Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Science. 2018 Sep 21;361(6408):1259-1262. doi: 10.1126/science.aas9129. Epub 2018 Aug 30.
The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.
RNA 指导的内切酶 Cas9 可切割其靶 DNA，是一种强大的基因组编辑工具。然而，广泛使用的 Cas9 酶（SpCas9）需要 NGG 前导间隔基序（PAM）进行靶标识别，从而限制了可靶向的基因组位点。在这里，我们报告了一种经过合理设计的 SpCas9 变体（SpCas9-NG），它可以识别宽松的 NG PAMs。晶体结构表明，与第三个核苷酸的碱基特异性相互作用的丧失被新引入的非碱基特异性相互作用所补偿，从而能够识别 NG PAM。我们表明，SpCas9-NG 在携带 NG PAMs 的人类细胞内的内源性靶标位点诱导插入缺失。此外，我们发现 SpCas9-NG 与激活诱导胞嘧啶脱氨酶（AID）的融合介导了具有 NG PAMs 的靶标位点的 C 到 T 的转换。
