Discovery and Characterization of Novel Type V Cas12f Nucleases with Diverse Protospacer Adjacent Motif Preferences

Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type V Cas12f effectors present a solution given their small size. In this study, we describe a novel set of miniature (<490AA) Cas12f nucleases that cleave double-stranded DNA in human cells. We determined their optimal trans-activating RNA empirically through rational modifications, which resulted in an optimal single guide RNA. We show that these nucleases have broad protospacer adjacent motif (PAM) preferences, allowing for expanded genome targeting. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.