Structural insights into a high fidelity variant of SpCas9
Structural insights into a high fidelity variant of SpCas9
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DOI：
10.1038/s41422-018-0131-6
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发表时间：
2019-03-01
期刊：
影响因子：
44.1
通讯作者：
Huang, Zhiwei
中科院分区：
文献类型：
--
作者：
Guo, Minghui;Ren, Kuan;Huang, Zhiwei
CRISPR-Cas9系统的RNA引导的核酸内切酶，包括最广泛使用的来自化脓性链球菌（Streptococcus pyogenes）的Cas9（SpCas 9），正在成为模式生物中强大的基因组编辑工具，并为治疗应用带来巨大的希望。已经采用许多策略来克服由SpCas 9的脱靶效应及其对原型间隔区邻近基序（PAM）序列的严格要求引起的限制。然而，这些战略背后的结构机制仍然不明确。在这里，我们展示了SpCas 9变体xCas 9 3.7的晶体结构，它具有广泛的PAM相容性和高DNA靶向特异性，与单向导RNA及其双链DNA靶标复合。结构比较显示，盐桥稳定的R1335对于SpCas 9对PAM序列的严格选择是关键的。通过xCas 9 3.7中的E1219 V突变对该残基的不受限制的旋转异构化降低了PAM识别的严格性，并允许SpCas 9识别多个PAM序列，如生化数据进一步支持的。与野生型（WT）SpCas 9中的那些相比，xCas 9 3.7中的REC 2和REC 3结构域经历显著的构象变化，导致与DNA底物的接触减少。被工程化以显示与DNA的较少相互作用并具有构象上更灵活的REC 2和REC 3结构域的SpCas 9突变体在生物化学和细胞测定中显示出对DNA底物的增强的特异性。总之，我们的发现揭示了xCas 9 3.7的扩大的PAM相容性和高DNA保真度的结构机制，这可以帮助更有效的SpCas 9变体和可能的其他Cas9直系同源物的合理工程化。
The RNA-guided endonucleases of the CRISPR-Cas9 system, including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), are becoming a robust genome editing tool in model organisms and hold immense promise for therapeutic applications. Many strategies have been employed to overcome the limitations caused by SpCas9's off-target effects and its stringent requirement for the protospacer adjacent motif (PAM) sequence. However, the structural mechanisms underlying these strategies remain undefined. Here, we present crystal structure of a SpCas9 variant, xCas9 3.7 that has broad PAM compatibility and high DNA targeting specificity, in complex with a single-guide RNA and its double-stranded DNA targets. Structural comparison revealed that salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence by SpCas9. Unrestricted rotamerization of this residue by the E1219V mutation in xCas9 3.7 lessens the stringency for PAM recognition and allows SpCas9 to recognize multiple PAM sequences as further supported by biochemical data. Compared to those in wild-type (WT) SpCas9, REC2 and REC3 domains in xCas9 3.7 undergo striking conformational changes, leading to reduced contact with DNA substrate. SpCas9 mutants engineered to display less interaction with DNA and have conformationally more flexible REC2 and REC3 domains display enhanced specificity for DNA substrates in both biochemical and cellular assays. Taken together, our findings reveal the structural mechanisms underlying the broadened PAM compatibility and high DNA fidelity of xCas9 3.7, which can assist rational engineering of more efficient SpCas9 variants and probably other Cas9 orthologs.