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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Primary datasets are available in Supplementary Table 2. Any other data that support this study are available from the corresponding author upon reasonable request.
The script used to determine the SpRYgest rate constants is provided as Supplementary Note 9.
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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.
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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Table of contents for Supplementary Information, titles of Supplementary Tables 1–7, Supplementary Notes 1–9, Supplementary Figs. 1–18 and Supplementary References
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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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