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Nat Methods. 2016 Jan;13(1):41-50. doi: 10.1038/nmeth.3684.

Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery.

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  • 1Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
  • 2Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.


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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