Journal
NATURE BIOTECHNOLOGY
Volume 32, Issue 3, Pages 279-284Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.2808
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Funding
- National Institutes of Health (NIH) Director's Pioneer Award [DP1 GM105378]
- NIH [R01 GM088040, NIH P50 HG005550]
- Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award
- US Army Research Laboratory
- US Army-Research Office [W911NF-11-2-0056]
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Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools(1-3). CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target(4-6). Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.
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