Never-germinating Arabidopsis seeds with LbCas12a-induced mutations in 6 clade A type 2C protein phosphatase genes

Cas12 nucleases, such as Cas12a, Cas12i, and Cas12f, are genome-editing tools that possess several unique attributes. However, the potential of various Cas12 variants for multiplex genome editing in Arabidopsis (Arabidopsis thaliana) remains insufficiently characterized. In this report, we systematically evaluated 18 additional targets and demonstrated that the LbCas12a variant carrying D156R and E795L mutations exhibits minimal target bias. Furthermore, we achieved an editing efficiency of at least 73.8% (45/61) in generating T1 homozygous sextuple mutants, with more than half of these mutants exhibiting a complete seed germination arrest phenotype. Comparative analysis of 7 LbCas12a variants revealed that the optimization of nuclear localization sequences, rather than codon usage, is fundamental for improved editing efficiency, and that the E795L mutation had synergistic effects with other mutations in highly efficient LbCas12a variants. Further investigation into 1 Cas12i3 and 2 AsCas12f variants showed that the Cas12i3 variant also exhibits sufficiently high editing efficiency in Arabidopsis, although additional refinements were required to mitigate its target bias. Collectively, in this study, we developed the most efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) tool for multiplex genome editing in Arabidopsis, as demonstrated by the highly efficient generation of never-germinating seeds harboring mutations in 6 clade A type 2C protein phosphatase genes.