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Title: Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production
Summary

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,mir390mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild‐typeMIR390. Small RNA sequencing showed the two base pair deletion inmir390substantially 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 generatedmir160anull mutants with fragment deletions, at a higher efficiency than a single‐guide RNA. The difference between the phenotypic severity ofmiR160amutants 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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PAR ID:
10457194
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
Plant Biotechnology Journal
Volume:
18
Issue:
7
ISSN:
1467-7644
Page Range / eLocation ID:
p. 1526-1536
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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