High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes.
High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes.
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DOI：
10.1534/genetics.115.179382
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发表时间：
2015-09
期刊：
影响因子：
3.3
通讯作者：
Seydoux G
中科院分区：
文献类型：
--
作者：
Paix A;Folkmann A;Rasoloson D;Seydoux G
由 RNA 编程的核酸酶 Cas9 诱导的断裂的同源定向修复 (HDR) 已成为多种生物体中基因组编辑的流行方法。大多数 HDR 方案依赖于基于质粒的 Cas9 表达和基因特异性向导 RNA。在此，我们报告将体外组装的 Cas9-CRISPR RNA (crRNA) 反式激活 crRNA (tracrRNA) 核糖核蛋白复合物直接注射到秀丽隐杆线虫的性腺中，可产生高频率的 HDR 编辑。基于我们早期的发现，即具有 35 个碱基同源性的 PCR 片段是有效的修复模板，我们开发了一种完全免克隆的协议，用于无需选择即可生成无缝 HDR 编辑。与 co-CRISPR 方法相结合，该协议足够稳健，可与低效指导 RNA 一起使用并生成复杂的编辑，包括 ORF 替换和用荧光蛋白同时标记两个基因。
Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro–assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.