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Improving CRISPR-Cas nuclease specificity using truncated guide RNAs

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Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools1,2,3. 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 target4,5,6. 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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Figure 1: On-target genome editing activities of truncated gRNAs and Cas9 nuclease in human cells.
Figure 2: tru-gRNAs exhibit enhanced specificities and function efficiently with Cas9 nuclease and paired Cas9 nickases in human cells.

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  • 29 January 2014

    In the version of this article initially published online, two nearly identical versions of a sentence were included in the abstract (“In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Also, use of truncated gRNAs can lead to further reductions in off-target mutations induced by pairs of Cas9 variants that nick DNA (paired nickases).”) and the name and reference citation for a plasmid in the Online Methods was incorrect. The correct sentence is “In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases)” and the plasmid name and reference are “pJDS246 (ref. 4),” not “pJDS2464.” These errors have been corrected for the print, PDF and HTML versions of this article.


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We thank M. Maeder, S. Tsai and J. Angstman for helpful discussions and comments on the manuscript and J. Foden for technical assistance. This work was funded by a National Institutes of Health (NIH) Director's Pioneer Award (DP1 GM105378), NIH R01 GM088040, NIH P50 HG005550, and the Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award. This material is based upon work supported by, or in part by, the US Army Research Laboratory and the US Army Research Office under grant number W911NF-11-2-0056.

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Authors and Affiliations



Y.F., J.D.S., D.R. and J.K.J. conceived of and designed experiments. Y.F., J.D.S. and V.M.C. performed experiments. D.R. developed the updated version of ZiFiT software. Y.F., J.D.S. and J.K.J. wrote the paper.

Corresponding authors

Correspondence to Jeffry D Sander or J Keith Joung.

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Competing interests

J.K.J. has financial interests in Editas Medicine and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. J.K.J. and J.D.S. have filed a patent application on the tru-gRNA/tru-RGN technology. J.K.J. and J.D.S. are consultants for Editas Medicine, a company focused on developing genome-editing therapeutics. Following the completion of this work, D.R. has become an employee of Biogen Idec.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–6 and Supplementary Tables 1–6 (PDF 1458 kb)

Supplementary Table 7

Primers used to construct gRNA expression vectors (XLSX 26 kb)

Supplementy Table 8

Primers used to amplify genomic loci for T7EI and deep sequencing assays (XLSX 62 kb)

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Fu, Y., Sander, J., Reyon, D. et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32, 279–284 (2014).

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