TY - JOUR
T1 - Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9
AU - Singh, Digvijay
AU - Sternberg, Samuel H.
AU - Fei, Jingyi
AU - Doudna, Jennifer A.
AU - Ha, Taekjip
N1 - Funding Information:
The project was supported by grants from the National Science Foundation (PHY-1430124 to T.H. and 1244557 to J.A.D.) and National Institutes of Health (GM065367; GM112659 to T.H.); T.H. and J.A.D. are investigators with the Howard Hughes Medical Institute.
Publisher Copyright:
© 2016 The Author(s).
PY - 2016/9/14
Y1 - 2016/9/14
N2 - Binding specificity of Cas9-guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s-1 to >2 s-1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s-1), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.
AB - Binding specificity of Cas9-guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s-1 to >2 s-1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s-1), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.
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U2 - 10.1038/ncomms12778
DO - 10.1038/ncomms12778
M3 - Article
C2 - 27624851
AN - SCOPUS:84987837936
VL - 7
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
M1 - 12778
ER -