Targeted mutagenesis in Arabidopsis thaliana using CRISPR-Casi2b/C2ci^°°

Casi2b/C2ci is a newly identified class 2 CRISPR endonuclease that was recently engineered for targeted genome editing in mammals and rice.To explore the potential applications of the CRISPR-Casi2b system in the dicot Arabidopsis thaliana,we selected BvCasi2b and BhCasi2b v4 for analysis.We successfully used both endonucleases to induce mutations,perform multiplex genome editing,and create large deletions at multiple loci.No significant mutations were detected at potential off-target sites.Analysis of the insertion/deletion frequencies and patterns of mutants generated via targeted gene mutagenesis highlighted the potential utility of CRISPRCasi2b systems for genome editing in Arabidopsis.