Defective heart chamber growth and myofibrillogenesis after knockout of adprhl1 gene function by targeted disruption of the ancestral catalytic active site
Fig 3
Adprhl1 gRNAs—Position, sequence and activity in embryos.
A: Diagram showing the hybridization position of gRNAs in relation to exons of the X. laevis adprhl1 locus. Separate gRNAs for S- and L-homeologous alleles were prepared if sequence differences existed between the two at the selected location. Arrows indicate the 5’-3’ direction of each gRNA. Red arrows denote gRNAs whose activities were further examined by sequencing mutant allele DNA. Black arrow gRNAs have their effects on embryo development presented in this figure while grey gRNAs feature in S8 Fig. The table lists the adprhl1-specific sequence of each gRNA along with its protospacer-associated motif (PAM). Mismatched bases of control gRNAs are coloured red. Mismatched 5’-bases added to enable gRNA transcription from plasmid template DNA are coloured blue. Note -e5-1 (S and L) targeting exon 5 gave poor synthesis yields (Materials and Methods 2.2). B: Effect of adprhl1 gRNAs plus Cas9 on embryo development. Parts-of-whole charts showing the frequency of stage 44 tadpole phenotypes that occurred after injection of gRNA along with Cas9 protein into one-cell stage embryos. Red rectangles surround charts for the principal gRNAs whose activities were also examined by DNA sequencing. Green rectangles denote control gRNAs and also a chart presenting the cumulative total for non-injected sibling tadpoles assessed from the experiments. The lower right chart shows the consequence of injecting a mixture of gRNAs to target adjacent intron 2—exon 3 regions of the adprhl1 gene (additional combinatorial gRNA experiments are shown in S8 Fig). Heart defects were detected at higher frequency using the gAdprhl1-e3-1 and in particular the -e6-1 gRNA. Images showing representative tadpoles after gAdprhl1-e6-1 mutation are shown in Fig 6.