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An efficient CRISPR-Cas12a-mediated MicroRNA knockout strategy in plants
Wednesday, 2024/10/23 | 08:03:14

Xuelian ZhengXu TangYuechao WuXiaoqin ZhengJianping ZhouQinqin HanYalan TangXinxuan FuJiao DengYibo WangDanning WangShuting ZhangTao ZhangYiping QiYong Zhang

First published: 14 October 2024; https://doi.org/10.1111/pbi.14484

Summary

In recent years, the CRISPR-Cas9 nuclease has been used to knock out MicroRNA (miRNA) genes in plants, greatly promoting the study of miRNA function. However, due to its propensity for generating small insertions and deletions, Cas9 is not well-suited for achieving a complete knockout of miRNA genes. By contrast, CRISPR-Cas12a nuclease 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 a 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 profiling 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.

 

See https://onlinelibrary.wiley.com/doi/10.1111/pbi.14484

 

Figure 1

Cas12a is more efficient than Cas9 in generating null alleles of OsMIR390. (a) Comparison of the effects of different CRISPR-Cas nucleases in gene editing of miRNAs. Cas12a is more likely to produce larger deletions, causing damage to secondary structure of pre-miRNA, unable to produce mature miRNA and resulting in complete loss-function of miRNA. The Cas12a nuclease outperforms the Cas9 nuclease for miRNA gene editing. (b) Secondary structure of primary OsMIR390. Matured osa-miRNA 5P sequence is in red, matured osa-miRNA 3P sequence is in blue, CRISPR-Cas12a targeted site and CRISPR-Cas9 targeted site are indicated by arrows. (c) Comparison of the effects of knockout OsMIR390 with Cas9 and Cas12a on the efficiency of regeneration and transformation in rice. (d) Knockout of OsMIR390 with Cas12a significantly inhibited the regeneration of rice calli. (e) Cas12a-generated OsMIR390 T0 mutants were all heterozygous, with the genotypes of >3 nt deletion. (f) Cas9-generated OsMIR390 T0 mutants were heterozygous and homozygous, with the genotypes of 1 nt insertion. (g) Comparison of pre-miRNA prediction secondary structures of OsMIR390 mutants generated by Cas9 and Cas12a. (h) Comparison of seeds germination for 3 days of OsMIR390 T1 mutants generated by Cas9 and Cas12a. The T1 generation heterozygous seeds of the OsMIR390 mutants germinated normally like WT; Cas12a-generated T1 homozygous seeds cannot germinate. Few of Cas9-genetated T1 homozygous seeds can germinate, but only produce radicle without any bud. (i) Comparison of 14-day seedlings of OsMIR390 T1 mutants generated by Cas9 and Cas12a. The T1 generation heterozygous seedlings of the OsMIR390 mutants grow normally as well as WT. Cas12a-generated T1 seeds did not grow into seedlings, and few of Cas9-generated T1 homozygous seeds had elongated lateral roots without any shoot.

 

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