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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.

Author information

1
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
2
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
3
Toolgen, Seoul 08501, Republic of Korea.
4
Bloomberg School of Public Health, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
5
Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
6
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
7
Howard Hughes Medical Institute, Baltimore, MD 21205, USA.

Abstract

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.

PMID:
31713616
DOI:
10.1093/nar/gkz1058

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