NEW YORK — A CRISPR-Cas variant engineered to no longer need a protospacer adjacent motif (PAM) can be harnessed to make cuts at any DNA base in vitro, a new study reports. Its ability to make precise breaks anywhere could be applied to a number of DNA engineering applications.
Researchers from Massachusetts General Hospital engineered the nearly PAMless variant, which they dubbed SpRY, as they reported in 2020. Typically, wild-type CRISPR-Cas9 or -Cas12 nucleases require the recognition of a PAM near the target site, which limits where the enzymes may cleave. Likewise, restriction enzymes can only make cuts at certain DNA motifs.
"For in vitro applications, this means that as researchers we don't have complete flexibility to cut DNA at any sequence, since the enzymes that we use as tools are beholden to these short sequence motifs," senior author Benjamin Kleinstiver, an assistant investigator at MGH, said in an email.
SpRY, by contrast, has more relaxed requirements. In a new study appearing in Nature Biotechnology on Thursday, Kleinstiver and his colleagues reported that SpRY DNA digest, or SpRYgests, can make cuts at nearly any DNA sequence in vitro with a guide RNA. The researchers further optimized the SpRYgest process, including the gRNA production, to improve workflows for its wider adoption.
As part of their analysis, the researchers first compared the abilities of wild-type SpCas9 and SpRY to create double-stranded breaks at 20 different target locations along a DNA substrate with varying PAM motifs. With 20 guide RNAs, wild-type SpCas9 could digest four of these sites to near completion, while SpRY could do so for 19 of the sites.
They additionally compared the activity of wild-type SpCas9, SpRY, and SpG, a SpCas9 variant engineered to target certain PAMs, using 64 additional gRNAs targeting a range of sites. Their findings underscored wild-type SpCas9's preference for NGG PAMs and SpG's preference for NGN. SpRY, meanwhile, could digest 59 of the 64 sites to near completion, suggesting it was indeed nearly PAMless.
SpRYgests could be used in a range of applications. For instance, they could be employed alongside isothermal assembly methods in molecular cloning, such as when parts of plasmids are interchanged. According to Kleinstiver, they could simplify and improve the precision of the approach since SpRYgests allow DNA cuts to be made at any spot specified by a gRNA, rather than having to identify a nearby restriction enzyme target site. Likewise, SpRYgests could be applied to generate saturation mutagenesis libraries, to deplete unwanted sequences from next-generation sequencing libraries, and to target enrichment in sequencing protocols, as the researchers noted in their paper.
To enable SpRYgests to be more widely used, the researchers also made improvements to the SpRY protein purification gRNA synthesis protocol. In particular, they combined the template and the in vitro transcription (IVT) steps and shortened the IVT reaction time to less than four hours. This one-pot gRNA synthesis approach reduces hands-on time, decreases cost, and makes the SpRYgest workflow more similar to that of other molecular cloning approaches, the researchers noted.
These optimizations, Kleinstiver noted, should make SpRYgest "straightforward to implement."
He added that the full method is included in the paper. "The oligonucleotides can be purchased from vendors, the gRNA transcription kits are already commercially available, and we hope that soon the protein will be available from vendors as well (but in the meantime, have provided details to purify the SpRY enzyme)," he said. "We hope that the simplicity of this method will permit users to incorporate SpRYgests into their typical molecular cloning workflows without too many challenges."