Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis.
Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis.
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DOI：
10.1038/s41594-018-0051-7
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发表时间：
2018-04
影响因子：
16.8
通讯作者：
Ha T
中科院分区：
文献类型：
--
作者：
Singh D;Wang Y;Mallon J;Yang O;Fei J;Poddar A;Ceylan D;Bailey S;Ha T
Cas9（来自化脓性链球菌）与向导rna复合物靶向互补DNA进行切割。在这里，我们采用单分子FRET分析来研究两种工程cas9 （eCas9和Cas9-HF1）特异性增强的机制。DNA解绕实验表明，错配通过重新平衡解绕-复绕平衡来影响裂解反应。pam -远端错配的增加促进了倒绕，相关的解理损伤表明解理是从解绕状态进行的。经过改造的cas9更容易因不匹配而减少未缠绕状态的基因。工程cas9的内在裂解率要低得多，这防止了瞬时解绕脱靶的裂解。工程cas9需要大约一个额外的碱基对匹配才能稳定结合，从而将它们从原本会隔离它们的位点中解放出来。因此，经过工程改造的cas9通过抑制DNA与部分匹配序列的稳定结合，(2)使DNA解旋对错配更加敏感，以及(3)减缓内在裂解反应来实现其改进的特异性。
Cas9 (from S. pyogenes) in complex with a guide-RNA targets complementary DNA for cleavage. Here, we developed single molecule FRET analysis to study the mechanisms of specificity enhancement of two engineered Cas9s (eCas9 and Cas9-HF1). DNA unwinding assay showed that mismatches affect cleavage reactions through rebalancing the unwinding-rewinding equilibrium. Increasing PAM-distal mismatches facilitate rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily with mismatches. Intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. Engineered Cas9s require about one additional base-pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity (1) by inhibiting stable DNA binding to partially matching sequences, (2) by making DNA unwinding more sensitive to mismatches, and (3) by slowing down intrinsic cleavage reaction.
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DOI：
10.1038/nrg2749
发表时间：
2010-03
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
通讯作者：
--
影响因子：
14.9
作者：
Josephs EA;Kocak DD;Fitzgibbon CJ;McMenemy J;Gersbach CA;Marszalek PE
通讯作者：
Marszalek PE
影响因子：
8.8
作者：
Rutkauskas, Marius;Sinkunas, Tomas;Seidel, Ralf
通讯作者：
Seidel, Ralf
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
16.6
作者：
Singh D;Sternberg SH;Fei J;Doudna JA;Ha T
通讯作者：
Ha T