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.

Precise DNA Cloning via PAMless CRISPR-SpRYgests