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Nat Biotechnol. 2014 Mar;32(3):279-284. doi: 10.1038/nbt.2808. Epub 2014 Jan 26.

Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.

Fu Y#1,2,3,4, Sander JD#1,2,3,4, Reyon D1,2,3,4, Cascio VM1,2,3, Joung JK1,2,3,4.

Author information

1
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.
2
Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
3
Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.
4
Department of Pathology, Harvard Medical School, Boston, MA 02115 USA.
#
Contributed equally

Abstract

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. 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. 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.

PMID:
24463574
PMCID:
PMC3988262
DOI:
10.1038/nbt.2808
[Indexed for MEDLINE]
Free PMC Article

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