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GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

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Abstract

CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.

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Figure 1: Design, optimization and application of the GUIDE-seq method.
Figure 2: Sequences of off-target sites identified by GUIDE-seq for ten RGNs.
Figure 3: Analysis of RGN-induced, off-target sequence characteristics.
Figure 4: Comparisons of GUIDE-seq with computational prediction or ChIP-seq methods for identifying RGN off-target sites.
Figure 5: Large-scale structural alterations induced by RGNs.
Figure 6: GUIDE-seq profiles of RGNs directed by tru-gRNAs.

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Change history

  • 25 June 2015

    In the version of the supplementary file originally posted online, the primer labels 'Nuclease_off_+_GSP1' and 'Nuclease_off_-_GSP1' were switched in Supplementary Table 4, and the discovery thermocycling conditions were missing from the Supplementary Methods. The errors have been corrected in this file as of 25 June 2015.

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Acknowledgements

We thank J. Angstman, B. Kleinstiver, Y. Fu, J. Gehrke and R. Cottman for helpful comments on the manuscript and M. Maeder 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 R01 AR063070, and the Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award. S.Q.T. was supported by NIH F32 GM105189. 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. Links to software and resources for analyzing GUIDE-seq data will be made available at: http://www.jounglab.org/guideseq.

Author information

Authors and Affiliations

Authors

Contributions

S.Q.T. and J.K.J. conceived of the GUIDE-seq method. S.Q.T., Z.Z., A.J.I., L.P.L. and J.K.J. planned experiments. S.Q.T., Z.Z., N.T.N., M.L., N.W. and C.K. performed experiments. S.Q.T., Z.Z., V.V.T., V.T. and M.J.A. performed bioinformatics and computational analysis of the data. S.Q.T. and J.K.J. wrote the paper.

Corresponding authors

Correspondence to Shengdar Q Tsai or J Keith Joung.

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Competing interests

J.K.J. is a consultant for Horizon Discovery. 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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1, 3 and 4, Supplementary Results, Supplementary Discussion and Supplementary Protocol (PDF 5037 kb)

Supplementary Table 2

Genomic locations of all GUIDE-Seq detected RGN-induced cleavage sites (XLSX 53 kb)

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Tsai, S., Zheng, Z., Nguyen, N. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33, 187–197 (2015). https://doi.org/10.1038/nbt.3117

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