Expanding the targeting space of Cas9 CRISPR-Cas9 associates with a guide RNA to target and cleave a specific DNA site next to a protospacer adjacent motif (PAM). Streptococcus pyogenes Cas9 (SpCas9), the one most often used for genome editing, only recognizes the NGG sequence (where N is any nucleobase) as the PAM, which restricts regions in the genome that can be targeted. To address this limitation, Nishimasu et al. created a SpCas9 variant that recognizes NG rather than NGG. The SpCas9-NG variant increased the targeting range, had a specificity similar to that of the wild-type enzyme, and could be used with a base editor. Thus, SpCas9-NG is a powerful addition to the CRISPR-Cas9 genome engineering toolbox and will be useful in a broad range of applications, from basic research to clinical therapeutics. Science, this issue p. 1259 An engineered CRISPR-Cas9 nuclease increases the range of genomic sequences that can be targeted in Cas9-mediated genome engineering. The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Streptococcus pyogenes 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.

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具有扩展靶向空间的工程化 CRISPR-Cas9 核酸酶

扩展 Cas9 的靶向空间 CRISPR-Cas9 与引导 RNA 相关联，以靶向和切割原始间隔区相邻基序 (PAM) 旁边的特定 DNA 位点。化脓性链球菌 Cas9 (SpCas9) 是最常用于基因组编辑的一种，它仅将 NGG 序列（其中 N 是任何核碱基）识别为 PAM，从而限制了基因组中可以靶向的区域。为了解决这一限制，Nishimasu 等人。创建了识别 NG 而不是 NGG 的 SpCas9 变体。SpCas9-NG 变体增加了靶向范围，具有与野生型酶相似的特异性，并且可以与碱基编辑器一起使用。因此，SpCas9-NG 是 CRISPR-Cas9 基因组工程工具箱的强大补充，并将用于从基础研究到临床治疗的广泛应用。科学，这个问题 p。1259 一种工程化的 CRISPR-Cas9 核酸酶增加了可在 Cas9 介导的基因组工程中靶向的基因组序列范围。RNA 引导的核酸内切酶 Cas9 切割其目标 DNA，是一种强大的基因组编辑工具。然而，广泛使用的化脓性链球菌 Cas9 酶 (SpCas9) 需要 NGG protospacer 相邻基序 (PAM) 进行目标识别，从而限制了可靶向的基因组位点。在这里，我们报告了一种合理设计的 SpCas9 变体（SpCas9-NG），它可以识别松弛的 NG PAM。晶体结构表明，与第三个核碱基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，从而实现了 NG PAM 识别。我们表明 SpCas9-NG 在人类细胞中带有 NG PAM 的内源性靶位点诱导插入缺失。此外，