Abstract

CRISPR-Cas systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact Cas{Phi} nuclease is able to generate stably inherited gene edits in Arabidopsis, and that Cas{Phi} 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 Cas{Phi} displays higher editing efficiency when the target locus is not DNA methylated, suggesting that Cas{Phi} is sensitive to chromatin environment. Importantly, two Cas{Phi} protein variants, vCas{Phi} and nCas{Phi}, both showed much higher editing efficiency relative to the wildtype Cas{Phi} enzyme, and yielded more offspring plants with inherited edits. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that Cas{Phi} is highly specific. The hypercompact size, T-rich minimal PAM and wide range of working temperatures make Cas{Phi} an excellent supplement to existing plant genome editing systems. Significance StatementPlant genome engineering with CRISPR-Cas systems is frequently used in both research and agriculture. Here, we demonstrate that the hypercompact Cas{Phi}-2 nuclease is able to generate heritable gene edits in Arabidopsis. Two Cas{Phi} protein variants vCas{Phi} and nCas{Phi} increased the editing efficiency in plants. Cas{Phi} also has a wide range of working temperatures and the editing by Cas{Phi} is highly specific. We also observed that editing by Cas{Phi} is sensitive to chromatin environment. The hypercompact size, T-rich minimal PAM and wide range of working temperatures make Cas{Phi} an excellent supplement to existing plant genome editing systems.