Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas
Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas
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DOI：
10.1016/j.molcel.2019.03.014
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发表时间：
2019-06-06
期刊：
影响因子：
16
通讯作者：
Zhang, Yan
中科院分区：
文献类型：
--
作者：
Dolan, Adam E.;Hou, Zhonggang;Zhang, Yan
CRISPR-Cas系统使微生物获得性免疫成为可能，并提供真核基因组编辑工具。这些工具采用II型或V型CRISPR的单一效应酶来产生RNA引导的精确基因组断裂。在这里，我们证明了使用I型CRISPR-Cas在人类胚胎干细胞和HAP 1细胞中有效引入一系列长距离染色体缺失的可行性。I型CRISPR系统依赖于多亚基核糖核蛋白（RNP）复合物Cascade来鉴定DNA靶标，并依赖于解旋酶-核酸酶Cas 3来降解DNA原核酶。随着T. fusca Cascade和Cas 3，我们获得了13%-60%的编辑效率。基于长距离PCR和基于高通量测序的损伤分析显示，在靶位点的上游产生了多种缺失，范围从几百个碱基对到100千个碱基。这些结果突出了I型CRISPR-Cas用于真核生物中的远程基因组操作和缺失筛选的潜在效用。
CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilo-bases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.