Figure 4. Impact of CRISPR/Cas9 mutations on miRNA processing.

(A) Schematic representation of a pri-miRNA hairpin highlighting the position of important secondary structures and sgRNA-mediated mutagenesis sites. (B) Computational analysis of predicted RNA hairpin structures of wild type and mutant miRNA genome sequences analyzed. Percentile is indicative of the number of miRNA mutants falling in the indicated categories compared to the wild type structures. (C) Schematic representation of zebrafish miRNA biogenesis assay (top). Northern blot of miR-125 mature sequence expression obtained upon biogenesis assay of miR-125-a1, -a2, -b1, -b2 and -b3 mutants and wild type pri-miRNA genes (bottom). (D) Chart shows the quantification (pixel intensity) of northern blots in (C) normalized to the respective total RNA and the ectopic expression of wild type pri-miRNA injection. (E) Predicted RNA structure obtained with the ViennaRNA Software Package2 miRBase v21³⁶ of the wild type and mutant sequence. Colored in red are the sequences of the respective mature miRNAs. (F) Average level of mature miRNA expression of the indicated miRNAs. qRT-PCRs were performed on adult fin clipped F2 miRNA founders genotyped accordingly (n = 3). Bar plots show mean + S.E.M. and significance calculations were relative to wild type embryos. n.s. (not significant, p > 0.05).