Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila
Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila
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DOI：
10.1073/pnas.1405500111
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发表时间：
2014-07-22
影响因子：
11.1
通讯作者：
Bullock, Simon L.
中科院分区：
文献类型：
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作者：
Port, Fillip;Chen, Hui-Min;Bullock, Simon L.
II型成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关（Cas）系统最近已成为一种对各种生物体基因组进行操作的强大方法。在此，我们报道了一个用于黑腹果蝇高效基因组工程的工具箱，它由转基因Cas9品系和通用的引导RNA（gRNA）表达质粒组成。系统评估揭示了具有普遍存在或生殖系受限活性模式的Cas9品系。我们还证明了由不同的U6小核RNA启动子表达的相同gRNA具有不同的活性，其中先前未经测试的U6:3启动子效果最强。Cas9和gRNA的适当组合能够以25 - 100%的传递率靶向必需基因和非必需基因。我们还证明我们优化的CRISPR/Cas工具可用于基于交错切口的诱变。此外，与寡核苷酸或长双链供体模板相结合，我们的试剂能够通过同源定向修复进行精确的基因组编辑，其编辑率使得选择标记不再必要。最后，我们展示了CRISPR/Cas介导技术的一种新应用，即在以普遍存在或组织受限方式表达的Cas9进行高效双等位基因靶向后，揭示体细胞中的功能丧失表型。我们的CRISPR/Cas工具将有助于快速评估特定基因的突变表型，并以单核苷酸精度对基因组进行精确修饰。我们的结果也为利用CRISPR/Cas进行高通量基因筛选铺平了道路。
The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germline-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6: 3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.