Rapid reverse genetic screening using CRISPR in zebrafish.
Rapid reverse genetic screening using CRISPR in zebrafish.
复制标题
在斑马鱼中使用CRISPR快速反向遗传筛查。
DOI：
10.1038/nmeth.3360
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发表时间：
2015-06
期刊：
影响因子：
48
通讯作者：
Moens CB
中科院分区：
文献类型：
--
作者：
Shah AN;Davey CF;Whitebirch AC;Miller AC;Moens CB
识别生物过程中涉及的基因对于理解生命的分子构件至关重要。基因工程CRISPR(聚簇规则间隔短回文重复序列)有效地突变特定基因，再加上斑马鱼(Danio Rerio)的可及性，为筛选脊椎动物生物学过程中涉及的基因提供了机会。注射编码Cas9的mRNA和经过改造的单引导RNA(SgRNA)可以在注射的胚胎中导致双等位基因突变，这些突变表现为已知的突变表型。我们发现，提高CRISPR效率和多路复用sgRNAs允许已知突变体在许多表型上的表型复制。我们进行了概念验证筛选，通过交叉、多重池注射检查了48个基因座，并确定了两个与电突触形成有关的新基因。通过对目的基因的深度测序，我们发现90%的基因得到了有效筛选。我们得出结论，CRISPR可以在脊椎动物系统中作为一种强大的反向遗传筛选策略。
Identifying genes involved in biological processes is critical for understanding the molecular building blocks of life. The effectiveness of engineered CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to efficiently mutate specific loci coupled with the accessibility of zebrafish (Danio rerio) provides an opportunity to screen for genes involved in vertebrate biological processes. Injection of Cas9-encoding mRNA and an engineered, single guide RNA (sgRNA) can cause biallelic mutations in injected embryos that phenocopy known mutant phenotypes. We found that increasing CRISPR efficiency and multiplexing sgRNAs allowed for phenocopy of known mutants across many phenotypes. We performed a proof-of-concept screen examining 48 loci by intersecting, multiplexed pool injections, and identified two new genes involved in electrical synapse formation. By deep-sequencing target loci we found that 90% of the genes were effectively screened. We conclude that CRISPR can be used as a powerful reverse genetic screening strategy in vivo in a vertebrate system.