Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
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DOI：
10.1038/nature14592
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发表时间：
2015-07-23
期刊：
影响因子：
64.8
通讯作者：
Joung JK
中科院分区：
文献类型：
--
作者：
Kleinstiver BP;Prew MS;Tsai SQ;Topkar VV;Nguyen NT;Zheng Z;Gonzales AP;Li Z;Peterson RT;Yeh JR;Aryee MJ;Joung JK
尽管CRISPR-Cas9核酸酶广泛用于基因组编辑，但Cas9可以识别的序列范围受到对特定前间区序列邻近基序（PAM）的需要的限制。因此，通常难以以各种基因组编辑应用所需的精确度靶向双链断裂（DSB）。工程化具有有目的地改变的PAM特异性的Cas9衍生物的能力将解决这一限制。在这里，我们表明，常用的化脓性链球菌Cas9（SpCas 9）可以被修改，以识别替代PAM序列使用结构信息，细菌选择为基础的定向进化，和组合设计。这些改变的PAM特异性变体使得能够对目前野生型SpCas 9不可靶向的斑马鱼和人类细胞中的内源性基因位点进行稳健编辑，并且如通过GUIDE-Seq分析判断的，它们的全基因组特异性与野生型SpCas 9相当。此外，我们鉴定并表征了另一种SpCas 9变体，其在人细胞中表现出改善的特异性，对具有非典型NAG和NGA PAM和/或错配间隔区的脱靶位点具有更好的区分力。我们还发现两种较小的Cas9直系同源物，嗜热链球菌Cas9（St 1Cas 9）和金黄色葡萄球菌Cas9（SaCas 9），在细菌选择系统和人类细胞中有效地发挥作用，这表明我们的工程策略可以扩展到其他物种的Cas9。我们的发现提供了广泛有用的SpCas 9变体，更重要的是，建立了工程化具有改变和改善的PAM特异性的广泛Cas9的可行性。
Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-Seq analysis. In addition, we identified and characterized another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also found that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.