Derepression of specific miRNA-target genes in rice using CRISPR/Cas9

Abstract

MicroRNAs (miRNAs) target specific mRNA molecules based on sequence complementarity for their degradation or repression of translation, thereby regulating various developmental and physiological processes in eukaryotic organisms. Expressing the target mimicry (MIM) and short tandem target mimicry (STTM) can block endogenous activity of mature miRNAs and eliminate the inhibition of their target genes, resulting in phenotypic changes due to higher expression of the target genes. Here, we report a strategy to achieve derepression of interested miRNA-target genes through CRISPR/Cas9-based generation of in-frame mutants within the miRNA-complementary sequence of the target gene. We show that two rice genes, OsGRF4 (GROWTH REGULATING FACTOR 4) and OsGRF8 carrying in-frame mutants with disruption of the miR396 recognition sites, escape from miR396-mediated post-transcriptional silencing, resulting in enlarged grain size and increase in brown planthopper (BPH) resistance, in their respective transgenic rice lines. These results demonstrate that CRISPR/Cas9-mediated disruption of miRNA target sites can be effectively employed to precisely derepress particular target genes of functional importance for trait improvement in plants.

Keywords: CRISPR/Cas9; de-repression; in-frame; miRNAs; rice.

Fig. 1.

Development of large grains through CRISPR/Cas9-mediated miR396-resistant mutants of the OsGRF4 allele. (A) Gene structure of OsGRF4 in ‘S143’ (WT) and the miR396 target site. The sgRNA targeting the flanking sequences of the recognition site is indicated by red letters. The miR396 recognition sequence is marked in grey background. (B) Mutations in T₀ plants harbouring in-frame osgrf4 and frame-shift osgrf4 variants. The introduced deletions and insertions are indicated by black dashes and red letters, respectively. Numbers on the right side indicate the lengths of indels compared with WT. –: deletion; +: insertion; combined mutations are distinguished by ‘/’. (C) Grain morphology of T₀ mutant plants and the corresponding WT. Scale bar: 0.5 cm. (D) Expression of OsGRF4 in young panicles of T₀ mutant plants. Data are means ± SD. **P < 0.01 compared with WT using Student’s t-test. NS: no significant difference.

Fig. 2.

Segregation analysis of T₁ progeny of in-fame grf4-#25 mutant. (A) Alignment of sequences of the indels site in T₀ and T₁ progeny of grf4-#25. †Bi-allelic sequences were decoded from sequencing chromatograms using an online program DSDecode (http://dsdecode.scgene.com/). (B) Examples of sequencing chromatograms from different mutants presented in (A). (C) Grain phenotypes of segregations in T₁ progeny of grf4-#25. Scale bar: 1 cm.

Fig. 3.

Phenotypes and grain yield analysis of T₂ generation mutants. (A) Grain morphology of osgrf4 mutants and the wild type ‘S143’ (WT). Scale bar: 1 cm. (B) Morphological phenotypes of osgrf4 mutants and the corresponding WT. Scale bar: 20 cm. (C-G) Statistical data of the grain length (C), grain width (D), 1000-grain weight (E), plant height (F), till number (G), and grain yield per plant (H) of the WT and osgrf4 variants. Values are means ± SD. **P < 0.01 compared with WT using Student’s t-test. NS: no significant difference.

Fig. 4.

Development of BPH resistance rice through CRISPR/Cas9-mediated miR396-resistant mutants of the OsGRF8 allele. (A) Gene structure of OsGRF8 in ‘S143’ (WT) and the miR396 target site. The sgRNA targeting the flanking sequences of the recognition site is indicated by red letters. (B) Mutations in T₀ plants harbouring in-frame Osgrf8 and frame-shift Osgrf8 variants. The miR396 recognition sequence was marked in grey background. The introduced deletions and insertions are indicated by black dashes and red letters, respectively. Numbers on the right side indicate the lengths of indels compared with WT. –: deletion; +: insertion; combined mutations are distinguished by ‘/’. (C) Expression of OsGRF8 in leaves of T₀ mutant plants compared with WT. (D) Individual tests to determine the BPH resistance of the WT, in-frame variants and frame-shift mutants. Scale bar: 5 cm. (E) Statistical analysis of the survival rates of WT plants and Osgrf8 mutants after BPH feeding (n=3). (F) Expression of OsF3H in leaves of WT and in-frame Osgrf8 plants. (G) Quantitative determination of flavonoid content in the WT and in-frame Osgrf8 mutants (n=3). Data are mean ± SD. **P<0.01 compared with WT using Student’s t-test.