瑞士苏黎世大学Martin Jinek研究团队揭示Cas9脱靶活性的结构基础。相关论文于2022年10月27日发表在《细胞》杂志上。

研究人员报告了Cas9与真正的非靶标底物结合的结晶学结构，揭示了非靶标结合是由向导:非靶标异二聚体中的一系列非经典碱基配对相互作用促成的。含有相对于引导RNA的单核苷酸缺失的非靶标底物，可通过碱基跳过或多个非经典碱基对而不是RNA隆起的形成来适应。最后，PAM远端错配导致双链不配对，并诱发Cas9 REC叶的构象变化，扰乱了其构象激活。总之，这些见解为Cas9的脱靶活性提供了结构上的依据，并有助于改进向导RNA的合理设计和脱靶预测算法。

据了解，CRISPR相关基因组编辑核酸酶Cas9的靶标DNA特异性，是由其向导RNA中的20个核苷酸片段的互补性决定的。然而，Cas9可以结合并切割部分互补的非目标序列，这引起了人们对其在临床应用中的安全性的关注。

附：英文原文

Title: Structural basis for Cas9 off-target activity

Author: Martin Pacesa, Chun-Han Lin, Antoine Cléry, Aakash Saha, Pablo R. Arantes, Katja Bargsten, Matthew J. Irby, Frédéric H.-T. Allain, Giulia Palermo, Peter Cameron, Paul D. Donohoue, Martin Jinek

Issue&Volume: 2022/10/27

Abstract: The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

DOI: 10.1016/j.cell.2022.09.026

Source: https://www.cell.com/cell/fulltext/S0092-8674(22)01198-9