Recently, Plant Biotechnology Journal published the paper "An efficient CRISPR-Cas12a mediated MicroRNA knockout strategy in plants" written by Daniel Zhang's research group and his collaborative team at Southwest University/University of Electronic Science and Technology of China. By comparing the effects of CRISPR-Cas12a and CRISPR-Cas9 editing systems on knocking out the rice non-coding gene OsMIR390, this work confirmed that Cas12a is better than Cas9 to effectively knock out the MicroRNA (miRNA) gene and generate reliable loss-of-function mutants. Furthermore, the whole rice miRNA was knocked out and edited, and a number of loss-of-function mutants related to development, grain traits and quality were identified, revealing the new functions of these miRNAs in growth and development, grain size and quality regulation. The effective plant miRNA knockout strategy based on CRISPR-Cas12a reported by the research team provides a clear editing tool, editing strategy and new germplasm material for rice miRNA for in-depth analysis of plant miRNA biological functions and molecular breeding events.
MicroRNA（miRNA）是真核生物中一类非编码内源性小RNA，在细胞内miRNA通过RNA切割或翻译抑制来调节基因的表达。 2024年的诺贝尔生理学或医学奖授予美国科学家 Victor Ambros 和 Gary Ruvkun，以表彰他们在发现miRNA及其在基因调控中的作用方面做出的开创性贡献。 广泛的研究表明，植物miRNA在调节植物生命的各个方面发挥重要作用，包括植物形态发生、信号转导、代谢、营养吸收、激素调节以及生物和非生物胁迫反应(Chen and Rechavi, 2021; Chi et al., 2012; Fabian and Sonenberg, 2012; Lytle et al., 2007; Voinnet, 2009; Voinnet, 2022)。 在植物miRNA研究中常使用靶标模拟（target mimic，TM）和短串联靶标模拟（short tandem targets mimic，STTM）等干扰miRNA内源活性（Franco-Zorrilla et al., 2007; Yan et al., 2012; Zhang et al., 2017）。 TALEN与CRISPR-Cas9等基因组编辑工具也已被用于植物miRNA的敲除(Bi et al., 2020; Cheng et al., 2021; Deng et al., 2022; He et al., 2024; Miao et al., 2020; Tang and Zhang, 2023; Wang et al., 2021; Zhou et al., 2017; Zhou et al., 2021; Zhou et al., 2022)。 但由于Cas9倾向于产生1bp-3bp的核苷酸插入或缺失，可能无法彻底实现植物miRNA的功能缺失。 与之相比，CRISPR-Cas12a核酸酶能够产生较大的缺失，可以显著破坏pre-miRNA的二级结构，阻止成熟miRNA的产生，因此在敲除miRNA基因获得功能缺失型突变体方面具有明显优势（图1A）(He et al., 2024; Hui et al., 2024; Liu et al., 2024; Tang and Zhang, 2023; Zhou et al., 2017)。 西南大学/电子科技大学张勇教授及其合作团队确认了CRISPR-Cas12a介导的有效miRNA敲除编辑系统及策略，可以有效创制植物全基因组功能缺失型miRNA敲除编辑突变体，极大促进了植物miRNA基因功能鉴定及育种应用的相关研究。 全文主要研究结果如下：
1. Cas12a is more efficient than Cas9 in generating miRNA loss-of-function knockout mutants
The researchers compared and tested the effects of the CRISPR-Cas9 and CRISPR-Cas12a systems in knocking out the OsMIR390 gene in rice. By designing sgRNA (for SpCas9) and crRNA (for LbCas12a) in the structure of the pre-miR390 stem loop (Fig. 1B), it was found that the regeneration efficiency of rice decreased after directional editing of OsMIR390 gene compared with the control, and the regeneration efficiency of Cas12a was significantly lower than that of Cas9 (Fig. 1C and 1D). The results of genotyping showed that the osmir390 mutant plants produced by Cas12a were all heterozygous with a deletion of >4 bp (Fig. 1E), while the osmir390 mutant plants produced by Cas9 were heterozygous and homozygous, and the mutation type was mostly 1 bp insertion (Fig. 1F). Prediction of the stem-loop structure of the mutant pre-miR390 suggests that the base deletion generated by Cas12a severely alters the stem-loop structure of the pre-miRNA, which may affect its processing and maturation (Fig. 1G). The detection of seed germination and growth of T1 generation of heterozygous mutants showed that the isolation of mutant progeny produced by the two nucleases followed Mendel's law of inheritance in a ratio of 1:2:1. None of the homozygous progeny isolated from Cas12a-osmir390 mutant could germinate and grow normally, while there was no difference between the heterozygous progeny and the isolated WT. The heterozygous progeny of the Cas9-osmir390 mutant also germinated normally, while a small number of seeds in the homozygous progeny germinated and produced elongated lateral roots (Figs. 1H, 1I), although these homozygous Cas9-osmir390 mutants did not bud and did not eventually develop into seedlings. These results suggest that Cas9 nuclease can successfully knock out miRNA genes when editing miRNA genes in a targeted manner, but may still retain part of the miRNA function. The above results indicate that the Cas12a editing system is the preferred tool for miRNA gene knockout to obtain loss-of-function mutants compared with the Cas9 editing system.
Fig.1 Comparison of loss-of-function mutants from Cas12a and Cas9 knockout miRNA genes
2. CRISPR-Cas12a系统高效特异地介导水稻OsMIRNA基因敲除
In order to verify the broad adaptability of CRISPR-Cas12a in miRNA non-coding gene editing, the researchers selected 9 OsMIRNA genes with different expression patterns in rice (Fig. 2A) and designed crRNAs for their pre-miRNA secondary stem-loop structure (Fig. 2B), and successfully obtained 9 plants with directional knockout of OsMIRNA genes. The CRISPR-Cas12a system achieved an editing efficiency of 50.0%-100.0% at 9 OsMIRNA loci and a mutation efficiency of 18.75%-88.89% for biallelic mutations (Fig. 2C). The genotyping results of 128 T0 mutants showed that Cas12a nuclease tended to produce large deletions at the target sites of 9 OsMIRNA genes, among which the deletion ratio of 6bp-20 bp was more than 77.8%, and the deletion ratio of 1bp-5bp was only 6.2% (Fig. 2D). Cas9 nuclease-mediated mutation analysis of the OsMIRNA gene showed that the vast majority (76.5%) of the mutations were insertions, deletions, or substitutions of 1bp-3bp (Fig. 2D) (Zhou et al., 2017). Small RNA sequencing (sRNA-seq) of the knockout mutants of the OsMIR394, OsMIR3979, and OsMIR5794 genes showed that the corresponding mature miRNAs had been completely knocked out in the mutants and had no effect on adjacent miRNA gene expression, including the same transcript, (Figure 2E, with S6). Further comparison of the expression changes of all pri-miRNAs and mature miRNAs in the three OsMIRNA mutants and WT showed that more than 90% of the miRNA expression in the OsMIR394, OsMIR3979, and OsMIR5794 mutants did not change (Fig. 2F). These results have clearly confirmed that CRISPR-Cas12a can be specifically and effectively applied to the creation of plant genome-wide miRNA knockout editing mutant material libraries.
Figure 2. Characterization of CRISPR-Cas12a-mediated OsMIRNA gene knockout in rice
3. Effect of knockout of OsMIRNA gene on yield traits based on CRISPR-Cas12a system
Seed phenotypic analysis of homozygous T3 mutants of five OsMIR3 (OsMIR394, OsMIR827, OsMIR3979, OsMIR5789 and OsMIR5794) showed that the seed length, grain width, grain thickness (Fig. 3A-F) and 1000-grain weight (Fig. 3G) of the knockout mutants of OsMIR394, OsMIR827 and OsMIR3979 were significantly changed. The length and number of epidermal cells in the seeds of OsMIR394, OsMIR827 and OsMIR3979 knockout mutants were also changed in the longitudinal and transverse directions by scanning electron microscopy (Fig. 3H-J). These results revealed the role and molecular function of OsMIR394, OsMIR827 and OsMIR3979 miRNA genes in regulating seed grain development.
Fig.3 Grain phenotype of rice with CRISPR-Cas12a-mediated OsMIRNA knockout
4. Effect of knockout of OsMIRNA gene on quality traits based on CRISPR-Cas12a system
The researchers also analyzed the appearance quality, total starch and amylose content of the T3 homozygous seeds of the five OsMIR3 knockout mutants, and the results showed that the transparency and chalkiness of the OsMIR3979 and OsMIR5794 knockout mutants changed significantly (Fig. 4A-C). The total starch content, amylose and amylose content of the OsMIR5789 and OsMIR5794 knockout mutants also changed compared to WT (Fig. 4D-F). The structure of starch granules in cross-section of seeds was analyzed by scanning electron microscopy, and the results showed that the starch granules of OsMIR3979 and OsMIR5794 knockout mutants were loosely arranged, with weak crystalline sensitivity and obvious granularity, which were significantly different from those of the wild type (Fig. 4G). These results indicate the role of these OsMIRNA genes in regulating starch synthesis and seed quality formation.
Figure 4. CRISPR-Cas12a-mediated quality analysis of OsMIRNA knockout rice
5. Effect of knockout of OsMIRNA gene on growth and development based on CRISPR-Cas12a system
The researchers also analyzed the seed germination and seedling growth of the five OsMIRNA knockout mutants, and the OsMIR827, OsMIR3979, and OsMIR5794 knockout mutants showed differences with WT in seed germination rate and early germination rate (Fig. 5A6B). The seedling height and root length of OsMIR394, OsMIR827, OsMIR3979, OsMIR5789 and OsMIR5794 knockout mutant seedlings also changed significantly compared with WT (Fig. 5C-E), revealing the potential role of these OsMIRNA genes in regulating seed germination and early seedling growth.
Figure 5. CRISPR-Cas12a-mediated OsMIRNA knockout rice seedling growth and development
The effective miRNA editing strategy based on the CRISPR-Cas12a gene editing system developed in this study not only demonstrated its effectiveness in the creation of plant-wide miRNA knockout editing mutant materials, but also revealed the new functions of multiple rice OsMIRNA genes in regulating plant development, providing clear editing tools, editing strategies and new rice miRNA germplasm materials for in-depth analysis of plant miRNA biological functions and molecular breeding events.
Associate Professor Zheng Xuelian and Professor Tang Xu of Southwest University and Western (Chongqing) Science City Germplasm Creation Science Center, Wu Yuechao, Ph.D. student of Yangzhou University, and Professor Zheng Xiaoqin of Chengdu University are the co-first authors of the paper, School of Life Sciences, Southwest University/ Professor Daniel Zhang (http://smkxxy.swu.edu.cn/info/1014/2396.htm;https://scholar.google.com/citations?user=U3PtmKoAAAAJ&hl=en) from the School of Life Science and Technology, University of Electronic Science and Technology of China, Professor Yiping Qi from the University of Maryland, and Professor Tao Zhang from Yangzhou University are the co-corresponding authors of the study. The research work has been funded by the Major Biological Breeding Project of the Ministry of Agriculture and the National Natural Science Foundation of China.
Paper Links:
https://onlinelibrary.wiley.com/doi/full/10.1111/pbi.14484
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