ABSTRACT Circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) is a sensitive and unbiased method for defining the genome-wide activity (on-target and

off-target) of CRISPR–Cas9 nucleases by selective sequencing of nuclease-cleaved genomic DNA (gDNA). Here, we describe a detailed experimental and analytical protocol for CIRCLE-seq. The

principle of our method is to generate a library of circularized gDNA with minimized numbers of free ends. Highly purified gDNA circles are treated with CRISPR–Cas9 ribonucleoprotein

complexes, and nuclease-linearized DNA fragments are then ligated to adapters for high-throughput sequencing. The primary advantages of CIRCLE-seq as compared with other in vitro methods for

reference genomes. The entire protocol can be completed in 2 weeks, including time for gRNA cloning, sequence verification, in vitro transcription, library preparation, and sequencing.

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OTHERS TRACKING-SEQ REVEALS THE HETEROGENEITY OF OFF-TARGET EFFECTS IN CRISPR–CAS9-MEDIATED GENOME EDITING Article 02 July 2024 DEFINING GENOME-WIDE CRISPR–CAS GENOME-EDITING NUCLEASE

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Malinin for helpful comments and suggestions on the manuscript. This work was supported by St. Jude Children’s Research Hospital and ALSAC, St. Jude Children’s Research Hospital

Collaborative Research Consortium on Novel Gene Therapies for Sickle Cell Disease (SCD), the Doris Duke Charitable Foundation (2017093), National Institutes of Health (NIH) grant U01HL145793

(to S.Q.T.), an NIH Director’s Pioneer Award (DP1GM105378) (to J.K.J.), NIH grants R35GM118158 and NIH R01GM107427 (to J.K.J.), and the Desmond and Ann Heathwood MGH Research Scholar Award

(to J.K.J.). AUTHOR INFORMATION Author notes * Nhu T. Nguyen Present address: Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital and Harvard Medical

School, Boston, MA, USA AUTHORS AND AFFILIATIONS * Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA Cicera R. Lazzarotto, Xing Tang & Shengdar Q. Tsai *

Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA Nhu T. Nguyen, Jose

Malagon-Lopez, Jimmy A. Guo, Martin J. Aryee & J. Keith Joung * Department of Pathology, Harvard Medical School, Boston, MA, USA Jose Malagon-Lopez, Martin J. Aryee & J. Keith Joung

* Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA Jose Malagon-Lopez & Martin J. Aryee Authors * Cicera R. Lazzarotto View author publications You

can also search for this author inPubMed Google Scholar * Nhu T. Nguyen View author publications You can also search for this author inPubMed Google Scholar * Xing Tang View author

publications You can also search for this author inPubMed Google Scholar * Jose Malagon-Lopez View author publications You can also search for this author inPubMed Google Scholar * Jimmy A.

Guo View author publications You can also search for this author inPubMed Google Scholar * Martin J. Aryee View author publications You can also search for this author inPubMed Google

Scholar * J. Keith Joung View author publications You can also search for this author inPubMed Google Scholar * Shengdar Q. Tsai View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS C.R.L. and S.Q.T. wrote the manuscript with input from all authors. N.T.N. and S.Q.T. developed the original experimental protocol in the J.K.J. lab.

C.R.L. in the S.Q.T. lab and J.A.G. in the J.K.J. lab further optimized the protocol. X.T., J.M.-L., M.J.A. and S.Q.T. contributed to the CIRCLE-seq software analysis pipeline. C.R.L.

performed experiments and data analysis. CORRESPONDING AUTHOR Correspondence to Shengdar Q. Tsai. ETHICS DECLARATIONS COMPETING INTERESTS J.K.J. has financial interests in Beam Therapeutics,

Blink Therapeutics, Editas Medicine, Encadia, Monitor Biotechnologies (formerly Beacon Genomics), Pairwise Plants, Poseida Therapeutics and Transposagen Biopharmaceuticals. S.Q.T. and

M.J.A. have financial interests in Monitor Biotechnologies. M.J.A. and 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 S.Q.T. are co-inventors on a patent describing the CIRCLE-seq method that has been licensed to Monitor Biotechnologies. J.K.J.

is a member of the Board of Directors of the American Society of Gene & Cell Therapy. The remaining authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE:

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RELATED LINKS KEY REFERENCES USING THIS PROTOCOL 1. Tsai, S. Q. et al.

_Nat. Methods_ 14, 607–614 (2017): https://doi.org/10.1038/nmeth.4278 2. Akcakaya, P. et al. _Nature_ 561, 416–419 (2018): https://doi.org/10.1038/s41586-018-0500-9 RIGHTS AND PERMISSIONS

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