Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system
Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system
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DOI：
10.1073/pnas.1308335110
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发表时间：
2013-08-20
影响因子：
11.1
通讯作者：
Chen, Wenbiao
中科院分区：
文献类型：
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作者：
Jao, Li-En;Wente, Susan R.;Chen, Wenbiao
长期以来一直在寻求一种简单而坚固的方法，用于斑马鱼中的靶向诱变。先前的方法会在F0动物的生殖系中产生单相关突变，通常会延迟对F2代生成的突变的纯合性。 F0中强大的双重突变产生将直接在注射动物中进行表型分析。最近，II型原核生物群集定期间隔短的短质体重复序列（CRISPR）/与CRISPR相关的蛋白质（CAS）系统已被改编为作为靶向基因组诱变工具。在这里，我们报告了斑马鱼中改进的CRISPR/CAS系统，并具有自定义指南RNA和斑马鱼密码子优化的Cas9蛋白，该蛋白有效地针对了记者转基因TG（-5.1MNX1：EGFP）和四个内源性位点（Tyr，Golden，Mitfa和ddx19） ）。诱变率达到75-99％，表明大多数细胞含有双重突变。在五个靶向病例中的四个中观察到了隐性无效的表型，从而支持高质基因破坏率。我们还观察到CAS9诱导的突变的有效种系传输。最后，可以同时将五个基因组基因座靶向，从而导致同一注射鱼类的多功能表型。该CRISPR/CAS9系统代表了斑马鱼中一种高效且可扩展的基因敲除方法，并且具有在其他模型生物中应用的潜力。
A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.