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Precise DNA cleavage using CRISPR-SpRYgests

Nature Biotechnology volume 41, pages 409–416 (2023)Cite this article

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Abstract

Methods for in vitro DNA cleavage and molecular cloning remain unable to precisely cleave DNA directly adjacent to bases of interest. Restriction enzymes (REs) must bind specific motifs, whereas wild-type CRISPR–Cas9 or CRISPR–Cas12 nucleases require protospacer adjacent motifs (PAMs). Here we explore the utility of our previously reported near-PAMless SpCas9 variant, named SpRY, to serve as a universal DNA cleavage tool for various cloning applications. By performing SpRY DNA digests (SpRYgests) using more than 130 guide RNAs (gRNAs) sampling a wide diversity of PAMs, we discovered that SpRY is PAMless in vitro and can cleave DNA at practically any sequence, including sites refractory to cleavage with wild-type SpCas9. We illustrate the versatility and effectiveness of SpRYgests to improve the precision of several cloning workflows, including those not possible with REs or canonical CRISPR nucleases. We also optimize a rapid and simple one-pot gRNA synthesis protocol to streamline SpRYgest implementation. Together, SpRYgests can improve various DNA engineering applications that benefit from precise DNA breaks.

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Fig. 1: Characterization of SpRYgest in vitro cleavage efficiencies.
Fig. 2: Molecular cloning via SpRYgest.
Fig. 3: Rapid generation of saturation mutagenesis libraries via SpRYgest.
Fig. 4: Optimization of rapid, efficient and specific SpRYgest reactions.

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Data availability

Primary datasets are available in Supplementary Table 2. Any other data that support this study are available from the corresponding author upon reasonable request.

Code availability

The script used to determine the SpRYgest rate constants is provided as Supplementary Note 9.

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Acknowledgements

We thank D. R. Edgell for helpful suggestions and S. Mahendraker for assistance developing the web version of SpOT-check. K.A.C. was supported by a Massachusetts General Hospital (MGH) Fund for Medical Discovery Fundamental Research Fellowship Award. B.P.K. acknowledges support from an MGH Executive Committee on Research Howard M. Goodman Fellowship and National Institutes of Health (NIH) grant P01 HL142494. R.T.W. is supported by the National Science Foundation Graduate Research Fellowship Program under grant 1745302. L.P. is partially supported by NIH R35 HG010717.

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Authors and Affiliations

  1. Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

    Kathleen A. Christie, Jimmy A. Guo, Rachel A. Silverstein, Roman M. Doll, Hannah E. Stutzman, Linyuan Ma & Benjamin P. Kleinstiver

  2. Department of Pathology, Massachusetts General Hospital, Boston, MA, USA

    Kathleen A. Christie, Jimmy A. Guo, Rachel A. Silverstein, Roman M. Doll, Hannah E. Stutzman, Linyuan Ma & Benjamin P. Kleinstiver

  3. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Kathleen A. Christie, Jiecong Lin, Linyuan Ma, Luca Pinello & Benjamin P. Kleinstiver

  4. Biological and Biomedical Sciences Program, Harvard University, Boston, MA, USA

    Jimmy A. Guo & Rachel A. Silverstein

  5. Molecular Biosciences/Cancer Biology Program, Heidelberg University and German Cancer Research Center (DKFZ), Heidelberg, Germany

    Roman M. Doll

  6. New England Biolabs, Ipswich, MA, USA

    Megumu Mabuchi & G. Brett Robb

  7. Molecular Pathology Unit, Massachusetts General Hospital, Boston, MA, USA

    Jiecong Lin & Luca Pinello

  8. Center for Cancer Research, Massachusetts General Hospital Charlestown, Boston, MA, USA

    Jiecong Lin & Luca Pinello

  9. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Russell T. Walton

  10. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Russell T. Walton & Luca Pinello

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Contributions

K.A.C. and B.P.K. conceived of and designed the study. K.A.C., J.A.G., R.A.S., R.M.D., H.E.S. and L.M. performed experiments. M.M. and G.B.R. expressed and purified SpRY and SpRY-HF1. R.A.S. designed and wrote SpOT-check. R.M.D. and R.T.W. assisted with rate constant determination. J.L. and L.P. performed the predictive modeling. All authors analyzed data. K.A.C. and B.P.K. wrote the manuscript draft and finalized the manuscript, with input from all authors.

Corresponding author

Correspondence to Benjamin P. Kleinstiver.

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Competing interests

K.A.C., R.T.W. and B.P.K are inventors on patents and/or patent applications filed by Mass General Brigham that describe genome engineering technologies, including for the development of SpRY (R.T.W. and B.P.K.). B.P.K. is a consultant for EcoR1 Capital and is an advisor to Acrigen Biosciences, Life Edit Therapeutics and Prime Medicine. L.P. has financial interests in Edilytics and SeQure Dx, Inc. L.P.ʼs interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. M.M. and G.B.R. are employees of the Research Department at New England Biolabs (NEB). NEB is a commercial supplier of molecular biology reagents, including some that were used in this work. The remaining authors declare no competing interests.

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Christie, K.A., Guo, J.A., Silverstein, R.A. et al. Precise DNA cleavage using CRISPR-SpRYgests. Nat Biotechnol 41, 409–416 (2023). https://doi.org/10.1038/s41587-022-01492-y

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