Compact CRISPR-CasX (Cas12e) nucleases enable genome editing and generation of mutant lines in rice
Keywords:
CRISPR-Cas, CasX (Cas12e), compact Cas nuclease, genome editing, stable mutation, riceAbstract
The diversity and large molecular size of CRISPR-associated nucleases often limit their application in plant genome engineering. CasX (also known as Cas12e) is a recently identified RNA-guided nuclease that features a compact protein size (<1000 amino acids) and a relaxed PAM preference (5′-TTCN-3′). Here, we explored the use of two CasX orthologs, DpbCasX and PlmCasX, for targeted genome modification in rice. Both wild-type nucleases exhibited detectable but modest activity in protoplast assays. Structure-guided engineering led to the generation of DpbCasX_RRR and PlmCasX_RRR variants, each containing amino-acid substitutions predicted to enhance DNA binding. These variants displayed improved editing activity, with PlmCasX_RRR showing consistently higher performance. Mutation profiling indicated a tendency toward multi-nucleotide deletions located distal to the PAM. Furthermore, stable rice transformants expressing PlmCasX_RRR transmitted targeted mutations to progeny, producing stable knockout phenotypes. These results establish CasX as a compact and functional nuclease for plant genome editing, broadening the range of CRISPR tools available for crop research and improvement.References
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