Engineering and comparison of cas12a‐based genome editing systems in plants

SUMMARY While Cas9 and Cas12a are both RNA‐guided endonucleases used for genome editing, only Cas12a is able to process pre‐crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9‐based genome editing systems thus providing an attractive alternative for generating site‐specific mutations in plants. Here we aimed to improve the efficiency of the cas12a ‐based generation of two double‐strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a ‐based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana , Lotus japonicus , and Nicotiana benthamiana . We detected the highest cas12a ‐based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re‐coded cas12a , adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a ‐based genome editing systems for plant genetic research.