Harnessing type I CRISPR-Cas systems for genome engineering in human cells
Harnessing type I CRISPR-Cas systems for genome engineering in human cells
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DOI：
10.1038/s41587-019-0310-0
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发表时间：
2019-12-01
影响因子：
46.9
通讯作者：
Sternberg, Samuel H.
中科院分区：
文献类型：
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作者：
Cameron, Peter;Coons, Mary M.;Sternberg, Samuel H.
I 型 CRISPR-Cas 系统是细菌和古细菌中最丰富的适应性免疫系统(1,2)。靶点干扰依赖于称为 Cascade(3,4) 的多亚基、RNA 引导复合物，该复合物招募反式作用解旋酶-核酸酶 Cas(3) 来进行靶点降解 (5-7)。由于多组分级联复合体的异源表达相对困难，I 型系统很少用于真核基因组工程应用。在这里，我们将 Cascade 与二聚化依赖性非特异性 FokI 核酸酶结构域 (8-11) 融合，并在多种人类细胞系中实现 RNA 引导的基因编辑，具有高特异性和高达 50% 的效率。 FokI-Cascade 可以通过优化的双组分表达系统进行重建，该系统在单个多顺反子载体上编码 CRISPR 相关 (Cas) 蛋白，在单独的质粒上编码指导 RNA (gRNA)。完整 Cascade-Cas3 复合物在人类细胞中的表达导致长度高达 200 kb 的靶向删除。我们的工作表明，高度丰富、以前未开发的 I 型 CRISPR-Cas 系统可用于真核细胞的基因组工程应用。
Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea(1,2). Target interference relies on a multi-subunit, RNA-guided complex called Cascade(3,4), which recruits a trans-acting helicase-nuclease, Cas(3), for target degradation(5-7). Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain(8-11) and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to similar to 50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to similar to 200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.