TY - JOUR
T1 - Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis
AU - Singh, Digvijay
AU - Wang, Yanbo
AU - Mallon, John
AU - Yang, Olivia
AU - Fei, Jingyi
AU - Poddar, Anustup
AU - Ceylan, Damon
AU - Bailey, Scott
AU - Ha, Taekjip
N1 - Funding Information:
The project was supported by grants from the National Science Foundation (PHY-1430124 to T.H.) and National Institutes of Health (GM065367; GM112659 to T.H. and GM097330 to S.B.); T.H. is supported by the Howard Hughes Medical Institute. J.M. is supported by the National Institutes of Health Chemical Biology Interface training program (T32GM080189). We thank J. A. Doudna and S. H. Sternberg for useful early discussions about the design of experiments. We also thank S. H. Sternberg and J. S. Chen of the Doudna laboratory (University of California-Berkeley) for generously providing Cas9 stocks and EngCas9 expression plasmids, respectively.
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/4/1
Y1 - 2018/4/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41594-018-0051-7
DO - 10.1038/s41594-018-0051-7
M3 - Article
C2 - 29622787
AN - SCOPUS:85045098668
SN - 1545-9993
VL - 25
SP - 347
EP - 354
JO - Nature Structural and Molecular Biology
JF - Nature Structural and Molecular Biology
IS - 4
ER -