Genome editing in plants using the compact editor CasF

CRISPR-Cas systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasF nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasF guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele we show that CasF displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasF is sensitive to chromatin environment. Importantly, two CasF protein variants, vCasF and nCasF, both showed much higher editing efficiency relative to the wildtype CasF enzyme, and yielded more offspring plants with inherited edits. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasF is highly specific. The hypercompact size, flexible PAM and wide range of working temperatures make CasF an excellent addition to existing plant genome editing systems. Overall design: This dataset includes amplicon sequencing data to evaluate the editing efficiencies of target loci by CasF, and whole genome sequencing to assess the off-target editing level by CasF in plants.