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 defining genome-wide genome editing activity are (i) high enrichment for sequencing nuclease-cleaved gDNA/low background, enabling sensitive detection with low sequencing depth requirements; and (ii) the fact that paired-end reads can contain complete information on individual nuclease cleavage sites, enabling use of CIRCLE-seq in species without high-quality 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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Acknowledgements
We thank N. 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.).
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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.
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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.
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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
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Lazzarotto, C.R., Nguyen, N.T., Tang, X. et al. Defining CRISPR–Cas9 genome-wide nuclease activities with CIRCLE-seq. Nat Protoc 13, 2615–2642 (2018). https://doi.org/10.1038/s41596-018-0055-0
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DOI: https://doi.org/10.1038/s41596-018-0055-0
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