Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.
Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
Nature. 2018 Apr 5;556(7699):57-63. doi: 10.1038/nature26155. Epub 2018 Feb 28.
A key limitation of the use of the CRISPR-Cas9 system 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), the required PAM sequence is NGG. No natural or engineered Cas9 variants that have been shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we use phage-assisted continuous evolution 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 our knowledge, among Cas9 proteins that are active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and cytidine and adenine base editing. Notably, 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.
CRISPR-Cas9 系统在基因组编辑和其他应用中的一个关键限制是，在靶位点必须存在一个邻近基序（PAM）。对于最常用的来自酿脓链球菌的 Cas9（SpCas9），所需的 PAM 序列是 NGG。在已证明在哺乳动物细胞中有效发挥作用的天然或工程 Cas9 变体中，没有比 NGG 限制更小的 PAM。在这里，我们使用噬菌体辅助连续进化来进化出一种扩展的 PAM SpCas9 变体（xCas9），它可以识别广泛的 PAM 序列，包括 NG、GAA 和 GAT。据我们所知，xCas9 的 PAM 兼容性在在哺乳动物细胞中具有活性的 Cas9 蛋白中是报道的最广泛的，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嘧啶和腺嘌呤碱基编辑。值得注意的是，尽管 xCas9 的 PAM 兼容性得到了扩展，但它比 SpCas9 具有更高的 DNA 特异性，在所有测试的 NGG 靶位点上的全基因组脱靶活性都大大降低，并且在靶向非 NGG PAM 的基因组位点时几乎没有脱靶活性。这些发现扩展了 CRISPR 系统的 DNA 靶向范围，并证实 Cas9 编辑效率、PAM 兼容性和 DNA 特异性之间没有必要的权衡。
