An efficient CRISPR-Cas12a mediated miRNA knockout system in plants

In recent years, the CRISPR-Cas9 system has been widely used to knock out miRNA genes in plants, greatly promoting the study of miRNA function. However, because CRISPR-Cas9 generates small insertions and deletions, it is not efficient to generate full knockout of miRNA genes. By contrast, CRISPR-Cas12a generates larger deletions, which could significantly disrupt the secondary structure of pre-miRNA and prevent the production of mature miRNAs. Through the case study of OsMIR390 in rice, we confirmed that Cas12a is a more efficient tool than Cas9 in generating knockout mutants of an miRNA gene. To further demonstrate CRISPR-Cas12a mediated knockout of miRNA genes in rice, we targeted nine OsMIRNA genes that have different spaciotemporal expression and have not been previously investigated via genetic knockout approaches. With CRISPR-Cas12a, up to 100% genome editing efficiency was observed at these miRNA loci. The resulting larger deletions suggest Cas12a robustly generated null alleles of miRNA genes. Transcriptome analysis of the miRNA mutants, as well as phenotypic analysis of the rice grains revealed the function of these miRNAs in controlling gene expression and regulating grain quality and seed development. This study established CRISPR-Cas12a as an efficient tool for genetic knockout of miRNA genes in plants.