Abstract
The simplicity of site-specific genome targeting by type II clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 nucleases, along with their robust activity profile, has changed the landscape of genome editing. These favorable properties have made the CRISPR-Cas9 system the technology of choice for sequence-specific modifications in vertebrate systems. For many applications, whether the focus is on basic science investigations or therapeutic efficacy, activity and precision are important considerations when one is choosing a nuclease platform, target site and delivery method. Here we review recent methods for increasing the activity and accuracy of Cas9 and assessing the extent of off-target cleavage events.
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Acknowledgements
We apologize for the omission of references that could not be included in this review because of space constraints. We thank L.J. Zhu for assistance with the calculation of mismatched off-target sites in the genome for a population of sgRNAs. This work was supported by the US National Institutes of Health (grant R01AI117839 to S.A.W. and J. Luban, and grant R01HL093766 to S.A.W. and N. Lawson).
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The authors have filed patent applications related to genome engineering technologies. S.A.W. is a consultant for Editas Medicine.
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Bolukbasi, M., Gupta, A. & Wolfe, S. Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery. Nat Methods 13, 41–50 (2016). https://doi.org/10.1038/nmeth.3684
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DOI: https://doi.org/10.1038/nmeth.3684
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