Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.
Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.
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DOI：
10.1038/nature26155
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发表时间：
2018-04-05
期刊：
影响因子：
64.8
通讯作者：
Liu DR
中科院分区：
文献类型：
--
作者：
Hu JH;Miller SM;Geurts MH;Tang W;Chen L;Sun N;Zeina CM;Gao X;Rees HA;Lin Z;Liu DR
使用CRISPR-Cas9蛋白进行基因组编辑和其他应用的一个关键限制是要求靶位点处存在前间区序列邻近基序（PAM）。对于最常用的来自化脓性链球菌的Cas9（SpCas 9），该PAM要求是NGG。没有显示在哺乳动物细胞中有效发挥功能的天然或工程化Cas9变体提供比NGG限制性更小的PAM。在这里，我们使用噬菌体辅助连续进化（PACE）来进化扩展的PAM SpCas 9变体（xCas 9），其可以识别广泛的PAM序列，包括NG、GAA和GAT。xCas 9的PAM相容性是迄今为止在哺乳动物细胞中有活性的Cas9中报道的最广泛的，并且支持在人类细胞中的应用，包括靶向转录激活、核酸酶介导的基因破坏以及胞苷和腺嘌呤碱基编辑。值得注意的是，尽管其扩大了PAM相容性，但xCas 9具有比SpCas 9大得多的DNA特异性，在测试的所有NGG靶位点处具有显著较低的全基因组脱靶活性，以及当用非NGG PAM靶向基因组位点时具有最小的脱靶活性。这些发现扩大了CRISPR系统的DNA靶向范围，并确定了Cas9编辑效率、PAM相容性和DNA特异性之间没有必要的权衡。
A key limitation to the use of CRISPR-Cas9 proteins for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), this PAM requirement is NGG. No natural or engineered Cas9 variants shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we used phage-assisted continuous evolution (PACE) to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA, and GAT. The PAM compatibility of xCas9 is the broadest reported to date among Cas9s active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and both cytidine and adenine base editing. Remarkably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity.
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影响因子：
46.9
作者：
Kim YB;Komor AC;Levy JM;Packer MS;Zhao KT;Liu DR
通讯作者：
Liu DR
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
64.8
作者：
Komor AC;Kim YB;Packer MS;Zuris JA;Liu DR
通讯作者：
Liu DR
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
14.8
作者：
Meng, Xiangdong;Wolfe, Scot A.
通讯作者：
Wolfe, Scot A.