Cas9 脱靶活性的结构基础。
Structural basis for Cas9 off-target activity.
机构信息
Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
Caribou Biosciences, 2929 Seventh Street Suite 105, Berkeley, CA 94710, USA.
出版信息
Cell. 2022 Oct 27;185(22):4067-4081.e21. doi: 10.1016/j.cell.2022.09.026.
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.
CRISPR 相关的基因组编辑核酸酶 Cas9 的靶 DNA 特异性由其指导 RNA 中的 20 个核苷酸片段的互补性决定。然而，Cas9 可以结合并切割部分互补的脱靶序列，这引起了人们对其在临床应用中的安全性的关注。在这里，我们报告了 Cas9 与真实的脱靶底物结合的晶体结构，揭示了脱靶结合是由指导：脱靶异源双链体中的一系列非canonical 碱基配对相互作用所允许的。相对于指导 RNA 含有单核苷酸缺失的脱靶底物通过碱基跳过或多个非canonical 碱基对得到容纳，而不是 RNA 突环形成。最后，PAM 远端错配导致双链体解配对，并诱导 Cas9 的 REC 结构域发生构象变化，扰乱其构象激活。总之，这些见解为 Cas9 的脱靶活性提供了结构基础，并有助于指导 RNA 和脱靶预测算法的改进理性设计。
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