Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.
Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.
复制标题
DOI：
10.1038/nbt.3404
复制
发表时间：
2015-12
影响因子：
46.9
通讯作者：
Joung JK
中科院分区：
文献类型：
--
作者：
Kleinstiver BP;Prew MS;Tsai SQ;Nguyen NT;Topkar VV;Zheng Z;Joung JK
CRISPR-Cas9核酸酶主要由RNA-DNA相互作用引导，但也需要cas9介导的原间隔器邻近基序（PAM）的识别。虽然具有潜在的特异性优势，但扩展的PAM序列限制了Cas9同源物在基因组编辑中的靶向范围。缓解这种限制的一种可能策略是放宽PAM中某些位置的特异性。本研究利用分子进化技术对金黄色葡萄球菌Cas9 （SaCas9）的NNGRRT PAM特异性进行了修饰。我们发现的一种变体KKH SaCas9在内源性人类靶位点显示出强大的基因组编辑活性，具有NNNRRT pam。重要的是，使用GUIDE-seq，我们发现野生型和KKH SaCas9在人类细胞中诱导了相当数量的脱靶效应。KKH SaCas9将SaCas9的瞄准范围增加了近2至4倍。我们的分子进化策略不需要结构信息，因此应该适用于广泛的Cas9同源物。
CRISPR-Cas9 nucleases are primarily guided by RNA-DNA interactions but also require Cas9-mediated recognition of a protospacer adjacent motif (PAM). While potentially advantageous for specificity, extended PAM sequences limit the targeting range of Cas9 orthologues for genome editing. One possible strategy to relieve this restriction is to relax specificities for certain positions within the PAM. Here we used molecular evolution to modify the NNGRRT PAM specificity of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, shows robust genome editing activities at endogenous human target sites with NNNRRT PAMs. Importantly, using GUIDE-seq, we show that both wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. KKH SaCas9 increased the targeting range of SaCas9 by nearly two- to four-fold. Our molecular evolution strategy does not require structural information and therefore should be applicable to a wide range of Cas9 orthologues.