Rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the cardiodeleter system.

IF 4.3 Q1 BIOCHEMICAL RESEARCH METHODS
Sean Keeley, Miriam Fernández-Lajarín, David Bergemann, Nicolette John, Lily Parrott, Brittany E Andrea, Juan Manuel González-Rosa
{"title":"Rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the cardiodeleter system.","authors":"Sean Keeley, Miriam Fernández-Lajarín, David Bergemann, Nicolette John, Lily Parrott, Brittany E Andrea, Juan Manuel González-Rosa","doi":"10.1016/j.crmeth.2025.101003","DOIUrl":null,"url":null,"abstract":"<p><p>CRISPR-Cas9 has accelerated loss-of-function studies in zebrafish, but creating tissue-specific mutant lines is still labor intensive. While some tissue-specific Cas9 zebrafish lines exist, standardized methods for gene targeting, including guide RNA (gRNA) delivery, are lacking, limiting broader use in the community. To tackle these limitations, we develop a cardiomyocyte-specific Cas9 line, the cardiodeleter, that efficiently generates biallelic mutations in combination with gene-specific gRNAs. We create transposon-based guide shuttles that deliver gRNAs targeting a gene of interest while permanently labeling cells susceptible to becoming mutant. We validate this modular approach by deleting five genes (ect2, tnnt2a, cmlc2, amhc, and erbb2), resulting in the loss of the corresponding protein or phenocopy of established mutants. We provide detailed protocols for generating guide shuttles, facilitating the adoption of these techniques in the zebrafish community. Our approach enables rapid generation of tissue-specific crispants and analysis of mosaic phenotypes, making it a valuable tool for cell-autonomous studies and genetic screening.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":"5 3","pages":"101003"},"PeriodicalIF":4.3000,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cell Reports Methods","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.crmeth.2025.101003","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

Abstract

CRISPR-Cas9 has accelerated loss-of-function studies in zebrafish, but creating tissue-specific mutant lines is still labor intensive. While some tissue-specific Cas9 zebrafish lines exist, standardized methods for gene targeting, including guide RNA (gRNA) delivery, are lacking, limiting broader use in the community. To tackle these limitations, we develop a cardiomyocyte-specific Cas9 line, the cardiodeleter, that efficiently generates biallelic mutations in combination with gene-specific gRNAs. We create transposon-based guide shuttles that deliver gRNAs targeting a gene of interest while permanently labeling cells susceptible to becoming mutant. We validate this modular approach by deleting five genes (ect2, tnnt2a, cmlc2, amhc, and erbb2), resulting in the loss of the corresponding protein or phenocopy of established mutants. We provide detailed protocols for generating guide shuttles, facilitating the adoption of these techniques in the zebrafish community. Our approach enables rapid generation of tissue-specific crispants and analysis of mosaic phenotypes, making it a valuable tool for cell-autonomous studies and genetic screening.

使用cardiodeleter系统在斑马鱼中快速稳健地生成心肌细胞特异性criscriss。
CRISPR-Cas9加速了斑马鱼的功能丧失研究,但创建组织特异性突变系仍然是劳动密集型的。虽然存在一些组织特异性Cas9斑马鱼品系,但缺乏包括引导RNA (gRNA)递送在内的基因靶向的标准化方法,限制了在社区中的广泛应用。为了解决这些限制,我们开发了一种心肌细胞特异性Cas9系,即心脏删除器,它可以有效地与基因特异性grna结合产生双等位基因突变。我们创造了以转座子为基础的引导穿梭子,在永久标记易突变的细胞的同时,将grna靶向感兴趣的基因。我们通过删除5个基因(ect2、tnnt2a、cmlc2、amhc和erbb2)来验证这种模块化方法,导致相应的蛋白质或已建立突变体的表型丢失。我们提供了生成引导梭子的详细协议,促进了这些技术在斑马鱼群落中的采用。我们的方法能够快速生成组织特异性crispants和马赛克表型分析,使其成为细胞自主研究和遗传筛选的宝贵工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Cell Reports Methods
Cell Reports Methods Chemistry (General), Biochemistry, Genetics and Molecular Biology (General), Immunology and Microbiology (General)
CiteScore
3.80
自引率
0.00%
发文量
0
审稿时长
111 days
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信