通过单分子 FRET 分析揭示了工程化 Cas9 特异性提高的机制。
Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis.
机构信息
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Bloomberg School of Public Health, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
出版信息
Nat Struct Mol Biol. 2018 Apr;25(4):347-354. doi: 10.1038/s41594-018-0051-7. Epub 2018 Apr 5.
Cas9 (from Streptococcus pyogenes) in complex with a guide RNA targets complementary DNA for cleavage. Here, we developed a single-molecule FRET analysis to study the mechanisms of specificity enhancement of two engineered Cas9s (eCas9 and Cas9-HF1). A DNA-unwinding assay showed that mismatches affect cleavage reactions through rebalancing the unwinding-rewinding equilibrium. Increasing PAM-distal mismatches facilitates rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily with mismatches. The intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. Engineered Cas9s require approximately one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity by inhibiting stable DNA binding to partially matching sequences, making DNA unwinding more sensitive to mismatches and slowing down the intrinsic cleavage reaction.
Cas9（来自酿脓链球菌）与指导 RNA 复合物靶向互补 DNA 进行切割。在这里，我们开发了一种单分子 FRET 分析方法来研究两种工程化 Cas9（eCas9 和 Cas9-HF1）特异性增强的机制。DNA 解旋实验表明，错配通过重新平衡解旋-重绕平衡来影响切割反应。增加 PAM 远端的错配有利于重绕，相关的切割损伤表明切割从解开状态开始。带有错配的工程 Cas9 更容易使解开状态失活。工程 Cas9 的固有切割速率要低得多，从而防止从瞬时解开的非靶标上进行切割。工程 Cas 9 大约需要额外一个碱基对匹配才能稳定结合，使它们不会与否则会将其隔离的位点结合。因此，工程 Cas9 通过抑制与部分匹配序列的稳定 DNA 结合来实现其改善的特异性，从而使 DNA 解旋对错配更加敏感，并减缓内在的切割反应。
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