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Nucleic Acids Res. 2019 Dec 16;47(22):11880-11888. doi: 10.1093/nar/gkz1058.

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding.

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Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Toolgen, Seoul 08501, Republic of Korea.
Bloomberg School of Public Health, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
Howard Hughes Medical Institute, Baltimore, MD 21205, USA.


Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5' end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5' end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.


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