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Precise DNA Cloning via PAMless CRISPR-SpRYgests

Christie, K. A.; Guo, J. A.; Silverstein, R. A.; Doll, R. M.; Mabuchi, M.; Stutzman, H. E.; Ma, L.; Robb, G. B.; Kleinstiver, B. P.

2022-01-12 biochemistry
10.1101/2022.01.11.474553 bioRxiv
Show 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.

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"Similar papers" are the closest papers from that journal in the model's embedding space. They show what the match is built on, but the ranking comes mostly from a classifier over the whole training set, not from these examples alone.