Mutation efficiency of the gRNAs for each candidate gene. Data for gata5 are shown in top left panel, api5 in top right panel, hspb7 in bottom left panel, and lmo2 in bottom right panel. In each panel, the upper gel images show the bands obtained following targeted PCR of genomic DNA extracted from four individual animals injected with the two most effective CRISPR gRNAs + Cas9 (based on 2 dpf morphological analysis), compared with the Cas9 injected control animals. The lower gel images show the same samples following T7E1 assay undertaken to reveal the cleavage of heteroduplex DNA. The chromatogram images in the middle of each panel show the result of Sanger sequencing undertaken on representative genomic DNA samples from the most effective gRNA + Cas9, per gene, compared with that from a representative Cas9-injected control animal. The lower scatter plot graphs in each panel show the indel size and frequency in the PCR products from most effective gRNA + Cas9 group (n = 4) per gene. Data points with same colour indicate the indels identified in the same individual embryo within the group. The size ranges of deletions between gRNA target sites are shaded. Indels with less than 5% frequency are presented by open circles. Note in all cases the most effective gRNA was the combined guide group (g#1,2,3) except for lmo2 which, due to high mortality in the g#1,2,3 group, the g#2-injected animals were selected for full analysis.
|
