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Precise DNA Cloning via PAMless CRISPR-SpRYgests

View ORCID ProfileKathleen A. Christie, View ORCID ProfileJimmy A. Guo, View ORCID ProfileRachel A. Silverstein, View ORCID ProfileRoman M. Doll, Megumu Mabuchi, View ORCID ProfileHannah E. Stutzman, View ORCID ProfileLinyuan Ma, View ORCID ProfileG. Brett Robb, View ORCID ProfileBenjamin P. Kleinstiver
doi: https://doi.org/10.1101/2022.01.11.474553
Kathleen A. Christie
1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
2Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
3Department of Pathology, Harvard Medical School, Boston, MA, 02115, USA
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Jimmy A. Guo
1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
2Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
4Biological and Biomedical Sciences Program, Harvard University, Boston, MA, 02115, USA
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Rachel A. Silverstein
1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
2Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
4Biological and Biomedical Sciences Program, Harvard University, Boston, MA, 02115, USA
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Roman M. Doll
1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
2Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
5Molecular Biosciences/Cancer Biology Program, Heidelberg University, and German Cancer Research Center, DKFZ, Heidelberg, Germany
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Megumu Mabuchi
6New England Biolabs, Ipswich, MA, 01938, USA
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Hannah E. Stutzman
1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
2Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
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Linyuan Ma
1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
2Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
3Department of Pathology, Harvard Medical School, Boston, MA, 02115, USA
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G. Brett Robb
6New England Biolabs, Ipswich, MA, 01938, USA
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Benjamin P. Kleinstiver
1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
2Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
3Department of Pathology, Harvard Medical School, Boston, MA, 02115, USA
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  • For correspondence: bkleinstiver@mgh.harvard.edu
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Abstract

While restriction enzymes (REs) remain the gold-standard for manipulating DNA in vitro, they have notable drawbacks including a dependence on short binding motifs that constrain their ability to cleave DNA substrates. Here we overcome limitations of REs by developing an optimized molecular workflow that leverages the PAMless nature of a CRISPR-Cas enzyme named SpRY to cleave DNA at practically any sequence. Using SpRY for DNA digests (SpRYgests), we establish a method that permits the efficient cleavage of DNA substrates at any base pair. We demonstrate the effectiveness of SpRYgests using more than 130 gRNAs, illustrating the versatility of this approach to improve the precision of and simplify several cloning workflows, including those not possible with REs. We also optimize a rapid and simple one-pot gRNA synthesis protocol, which reduces cost and makes the overall SpRYgest workflow comparable to that of RE digests. Together, SpRYgests are straightforward to implement and can be utilized to improve a variety of DNA engineering applications.

Competing Interest Statement

K.A.C. and B.P.K are inventors on patents and/or patent applications filed by Mass General Brigham that describe genome engineering technologies. B.P.K. is a consultant for Avectas Inc., EcoR1 capital, and ElevateBio, and is an advisor to Acrigen Biosciences and Life Edit Therapeutics.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted January 12, 2022.
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Precise DNA Cloning via PAMless CRISPR-SpRYgests
Kathleen A. Christie, Jimmy A. Guo, Rachel A. Silverstein, Roman M. Doll, Megumu Mabuchi, Hannah E. Stutzman, Linyuan Ma, G. Brett Robb, Benjamin P. Kleinstiver
bioRxiv 2022.01.11.474553; doi: https://doi.org/10.1101/2022.01.11.474553
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Precise DNA Cloning via PAMless CRISPR-SpRYgests
Kathleen A. Christie, Jimmy A. Guo, Rachel A. Silverstein, Roman M. Doll, Megumu Mabuchi, Hannah E. Stutzman, Linyuan Ma, G. Brett Robb, Benjamin P. Kleinstiver
bioRxiv 2022.01.11.474553; doi: https://doi.org/10.1101/2022.01.11.474553

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