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Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq


Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.

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Fig. 1: Schematic workflow of GUIDE-seq method.
Fig. 2: Principle of GUIDE-seq library preparation and paired-end analysis.
Fig. 3: Quality control steps in the GUIDE-seq protocol.
Fig. 4: GUIDE-seq example output.

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Data availability

Sequencing data from experiments originally described in Lazzarotto et al.8 are available in NCBI Sequence Read Archive PRJNA625995.

Code availability

Open-source GUIDE-seq analysis code is available at


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This work was supported by St. Jude Children’s Research Hospital and ALSAC, National Institutes of Health (NIH) Office Of The Director (OD) Somatic Cell Genome Editing (SCGE) initiative grant U01AI157189 (to S.Q.T.), St. Jude Children’s Research Hospital Collaborative Research Consortium on Novel Gene Therapies for Sickle Cell Disease (SCD), the Doris Duke Charitable Foundation (2017093 and 2020154) (to S.Q.T.), an (NIH) Directors Pioneer Award (DP1 GM105378) (to J.K.J.), NIH R01 GM088040 (to J.K.J.), NIH R01 AR063070 (to J.K.J.), and the Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award (to J.K.J.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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Authors and Affiliations



N.L.M., C.R.L., G.L., Y.L. and S.Q.T. drafted the manuscript. S.Q.T. and J.K.J. conceived the GUIDE-seq method. S.Q.T. and Z.Z. developed the GUIDE-seq protocol in the laboratories of J.K.J. and A.I. M.L. and N.N. performed experiments during original development of GUIDE-seq under supervision of S.Q.T. and Z.Z. S.Q.T., V.T. and M.A. contributed to development of the GUIDE-seq analysis software.

Corresponding author

Correspondence to Shengdar Q. Tsai.

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Competing interests

S.Q.T. is a co-inventor on patents covering the GUIDE-seq method. S.Q.T. is a member of the scientific advisory board of Kromatid and Twelve Bio. M.J.A. and J.K.J. hold equity in SeQure Dx, Inc. J.K.J. holds equity in Chroma Medicine. J.K.J. has financial interests in Beam Therapeutics, Editas Medicine, Excelsior Genomics, Pairwise Plants, Poseida Therapeutics, Transposagen Biopharmaceuticals, and Verve Therapeutics (f/k/a Endcadia). 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. is a co-inventor on various patents and patent applications that describe gene editing and epigenetic editing technologies, including the GUIDE-seq method.

Additional information

Peer review information Nature Protocols thanks Ciaran M. Lee and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Tsai, S. Q. et al. Nat. Biotechnol. 33, 187 (2015):

Lazzarotto, C. R. et al. Nat. Biotechnol. 38, 1317 (2020):

Kleinstiver P. B. et al. Nat. Biotechnol. 34 869 (2016):

Supplementary information

Reporting Summary

Supplementary Data

Example Illumina SampleSheet SampleSheet for NGS sequencing of GUIDE-seq samples. Demultiplexing is built into the pipeline and library’s indices shouldn’t be specified here. All sequence reads will be sorted into an Undetermined category used for further processing. However, it is important to add an extra eight As after index 2 to include reading of UMI required by the pipeline.

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Malinin, N.L., Lee, G., Lazzarotto, C.R. et al. Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq. Nat Protoc 16, 5592–5615 (2021).

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