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2017 ; 8
(ä): 1598
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CRISPR-Cas9 Based Genome Editing Reveals New Insights into MicroRNA Function and
Regulation in Rice
#MMPMID28955376
Zhou J
; Deng K
; Cheng Y
; Zhong Z
; Tian L
; Tang X
; Tang A
; Zheng X
; Zhang T
; Qi Y
; Zhang Y
Front Plant Sci
2017[]; 8
(ä): 1598
PMID28955376
show ga
MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in plant
development and stress responses. Loss-of-function analysis of miRNA genes has
been traditionally challenging due to lack of appropriate knockout tools. In this
study, single miRNA genes (OsMIR408 and OsMIR528) and miRNA gene families
(miR815a/b/c and miR820a/b/c) in rice were targeted by CRISPR-Cas9. We showed
single strand conformation polymorphism (SSCP) is a more reliable method than
restriction fragment length polymorphism (RFLP) for identifying CRISPR-Cas9
generated mutants. Frequencies of targeted mutagenesis among regenerated T0 lines
ranged from 48 to 89% at all tested miRNA target sites. In the case of miRNA528,
three independent guide RNAs (gRNAs) all generated biallelic mutations among
confirmed mutant lines. When targeted by two gRNAs, miRNA genes were readily to
be deleted at a frequency up to 60% in T0 rice lines. Thus, we demonstrate
CRISPR-Cas9 is an effective tool for knocking out plant miRNAs. Single-base pair
(bp) insertion/deletion mutations (indels) in mature miRNA regions can lead to
the generation of functionally redundant miRNAs. Large deletions at either the
mature miRNA or the complementary miRNA(*) were found to readily abolish miRNA
function. Utilizing mutants of OsMIR408 and OsMIR528, we find that knocking out a
single miRNA can result in expression profile changes of many other seemingly
unrelated miRNAs. In a case study on OsMIR528, we reveal it is a positive
regulator in salt stress. Our work not only provides empirical guidelines on
targeting miRNAs with CRISPR-Cas9, but also brings new insights into miRNA
function and complex cross-regulation in rice.