Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects.
Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects.
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DOI：
10.1089/crispr.2021.0076
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发表时间：
2022-04
期刊：
影响因子：
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通讯作者：
Zhicheng Zuo;Kesavan Babu;Chhandosee Ganguly;A. Zolekar;Sydney N Newsom;R. Rajan;Yu-Chieh Wang;Jin Liu
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文献类型：
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作者：
Zhicheng Zuo;Kesavan Babu;Chhandosee Ganguly;A. Zolekar;Sydney N Newsom;R. Rajan;Yu-Chieh Wang;Jin Liu
来自化脓性链球菌的rna引导Cas9核酸酶已成为一种重要的基因编辑工具。然而，其固有的脱靶活性是生物医学应用的主要挑战。与一些报道的专门针对单个结构域的工程策略不同，考虑到Cas9的特异性是由各个结构域协同决定的，我们合理地在酶的多个结构域中引入多个氨基酸替换，以创建潜在的高保真变体。我们还利用我们之前导出的活化Cas9复合体结构的原子模型来指导新的修饰。这种方法已经鉴定出具有增强的DNA切割特异性的HSC1.2 Cas9变体。尽管在体外切割实验中，与HSC1.2变体相关的增强特异性似乎与位置相关，但使用这种Cas9变体进行脱靶DNA编辑的频率远低于人类细胞中野生型Cas9的频率。通过分子动力学模拟研究了产生位置依赖效应的可能机制。我们的发现为利用结构和动态信息开发具有高特异性的基因编辑cas9样酶奠定了坚实的基础。
The RNA-guided Cas9 nuclease from Streptococcus pyogenes has become an important gene-editing tool. However, its intrinsic off-target activity is a major challenge for biomedical applications. Distinct from some reported engineering strategies that specifically target a single domain, we rationally introduced multiple amino acid substitutions across multiple domains in the enzyme to create potential high-fidelity variants, considering the Cas9 specificity is synergistically determined by various domains. We also exploited our previously derived atomic model of activated Cas9 complex structure for guiding new modifications. This approach has led to the identification of the HSC1.2 Cas9 variant with enhanced specificity for DNA cleavage. While the enhanced specificity associated with the HSC1.2 variant appeared to be position-dependent in the in vitro cleavage assays, the frequency of off-target DNA editing with this Cas9 variant is much less than that of the wild-type Cas9 in human cells. The potential mechanisms causing the observed position-dependent effect were investigated through molecular dynamics simulation. Our discoveries establish a solid foundation for leveraging structural and dynamic information to develop Cas9-like enzymes with high specificity in gene editing.