Multiplexed miRNA mutations induced efficient disruption of the miRNA family function.

(A) Sequences of miRNA loci mutated after multiplexed CRISPR/Cas9 injection. PCR fragments were cloned and sequenced from F0 embryos. Sequences were aligned using the T-Coffee multiple sequence alignment program. The alignments are colored according to their similarity to the wild type sequence (top sequence) (high = red to low = green). The two gRNAs designed for each miRNA hairpin arm are highlighted by a solid dark blue and light blue line. Arrows point to the PAM sequence. Dashed lines indicate insertions in the wild type (wt) sequence and dashed lines in the mutant (m) sequences indicate deletions. DNA sequences within the black box correspond to the mature miRNA. (B) The top panel shows the diagram of the miRNA sensor assay to establish miRNA loss of function. The bottom panel shows confocal images of the lateral trunk of 48 hpf embryos injected as indicated. Caudal is to the right. Arrows indicate the presence of cells with GFP derepression in embryos after the injection of the respective multiplexed sgRNAs and Cas9.