In this latest research, the team that previously engineered a nearly PAMless CRISPR–Cas9 variant, named SpRY, tested its utility to serve as a universal DNA cleavage tool.
By designing SpRY and guide RNA (gRNA) complexes that targeted more than 130 DNA sequences in laboratory experiments, the scientists surprisingly discovered that SpRY is PAMless in vitro and can effectively cleave DNA at any sequence programmed by the gRNA. The researhcers also showed that their technology can overcome limitations of restriction enzymes.
“We demonstrate that SpRY DNA digests – or SpRYgests – enable DNA cutting at practically any sequence, including a wide range that were previously untargetable with restriction enzymes or other CRISPR-Cas proteins,” said senior author Benjamin Kleinstiver, PhD, an assistant investigator at the Center for Genomic Medicine at Mass General Hospital and an assistant professor at Harvard Medical School. “This new method permits researchers to cut DNA in a test tube at any DNA location of choice. The new capabilities offered by SpRYgests will accelerate and reduce the cost of various basic research applications, including for studies that could have eventual clinical implications.”
The researchers envision that SpRYgests could be widely applicable to simplify typical molecular cloning approaches, for more complex cloning methods, for assembling next-generation sequencing libraries, and many others.
1. Christie, K.A., Guo, J.A., Silverstein, R.A. et al. Precise DNA cleavage using CRISPR-SpRYgests. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01492-y