Cas12a cleavage and trimming kinetics reveal mismatches as a tool to steer editing
Gene knockouts by CRISPR-Cas nucleases rely on targeted DNA cleavage and error-prone DNA repair: end-joining pathways can introduce insertions and deletions that assist in disrupting the coding sequence. However, only a fraction of edits achieves this, and an unfavorable array of repair outcomes typically requires switching to another editing technology. Key factors that influence repair are the…
CRISPR-Cas nucleases achieve gene knockouts through targeted DNA cleavage and error-prone DNA repair mechanisms. However, only a subset of edits result in these desired outcomes, often necessitating the switch to alternative editing technologies. The repair process is heavily influenced by the type and length of DNA ends generated following cleavage.
Researchers delved into the capabilities of Cas12a and its ability to produce varied ends, which could potentially be harnessed to control editing results. Utilizing kinetic modeling and nucleotide-resolution assays in vitro, they determined the cleavage sites and rates of Cas12a.
For the first time, they discovered that trimming - successive cleavage of a previously cut target - transpired roughly four times faster than the initial cleavage. Moreover, trimming introduced alternative DNA end structures that cellular repair systems could utilize. They then introduced specific mismatches to the guide RNA (gRNA) used in conjunction with Cas12a.
Despite the alterations, Cas12a retained its rapid target cleavage. Yet, the presence of mismatches influenced where in the target the cleavage occurred, as well as the speed of trimming compared to non-mismatched gRNAs. Leveraging these differences in cleavage dynamics, the researchers generated reprogrammed gRNAs, or rpgRNAs. These reprogrammed gRNAs maintained the high editing efficiency observed with conventional gRNAs while enabling the redirection of editing between in-frame and out-of-frame outcomes.
This innovation significantly improved gene knockout success rates across different genes, offering a streamlined method to tailor editing outcomes with CRISPR-Cas nucleases.
Written by urgent.news from bioRxiv's reporting — not their text. Machine-written — may contain errors; check the original before relying on it.
