Evaluation of editing efficiency of different CRISPR-Cas12f systems

CRISPR/Cas12f proteins belonging to the Type V-F family are reported to be only 1/4 to 1/3 the size of Cas9 protein molecules, providing a significant advantage in viral vector delivery. However, the CRISPR/Cas12f system for gene editing in plants has been reported to have lower editing activity, limiting its broader application in plant research. In this study, we compared the editing activities of OsCas12f, SpCas12f, and UnCas12f in three different systems: in vitro digestion, yeast, and transient expression in maize protoplasts. The results showed that the editing activities of OsCas12f and SpCas12f proteins were comparable in terms of in vitro digestion of Cas12f/sgRNA complexes, while no substrate digestion activity was detected for UnCas12f. In the yeast mutant eGFP expression restoration assay, OsCas12f exhibited an editing efficiency of over 95% at the two tested loci, which was comparable to Cas12i.3. On the other hand, SpCas12f achieved editing efficiencies of 1.63% and 3.20% at the two sites, respectively, representing the next highest effect. However, UnCas12f showed minimal editing activity. Furthermore, by transiently expressing maize protoplasts, we compared the editing efficiencies of OsCas12f and SpCas12f at endogenous maize loci. It was found that OsCas12f successfully mediated targeted editing at two loci with editing efficiencies of 2.72% and 1.97%, respectively, while SpCas12f only mediated targeted editing at one locus with an editing efficiency of 1.09%. Deletion of bases was the predominant type of mutation introduced by Cas12f proteins at the target loci, with deletion lengths ranging from -9 to -17 base pairs. These comprehensive results indicate that OsCas12f can serve as a versatile tool for developing plant microgene editors and related technologies.