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1
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Yang K, Cai L, Zhao Y, Cheng H, Zhou R. Optimization of genome editing by CRISPR ribonucleoprotein for high efficiency of germline transmission of Sox9 in zebrafish. N Biotechnol 2025; 86:47-54. [PMID: 39848539 DOI: 10.1016/j.nbt.2025.01.009] [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: 09/06/2024] [Revised: 01/17/2025] [Accepted: 01/20/2025] [Indexed: 01/25/2025]
Abstract
Primordial germ cells (PGCs) are the first germline stem cells to emerge during early embryonic development and are essential for the propagation and survival of species. Genome editing creates mutagenesis possibilities in vivo, but the generation of precise mutations in PGCs is still challenging. Here, we report an optimized approach for highly efficient genome editing via introducing biallelic variations in early embryos in zebrafish. We adopted an extended, GC-rich, and chemically modified sgRNA along with microinjection of the CRISPR ribonucleoprotein (RNP) complex into the yolk sac at the 1-cell stage. We found that genome editing of Sox9a generated a high proportion of heterozygotes with edited alleles in the F1 generation, indicating biallelic editing. Deep sequencing and mapping the edited cells from early embryos to future tissues revealed that the edited founder has a dominantly edited allele, sox9a M1, accounting for over 99 % of alleles in the testis. Specifically, all offspring of the founder inherited the edited allele, suggesting nearly complete editing of the alleles in early germline cells. Overall, the optimization delineates biallelic editing of sox9a in early embryos and transmission of edited alleles to offspring, thus presenting a method to create a desired genetic mutation line of Sox9a avoiding lengthy traditional crossbreeding.
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Affiliation(s)
- Kangning Yang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430072, China
| | - Le Cai
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430072, China
| | - Yu Zhao
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430072, China
| | - Hanhua Cheng
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430072, China.
| | - Rongjia Zhou
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430072, China.
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2
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Xie L, Jakutis G, Dooley CM, Guenther S, Kontarakis Z, Howard SP, Juan T, Stainier DYR. Induction of a transcriptional adaptation response by RNA destabilization events. EMBO Rep 2025; 26:2262-2279. [PMID: 40128410 PMCID: PMC12069562 DOI: 10.1038/s44319-025-00427-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2024] [Revised: 03/03/2025] [Accepted: 03/10/2025] [Indexed: 03/26/2025] Open
Abstract
Transcriptional adaptation (TA) is a cellular process whereby mRNA-destabilizing mutations are associated with the transcriptional upregulation of so-called adapting genes. The nature of the TA-triggering factor(s) remains unclear, namely whether an mRNA-borne premature termination codon or the subsequent mRNA decay process, and/or its products, elicits TA. Here, working with mouse Actg1, we first establish two types of perturbations that lead to mRNA destabilization: Cas9-induced mutations predicted to lead to mutant mRNA decay, and Cas13d-mediated mRNA cleavage. We find that both types of perturbations are effective in degrading Actg1 mRNA, and that they both upregulate Actg2. Notably, increased chromatin accessibility at the Actg2 locus was observed only in the Cas9-induced mutant cells but not in the Cas13d-targeted cells, suggesting that chromatin remodeling is not required for Actg2 upregulation. We further show that ribozyme-mediated Actg1 pre-mRNA cleavage also leads to a robust upregulation of Actg2, and that this upregulation is again independent of chromatin remodeling. Together, these data highlight the critical role of RNA destabilization events as a trigger for TA, or at least a TA-like response.
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Affiliation(s)
- Lihan Xie
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Hessen, 61231, Germany
| | - Gabrielius Jakutis
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Hessen, 61231, Germany
| | - Christopher M Dooley
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Hessen, 61231, Germany
| | - Stefan Guenther
- ECCPS Bioinformatics and Deep Sequencing Platform, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Hessen, 61231, Germany
| | - Zacharias Kontarakis
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Hessen, 61231, Germany
- Genome Engineering and Measurement Laboratory (GEML), Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland
- Functional Genomics Center Zürich, ETH Zürich/University of Zürich, Zürich, 8057, Switzerland
| | - Sarah P Howard
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Hessen, 61231, Germany
| | - Thomas Juan
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Hessen, 61231, Germany.
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Hessen, 61231, Germany.
- Excellence Cluster Cardio-Pulmonary Institute (CPI), Bad Nauheim, Giessen, Frankfurt, Germany.
- Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, 75 185, Sweden.
| | - Didier Y R Stainier
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Hessen, 61231, Germany.
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Hessen, 61231, Germany.
- Excellence Cluster Cardio-Pulmonary Institute (CPI), Bad Nauheim, Giessen, Frankfurt, Germany.
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3
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Duong TB, Fernandes AT, Ravisankar P, Talbot JC, Waxman JS. Tbx1-dependent and independent pathways promote six gene expression downstream of retinoic acid signaling to determine cardiomyocyte number in zebrafish. Dev Biol 2025; 524:17-28. [PMID: 40311730 DOI: 10.1016/j.ydbio.2025.04.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2024] [Revised: 03/26/2025] [Accepted: 04/28/2025] [Indexed: 05/03/2025]
Abstract
Tight regulation of retinoic acid (RA) levels is critical for normal heart development in vertebrates, with early RA signaling restricting the size of the cardiac progenitor field within the anterior lateral plate mesoderm (ALPM). However, the regulatory networks by which RA signaling limits the size of the cardiac progenitor field and consequently cardiomyocyte (CM) number are not fully understood. Here, we identified that the expression of the transcription factors six1b and six2a, whose orthologs regulate outflow tract (OFT) development in mice, are expanded within the ALPM of RA-deficient zebrafish embryos. At 48 h post-fertilization (hpf), RA-deficient six1b;six2a double mutants, but not single six1b or six2a mutants, had a reduction in the number of surplus CMs relative to RA-deficient wild-type embryos. The expansion of six1b, as well as fgf8a, within the ALPM were dependent on tbx1, a factor that is also expanded within the ALPM of RA-deficient zebrafish embryos. However, the restriction of six2a expression by RA was independent of Tbx1. Consistent with a bifurcation of pathways downstream of RA signaling, loss of function experiments demonstrates that tbx1 expansion alone does not contribute to the surplus CMs in RA-deficient embryos. Together, our data indicate that both Tbx1-dependent and independent pathways restrict Six dosage downstream of RA within the ALPM to pattern the CM progenitor field and establish proper heart size.
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Affiliation(s)
- Tiffany B Duong
- Molecular Genetics Graduate Program, University of Cincinnati College of Medicine, Cincinnati, OH, USA; Molecular Cardiovascular Biology Division and Heart Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
| | - Andrew T Fernandes
- Molecular Cardiovascular Biology Division and Heart Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Molecular and Developmental Biology Graduate Program, University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Padmapriyadarshini Ravisankar
- Molecular Cardiovascular Biology Division and Heart Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
| | - Jared C Talbot
- School of Biology and Ecology, University of Maine, Orono, ME, USA
| | - Joshua S Waxman
- Molecular Cardiovascular Biology Division and Heart Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
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4
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Juan T, Molina T, Xie L, Papadopoulou S, Cardoso B, Jha SG, Stainier DY. A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish. PLoS Genet 2025; 21:e1011594. [PMID: 39919116 PMCID: PMC11856399 DOI: 10.1371/journal.pgen.1011594] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2024] [Revised: 02/25/2025] [Accepted: 01/26/2025] [Indexed: 02/09/2025] Open
Abstract
Precise regulation of gene expression is essential to understand a wide range of biological processes. Control over gene expression can be achieved using site-directed recombinases and endonucleases whose efficiency is variable and dependent on the genomic context. Here, we develop a self-cleaving ribozyme-based tool to control mRNA levels of endogenous targets in zebrafish. Using an in vivo reporter strategy, we first show that inserting the T3H48 self-cleaving ribozyme in an intron enables rapid pre-mRNA cleavage, with up to 20-fold reduction in expression, and that this ribozyme displays superior activity compared with other ribozymes. We then inserted the T3H48 ribozyme in the second intron of the albino gene using a CRISPR/Cas9 strategy and observed a pigmentation phenotype similar to that in the mutant. Using a base-editing strategy to inactivate the ribozyme, we also show that this phenotype is reversible, illustrating the specificity of the approach. In addition, we generated a Flippase- and Cre-activatable version of the T3H48 ribozyme, called RiboFlip, to control the mRNA levels of the albino gene. RiboFlip activation induced mRNA knockdown and also recapitulated the albino mutant phenotype. Furthermore, we show that a Cre- and Dre-controllable Gal4/UAS reporter in the RiboFlip cassette can label knocked-down cells independently of the expression of the target gene. Altogether, we introduce the RiboFlip cassette as a flexible tool to control endogenous gene expression in a vertebrate model and as an alternative to existing conditional knockdown strategies.
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Affiliation(s)
- Thomas Juan
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
- Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany
- Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
| | - Tonatiuh Molina
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
| | - Lihan Xie
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
| | - Sofia Papadopoulou
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
| | - Bárbara Cardoso
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
| | - Shivam Govind Jha
- Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
| | - Didier Y.R. Stainier
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
- Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany
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5
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Zhang L, Zhou J. Zebrafish: A smart tool for heart disease research. JOURNAL OF FISH BIOLOGY 2024; 105:1487-1500. [PMID: 37824489 DOI: 10.1111/jfb.15585] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/22/2023] [Revised: 09/07/2023] [Accepted: 10/09/2023] [Indexed: 10/14/2023]
Abstract
The increasing prevalence of heart disease poses a significant threat to human survival and safety. However, the current treatments available for heart disease are quite limited. Therefore, it is important to utilize suitable animal models that can accurately simulate the physiological characteristics of heart disease. This would help improve our understanding of this disease and aid in the development of new treatment methods and drugs. Zebrafish heart not only exhibits similarities to mammalian hearts, but they also share ~70% of homologous genes with humans. Utilizing zebrafish as an alternative to expensive and time-consuming mammalian models offers numerous advantages. Zebrafish models can be easily established and maintained, and compound screening and genetic methods allow for the development of various economical and easily controlled zebrafish and zebrafish embryonic heart disease models in a short period of time. Consequently, zebrafish have become a powerful tool for exploring the pathological mechanisms of heart disease and identifying new effective genes. In this review, we summarize recent studies on different zebrafish models of heart disease. We also describe the techniques and protocols used to develop zebrafish models of myocardial infarction, heart failure, and congenital heart disease, including surgical procedures, forward and reverse genetics, and drug and combination screening. This review aims to promote the utilization of zebrafish models in investigating diverse pathological mechanisms of heart disease, enhancing our knowledge and comprehension of heart disease, and offering novel insights and objectives for exploring the prevention and treatment of heart disease.
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Affiliation(s)
- Lantian Zhang
- Education Branch, Chongqing Publishing Group, Chongqing, China
| | - Jinrun Zhou
- Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Qingdao, China
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6
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Hishida R, Ishiguro K, Yamanaka T, Toyokuni S, Matsui H. Homozygous slc25a20 zebrafish mutant reveals insights into carnitine-acylcarnitine translocase deficiency pathogenesis. Mol Genet Metab Rep 2024; 41:101165. [PMID: 39650084 PMCID: PMC11625244 DOI: 10.1016/j.ymgmr.2024.101165] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2024] [Revised: 11/15/2024] [Accepted: 11/18/2024] [Indexed: 12/11/2024] Open
Abstract
The SLC25A20 gene encodes carnitine-acylcarnitine translocase (CACT), facilitating the transport of long-chain acylcarnitine required for energy production via β-oxidation into the mitochondria. Loss-of-function mutations in this gene lead to CACT deficiency, a rare autosomal recessive disorder of fatty acid metabolism characterized by severe symptoms including cardiomyopathy, hepatic dysfunction, rhabdomyolysis, hypoketotic hypoglycemia, and hyperammonemia, often resulting in neonatal mortality. Here, we utilized CRISPR/Cas9 gene editing to isolate slc25a20 mutant zebrafish. Homozygous mutants displayed significant lethality, with the majority succumbing before reaching maturity. However, we identified a notably rare homozygous individual that survived into adulthood, prompting a histological examination. Firstly, we observed adipose tissue accumulation at various sites in the homozygous mutant. The mutant heart exhibited hypertrophy, along with degenerated myocardial and muscle cells containing numerous eosinophilic nuclei. Additionally, we found no large oil droplet vacuoles in the mutant liver; however, the hepatocytes displayed numerous small vacuoles resembling lipid droplets. Iron deposition was evident in the spleen and parts of the liver. Overall, our slc25a20 zebrafish mutant displayed tissue pathologies analogous to human CACT deficiency, suggesting its potential as a pathological model contributing to the elucidation of pathogenesis and the improvement/development of therapies for CACT deficiency.
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Affiliation(s)
- Ryuichi Hishida
- Department of Neuroscience of Disease, Brain Research Institute, Niigata University, Niigata 951-8585, Japan
| | - Kohei Ishiguro
- Department of Neuroscience of Disease, Brain Research Institute, Niigata University, Niigata 951-8585, Japan
| | - Tomoyuki Yamanaka
- Department of Neuroscience of Disease, Brain Research Institute, Niigata University, Niigata 951-8585, Japan
| | - Shinya Toyokuni
- Department of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
- Center for Low-temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
| | - Hideaki Matsui
- Department of Neuroscience of Disease, Brain Research Institute, Niigata University, Niigata 951-8585, Japan
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7
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Song B, Bae S. Genome editing using CRISPR, CAST, and Fanzor systems. Mol Cells 2024; 47:100086. [PMID: 38909984 PMCID: PMC11278801 DOI: 10.1016/j.mocell.2024.100086] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2024] [Revised: 06/14/2024] [Accepted: 06/18/2024] [Indexed: 06/25/2024] Open
Abstract
Genetic engineering technologies are essential not only for basic science but also for generating animal models for therapeutic applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system, derived from adapted prokaryotic immune responses, has led to unprecedented advancements in the field of genome editing because of its ability to precisely target and edit genes in a guide RNA-dependent manner. The discovery of various types of CRISPR-Cas systems, such as CRISPR-associated transposons (CASTs), has resulted in the development of novel genome editing tools. Recently, research has expanded to systems associated with obligate mobile element guided activity (OMEGA) RNAs, including ancestral CRISPR-Cas and eukaryotic Fanzor systems, which are expected to complement the conventional CRISPR-Cas systems. In this review, we briefly introduce the features of various CRISPR-Cas systems and their application in diverse animal models.
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Affiliation(s)
- Beomjong Song
- Department of Anatomy, College of Medicine, Soonchunhyang University, Cheonan 33151, Republic of Korea.
| | - Sangsu Bae
- Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Medical Research Center of Genomic Medicine Institute, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Cancer Research Institute, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Institute of Molecular Biology and Genetics, Seoul National University, Seoul 08826, Republic of Korea.
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8
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Pollitt EJG, Sánchez-Posada J, Snashall CM, Derrick CJ, Noël ES. Llgl1 mediates timely epicardial emergence and establishment of an apical laminin sheath around the trabeculating cardiac ventricle. Development 2024; 151:dev202482. [PMID: 38940292 PMCID: PMC11234374 DOI: 10.1242/dev.202482] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2023] [Accepted: 05/31/2024] [Indexed: 06/29/2024]
Abstract
During heart development, the embryonic ventricle becomes enveloped by the epicardium, which adheres to the outer apical surface of the heart. This is concomitant with onset of ventricular trabeculation, where a subset of cardiomyocytes lose apicobasal polarity and delaminate basally from the ventricular wall. Llgl1 regulates the formation of apical cell junctions and apicobasal polarity, and we investigated its role in ventricular wall maturation. We found that llgl1 mutant zebrafish embryos exhibit aberrant apical extrusion of ventricular cardiomyocytes. While investigating apical cardiomyocyte extrusion, we identified a basal-to-apical shift in laminin deposition from the internal to the external ventricular wall. We find that epicardial cells express several laminin subunits as they adhere to the ventricle, and that the epicardium is required for laminin deposition on the ventricular surface. In llgl1 mutants, timely establishment of the epicardial layer is disrupted due to delayed emergence of epicardial cells, resulting in delayed apical deposition of laminin on the ventricular surface. Together, our analyses reveal an unexpected role for Llgl1 in correct timing of epicardial development, supporting integrity of the ventricular myocardial wall.
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Affiliation(s)
- Eric J. G. Pollitt
- School of Biosciences and Bateson Centre, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Juliana Sánchez-Posada
- School of Biosciences and Bateson Centre, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Corinna M. Snashall
- School of Biosciences and Bateson Centre, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Christopher J. Derrick
- School of Biosciences and Bateson Centre, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
| | - Emily S. Noël
- School of Biosciences and Bateson Centre, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
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9
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Hammond FR, Lewis A, Pollara G, Tomlinson GS, Noursadeghi M, Kiss-Toth E, Elks PM. Tribbles1 is host protective during in vivo mycobacterial infection. eLife 2024; 13:e95980. [PMID: 38896446 PMCID: PMC11186633 DOI: 10.7554/elife.95980] [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: 01/10/2024] [Accepted: 05/28/2024] [Indexed: 06/21/2024] Open
Abstract
Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production, and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo, we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown/knockout exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of proinflammatory factors il-1β and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.
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Affiliation(s)
- Ffion R Hammond
- The Bateson Centre, School of Medicine and Population Health, Faculty of Health, University of SheffieldSheffieldUnited Kingdom
| | - Amy Lewis
- The Bateson Centre, School of Medicine and Population Health, Faculty of Health, University of SheffieldSheffieldUnited Kingdom
| | - Gabriele Pollara
- Division of Infection & Immunity, University College LondonLondonUnited Kingdom
| | - Gillian S Tomlinson
- Division of Infection & Immunity, University College LondonLondonUnited Kingdom
| | - Mahdad Noursadeghi
- Division of Infection & Immunity, University College LondonLondonUnited Kingdom
| | - Endre Kiss-Toth
- The Bateson Centre, School of Medicine and Population Health, Faculty of Health, University of SheffieldSheffieldUnited Kingdom
| | - Philip M Elks
- The Bateson Centre, School of Medicine and Population Health, Faculty of Health, University of SheffieldSheffieldUnited Kingdom
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Zhang C, Yang L, Zhang H, Wu F, Zhang Y, Zhang K, Wu C, Li R, Dong M, Zhao S, Song H. TAF1 is needed for the proliferation and maturation of thyroid follicle cells via Notch signaling. Am J Physiol Endocrinol Metab 2024; 326:E832-E841. [PMID: 38656129 DOI: 10.1152/ajpendo.00403.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Revised: 04/15/2024] [Accepted: 04/16/2024] [Indexed: 04/26/2024]
Abstract
Thyroid dysgenesis (TD) is the common pathogenic mechanism of congenital hypothyroidism (CH). In addition, known pathogenic genes are limited to those that are directly involved in thyroid development. To identify additional candidate pathogenetic genes, we performed forward genetic screening for TD in zebrafish, followed by positional cloning. The candidate gene was confirmed in vitro using the Nthy-ori 3.1 cell line and in vivo using a zebrafish model organism. We obtained a novel zebrafish line with thyroid dysgenesis and identified the candidate pathogenetic mutation TATA-box binding protein associated Factor 1 (taf1) by positional cloning. Further molecular studies revealed that taf1 was needed for the proliferation of thyroid follicular cells by binding to the NOTCH1 promoter region. Knockdown of TAF1 impaired the proliferation and maturation of thyroid cells, thereby leading to thyroid dysplasia. This study showed that TAF1 promoted Notch signaling and that this association played a pivotal role in thyroid development.NEW & NOTEWORTHY In our study, we obtained a novel zebrafish line with thyroid dysgenesis (TD) and identified the candidate pathogenetic mutation TATA-box binding protein associated Factor 1 (taf1). Further researches revealed that taf1 was required for thyroid follicular cells by binding to the NOTCH1 promoter region. Our findings revealed a novel role of TAF1 in thyroid morphogenesis.
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Affiliation(s)
- Caoxu Zhang
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Liu Yang
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Haiyang Zhang
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Fengyao Wu
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Yue Zhang
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Kaiwen Zhang
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Chenyang Wu
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Rui Li
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Mei Dong
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Shuangxia Zhao
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
| | - Huaidong Song
- Department of Molecular Diagnostics & Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People's Hospital, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China
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11
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Liu LL, Yin YQ, Ma KX, Xing JC, Ren XX, Huang JY, Liao M, Qi WB, Huang LH. Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library. Vet Microbiol 2024; 293:110099. [PMID: 38677125 DOI: 10.1016/j.vetmic.2024.110099] [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: 01/27/2024] [Revised: 04/15/2024] [Accepted: 04/20/2024] [Indexed: 04/29/2024]
Abstract
Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.
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Affiliation(s)
- Le-le Liu
- State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou 510642, China; Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China; National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou 510642, China; Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou 510642, China
| | - You-Qin Yin
- State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou 510642, China; Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China; National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou 510642, China; Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou 510642, China
| | - Kai-Xiong Ma
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China
| | - Jin-Chao Xing
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China
| | - Xing-Xing Ren
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China
| | - Jin-Yu Huang
- State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou 510642, China; Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China; National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou 510642, China; Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou 510642, China
| | - Ming Liao
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China; National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou 510642, China; Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou 510642, China
| | - Wen-Bao Qi
- State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou 510642, China; Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China; National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou 510642, China; Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou 510642, China.
| | - Li-Hong Huang
- State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou 510642, China; Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, Guangzhou 510642, China; National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou 510642, China; Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou 510642, China.
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12
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Ye X, Lin J, Chen Q, Lv J, Liu C, Wang Y, Wang S, Wen X, Lin F. An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line. MARINE BIOTECHNOLOGY (NEW YORK, N.Y.) 2024; 26:588-598. [PMID: 38652190 DOI: 10.1007/s10126-024-10320-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/19/2024] [Accepted: 04/18/2024] [Indexed: 04/25/2024]
Abstract
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293 T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.
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Affiliation(s)
- Xiaokang Ye
- Guangdong Provincial Key Laboratory of Marine Biotechnology, Institute of Marine Sciences, Shantou University, 243 Daxue Road, Shantou, 515063, China
| | - Jiali Lin
- College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China
| | - Qiuji Chen
- Guangdong Provincial Key Laboratory of Marine Biotechnology, Institute of Marine Sciences, Shantou University, 243 Daxue Road, Shantou, 515063, China
| | - Jiehuan Lv
- Guangdong Provincial Key Laboratory of Marine Biotechnology, Institute of Marine Sciences, Shantou University, 243 Daxue Road, Shantou, 515063, China
| | - Chunsheng Liu
- Guangdong Provincial Key Laboratory of Marine Biotechnology, Institute of Marine Sciences, Shantou University, 243 Daxue Road, Shantou, 515063, China
| | - Yuping Wang
- Guangdong Provincial Key Laboratory of Marine Biotechnology, Institute of Marine Sciences, Shantou University, 243 Daxue Road, Shantou, 515063, China
| | - Shuqi Wang
- Guangdong Provincial Key Laboratory of Marine Biotechnology, Institute of Marine Sciences, Shantou University, 243 Daxue Road, Shantou, 515063, China
| | - Xiaobo Wen
- College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China
| | - Fan Lin
- Guangdong Provincial Key Laboratory of Marine Biotechnology, Institute of Marine Sciences, Shantou University, 243 Daxue Road, Shantou, 515063, China.
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13
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Vasconcelos Komninakis S, Domingues W, Saeed Sanabani S, Angelo Folgosi V, Neves Barbosa I, Casseb J. CRISPR/CAS as a Powerful Tool for Human Immunodeficiency Virus Cure: A Review. AIDS Res Hum Retroviruses 2024; 40:363-375. [PMID: 38164106 DOI: 10.1089/aid.2022.0148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2024] Open
Abstract
Despite care and the availability of effective antiretroviral treatment, some human immunodeficiency virus (HIV)-infected individuals suffer from neurocognitive disorders associated with HIV (HAND) that significantly affect their quality of life. The different types of HAND can be divided into asymptomatic neurocognitive impairment, mild neurocognitive disorder, and the most severe form known as HIV-associated dementia. Little is known about the mechanisms of HAND, but it is thought to be related to infection of astrocytes, microglial cells, and macrophages in the human brain. The formation of a viral reservoir that lies dormant as a provirus in resting CD4+ T lymphocytes and in refuge tissues such as the brain contributes significantly to HIV eradication. In recent years, a new set of tools have emerged: the gene editing based on the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system, which can alter genome segments by insertion, deletion, and replacement and has great therapeutic potential. This technology has been used in research to treat HIV and appears to offer hope for a possible cure for HIV infection and perhaps prevention of HAND. This approach has the potential to directly impact the quality of life of HIV-infected individuals, which is a very important topic to be known and discussed.
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Affiliation(s)
- Shirley Vasconcelos Komninakis
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
| | - Wilson Domingues
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
| | - Sabri Saeed Sanabani
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
| | - Victor Angelo Folgosi
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
| | - Igor Neves Barbosa
- Institute of Genetic Biology at the Biological Institute of São Paulo University, São Paulo, São Paulo, Brazil
| | - Jorge Casseb
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
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14
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Lai S, Shiraishi H, Sebastian WA, Shimizu N, Umeda R, Ikeuchi M, Kiyota K, Takeno T, Miyazaki S, Yano S, Shimada T, Yoshimura A, Hanada R, Hanada T. Effect of nonsense-mediated mRNA decay factor SMG9 deficiency on premature aging in zebrafish. Commun Biol 2024; 7:654. [PMID: 38806677 PMCID: PMC11133409 DOI: 10.1038/s42003-024-06356-6] [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: 06/20/2023] [Accepted: 05/20/2024] [Indexed: 05/30/2024] Open
Abstract
SMG9 is an essential component of the nonsense-mediated mRNA decay (NMD) machinery, a quality control mechanism that selectively degrades aberrant transcripts. Mutations in SMG9 are associated with heart and brain malformation syndrome (HBMS). However, the molecular mechanism underlying HBMS remains unclear. We generated smg9 mutant zebrafish (smg9oi7/oi7) that have a lifespan of approximately 6 months or longer, allowing for analysis of the in vivo function of Smg9 in adults in more detail. smg9oi7/oi7 zebrafish display congenital brain abnormalities and reduced cardiac contraction. Additionally, smg9oi7/oi7 zebrafish exhibit a premature aging phenotype. Analysis of NMD target mRNAs shows a trend toward increased mRNA levels in smg9oi7/oi7 zebrafish. Spermidine oxidase (Smox) is increased in smg9oi7/oi7 zebrafish, resulting in the accumulation of byproducts, reactive oxygen species, and acrolein. The accumulation of smox mRNA due to NMD dysregulation caused by Smg9 deficiency leads to increased oxidative stress, resulting in premature aging.
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Affiliation(s)
- Shaohong Lai
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Hiroshi Shiraishi
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | | | - Nobuyuki Shimizu
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Ryohei Umeda
- Department of Neurophysiology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Mayo Ikeuchi
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Kyoko Kiyota
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Takashi Takeno
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Shuya Miyazaki
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Shinji Yano
- Institute for Research Management, Oita University, Yufu, Oita, Japan
| | - Tatsuo Shimada
- Oita Medical Technology School, Japan College of Judo-Therapy, Acupuncture & Moxibustion Therapy, Oita, Japan
| | - Akihiko Yoshimura
- Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan
| | - Reiko Hanada
- Department of Neurophysiology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Toshikatsu Hanada
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan.
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15
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Inoue M, Sebastian WA, Sonoda S, Miyahara H, Shimizu N, Shiraishi H, Maeda M, Yanagi K, Kaname T, Hanada R, Hanada T, Ihara K. Biallelic variants in LARS1 induce steatosis in developing zebrafish liver via enhanced autophagy. Orphanet J Rare Dis 2024; 19:219. [PMID: 38807157 PMCID: PMC11134648 DOI: 10.1186/s13023-024-03226-6] [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: 11/13/2023] [Accepted: 05/19/2024] [Indexed: 05/30/2024] Open
Abstract
BACKGROUND Biallelic pathogenic variants of LARS1 cause infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute hepatic failure with steatosis in infants. LARS functions as a protein associated with mTORC1 and plays a crucial role in amino acid-triggered mTORC1 activation and regulation of autophagy. A previous study demonstrated that larsb-knockout zebrafish exhibit conditions resembling ILFS. However, a comprehensive analysis of larsb-knockout zebrafish has not yet been performed because of early mortality. METHODS We generated a long-term viable zebrafish model carrying a LARS1 variant identified in an ILFS1 patient (larsb-I451F zebrafish) and analyzed the pathogenesis of the affected liver of ILFS1. RESULTS Hepatic dysfunction is most prominent in ILFS1 patients during infancy; correspondingly, the larsb-I451F zebrafish manifested hepatic anomalies during developmental stages. The larsb-I451F zebrafish demonstrates augmented lipid accumulation within the liver during autophagy activation. Inhibition of DGAT1, which converts fatty acids to triacylglycerols, improved lipid droplets in the liver of larsb-I451F zebrafish. Notably, treatment with an autophagy inhibitor ameliorated hepatic lipid accumulation in this model. CONCLUSIONS Our findings suggested that enhanced autophagy caused by biallelic LARS1 variants contributes to ILFS1-associated hepatic dysfunction. Furthermore, the larsb-I451F zebrafish model, which has a prolonged survival rate compared with the larsb-knockout model, highlights its potential utility as a tool for investigating the pathophysiology of ILFS1-associated liver dysfunction.
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Affiliation(s)
- Masanori Inoue
- Department of Pediatrics, Oita University Faculty of Medicine, Oita, Japan
| | | | - Shota Sonoda
- Department of Pediatrics, Oita University Faculty of Medicine, Oita, Japan
| | - Hiroaki Miyahara
- Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Aichi, Japan
| | - Nobuyuki Shimizu
- Department of Cell Biology, Oita University Faculty of Medicine, Oita, Japan
| | - Hiroshi Shiraishi
- Department of Cell Biology, Oita University Faculty of Medicine, Oita, Japan
| | - Miwako Maeda
- Department of Pediatrics, Oita University Faculty of Medicine, Oita, Japan
| | - Kumiko Yanagi
- Department of Genome Medicine, National Center for Child Health and Development, Tokyo, Japan
| | - Tadashi Kaname
- Department of Genome Medicine, National Center for Child Health and Development, Tokyo, Japan
| | - Reiko Hanada
- Department of Neurophysiology, Oita University Faculty of Medicine, Oita, Japan
| | - Toshikatsu Hanada
- Department of Cell Biology, Oita University Faculty of Medicine, Oita, Japan.
| | - Kenji Ihara
- Department of Pediatrics, Oita University Faculty of Medicine, Oita, Japan.
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16
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da Silva AR, Gunawan F, Boezio GLM, Faure E, Théron A, Avierinos JF, Lim S, Jha SG, Ramadass R, Guenther S, Looso M, Zaffran S, Juan T, Stainier DYR. egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis. SCIENCE ADVANCES 2024; 10:eadl0633. [PMID: 38748804 PMCID: PMC11095463 DOI: 10.1126/sciadv.adl0633] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/27/2023] [Accepted: 04/11/2024] [Indexed: 05/19/2024]
Abstract
Biomechanical forces, and their molecular transducers, including key mechanosensitive transcription factor genes, such as KLF2, are required for cardiac valve morphogenesis. However, klf2 mutants fail to completely recapitulate the valveless phenotype observed under no-flow conditions. Here, we identify the transcription factor EGR3 as a conserved biomechanical force transducer critical for cardiac valve formation. We first show that egr3 null zebrafish display a complete and highly penetrant loss of valve leaflets, leading to severe blood regurgitation. Using tissue-specific loss- and gain-of-function tools, we find that during cardiac valve formation, Egr3 functions cell-autonomously in endothelial cells, and identify one of its effectors, the nuclear receptor Nr4a2b. We further find that mechanical forces up-regulate egr3/EGR3 expression in the developing zebrafish heart and in porcine valvular endothelial cells, as well as during human aortic valve remodeling. Altogether, these findings reveal that EGR3 is necessary to transduce the biomechanical cues required for zebrafish cardiac valve morphogenesis, and potentially for pathological aortic valve remodeling in humans.
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Affiliation(s)
- Agatha Ribeiro da Silva
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
- Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany
| | - Felix Gunawan
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
- Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany
| | - Giulia L. M. Boezio
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
- Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany
| | - Emilie Faure
- Aix Marseille Université, INSERM, MMG, U1251, 13005 Marseille, France
| | - Alexis Théron
- Aix Marseille Université, INSERM, MMG, U1251, 13005 Marseille, France
- Service de Chirurgie Cardiaque, AP-HM, Hôpital de la Timone, 13005 Marseille, France
| | - Jean-François Avierinos
- Aix Marseille Université, INSERM, MMG, U1251, 13005 Marseille, France
- Service de Cardiologie, AP-HM, Hôpital de la Timone, 13005 Marseille, France
| | - SoEun Lim
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
| | - Shivam Govind Jha
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
| | - Radhan Ramadass
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
| | - Stefan Guenther
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
- Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany
- Bioinformatics and Deep Sequencing Platform, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
| | - Mario Looso
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
- Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany
- Bioinformatics Core Unit (BCU), Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
| | - Stéphane Zaffran
- Aix Marseille Université, INSERM, MMG, U1251, 13005 Marseille, France
| | - Thomas Juan
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
- Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany
| | - Didier Y. R. Stainier
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
- German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
- Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany
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17
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Dorner L, Stratmann B, Bader L, Podobnik M, Irion U. Efficient genome editing using modified Cas9 proteins in zebrafish. Biol Open 2024; 13:bio060401. [PMID: 38545958 PMCID: PMC10997048 DOI: 10.1242/bio.060401] [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: 03/05/2024] [Accepted: 03/12/2024] [Indexed: 04/07/2024] Open
Abstract
The zebrafish (Danio rerio) is an important model organism for basic as well as applied bio-medical research. One main advantage is its genetic tractability, which was greatly enhanced by the introduction of the CRISPR/Cas method a decade ago. The generation of loss-of-function alleles via the production of small insertions or deletions in the coding sequences of genes with CRISPR/Cas systems is now routinely achieved with high efficiency. The method is based on the error prone repair of precisely targeted DNA double strand breaks by non-homologous end joining (NHEJ) in the cell nucleus. However, editing the genome with base pair precision, by homology-directed repair (HDR), is by far less efficient and therefore often requires large-scale screening of potential carriers by labour intensive genotyping. Here we confirm that the Cas9 protein variant SpRY, with relaxed PAM requirement, can be used to target some sites in the zebrafish genome. In addition, we demonstrate that the incorporation of an artificial nuclear localisation signal (aNLS) into the Cas9 protein variants not only enhances the efficiency of gene knockout but also the frequency of HDR, thereby facilitating the efficient modification of single base pairs in the genome. Our protocols provide a guide for a cost-effective generation of versatile and potent Cas9 protein variants and efficient gene editing in zebrafish.
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Affiliation(s)
- Laura Dorner
- Max Planck Institute for Biology, RG Colour Pattern Evolution, Tuebingen, Max-Planck-Ring 5, 72076 Tuebingen, Germany
| | - Benedikt Stratmann
- Max Planck Institute for Biology, RG Colour Pattern Evolution, Tuebingen, Max-Planck-Ring 5, 72076 Tuebingen, Germany
| | - Laura Bader
- Max Planck Institute for Biology, RG Colour Pattern Evolution, Tuebingen, Max-Planck-Ring 5, 72076 Tuebingen, Germany
| | - Marco Podobnik
- Max Planck Institute for Biology, RG Colour Pattern Evolution, Tuebingen, Max-Planck-Ring 5, 72076 Tuebingen, Germany
| | - Uwe Irion
- Max Planck Institute for Biology, RG Colour Pattern Evolution, Tuebingen, Max-Planck-Ring 5, 72076 Tuebingen, Germany
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18
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Ikeuchi M, Inoue M, Miyahara H, Sebastian WA, Miyazaki S, Takeno T, Kiyota K, Yano S, Shiraishi H, Shimizu N, Hanada R, Yoshimura A, Ihara K, Hanada T. A pH imbalance is linked to autophagic dysregulation of inner ear hair cells in Atp6v1ba-deficient zebrafish. Biochem Biophys Res Commun 2024; 699:149551. [PMID: 38277730 DOI: 10.1016/j.bbrc.2024.149551] [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: 01/07/2024] [Accepted: 01/17/2024] [Indexed: 01/28/2024]
Abstract
V-ATPase is an ATP hydrolysis-driven proton pump involved in the acidification of intracellular organelles and systemic acid-base homeostasis through H+ secretion in the renal collecting ducts. V-ATPase dysfunction is associated with hereditary distal renal tubular acidosis (dRTA). ATP6V1B1 encodes the B1 subunit of V-ATPase that is integral to ATP hydrolysis and subsequent H+ transport. Patients with pathogenic ATP6V1B1 mutations often exhibit an early onset of sensorineural hearing loss. However, the mechanisms underlying this association remain unclear. We employed morpholino oligonucleotide-mediated knockdown and CRISPR/Cas9 gene editing to generate Atp6v1ba-deficient (atp6v1ba-/-) zebrafish as an ortholog model for ATP6V1B1. The atp6v1ba-/- zebrafish exhibited systemic acidosis and significantly smaller otoliths compared to wild-type siblings. Moreover, deficiency in Atp6v1ba led to degeneration of inner ear hair cells, with ultrastructural changes indicative of autophagy. Our findings indicate a critical role of ATP6V1B1 in regulating lysosomal pH and autophagy in hair cells, and the results provide insights into the pathophysiology of sensorineural hearing loss in dRTA. Furthermore, this study demonstrates that the atp6v1ba-/- zebrafish model is a valuable tool for further investigation into disease mechanisms and potential therapies for acidosis-related hearing impairment.
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Affiliation(s)
- Mayo Ikeuchi
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan; Department of Pediatrics, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Masanori Inoue
- Department of Pediatrics, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Hiroaki Miyahara
- Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Aichi, Japan
| | | | - Shuya Miyazaki
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Takashi Takeno
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Kyoko Kiyota
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan; Department of Pediatrics, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Shinji Yano
- Institute for Research Management, Oita University, Yufu, Oita, Japan
| | - Hiroshi Shiraishi
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Nobuyuki Shimizu
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Reiko Hanada
- Department of Neurophysiology, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Akihiko Yoshimura
- Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan
| | - Kenji Ihara
- Department of Pediatrics, Oita University Faculty of Medicine, Yufu, Oita, Japan
| | - Toshikatsu Hanada
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu, Oita, Japan.
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19
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Bischof J, Hierl M, Koller U. Emerging Gene Therapeutics for Epidermolysis Bullosa under Development. Int J Mol Sci 2024; 25:2243. [PMID: 38396920 PMCID: PMC10889532 DOI: 10.3390/ijms25042243] [Citation(s) in RCA: 12] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2023] [Revised: 02/01/2024] [Accepted: 02/11/2024] [Indexed: 02/25/2024] Open
Abstract
The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on the patient's skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to the functional impairment, reduction, or absence of the encoded protein within the skin's basement membrane zone connecting the epidermis to the underlying dermis. The major burden of affected families justifies the development of long-lasting and curative therapies operating at the genomic level. The landscape of causal therapies for EB is steadily expanding due to recent breakthroughs in the gene therapy field, providing promising outcomes for patients suffering from this severe disease. Currently, two gene therapeutic approaches show promise for EB. The clinically more advanced gene replacement strategy was successfully applied in severe EB forms, leading to a ground-breaking in vivo gene therapy product named beremagene geperpavec (B-VEC) recently approved from the US Food and Drug Administration (FDA). In addition, the continuous innovations in both designer nucleases and gene editing technologies enable the efficient and potentially safe repair of mutations in EB in a potentially permanent manner, inspiring researchers in the field to define and reach new milestones in the therapy of EB.
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Affiliation(s)
- Johannes Bischof
- EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria; (J.B.); (M.H.)
| | - Markus Hierl
- EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria; (J.B.); (M.H.)
- Department of Biosciences and Medical Biology, University of Salzburg, 5020 Salzburg, Austria
| | - Ulrich Koller
- EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria; (J.B.); (M.H.)
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20
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Cumplido N, Arratia G, Desvignes T, Muñoz-Sánchez S, Postlethwait JH, Allende ML. Hox genes control homocercal caudal fin development and evolution. SCIENCE ADVANCES 2024; 10:eadj5991. [PMID: 38241378 PMCID: PMC10798566 DOI: 10.1126/sciadv.adj5991] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Accepted: 12/19/2023] [Indexed: 01/21/2024]
Abstract
Ancient bony fishes had heterocercal tails, like modern sharks and sturgeons, with asymmetric caudal fins and a vertebral column extending into an elongated upper lobe. Teleost fishes, in contrast, developed a homocercal tail characterized by two separate equal-sized fin lobes and the body axis not extending into the caudal fin. A similar heterocercal-to-homocercal transition occurs during teleost ontogeny, although the underlying genetic and developmental mechanisms for either transition remain unresolved. Here, we investigated the role of hox13 genes in caudal fin formation as these genes control posterior identity in animals. Analysis of expression profiles of zebrafish hox13 paralogs and phenotypes of CRISPR/Cas9-induced mutants showed that double hoxb13a and hoxc13a mutants fail to form a caudal fin. Furthermore, single mutants display heterocercal-like morphologies not seen since Mesozoic fossil teleosteomorphs. Relaxation of functional constraints after the teleost genome duplication may have allowed hox13 duplicates to neo- or subfunctionalize, ultimately contributing to the evolution of a homocercal tail in teleost fishes.
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Affiliation(s)
- Nicolás Cumplido
- Millennium Institute Center for Genome Regulation, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | - Gloria Arratia
- University of Kansas, Department of Ecology and Evolutionary Biology and Biodiversity Institute, Lawrence, KS, USA
| | - Thomas Desvignes
- Institute of Neuroscience, University of Oregon, Eugene, OR, USA
| | - Salomé Muñoz-Sánchez
- Millennium Institute Center for Genome Regulation, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
| | | | - Miguel L. Allende
- Millennium Institute Center for Genome Regulation, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
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21
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Bhushan B, Singh K, Kumar S, Bhardwaj A. Advancements in CRISPR-Based Therapies for Genetic Modulation in Neurodegenerative Disorders. Curr Gene Ther 2024; 25:34-45. [PMID: 38738727 DOI: 10.2174/0115665232292246240426125504] [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: 01/01/2024] [Revised: 03/18/2024] [Accepted: 03/27/2024] [Indexed: 05/14/2024]
Abstract
Neurodegenerative disorders pose significant challenges in the realm of healthcare, as these conditions manifest in complex, multifaceted ways, often attributed to genetic anomalies. With the emergence of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, a new frontier has been unveiled in the quest for targeted, precise genetic manipulation. This abstract explores the recent advancements and potential applications of CRISPR-based therapies in addressing genetic components contributing to various neurodegenerative disorders. The review delves into the foundational principles of CRISPR technology, highlighting its unparalleled ability to edit genetic sequences with unprecedented precision. In addition, it talks about the latest progress in using CRISPR to target specific genetic mutations linked to neurodegenerative diseases like Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Parkinson's disease. It talks about the most important studies and trials that show how well and safely CRISPR-based therapies work. This shows how this technology can change genetic variants that cause diseases. Notably, the discussion emphasizes the challenges and ethical considerations associated with the implementation of CRISPR in clinical settings, including off-target effects, delivery methods, and long-term implications. Furthermore, the article explores the prospects and potential hurdles in the widespread application of CRISPR technology for treating neurodegenerative disorders. It touches upon the need for continued research, improved delivery mechanisms, and ethical frameworks to ensure responsible and equitable access to these groundbreaking therapies.
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Affiliation(s)
- Bharat Bhushan
- Department of Pharmacology, Institute of Pharmaceutical Research, GLA University, Mathura, Uttar Pradesh, India
| | - Kuldeep Singh
- Department of Pharmacology, Rajiv Academy for Pharmacy, Mathura, Uttar Pradesh, India
| | - Shivendra Kumar
- Department of Pharmacology, Rajiv Academy for Pharmacy, Mathura, Uttar Pradesh, India
| | - Anjali Bhardwaj
- Department of Pharmaceutics, Durga College of Pharmacy, Sambhal, Uttar Pradesh, India
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22
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Park JH, Kim H. Harnessing CRISPR/Cas9 for Enhanced Disease Resistance in Hot Peppers: A Comparative Study on CaMLO2-Gene-Editing Efficiency across Six Cultivars. Int J Mol Sci 2023; 24:16775. [PMID: 38069102 PMCID: PMC10706117 DOI: 10.3390/ijms242316775] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2023] [Revised: 11/20/2023] [Accepted: 11/23/2023] [Indexed: 12/18/2023] Open
Abstract
The Capsicum annuum Mildew Locus O (CaMLO2) gene is vital for plant defense responses against fungal pathogens like powdery mildew, a significant threat to greenhouse pepper crops. Recent advancements in genome editing, particularly using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, have unlocked unprecedented opportunities for modifying disease-resistant genes and improving crop characteristics. However, the application of CRISPR technology in pepper cultivars has been limited, and the regeneration process remains challenging. This study addresses these limitations by investigating the feasibility of using the validated CaMLO2 genetic scissors system in six commercial hot pepper cultivars. We assessed the gene-editing efficiency of the previously reported high-efficiency Cas9/CaMLO2single-guide RNA (sgRNA)1-ribonucleoprotein (RNP) and the low-efficiency Cas9/CaMLO2sgRNA2-RNP systems by extending their application from the bell pepper 'Dempsey' and the hot pepper 'CM334' to six commercial hot pepper cultivars. Across the six cultivars, CaMLO2sgRNA1 demonstrated an editing efficiency ranging from 6.3 to 17.7%, whereas CaMLO2sgRNA2 exhibited no editing efficiency, highlighting the superior efficacy of sgRNA1. These findings indicate the potential of utilizing the verified Cas9/CaMLO2sgRNA1-RNP system to achieve efficient gene editing at the CaMLO2 locus in different Capsicum annuum cultivars regardless of their cultivar genotypes. This study provides an efficacious genome-editing tool for developing improved pepper cultivars with CaMLO2-mediated enhanced disease resistance.
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Affiliation(s)
- Jae-Hyeong Park
- Interdisciplinary Graduate Program in BIT Medical Convergence, Kangwon National University, Chuncheon 24341, Republic of Korea;
| | - Hyeran Kim
- Interdisciplinary Graduate Program in BIT Medical Convergence, Kangwon National University, Chuncheon 24341, Republic of Korea;
- Department of Biological Sciences, College of Natural Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea
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23
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Hu Y, Liu L, Jiang Q, Fang W, Chen Y, Hong Y, Zhai X. CRISPR/Cas9: a powerful tool in colorectal cancer research. J Exp Clin Cancer Res 2023; 42:308. [PMID: 37993945 PMCID: PMC10664500 DOI: 10.1186/s13046-023-02901-z] [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: 09/14/2023] [Accepted: 11/14/2023] [Indexed: 11/24/2023] Open
Abstract
Colorectal cancer (CRC) is one of the most common malignant cancers worldwide and seriously threatens human health. The clustered regulatory interspaced short palindromic repeat/CRISPR-associate nuclease 9 (CRISPR/Cas9) system is an adaptive immune system of bacteria or archaea. Since its introduction, research into various aspects of treatment approaches for CRC has been accelerated, including investigation of the oncogenes, tumor suppressor genes (TSGs), drug resistance genes, target genes, mouse model construction, and especially in genome-wide library screening. Furthermore, the CRISPR/Cas9 system can be utilized for gene therapy for CRC, specifically involving in the molecular targeted drug delivery or targeted knockout in vivo. In this review, we elucidate the mechanism of the CRISPR/Cas9 system and its comprehensive applications in CRC. Additionally, we discussed the issue of off-target effects associated with CRISPR/Cas9, which serves to restrict its practical application. Future research on CRC should in-depth and systematically utilize the CRISPR/Cas9 system thereby achieving clinical practice.
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Affiliation(s)
- Yang Hu
- Department of Gastroenterology, The First People's Hospital of Jiande, Hangzhou, 311600, China
| | - Liang Liu
- Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
| | - Qi Jiang
- Department of Biological Repositories, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
| | - Weiping Fang
- Department of Gastroenterology, The First People's Hospital of Jiande, Hangzhou, 311600, China
| | - Yazhu Chen
- West China Hospital of Sichuan University, Chengdu, 610044, China.
| | - Yuntian Hong
- Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
| | - Xiang Zhai
- Department of Colorectal and Anal Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
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24
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Shin M, Yin HM, Shih YH, Nozaki T, Portman D, Toles B, Kolb A, Luk K, Isogai S, Ishida K, Hanasaka T, Parsons MJ, Wolfe SA, Burns CE, Burns CG, Lawson ND. Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish. Dev Cell 2023; 58:2614-2626.e7. [PMID: 37633272 PMCID: PMC10840978 DOI: 10.1016/j.devcel.2023.07.022] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2022] [Revised: 05/30/2023] [Accepted: 07/28/2023] [Indexed: 08/28/2023]
Abstract
The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.
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Affiliation(s)
- Masahiro Shin
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Hui-Min Yin
- Division of Basic and Translational Cardiovascular Research, Department of Cardiology, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA
| | - Yu-Huan Shih
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Takayuki Nozaki
- Technical Support Center for Life Science Research, Iwate Medical University, Shiwa, Iwate 028-3694, Japan
| | - Daneal Portman
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Benjamin Toles
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Amy Kolb
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Kevin Luk
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Sumio Isogai
- Department of Medical Education, Iwate Medical University, Shiwa, Iwate 028-3694, Japan
| | - Kinji Ishida
- Technical Support Center for Life Science Research, Iwate Medical University, Shiwa, Iwate 028-3694, Japan
| | - Tomohito Hanasaka
- Technical Support Center for Life Science Research, Iwate Medical University, Shiwa, Iwate 028-3694, Japan
| | - Michael J Parsons
- Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, Irvine, CA 92697, USA
| | - Scot A Wolfe
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
| | - Caroline E Burns
- Division of Basic and Translational Cardiovascular Research, Department of Cardiology, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA
| | - C Geoffrey Burns
- Division of Basic and Translational Cardiovascular Research, Department of Cardiology, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA
| | - Nathan D Lawson
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
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25
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Elizalde MJ, Gorelick DA. Mechanistic toxicology in light of genetic compensation. Toxicol Sci 2023; 197:kfad113. [PMID: 37941503 PMCID: PMC10823772 DOI: 10.1093/toxsci/kfad113] [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] [Indexed: 11/10/2023] Open
Abstract
Mechanistic toxicology seeks to identify the molecular and cellular mechanisms by which toxicants exert their deleterious effects. One powerful approach is to generate mutations in genes that respond to a particular toxicant, and then test how such mutations change the effects of the toxicant. CRISPR is a rapid and versatile approach to generate mutations in cultured cells and in animal models. Many studies use CRISPR to generate short insertions or deletions in a target gene and then assume that the resulting mutation, such as a premature termination codon, causes a loss of functional protein. However, recent studies demonstrate that this assumption is flawed. Cells can compensate for short insertion and deletion mutations, leading toxicologists to draw erroneous conclusions from mutant studies. In this review, we will discuss mechanisms by which a mutation in one gene may be rescued by compensatory activity. We will discuss how CRISPR insertion and deletion mutations are susceptible to compensation by transcriptional adaptation, alternative splicing, and rescue by maternally derived gene products. We will review evidence that measuring levels of messenger RNA transcribed from a mutated gene is an unreliable indicator of the severity of the mutation. Finally, we provide guidelines for using CRISPR to generate mutations that avoid compensation.
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Affiliation(s)
- Mary Jane Elizalde
- Department of Molecular & Cellular Biology, Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030, United States
| | - Daniel A Gorelick
- Department of Molecular & Cellular Biology, Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030, United States
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26
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Shimizu N, Shiraishi H, Hanada T. Zebrafish as a Useful Model System for Human Liver Disease. Cells 2023; 12:2246. [PMID: 37759472 PMCID: PMC10526867 DOI: 10.3390/cells12182246] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Revised: 08/31/2023] [Accepted: 09/08/2023] [Indexed: 09/29/2023] Open
Abstract
Liver diseases represent a significant global health challenge, thereby necessitating extensive research to understand their intricate complexities and to develop effective treatments. In this context, zebrafish (Danio rerio) have emerged as a valuable model organism for studying various aspects of liver disease. The zebrafish liver has striking similarities to the human liver in terms of structure, function, and regenerative capacity. Researchers have successfully induced liver damage in zebrafish using chemical toxins, genetic manipulation, and other methods, thereby allowing the study of disease mechanisms and the progression of liver disease. Zebrafish embryos or larvae, with their transparency and rapid development, provide a unique opportunity for high-throughput drug screening and the identification of potential therapeutics. This review highlights how research on zebrafish has provided valuable insights into the pathological mechanisms of human liver disease.
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Affiliation(s)
- Nobuyuki Shimizu
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu 879-5593, Oita, Japan;
| | | | - Toshikatsu Hanada
- Department of Cell Biology, Oita University Faculty of Medicine, Yufu 879-5593, Oita, Japan;
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27
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Shin U, Lee Y. Unraveling DNA Repair Processes In Vivo: Insights from Zebrafish Studies. Int J Mol Sci 2023; 24:13120. [PMID: 37685935 PMCID: PMC10487404 DOI: 10.3390/ijms241713120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2023] [Revised: 08/16/2023] [Accepted: 08/22/2023] [Indexed: 09/10/2023] Open
Abstract
The critical role of the DNA repair system in preserving the health and survival of living organisms is widely recognized as dysfunction within this system can result in a broad range of severe conditions, including neurodegenerative diseases, blood disorders, infertility, and cancer. Despite comprehensive research on the molecular and cellular mechanisms of DNA repair pathways, there remains a significant knowledge gap concerning these processes at an organismal level. The teleost zebrafish has emerged as a powerful model organism for investigating these intricate DNA repair mechanisms. Their utility arises from a combination of their well-characterized genomic information, the ability to visualize specific phenotype outcomes in distinct cells and tissues, and the availability of diverse genetic experimental approaches. In this review, we provide an in-depth overview of recent advancements in our understanding of the in vivo roles of DNA repair pathways. We cover a variety of critical biological processes including neurogenesis, hematopoiesis, germ cell development, tumorigenesis, and aging, with a specific emphasis on findings obtained from the use of zebrafish as a model system. Our comprehensive review highlights the importance of zebrafish in enhancing our understanding of the functions of DNA repair systems at the organismal level and paves the way for future investigations in this field.
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Affiliation(s)
- Unbeom Shin
- School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
| | - Yoonsung Lee
- Clinical Research Institute, Kyung Hee University Hospital at Gangdong, School of Medicine, Kyung Hee University, Seoul 05278, Republic of Korea
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28
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Kim C, Cnaani A, Kültz D. Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system. Sci Rep 2023; 13:12086. [PMID: 37495710 PMCID: PMC10371998 DOI: 10.1038/s41598-023-37928-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2022] [Accepted: 06/29/2023] [Indexed: 07/28/2023] Open
Abstract
MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions.
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Affiliation(s)
- Chanhee Kim
- Department of Animal Sciences, University of California, Davis, CA, 95616, USA
| | - Avner Cnaani
- Department of Poultry and Aquaculture, Institute of Animal Sciences, Agricultural Research Organization, Volcani Center, 7528809, Rishon LeZion, Israel
| | - Dietmar Kültz
- Department of Animal Sciences, University of California, Davis, CA, 95616, USA.
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29
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Spead O, Moreland T, Weaver CJ, Costa ID, Hegarty B, Kramer KL, Poulain FE. Teneurin trans-axonal signaling prunes topographically missorted axons. Cell Rep 2023; 42:112192. [PMID: 36857189 PMCID: PMC10131173 DOI: 10.1016/j.celrep.2023.112192] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Revised: 01/04/2023] [Accepted: 02/14/2023] [Indexed: 03/02/2023] Open
Abstract
Building precise neural circuits necessitates the elimination of axonal projections that have inaccurately formed during development. Although axonal pruning is a selective process, how it is initiated and controlled in vivo remains unclear. Here, we show that trans-axonal signaling mediated by the cell surface molecules Glypican-3, Teneurin-3, and Latrophilin-3 prunes misrouted retinal axons in the visual system. Retinotopic neuron transplantations revealed that pioneer ventral axons that elongate first along the optic tract instruct the pruning of dorsal axons that missort in that region. Glypican-3 and Teneurin-3 are both selectively expressed by ventral retinal ganglion cells and cooperate for correcting missorted dorsal axons. The adhesion G-protein-coupled receptor Latrophilin-3 signals along dorsal axons to initiate the elimination of topographic sorting errors. Altogether, our findings show an essential function for Glypican-3, Teneurin-3, and Latrophilin-3 in topographic tract organization and demonstrate that axonal pruning can be initiated by signaling among axons themselves.
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Affiliation(s)
- Olivia Spead
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Trevor Moreland
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Cory J Weaver
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Irene Dalla Costa
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | - Brianna Hegarty
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA
| | | | - Fabienne E Poulain
- Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA.
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30
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Morris EK, Daignault-Mill S, Stehbens SJ, Genovesi LA, Lagendijk AK. Addressing blood-brain-tumor-barrier heterogeneity in pediatric brain tumors with innovative preclinical models. Front Oncol 2023; 13:1101522. [PMID: 36776301 PMCID: PMC9909546 DOI: 10.3389/fonc.2023.1101522] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2022] [Accepted: 01/06/2023] [Indexed: 01/27/2023] Open
Abstract
Brain tumors represent the leading cause of disease-related mortality and morbidity in children, with effective treatments urgently required. One factor limiting the effectiveness of systemic therapy is the blood-brain-barrier (BBB), which limits the brain penetration of many anticancer drugs. BBB integrity is often compromised in tumors, referred to as the blood-brain-tumor-barrier (BBTB), and the impact of a compromised BBTB on the therapeutic sensitivity of brain tumors has been clearly shown for a few selected agents. However, the heterogeneity of barrier alteration observed within a single tumor and across distinct pediatric tumor types represents an additional challenge. Herein, we discuss what is known regarding the heterogeneity of tumor-associated vasculature in pediatric brain tumors. We discuss innovative and complementary preclinical model systems that will facilitate real-time functional analyses of BBTB for all pediatric brain tumor types. We believe a broader use of these preclinical models will enable us to develop a greater understanding of the processes underlying tumor-associated vasculature formation and ultimately more efficacious treatment options.
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Affiliation(s)
- Elysse K. Morris
- Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, Australia
| | - Sheena Daignault-Mill
- Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, Australia
| | - Samantha J. Stehbens
- Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, Australia
| | - Laura A. Genovesi
- The University of Queensland Frazer Institute, Faculty of Medicine, The University of Queensland, Brisbane, QLD, Australia,*Correspondence: Laura A. Genovesi, ; Anne K. Lagendijk,
| | - Anne K. Lagendijk
- Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, Australia,School of Biomedical Sciences, University of Queensland, St. Lucia, QLD, Australia,*Correspondence: Laura A. Genovesi, ; Anne K. Lagendijk,
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31
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CRISPR/Cas9-mediated knockin of IRES-tdTomato at Ins2 locus reveals no RFP-positive cells in mouse islets. Funct Integr Genomics 2023; 23:42. [PMID: 36652148 PMCID: PMC9849276 DOI: 10.1007/s10142-023-00960-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2022] [Revised: 12/30/2022] [Accepted: 01/02/2023] [Indexed: 01/19/2023]
Abstract
Using the CRISPR/Cas9 genomic editing technology, we constructed a transgenic mouse model to express specific fluorescent protein in pancreatic β cells, which harbor tdTomato exogenous gene downstream of the Ins2 promoter in C57BL/6 J mice. The Ins2-specific single-guide RNA-targeted exon2 was designed for the CRISPR/Cas9 system and Donor vector was constructed at the same time. Then Cas9, sgRNA, and Donor vector were microinjected in vitro into the mouse zygotes that were implanted into pseudo-pregnant mice. We obtained homozygotes through mating heterozygotes, and verified the knockin effect through genotype identification, in vivo imaging, and frozen section. Six F0 mice and stable inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed that the knockin group had no change in the Ins2 exon compared with the control group, while only the base sequence of tdTomato was added and no base mutation occurred. However, in vivo imaging and frozen section did not observe the expression of red fluorescent protein (RFP), and the protein expression of knockin gene tdTomato was negative. As a result, the expressions of tdTomato protein and fluorescence intensity were low and the detection threshold was not reached. In the CRISP/Cas9 technique, the exogenous fragment of IRES connection would affect the transcription level of the preceding gene, which in turn would lead to low-level expression of the downstream gene and affect the effect of gene insertion.
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32
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Juan T, Ribeiro da Silva A, Cardoso B, Lim S, Charteau V, Stainier DYR. Multiple pkd and piezo gene family members are required for atrioventricular valve formation. Nat Commun 2023; 14:214. [PMID: 36639367 PMCID: PMC9839778 DOI: 10.1038/s41467-023-35843-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2022] [Accepted: 01/04/2023] [Indexed: 01/15/2023] Open
Abstract
Cardiac valves ensure unidirectional blood flow through the heart, and altering their function can result in heart failure. Flow sensing via wall shear stress and wall stretching through the action of mechanosensors can modulate cardiac valve formation. However, the identity and precise role of the key mechanosensors and their effectors remain mostly unknown. Here, we genetically dissect the role of Pkd1a and other mechanosensors in atrioventricular (AV) valve formation in zebrafish and identify a role for several pkd and piezo gene family members in this process. We show that Pkd1a, together with Pkd2, Pkd1l1, and Piezo2a, promotes AV valve elongation and cardiac morphogenesis. Mechanistically, Pkd1a, Pkd2, and Pkd1l1 all repress the expression of klf2a and klf2b, transcription factor genes implicated in AV valve development. Furthermore, we find that the calcium-dependent protein kinase Camk2g is required downstream of Pkd function to repress klf2a expression. Altogether, these data identify, and dissect the role of, several mechanosensors required for AV valve formation, thereby broadening our understanding of cardiac valvulogenesis.
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Affiliation(s)
- Thomas Juan
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany. .,German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany. .,Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany.
| | - Agatha Ribeiro da Silva
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany.,German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
| | - Bárbara Cardoso
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany.,German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
| | - SoEun Lim
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany.,German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany
| | - Violette Charteau
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany.,German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany.,Institute for Molecules and Materials (IMM), Department of Biomolecular Chemistry, Radboud University, Nijmegen, The Netherlands
| | - Didier Y R Stainier
- Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany. .,German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Bad Nauheim, Germany. .,Cardio-Pulmonary Institute (CPI), Bad Nauheim, Germany.
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Yan CY, Wu FY, Sun F, Fang Y, Zhang RJ, Zhang CR, Zhang CX, Wang Z, Yang RM, Yang L, Dong M, Zhang QY, Ye XP, Song HD, Zhao SX. The isl2a transcription factor regulates pituitary development in zebrafish. Front Endocrinol (Lausanne) 2023; 14:920548. [PMID: 36824359 PMCID: PMC9941339 DOI: 10.3389/fendo.2023.920548] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2022] [Accepted: 01/25/2023] [Indexed: 02/10/2023] Open
Abstract
BACKGROUND ISL LIM homeobox 2, also known as insulin gene enhancer protein ISL-2 (ISL2), is a transcription factor gene that participates in a wide range of developmental events. However, the role of ISL2 in the hypothalamus-pituitary-thyroid axis is largely unknown. In the present study, we characterized the expression patterns of ISL2 and revealed its regulative role during embryogenesis using zebrafish. METHODS We used the CRISPR/Cas9 system to successfully establish homozygous ISL2-orthologue (isl2a and isl2b) knockout zebrafish. Moreover, we utilized these knockout zebrafish to analyze the pituitary and thyroid phenotypes in vivo. For further molecular characterization, in situ hybridization and immunofluorescence were performed. RESULTS The isl2a mutant zebrafish presented with thyroid hypoplasia, reduced whole-body levels of thyroid hormones, increased early mortality, gender imbalance, and morphological retardation during maturity. Additionally, thyrotropes, a pituitary cell type, was notably decreased during development. Importantly, the transcriptional levels of pituitary-thyroid axis hormones-encoding genes, such as tshba, cga, and tg, were significantly decreased in isl2a mutants. Finally, the thyroid dysplasia in isl2a mutant larvae may be attributed to a reduction in proliferation rather than changes in apoptosis. CONCLUSIONS In summary, isl2a regulates the transcriptional levels of marker genes in hypothalamus-pituitary-thyroid axis, and isl2a knockout causing low thyroid hormone levels in zebrafish. Thus, isl2a identified by the present study, is a novel regulator for pituitary cell differentiation in zebrafish, resulting in thyroid gland hypoplasia and phenotypes of hypothyroidism.
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Affiliation(s)
- Chen-Yan Yan
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
- Geriatric Medicine Center, Department of Endocrinology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, China
| | - Feng-Yao Wu
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Feng Sun
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Ya Fang
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Rui-Jia Zhang
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Chang-Run Zhang
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Cao-Xu Zhang
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Zheng Wang
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Rui-Meng Yang
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Liu Yang
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Mei Dong
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Qian-Yue Zhang
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Xiao-Ping Ye
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
| | - Huai-Dong Song
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
- *Correspondence: Shuang-Xia Zhao, ; Huai-Dong Song,
| | - Shuang-Xia Zhao
- Department of Molecular Diagnostics and Endocrinology, The Core Laboratory in Medical Center of Clinical Research, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai, China
- *Correspondence: Shuang-Xia Zhao, ; Huai-Dong Song,
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Xing D, Li S, Shang M, Wang W, Zhang Q, Wang J, Hasin T, Hettiarachchi D, Alston V, Bern L, Parrales AP, Lu C, Coogan M, Johnson A, Qin Z, Su B, Dunham R. A New Strategy for Increasing Knock-in Efficiency: Multiple Elongase and Desaturase Transgenes Knock-in by Targeting Long Repeated Sequences. ACS Synth Biol 2022; 11:4210-4219. [PMID: 36332126 DOI: 10.1021/acssynbio.2c00252] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
CRISPR/Cas9-mediated knock-in (KI) has a wide application in gene therapy, gene function study, and transgenic breeding programs. Unlike gene therapy, which requires accurate KI to correct gene mutation, transgenic breeding programs can accept robust KI as long as integration does not interrupt normal gene functions and result in any negative pleiotropic effects. High KI efficiency is required to reduce the breeding cost and shorten the breeding period, especially in transferring multiple foreign genes to a single individual. To elevate the KI efficacy and achieve multiple gene KIs simultaneously, we introduced a new strategy that enables transgene integration into numerous sites of the genome by targeting long repeated sequences (LRSs). Using this simple strategy, for the first time we successfully generated transgenic fish carrying the masu salmon (Oncorhynchus masou) elovl2 gene and rabbitfish (Siganus canaliculatus) Δ4 fad and Δ6 fad genes, and achieved robust target KI of elovl2 and Δ6 fad genes at multiple sites of LRS1 and LRS3, respectively, in the initial generation. This demonstrated that donor plasmid homology arms, which were nearly identical but not completely the same as the genome sequence, still led to on-target KI. Although the target KI efficiencies at LRS1, LRS2, and LRS3 sites were still relatively low in the current study, it is very promising that 100% KI efficiency in the future could be realized and perfected by selection of better LRSs and optimization of sgRNAs.
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Affiliation(s)
- De Xing
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Shangjia Li
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Mei Shang
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Wenwen Wang
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Qin Zhang
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Jinhai Wang
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Tasnuba Hasin
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Darshika Hettiarachchi
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Veronica Alston
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Logan Bern
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Abel Paladines Parrales
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Cuiyu Lu
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Michael Coogan
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Andrew Johnson
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Zhenkui Qin
- Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao266003, China
| | - Baofeng Su
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
| | - Rex Dunham
- School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, Alabama36849, United States
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Carrington B, Ramanagoudr-Bhojappa R, Bresciani E, Han TU, Sood R. A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening. BMC Genomics 2022; 23:810. [PMID: 36476416 PMCID: PMC9730659 DOI: 10.1186/s12864-022-08971-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Accepted: 10/26/2022] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Genome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods. RESULTS: Here, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1-5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene. CONCLUSIONS We have established a robust pipeline for the generation of zebrafish models with precise integration of small DNA sequences and point mutations at the desired sites in the genome. Our screening method is very efficient and easy to implement as it is PCR-based and only requires access to a capillary sequencer.
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Affiliation(s)
- Blake Carrington
- Translational and Functional Genomics Branch, Zebrafish Core, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Ramanagouda Ramanagoudr-Bhojappa
- Cancer Genetics Unit, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Erica Bresciani
- Oncogenesis and Development Section, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Tae-Un Han
- Molecular Neurogenetics Section, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Raman Sood
- Translational and Functional Genomics Branch, Zebrafish Core, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
- Oncogenesis and Development Section, Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
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Cho HJ, Lee WS, Jeong J, Lee JS. A review on the impacts of nanomaterials on neuromodulation and neurological dysfunction using a zebrafish animal model. Comp Biochem Physiol C Toxicol Pharmacol 2022; 261:109428. [PMID: 35940544 DOI: 10.1016/j.cbpc.2022.109428] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/04/2022] [Revised: 07/28/2022] [Accepted: 08/03/2022] [Indexed: 11/20/2022]
Abstract
Nanomaterials have been widely employed from industrial to medical fields due to their small sizes and versatile characteristics. However, nanomaterials can also induce unexpected adverse effects on health. In particular, exposure of the nervous system to nanomaterials can cause serious neurological dysfunctions and neurodegenerative diseases. A number of studies have adopted various animal models to evaluate the neurotoxic effects of nanomaterials. Among them, zebrafish has become an attractive animal model for neurotoxicological studies due to several advantages, including the well-characterized nervous system, efficient genome editing, convenient generation of transgenic lines, high-resolution in vivo imaging, and an array of behavioral assays. In this review, we summarize recent studies on the neurotoxicological effects of nanomaterials, particularly engineered nanomaterials and nanoplastics, using zebrafish and discuss key findings with advantages and limitations of the zebrafish model in neurotoxicological studies.
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Affiliation(s)
- Hyun-Ju Cho
- Microbiome Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea
| | - Wang Sik Lee
- Environmental Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea
| | - Jinyoung Jeong
- Environmental Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea; KRIBB School, University of Science and Technology, Yuseong-gu, Daejeon, 34141, Republic of Korea.
| | - Jeong-Soo Lee
- Microbiome Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea; KRIBB School, University of Science and Technology, Yuseong-gu, Daejeon, 34141, Republic of Korea.
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Qiang J, Cao ZM, Zhu HJ, Tao YF, He J, Xu P. Knock-down of amh transcription by antisense RNA reduces FSH and increases follicular atresia in female Oreochromis niloticus. Gene 2022; 842:146792. [PMID: 35961433 DOI: 10.1016/j.gene.2022.146792] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2021] [Revised: 07/25/2022] [Accepted: 08/05/2022] [Indexed: 11/28/2022]
Abstract
Anti-Müllerian hormone (Amh) plays an important role in regulating gonad development in teleosts. However, little is known about the effects of Amh on follicle development. In this study, we transfected the vector containing antisense RNA fragments of the amh gene to produce Nile tilapia, Oreochromis niloticus, with knocked-down Amh function in vivo. The results confirmed that the antisense RNA effectively inhibited amh transcription and Amh protein expression in female tilapia ovarian tissue. At 180 days of age, compared with control fish, female tilapia with knocked-down Amh function showed significantly increased growth and significantly decreased ovary weight and gonadosomatic index (P < 0.05). Female fish in the control group had ruddy-colored external genitalia, eggs extruded from the abdomen when gently squeezed, and most oocytes were developmental stage V. In contrast, the external genitalia of female fish with knocked-down Amh function did not have the ruddy color, no eggs extruded from the abdomen when squeezed, most oocytes were at developmental stages II and III, and considerable follicular atresia was apparent. At 180 days of age, the transcript levels of amhrII, cyp19a1a, foxl2 and sox9b in ovarian tissue, and the titers of luteinizing hormone, follicle stimulating hormone, and estradiol in the serum, were significantly lower in fish with knocked-down Amh function than in control fish (P < 0.05). We concluded that decreased serum hormone levels and an abnormal AMH signal delayed development and caused follicular degeneration in Nile tilapia with knocked-down Amh function. These findings show that antisense RNA is a feasible approach for gene silencing in fish, and represents an accurate and effective strategy to study gene function.
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Affiliation(s)
- Jun Qiang
- Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, Jiangsu, China.
| | - Zhe-Ming Cao
- Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, Jiangsu, China
| | - Hao-Jun Zhu
- Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, Jiangsu, China
| | - Yi-Fan Tao
- Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, Jiangsu, China
| | - Jie He
- Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, Jiangsu, China
| | - Pao Xu
- Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, Jiangsu, China.
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Livne H, Avital T, Ruppo S, Harazi A, Mitrani-Rosenbaum S, Daya A. Generation and characterization of a novel gne Knockout Model in Zebrafish. Front Cell Dev Biol 2022; 10:976111. [PMID: 36353515 PMCID: PMC9637792 DOI: 10.3389/fcell.2022.976111] [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: 06/23/2022] [Accepted: 09/14/2022] [Indexed: 12/04/2022] Open
Abstract
GNE Myopathy is a rare, recessively inherited neuromuscular worldwide disorder, caused by a spectrum of bi-allelic mutations in the human GNE gene. GNE encodes a bi-functional enzyme responsible for the rate-limiting step of sialic acid biosynthesis pathway. However, the process in which GNE mutations lead to the development of a muscle pathology is not clear yet. Cellular and mouse models for GNE Myopathy established to date have not been informative. Further, additional GNE functions in muscle have been hypothesized. In these studies, we aimed to investigate gne functions using zebrafish genetic and transgenic models, and characterized them using macroscopic, microscopic, and molecular approaches. We first established transgenic zebrafish lineages expressing the human GNE cDNA carrying the M743T mutation, driven by the zebrafish gne promoter. These fish developed entirely normally. Then, we generated a gne knocked-out (KO) fish using the CRISPR/Cas9 methodology. These fish died 8–10 days post-fertilization (dpf), but a phenotype appeared less than 24 h before death and included progressive body axis curving, deflation of the swim bladder and decreasing movement and heart rate. However, muscle histology uncovered severe defects, already at 5 dpf, with compromised fiber organization. Sialic acid supplementation did not rescue the larvae from this phenotype nor prolonged their lifespan. To have deeper insights into the potential functions of gne in zebrafish, RNA sequencing was performed at 3 time points (3, 5, and 7 dpf). Genotype clustering was progressive, with only 5 genes differentially expressed in gne KO compared to gne WT siblings at 3 dpf. Enrichment analyses of the primary processes affected by the lack of gne also at 5 and 7 dpf point to the involvement of cell cycle and DNA damage/repair processes in the gne KO zebrafish. Thus, we have established a gne KO zebrafish lineage and obtained new insights into gne functions. This is the only model where GNE can be related to clear muscle defects, thus the only animal model relevant to GNE Myopathy to date. Further elucidation of gne precise mechanism-of-action in these processes could be relevant to GNE Myopathy and allow the identification of novel therapeutic targets.
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Affiliation(s)
- Hagay Livne
- Faculty of Marine Sciences, Ruppin Academic Center, Michmoret, Israel
- Goldyne Savad Institute of Gene Therapy, Hadassah Medical Center, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Tom Avital
- Faculty of Marine Sciences, Ruppin Academic Center, Michmoret, Israel
| | - Shmuel Ruppo
- Info-CORE, Bioinformatics Unit of the I-CORE, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Avi Harazi
- Faculty of Marine Sciences, Ruppin Academic Center, Michmoret, Israel
- Goldyne Savad Institute of Gene Therapy, Hadassah Medical Center, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Stella Mitrani-Rosenbaum
- Goldyne Savad Institute of Gene Therapy, Hadassah Medical Center, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Alon Daya
- Faculty of Marine Sciences, Ruppin Academic Center, Michmoret, Israel
- *Correspondence: Alon Daya,
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Coogan M, Alston V, Su B, Khalil K, Elaswad A, Khan M, Johnson A, Xing D, Li S, Wang J, Simora RMC, Lu C, Page-McCaw P, Chen W, Michel M, Wang W, Hettiarachchi D, Hasin T, Butts IAE, Cone RD, Dunham RA. Improved Growth and High Inheritance of Melanocortin-4 Receptor (mc4r) Mutation in CRISPR/Cas-9 Gene-Edited Channel Catfish, Ictalurus punctatus. MARINE BIOTECHNOLOGY (NEW YORK, N.Y.) 2022; 24:843-855. [PMID: 35943638 DOI: 10.1007/s10126-022-10146-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/14/2022] [Accepted: 07/11/2022] [Indexed: 06/15/2023]
Abstract
Effects of CRISPR/Cas9 knockout of the melanocortin-4 receptor (mc4r) gene in channel catfish, Ictalurus punctatus, were investigated. Three sgRNAs targeting the channel catfish mc4r gene in conjunction with Cas9 protein were microinjected in embryos and mutation rate, inheritance, and growth were studied. Efficient mutagenesis was achieved as demonstrated by PCR, Surveyor® assay, and DNA sequencing. An overall mutation rate of 33% and 33% homozygosity/bi-allelism was achieved in 2017. Approximately 71% of progeny inherited the mutation. Growth was generally higher in MC4R mutants than controls (CNTRL) at all life stages and in both pond and tank environments. There was a positive relationship between zygosity and growth, with F1 homozygous/bi-allelic mutants reaching market size 30% faster than F1 heterozygotes in earthen ponds (p = 0.022). At the stocker stage (~ 50 g), MC4R × MC4R mutants generated in 2019 were 40% larger than the mean of combined CNTRL × CNTRL families (p = 0.005) and 54% larger than F1 MC4R × CNTRL mutants (p = 0.001) indicating mutation may be recessive. With a high mutation rate and inheritance of the mutation as well as improved growth, the use of gene-edited MC4R channel catfish appears to be beneficial for application on commercial farms.
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Affiliation(s)
- Michael Coogan
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA.
| | - Veronica Alston
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - Baofeng Su
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - Karim Khalil
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - Ahmed Elaswad
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
- Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt
| | - Mohd Khan
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
- Department of Fisheries Biology and Genetics, Agricultural University, Mymensingh, 2202, Bangladesh
| | - Andrew Johnson
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - De Xing
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - Shangjia Li
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - Jinhai Wang
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - Rhoda M C Simora
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
- College of Fisheries and Ocean Sciences, University of the Philippines Visayas, 5023, Miagao, Iloilo, Philippines
| | - Cuiyu Lu
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - Patrick Page-McCaw
- Department of Molecular and Integrative Physiology, Vanderbilt University, Nashville, TN, 37203-5721, USA
| | - Wenbiao Chen
- Department of Molecular and Integrative Physiology, Vanderbilt University, Nashville, TN, 37203-5721, USA
| | - Max Michel
- Department of Molecular and Integrative Physiology, Vanderbilt University, Nashville, TN, 37203-5721, USA
| | - Wenwen Wang
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | | | - Tasnuba Hasin
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - Ian A E Butts
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
| | - Roger D Cone
- Department of Molecular and Integrative Physiology, Vanderbilt University, Nashville, TN, 37203-5721, USA
| | - Rex A Dunham
- Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, USA
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CRISPR/Cas9 system: a reliable and facile genome editing tool in modern biology. Mol Biol Rep 2022; 49:12133-12150. [PMID: 36030476 PMCID: PMC9420241 DOI: 10.1007/s11033-022-07880-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Accepted: 08/17/2022] [Indexed: 11/10/2022]
Abstract
Genome engineering has always been a versatile technique in biological research and medicine, with several applications. In the last several years, the discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 technology has swept the scientific community and revolutionised the speed of modern biology, heralding a new era of disease detection and rapid biotechnology discoveries. It enables successful gene editing by producing targeted double-strand breaks in virtually any organism or cell type. So, this review presents a comprehensive knowledge about the mechanism and structure of Cas9-mediated RNA-guided DNA targeting and cleavage. In addition, genome editing via CRISPR-Cas9 technology in various animals which are being used as models in scientific research including Non-Human Primates Pigs, Dogs, Zebra, fish and Drosophila has been discussed in this review. This review also aims to understand the applications, serious concerns and future perspective of CRISPR/Cas9-mediated genome editing.
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Zhdanova PV, Lomzov AA, Prokhorova DV, Stepanov GA, Chernonosov AA, Koval VV. Thermodynamic Swings: How Ideal Complex of Cas9-RNA/DNA Forms. Int J Mol Sci 2022; 23:8891. [PMID: 36012157 PMCID: PMC9408429 DOI: 10.3390/ijms23168891] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2022] [Revised: 08/02/2022] [Accepted: 08/09/2022] [Indexed: 12/26/2022] Open
Abstract
Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR-Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR-Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex's components of Cas9-RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9-RNA/DNA formation from the Cas9-RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR-Cas9 system.
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Affiliation(s)
- Polina V. Zhdanova
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia
- Department of Natural Sciences, Novosibirsk State University, 630090 Novosibirsk, Russia
| | - Alexander A. Lomzov
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia
- Department of Natural Sciences, Novosibirsk State University, 630090 Novosibirsk, Russia
| | - Daria V. Prokhorova
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia
| | - Grigory A. Stepanov
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia
| | - Alexander A. Chernonosov
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia
| | - Vladimir V. Koval
- Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (SB RAS), 630090 Novosibirsk, Russia
- Department of Natural Sciences, Novosibirsk State University, 630090 Novosibirsk, Russia
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Yan M, Li B, Wang J, Bai Y, Ke Q, Zhou T, Xu P. Disruption of mstn Gene by CRISPR/Cas9 in Large Yellow Croaker (Larimichthys crocea). MARINE BIOTECHNOLOGY (NEW YORK, N.Y.) 2022; 24:681-689. [PMID: 35896844 DOI: 10.1007/s10126-022-10135-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/05/2022] [Accepted: 06/07/2022] [Indexed: 06/15/2023]
Abstract
The large yellow croaker (Larimichthys crocea) plays an economically vital role in the marine aquaculture in China. Suffering from infection of bacteria and protozoon, effect of extreme weather and stress from high-density farming, genome editing is thought to be an important tool applied to L. croea for enhancing commercial traits such as growth rate, disease resistance, and nutrition component. In this study, we identified two mstn genes in L. croea and investigated the different phylogenetic clades, gene structures, and conserved syntenic relationships. To obtain fast-growing large yellow croaker, we specially selected two validated targets for mstnb knockout, which was homologous to mammalian myostatin gene (MSTN) and downregulated skeletal muscle growth and development. Five significant mutation types were generated in two mosaic mutants by transferring specific CRISPR/Cas9 RNPs (ribonucleoprotein) into the one-cell fertilized embryos based on CRISPR/Cas9 technology. Subsequently, we also elucidated the obstacles and possible measures to improve the success rate of inducing modified large yellow croaker. Our results would provide valuable method and reference for facilitating genome editing programs of the large yellow croaker in the future.
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Affiliation(s)
- Mengzhen Yan
- Fujian Key Laboratory of Genetics and Breeding of Marine Organisms, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China
| | - Bijun Li
- Fujian Key Laboratory of Genetics and Breeding of Marine Organisms, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China
| | - Jiaying Wang
- Fujian Key Laboratory of Genetics and Breeding of Marine Organisms, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China
| | - Yulin Bai
- Fujian Key Laboratory of Genetics and Breeding of Marine Organisms, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China
| | - Qiaozhen Ke
- Fujian Key Laboratory of Genetics and Breeding of Marine Organisms, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China
- State Key Laboratory of Large Yellow Croaker Breeding, Ningde Fufa Fisheries Company Limited, Ningde, China
| | - Tao Zhou
- Fujian Key Laboratory of Genetics and Breeding of Marine Organisms, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China
- State Key Laboratory of Large Yellow Croaker Breeding, Ningde Fufa Fisheries Company Limited, Ningde, China
| | - Peng Xu
- Fujian Key Laboratory of Genetics and Breeding of Marine Organisms, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China.
- State Key Laboratory of Large Yellow Croaker Breeding, Ningde Fufa Fisheries Company Limited, Ningde, China.
- State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, China.
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Petel Légaré V, Rampal CJ, Gurberg TJN, Harji ZA, Allard-Chamard X, Rodríguez EC, Armstrong GAB. Development of an endogenously myc-tagged TARDBP (TDP-43) zebrafish model using the CRISPR/Cas9 system and homology directed repair. Comp Biochem Physiol B Biochem Mol Biol 2022; 261:110756. [PMID: 35580804 DOI: 10.1016/j.cbpb.2022.110756] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2022] [Revised: 05/08/2022] [Accepted: 05/11/2022] [Indexed: 11/25/2022]
Abstract
Many of the modern advances in cellular biology have been made by the expression of engineered constructs with epitope tags for subsequent biochemical investigations. While the utility of epitope tags has permitted insights in cellular and animal models, these are often expressed using traditional transgenic approaches. Using the CRISPR/Cas9 system and homology directed repair we recombine a single myc epitope sequence following the start codon of the zebrafish ortholog of TARDBP (TDP-43). TDP-43 is an RNA binding protein that is involved in the neurodegenerative disease amyotrophic lateral sclerosis and frontotemporal dementia. We report that zebrafish expressing the myc-tardbp engendered allele produced a stable protein that was detected by both western blot and immunofluorescence. Furthermore, both heterozygous and homozygous carriers of the myc-tardbp allele developed to sexual maturity. We propose that the methodology used here will be useful for zebrafish researchers and other comparative animal biologists interested in developing animal models expressing endogenously tagged proteins.
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Affiliation(s)
- Virginie Petel Légaré
- Department of Neurology and Neurosurgery, Montreal Neurological Institute, Faculty of Medicine, McGill University. https://twitter.com/virginiepet
| | - Christian J Rampal
- Department of Neurology and Neurosurgery, Montreal Neurological Institute, Faculty of Medicine, McGill University. https://twitter.com/ChristianRampal
| | - Tyler J N Gurberg
- Department of Neurology and Neurosurgery, Montreal Neurological Institute, Faculty of Medicine, McGill University
| | - Ziyaan A Harji
- Department of Neurology and Neurosurgery, Montreal Neurological Institute, Faculty of Medicine, McGill University. https://twitter.com/ziyaanharji
| | - Xavier Allard-Chamard
- Department of Neurology and Neurosurgery, Montreal Neurological Institute, Faculty of Medicine, McGill University
| | - Esteban C Rodríguez
- Department of Neurology and Neurosurgery, Montreal Neurological Institute, Faculty of Medicine, McGill University
| | - Gary A B Armstrong
- Department of Neurology and Neurosurgery, Montreal Neurological Institute, Faculty of Medicine, McGill University.
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44
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Treatment strategies for HIV infection with emphasis on role of CRISPR/Cas9 gene: Success so far and road ahead. Eur J Pharmacol 2022; 931:175173. [DOI: 10.1016/j.ejphar.2022.175173] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2022] [Revised: 07/15/2022] [Accepted: 07/21/2022] [Indexed: 11/20/2022]
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Chen XZ, Guo R, Zhao C, Xu J, Song H, Yu H, Pilarsky C, Nainu F, Li JQ, Zhou XK, Zhang JY. A Novel Anti-Cancer Therapy: CRISPR/Cas9 Gene Editing. Front Pharmacol 2022; 13:939090. [PMID: 35935840 PMCID: PMC9353945 DOI: 10.3389/fphar.2022.939090] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2022] [Accepted: 06/14/2022] [Indexed: 11/27/2022] Open
Abstract
Cancer becomes one of the main causes of human deaths in the world due to the high incidence and mortality rate and produces serious economic burdens. With more and more attention is paid on cancer, its therapies are getting more of a concern. Previous research has shown that the occurrence, progression, and treatment prognosis of malignant tumors are closely related to genetic and gene mutation. CRISPR/Cas9 has emerged as a powerful method for making changes to the genome, which has extensively been applied in various cell lines. Establishing the cell and animal models by CRISPR/Cas9 laid the foundation for the clinical trials which possibly treated the tumor. CRISPR-Cas9-mediated genome editing technology brings a great promise for inhibiting migration, invasion, and even treatment of tumor. However, the potential off-target effect limits its clinical application, and the effective ethical review is necessary. The article reviews the molecular mechanisms of CRISPR/Cas9 and discusses the research and the limitation related to cancer clinical trials.
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Affiliation(s)
- Xin-Zhu Chen
- Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, The NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
- The First Affiliated Hospital, Hainan Medical University, Haikou, China
| | - Rong Guo
- State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
| | - Cong Zhao
- Department of Cellular and Molecular Biology, Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Beijing, China
| | - Jing Xu
- Department of Biochemistry and Molecular Biology, School of Integrated Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei, China
| | - Hang Song
- Department of Biochemistry and Molecular Biology, School of Integrated Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei, China
| | - Hua Yu
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China
| | - Christian Pilarsky
- Department of Surgery, University Hospital of Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg (FAU), Erlangen, Germany
| | - Firzan Nainu
- Faculty of Pharmacy, Hasanuddin University, Makassar, Indonesia
| | - Jing-Quan Li
- The First Affiliated Hospital, Hainan Medical University, Haikou, China
| | - Xin-Ke Zhou
- Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, The NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
| | - Jian-Ye Zhang
- Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, The NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
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Zhao L, Sun X, Wang X, Qin S, Kong Y, Li M. Bombyx mori Vps13d is a key gene affecting silk yield. PLoS One 2022; 17:e0270840. [PMID: 35797274 PMCID: PMC9262180 DOI: 10.1371/journal.pone.0270840] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Accepted: 06/20/2022] [Indexed: 11/18/2022] Open
Abstract
Bombyx mori is an important economic insect, its economic value mainly reflected in the silk yield. The major functional genes affecting the silk yield of B. mori have not been determined yet. Bombyx mori vacuolar protein sorting-associated protein 13d (BmVps13d) has been identified, but its function is not reported. In this study, BmVps13d protein shared 30.84% and 34.35% identity with that of in Drosophila melanogaster and Homo. sapiens, respectively. The expressions of BmVps13d were significantly higher in the midgut and silk gland of JS (high silk yield) than in that of L10 (low silk yield). An insertion of 9 bp nucleotides and two deficiencies of adenine ribonucleotides in the putative promoter region of BmVps13d gene in L10 resulted in the decline of promoter activity was confirmed using dual luciferase assay. Finally, the functions of BmVps13d in B. mori were studied using the CRISPR/Cas9 system, and the mutation of BmVps13d resulted in a 24.7% decline in weight of larvae, as well as a 27.1% (female) decline and a 11.8% (male) decline in the silk yield. This study provides a foundation for studying the molecular mechanism of silk yield and breeding the silkworm with high silk yield.
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Affiliation(s)
- Luochao Zhao
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China
| | - Xia Sun
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China
- The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, 212018, Jiangsu, China
| | - Xueyang Wang
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China
- The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, 212018, Jiangsu, China
| | - Sheng Qin
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China
- The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, 212018, Jiangsu, China
| | - Yunhui Kong
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China
- The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, 212018, Jiangsu, China
| | - Muwang Li
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China
- The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, 212018, Jiangsu, China
- * E-mail:
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Krueger LA, Morris AC. Generation of a zebrafish knock-in line expressing MYC-tagged Sox11a using CRISPR/Cas9 genome editing. Biochem Biophys Res Commun 2022; 608:8-13. [PMID: 35378361 PMCID: PMC9050874 DOI: 10.1016/j.bbrc.2022.03.103] [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: 03/17/2022] [Accepted: 03/21/2022] [Indexed: 11/02/2022]
Abstract
Advances in CRISPR-Cas9 genome editing technology have strengthened the role of zebrafish as a model organism for genetics and developmental biology. These tools have led to a significant increase in the production of loss-of-function mutant zebrafish lines. However, the generation of precisely edited knock-in lines has remained a significant challenge in the field due to the decreased efficiency of homology directed repair (HDR). In this study, we overcame some of these challenges by combining available design tools and synthetic, commercially available CRISPR reagents to generate a knock-in line carrying an in-frame MYC epitope tag at the sox11a locus. Zebrafish Sox11a is a transcription factor with critical roles in organogenesis, neurogenesis, craniofacial, and skeletal development; however, only a few direct molecular targets of Sox11a have been identified. Here, we evaluate the knock-in efficiency of various HDR donor configurations and demonstrate the successful expression and localization of the resulting knock-in allele. Our results provide an efficient, streamlined approach to knock-in experiments in zebrafish, which will enable expansion of downstream experimental applications that have previously been difficult to perform. Moreover, the MYC-Sox11a line we have generated will allow further investigation into the function and direct targets of Sox11a.
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Affiliation(s)
- Laura A Krueger
- Department of Biology, University of Kentucky, Lexington, KY, 40506-0225, USA
| | - Ann C Morris
- Department of Biology, University of Kentucky, Lexington, KY, 40506-0225, USA.
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Wang X, Yi XL, Hou CX, Wang XY, Sun X, Zhang ZJ, Qin S, Li MW. Map-based cloning and functional analysis revealed ABCC2 is responsible for Cry1Ac toxin resistance in Bombyx mori. ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY 2022; 110:e21886. [PMID: 35307854 DOI: 10.1002/arch.21886] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/30/2022] [Revised: 02/22/2022] [Accepted: 03/08/2022] [Indexed: 06/14/2023]
Abstract
Bt toxins are parasporal crystals produced by Bacillus thuringiensis (Bt). They have specific killing activity against various insects and have been widely used to control agricultural pests. However, their widespread use has developed the resistance of many target insects. To maintain the sustainable use of Bt products, the resistance mechanism of insects to Bt toxins must be fully clarified. In this study, Bt-resistant and Bt-susceptible silkworm strains were used to construct genetic populations, and the genetic pattern of silkworm resistance to Cry1Ac toxin was determined. Sequence-tagged site molecular marker technology was used to finely map the resistance gene and to draw a molecular genetic linkage map, and the two closest markers were T1590 and T1581, indicating the resistance gene located in the 155 kb genetic region. After analyzing the sequence of the predicted gene in the genetic region, an ATP binding cassette transporter (ABCC2) was identified as the candidate gene. Molecular modeling and protein-protein docking result showed that a tyrosine insertion in the mutant ABCC2 might be responsible for the interaction between Cry1Ac and ABCC2. Moreover, CRISPR/Cas9-mediated genome editing technology was used to knockout ABCC2 gene. The homozygous mutant ABCC2 silkworm was resistant to Cry1Ac toxin, which indicated ABCC2 is the key gene that controls silkworm resistance to Cry1Ac toxin. The results have laid the foundation for elucidating the molecular resistance mechanism of silkworms to Cry1Ac toxin and could provide a theoretical basis for the biological control of lepidopteran pests.
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Affiliation(s)
- Xin Wang
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China
| | - Xiao-Li Yi
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China
| | - Cheng-Xiang Hou
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China
- Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, Jiangsu, China
| | - Xue-Yang Wang
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China
- Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, Jiangsu, China
| | - Xia Sun
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China
- Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, Jiangsu, China
| | - Zhong-Jie Zhang
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China
| | - Sheng Qin
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China
- Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, Jiangsu, China
| | - Mu-Wang Li
- Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China
- Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, Jiangsu, China
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Shin NR, Shin YH, Kim HS, Park YD. Function Analysis of the PR55/ B Gene Related to Self-Incompatibility in Chinese Cabbage Using CRISPR/Cas9. Int J Mol Sci 2022; 23:ijms23095062. [PMID: 35563453 PMCID: PMC9102814 DOI: 10.3390/ijms23095062] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Revised: 04/29/2022] [Accepted: 04/29/2022] [Indexed: 02/06/2023] Open
Abstract
Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50–70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T2 gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding.
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50
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Canzian J, Gonçalves FLS, Müller TE, Franscescon F, Santos LW, Adedara IA, Rosemberg DB. Zebrafish as a potential non-traditional model organism in translational bipolar disorder research: Genetic and behavioral insights. Neurosci Biobehav Rev 2022; 136:104620. [PMID: 35300991 DOI: 10.1016/j.neubiorev.2022.104620] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Revised: 02/16/2022] [Accepted: 03/10/2022] [Indexed: 01/14/2023]
Abstract
Bipolar disorder (BD) is a severe and debilitating illness that affects 1-2% of the population worldwide. BD is characterized by recurrent and extreme mood swings, including mania/hypomania and depression. Animal experimental models have been used to elucidate the mechanisms underlying BD and different strategies have been proposed to assess BD-like symptoms. The zebrafish (Danio rerio) has been considered a suitable vertebrate system for modeling BD-like responses, due to the genetic tractability, molecular/physiological conservation, and well-characterized behavioral responses. In this review, we discuss how zebrafish-based models can be successfully used to understand molecular, biochemical, and behavioral alterations paralleling those found in BD. We also outline some advantages and limitations of this aquatic species to examine BD-like phenotypes in translational neurobehavioral research. Overall, we reinforce the use of zebrafish as a promising tool to investigate the neural basis associated with BD-like behaviors, which may foster the discovery of novel pharmacological therapies.
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Affiliation(s)
- Julia Canzian
- Laboratory of Experimental Neuropsychobiology, Department of Biochemistry and Molecular Biology, Natural and Exact Sciences Center, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil; Graduate Program in Biological Sciences: Toxicological Biochemistry, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil
| | - Falco L S Gonçalves
- Laboratory of Experimental Neuropsychobiology, Department of Biochemistry and Molecular Biology, Natural and Exact Sciences Center, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil
| | - Talise E Müller
- Graduate Program in Biological Sciences: Toxicological Biochemistry, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil
| | - Francini Franscescon
- Laboratory of Experimental Neuropsychobiology, Department of Biochemistry and Molecular Biology, Natural and Exact Sciences Center, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil; Graduate Program in Biological Sciences: Toxicological Biochemistry, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil
| | - Laura W Santos
- Laboratory of Experimental Neuropsychobiology, Department of Biochemistry and Molecular Biology, Natural and Exact Sciences Center, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil
| | - Isaac A Adedara
- Laboratory of Experimental Neuropsychobiology, Department of Biochemistry and Molecular Biology, Natural and Exact Sciences Center, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil; Drug Metabolism and Toxicology Research Laboratories, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria.
| | - Denis B Rosemberg
- Laboratory of Experimental Neuropsychobiology, Department of Biochemistry and Molecular Biology, Natural and Exact Sciences Center, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil; Graduate Program in Biological Sciences: Toxicological Biochemistry, Federal University of Santa Maria, 1000 Roraima Avenue, Santa Maria, RS 97105-900, Brazil; The International Zebrafish Neuroscience Research Consortium (ZNRC), 309 Palmer Court, Slidell, LA 70458, USA.
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