Zheng Xuelian, Tang Xu, Wu Yuechao, Zheng Xiaoqin, Zhou Jianping, Han Qinqin, Tang Yalan, Fu Xinxuan, Deng Jiao, Wang Yibo, Wang Danning, Zhang Shuting, Zhang Tao, Qi Yiping, Zhang Yong
Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Chongqing Key Laboratory of Tree Germplasm Innovation and Utilization, School of Life Sciences, Southwest University, Chongqing, China.
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China.
Plant Biotechnol J. 2025 Jan;23(1):128-140. doi: 10.1111/pbi.14484. Epub 2024 Oct 14.
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.
近年来，CRISPR-Cas9核酸酶已被用于敲除植物中的微小RNA（miRNA）基因，极大地促进了miRNA功能的研究。然而，由于其倾向于产生小的插入和缺失，Cas9不太适合实现miRNA基因的完全敲除。相比之下，CRISPR-Cas12a核酸酶会产生更大的缺失，这可能会显著破坏前体miRNA的二级结构并阻止成熟miRNA的产生。通过对水稻中OsMIR390的案例研究，我们证实Cas12a在产生miRNA基因敲除突变体方面是比Cas9更有效的工具。为了进一步证明CRISPR-Cas12a介导的水稻miRNA基因敲除，我们针对九个具有不同时空表达且此前未通过基因敲除方法研究过的OsMIRNA基因。使用CRISPR-Cas12a，在这些miRNA位点观察到高达100%的基因组编辑效率。产生的更大缺失表明Cas12a有力地产生了miRNA基因的无效等位基因。对miRNA突变体的转录组分析以及对水稻籽粒的表型分析揭示了这些miRNA在控制基因表达以及调节籽粒品质和种子发育方面的功能。这项研究确立了CRISPR-Cas12a作为植物中miRNA基因遗传敲除的有效工具。
