery, and identifies opportunities for strategies to improve the therapeutic index of cancer drugs, and consequently, clinical outcomes.
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Affiliation(s)
- Mandana T. Manzari
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- These authors have contributed equally to this work
| | - Yosi Shamay
- Faculty of Biomedical Engineering, Technion-Israel Institute of Technology, Haifa, Israel
- These authors have contributed equally to this work
| | - Hiroto Kiguchi
- Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA
- These authors have contributed equally to this work
| | - Neal Rosen
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Weill Cornell Medical College, New York, NY, USA
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer, New York, NY, USA
| | - Maurizio Scaltriti
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer, New York, NY, USA
- Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Daniel A. Heller
- Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Weill Cornell Medical College, New York, NY, USA
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Karuppusamy KV, Babu P, Thangavel S. The Strategies and Challenges of CCR5 Gene Editing in Hematopoietic Stem and Progenitor Cells for the Treatment of HIV. Stem Cell Rev Rep 2021; 17:1607-1618. [PMID: 33788143 DOI: 10.1007/s12015-021-10145-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/24/2021] [Indexed: 12/29/2022]
Abstract
HIV infection continues to be a serious health issue with an alarming global spread, owing to the fact that attempts at developing an effective vaccine or a permanent cure remains futile. So far, the only available treatment for the clinical management of HIV is the combined Anti-Retroviral Therapy (cART), but the long-term cART is associated with metabolic changes, organ damages, and development and transmission of drug resistant HIV strains. Thus, there is a need for the development of one-time curative treatment for HIV infection. The allogeneic transplantation with the Hematopoietic Stem and Progenitor cells (HSPCs) having 32 bp deletion in Chemokine receptor 5 gene (CCR5 Δ32) demonstrated successful HIV remission in the Berlin and London patients, and highlighted that transplantation of CCR5 null HSPCs is a promising approach for a long- term HIV remission. The advent of gene editing technologies offers a new choice of generating ex vivo CCR5 ablated allogeneic or autologous HSPCs for stem cell transplantation into HIV patients. Many groups are attempting CCR5 disruption in HSPCs using various gene-editing strategies. At least two such studies, involving CCR5 gene editing in HSPCs have entered the clinical trials. This review aims to outline the strategies taken for CCR5 gene editing and discuss the challenges associated with the development of CCR5 manipulated HSPCs for the gene therapy of HIV infection.
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Affiliation(s)
- Karthik V Karuppusamy
- Centre for Stem Cell Research (A unit of inStem, Bengaluru), Christian Medical College, Vellore, Tamil Nadu, India.,Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Prathibha Babu
- Centre for Stem Cell Research (A unit of inStem, Bengaluru), Christian Medical College, Vellore, Tamil Nadu, India.,Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Saravanabhavan Thangavel
- Centre for Stem Cell Research (A unit of inStem, Bengaluru), Christian Medical College, Vellore, Tamil Nadu, India. .,Manipal Academy of Higher Education, Manipal, Karnataka, India.
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50
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Liu J, Wang S, Wang H, Luo B, Cai Y, Li X, Zhang Y, Wang X. Rapid generation of tomato male-sterile lines with a marker use for hybrid seed production by CRISPR/Cas9 system. MOLECULAR BREEDING : NEW STRATEGIES IN PLANT IMPROVEMENT 2021; 41:25. [PMID: 37309421 PMCID: PMC10236056 DOI: 10.1007/s11032-021-01215-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/22/2020] [Accepted: 02/15/2021] [Indexed: 06/13/2023]
Abstract
Owing to their superior agronomic performance, the hybrids of vegetable crops are currently applied extensively. However, effective hybrid production requires a laborious manual emasculation to ensure the purity of hybrid seeds in tomato because of the lack of an effective male sterility system. Here, we created two types of tomato nuclear male-sterile lines with different screening markers in a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Co-knockouts of male sterile 1035 (Ms1035) and glutathione S-transferase (GSTAA) created a male-sterile line marked by a green hypocotyl. The Ms1035 biallelic mutation was introduced into the woolly tomato background, resulting in the linkage of male sterility and a non-woolly phenotype. Two types of male-sterile lines were easily selected at the seedling stage by hypocotyl color or trichome density and further showed high seed purity during hybrid seed production. Our work established the procedure for a rapid transfer of the male-sterile phenotype to the parents of hybrids without extra-modification by the CRISPR/Cas9 system that can be practically applied to hybrid seed production in tomato. This method will be the basis and example for sterile parent creation of multiple crops for hybrid production with the CRISPR/Cas9 system. Supplementary Information The online version contains supplementary material available at 10.1007/s11032-021-01215-2.
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Affiliation(s)
- Jianwei Liu
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, 712100 Shaanxi China
| | - Shufen Wang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, 712100 Shaanxi China
| | - Hao Wang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, 712100 Shaanxi China
| | - Bote Luo
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, 712100 Shaanxi China
| | - Yiyong Cai
- Xi’an Jinpeng Seedlings Co., Ltd., Yangling, China
| | - Xiaodong Li
- Xi’an Jinpeng Seedlings Co., Ltd., Yangling, China
| | - Yanfeng Zhang
- Hybrid Rapeseed Research Center of Shaanxi Province, Yangling, China
| | - Xiaofeng Wang
- State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, 712100 Shaanxi China
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