Fig. 2

Generation of the attP knock-in fish lines. (A) Schematic of the SpCas9 target locus for tyr_1 and its corresponding single-stranded oligonucleotide (ssODN). The tyr_1 site is in the first exon (Ex 1) of the tyrosinase gene. Its target sequence is shown, and the spacer adjacent motif (PAM) is highlighted in gray. The cleavage site of SpCas9 is indicated by a pair of scissors. The ssODN used for attP knock-in harbors attP encompassed by two homology arms. The sequences for the homology arms were complementary to the non-target strand of SpCas9 and were 36 nts long on the PAM-distal side and 91 nts on the PAM-proximal side. Complete sequences of the ssODNs for attP knock-in at both tyr_1 and tyr_2 sites can be found in Table S4. (B) Schematic of the attP knock-in at the tyr_1 locus (designated as the attPtyr_1 allele) and the primers for PCR analysis. The thick black line indicates the overlapping sequence with the ssODN. To detect the attP insertion, nested PCR was performed using tyr-specific F1 and R1 primers, followed by F2 and R2 primers that recognize attP and the tyr gene, respectively. (C) Representative agarose gel analysis of the nested PCR from single embryos microinjected with SpCas9 protein, the tyr_1 gRNA, and the ssODN. The expected product size is 110 bps as indicated by a red arrow. M, size marker; 1-12, injected embryos; C, an uninjected control embryo. (D) Representative agarose gel analysis of the nested PCR from the attPtyr_1 founder screen. Twelve pools of embryos (five embryos per pool) from each potential founder were analyzed. The 110-bp band (red arrow) indicated the presence of the attPtyr_1 allele. The pools containing the attP knock-in alleles are labeled by red numbers on top. (E-H). Sequences of the attP knock-in at the tyr_1 (E), tyr_2 (F), gfap (G), and kcnh6a (H) targeted loci in the F1 fish. Endogenous gene sequences are in black letters. Insertions are in red letters. The attP sequences are in uppercase. For attPtyr_1 and attPtyr_2, in-frame stop codons are underlined. For attPgfap and attPkcnh6a, the lowercase letters are sequences added to keep the insertions in-frame for unperturbed translation of the endogenous proteins.