Genome editing in plants using the compact editor Cas?

Clustered regularly interspaced short palindromic repeats and CRISPR-associated pro-teins (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 wild-type Cas phi enzyme. Consistently, vCas phi and nCas phi yielded offspring plants with inherited edits at much higher rates compared to WTCas phi. 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 protospacer adjacent motif (PAM), and wide range of working temperatures make Cas phi an excellent supplement to existing plant genome editing systems.

Automated summary

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems are important tools for plant genome engineering. In this study, the hypercompact Cas phi nuclease is shown to generate stably inherited gene edits in Arabidopsis. The Cas phi protein variants, vCas phi and nCas phi, exhibit higher editing efficiency and no off-target editing was observed. The characteristics of Cas phi, such as its small size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures, make it an excellent supplement to existing plant genome editing systems.