Deletion of the furina 3′UTR YBE leads to abnormal LR patterning. (A) Schematic of the furina X1 3′UTR genomic region on chromosome 7. Exon 16 contains the 3′ end of the CDS with the stop codon (stop sign) and the entire 3375 bp of the 3′UTR. The red dotted line shows the sequences deleted in furina Δ3′UTR crispants by the two gRNAs (green arrows). The position of primers used for the simultaneous detection of 3′UTR deletion alleles (primer F and primer 2R) with wild-type alleles (primer F and primer 1R) in three primer genotyping PCRs are indicated. (B) Gel image showing furina Δ3′UTR crispant genotyping PCRs using genomic DNA from 1 dpf embryos. The expected sizes of the wild-type (wt) and the Δ3′UTR deletion mutant PCR products are shown next to the 100 bp DNA ladder (far-left lane). Mutant bands (cyan arrowheads) together with wild-type bands are only observed with the crispant samples. The non-injected control (Nij) only shows the wild-type band, and no product is detected in the no template control lane (NTC). (C) Stacked bar chart showing the percentage of embryos falling into different categories for spaw expression by WISH on 19-21 som embryos: non-injected wild-type embryos, Cas9 protein only-injected embryos, Cas9 RNPs loaded with the two targeting gRNAs (gRNAs 1+2), and control Cas9 RNPs loaded with gRNA2 and a non-cutting control gRNA (gRNAc). n=number of embryos. *P<0.05 by Fisher's exact test. (D) Images of spaw expression by WISH in 19-21 som embryos showing the various categories shown in the bar graph in C.
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