Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; email:
California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720.
Annu Rev Biophys. 2017 May 22;46:505-529. doi: 10.1146/annurev-biophys-062215-010822. Epub 2017 Mar 30.
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.
许多细菌的规律成簇间隔短回文重复（CRISPR）-CRISPR 相关（Cas）系统利用双 RNA 指导的 DNA 内切酶 Cas9 通过在靶 DNA 中引入特异性双链断裂来抵御入侵的噬菌体和可转移质粒。靶标识别严格要求靶位点侧翼有短的原间隔序列邻近基序（PAM），随后 R 环的形成和链的断裂是由向导 RNA 和靶 DNA 之间的互补碱基配对、Cas9-DNA 相互作用和相关构象变化驱动的。通过合成单链向导 RNA（sgRNA）模拟天然的双转录激活 CRISPR RNA（tracrRNA）-CRISPR RNA（crRNA）结构，简化了 CRISPR-Cas9 作为 RNA 可编程 DNA 靶向和编辑平台的使用。本综述旨在深入了解 Cas9 介导的 RNA 指导的 DNA 靶向和切割的机制和结构。生化和结构研究的分子见解为理性工程提供了一个框架，旨在改变催化功能、向导 RNA 特异性和 PAM 要求，并降低 Cas9 基于治疗遗传疾病的脱靶活性。
