Scheme for generating gas6 mutant line. a Schematic showing the 20 nucleotide (orange text) target site in exon 1 of gas6. CAA represents the PAM sequence (blue box) and ATG (green box) is the start codon. XcmI target sequence is indicated by the dotted red line, the red arrow denotes the cut site. b sgRNA and Cas9 mRNA was injected into 1-cell stage embryos. Injected embryos were raised to 24hpf and genomic DNA was extracted from a pool of embryos. XcmI digest of PCR products amplified from genomic DNA (extracted from injected embryos) reveal the presence of a mutation (red box in gel). c Injected embryos were raised to give rise to F0 adults. These fish were crossed with WT adults to raise the F1 generation. At 3 months age, genomic DNA was extracted from fin-clips from individual F1 fish and genotyped as in panel B. d Sequencing of F1 genomic DNA revealed transmission of four different mutant alleles (um296, um297, um298, um299), each with a different 4 nucleotide deletions (orange dashes). Each mutant allele codes for 96 out of frame amino acids (gray boxes) followed by a premature stop codon. e One quarter of the embryos collected from a cross of two heterozygous parents lack gas6 expression in r5/r6 (i). XcmI digest of PCR products amplified from genomic DNA extracted from embryos lacking gas6 expression were homozygous for mutant gas6 allele (iii, lane 5). cDNA was synthesized from total RNA extracted from WT and homozygous gas6 mutant fish. Quantitative RT-PCR using two different primer pairs (targeting the N and C termini, respectively) shows that homozygous gas6 mutants have significantly lower levels of gas6 mRNA (iv)
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