Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production
文献类型： 外文期刊
作者： Bi, Honghao 1 ; Fei, Qili 2 ; Li, Riqing 1 ; Liu, Bo 1 ; Xia, Rui 5 ; Char, Si Nian 1 ; Meyers, Blake C. 3 ; Yang, Bing 1 ;
作者机构： 1.Iowa State Univ, Dept Genet Dev & Cell Biol, Ames, IA 50011 USA
2.Univ Delaware, Dept Plant & Soil Sci, Delaware Biotechnol Inst, Newark, DE 19717 USA
3.Donald Danforth Plant Sci Ctr, St Louis, MO 63132 USA
4.Univ Missouri, Div Plant Sci, Columbia, MO 65211 USA
5.South China Agr Univ, Coll Hort, State Key Lab Conservat & Utilizat Subtrop Agrobi, Guangzhou, Guangdong, Peoples R China
6.Chinese Acad Agr Sci, Agr Genom Inst Shenzhen, Shenzhen, Guangdong, Peoples R China
关键词： rice; Arabidopsis; TALENs; CRISPR; Cas9; gene editing; miRNA
期刊名称：PLANT BIOTECHNOLOGY JOURNAL （ 影响因子：9.803； 五年影响因子：9.555 ）
ISSN： 1467-7644
年卷期：
页码：
收录情况： SCI
摘要： MicroRNAs (miRNAs) are 20-24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta-siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences. We used both TALENs (transcription activator-like effector nucleases) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to introduce heritable base pair mutations in mature miRNA sequences. For rice, TALEN constructs were built targeting five different mature miRNA sequences and yielding heritable mutations. Among the resulting mutants, mir390 mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild-type MIR390. Small RNA sequencing showed the two base pair deletion in mir390 substantially interfered with miR390 biogenesis. In Arabidopsis, CRISPR/Cas9-mediated editing of the miR160* strand confirmed that the asymmetric structure of miRNA is not a necessary determinant for secondary siRNA production. CRISPR/Cas9 with double-guide RNAs successfully generated mir160a null mutants with fragment deletions, at a higher efficiency than a single-guide RNA. The difference between the phenotypic severity of miR160a mutants in Col-0 versus Ler backgrounds highlights a diverged role for miR160a in different ecotypes. Overall, we demonstrated that TALENs and CRISPR/Cas9 are both effective in modifying miRNA precursor structure, disrupting miRNA processing and generating miRNA null mutant plants.
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