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Fig. 4 Mutagenesis of aldh1a2. a. Diagram of the aldh1a2 locus. Exons drawn to scale, introns not to scale. Below each intron-exon junction reading frame phase is indicated. b. Two highly active intronic aldh1a2 sgRNAs, aldh1a2 sgRNA1 and aldh1a2 sgRNA4, flank exon 8. Sequence corresponding to single guide RNA is shown in blue, PAM motif is in bold, and expected Cas9 cut site is indicated by a red x. c. Two independent aldh1a2 exon 8 deletion alleles recovered after co-injection of aldh1a2 sgRNA1 and aldh1a2 sgRNA4 along with nCas9n mRNA. For Sanger sequencing of the alleles, see S3 Fig. d, e. Deletion of exon 8 of aldh1a2 results in expected loss-of-function phenotype. F1 fish heterozygous for aldh1a2tpl137 and aldh1a2tpl138 deletions were crossed to each other. d. Images of 3 dpf embryos displaying wild type (top) and the expected aldh1a2 loss of function phenotypes: lack of pectoral fins, shortened hindbrain brain and cardiac edema. In addition, most of the 3 dpf embryos displaying these phenotypes had curved tails (d, bottom) consistent with uneven left/right somite numbers. e. aldh1a2tpl137/tpl138 trans-heterozygotes lack pectoral fin buds as revealed by loss of tbx18 expression at 32 hpf. f. Expression of tcf21 persists in the first and second branchial arches of aldh1a2tpl137/tpl138 trans-heterozygotes at 32 hpf. g. Diagram of the HDR template oligonucleotide used to knock in the loxP into aldh1a2 sgRNA1 target site. h. Sequencing of the precise loxP knockin allele. Additional knock-in alleles recovered from other F0 families are shown in S5 Fig. i. Genotyping of 16 phenotypically normal 3 dpf embryos from a cross between F1 fish heterozygous for aldh1a2tpl139 loxP knock-in results in Mendelian ratios of aldh1a2tpl139/tpl139 (red arrows), aldh1a2tpl139/wt (yellow arrows) and aldh1a2wt/wt embryos. PCR fragments from two of the embryos were sequenced to further confirm homozygosity for the loxP site.