School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China.
Nucleic Acids Res. 2021 Dec 2;49(21):12433-12444. doi: 10.1093/nar/gkab1139.
Streptococcus pyogenes Cas9 (SpCas9), a programmable RNA-guided DNA endonuclease, has been widely repurposed for biological and medical applications. Critical interactions between SpCas9 and DNA confer the high specificity of the enzyme in genome engineering. Here, we unveil that an essential SpCas9-DNA interaction located beyond the protospacer adjacent motif (PAM) is realized through electrostatic forces between four positively charged lysines among SpCas9 residues 1151-1156 and the negatively charged DNA backbone. Modulating this interaction by substituting lysines with amino acids that have distinct charges revealed a strong dependence of DNA target binding and cleavage activities of SpCas9 on the charge. Moreover, the SpCas9 mutants show markedly distinguishable DNA interaction sites beyond the PAM compared with wild-type SpCas9. Functionally, this interaction governs DNA sampling and participates in protospacer DNA unwinding during DNA interrogation. Overall, a mechanistic and functional understanding of this vital interaction explains how SpCas9 carries out efficient DNA interrogation.
化脓链球菌 Cas9（SpCas9）是一种可编程的 RNA 指导的 DNA 内切酶，已被广泛应用于生物学和医学领域。SpCas9 与 DNA 之间的关键相互作用赋予了该酶在基因组工程中的高度特异性。在这里，我们揭示了 SpCas9 与 DNA 的一个重要相互作用是通过 SpCas9 残基 1151-1156 之间的四个带正电荷的赖氨酸与带负电荷的 DNA 骨架之间的静电力实现的，而该相互作用位于原间隔基序（PAM）之外。通过用具有不同电荷的氨基酸取代赖氨酸来调节这种相互作用，揭示了 SpCas9 的 DNA 靶标结合和切割活性强烈依赖于电荷。此外，与野生型 SpCas9 相比，SpCas9 突变体在 PAM 之外显示出明显不同的 DNA 相互作用位点。从功能上讲，这种相互作用控制着 DNA 的采样，并参与 DNA 检测过程中原间隔 DNA 的解旋。总的来说，对这种重要相互作用的机制和功能的理解解释了 SpCas9 如何进行有效的 DNA 检测。
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