A team led by researchers at Massachusetts General Hospital has overcome a major constraint to cutting and editing DNA with CRISPR-Cas enzymes and other technologies. Although CRISPR-Cas tools can be programmed to target and cut virtually any DNA sequence, a major constraint in their targeting is the need to first recognize a short sequence flanking the target called the protospacer adjacent motif (PAM). Therefore, DNA could previously only be cut at sites flanking this specific motif. In this latest research, the team that previously designed a nearly PAM-free CRISPR-Cas9 variant named SpRY tested its utility as a universal DNA cleavage tool. By designing SpRY and guide RNA (gRNA) complexes targeting more than 130 DNA sequences in laboratory experiments, the scientists surprisingly found that SpRY is PAM-free in vitro and can efficiently cleave DNA at any sequence programmed by the gRNA. The researchers also showed that their technology can overcome the limitations of restriction enzymes. 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 more.
