Fig. 3

Recapitulation of mutant and morpholino phenotypes in crispants. (A-G) Mutagenesis of golden (gol). (A) wild-type control. (B,C) Targeting with Cas9 mRNA causes mosaic pigment cell phenotypes (B), while solubilized RNP injections can result in zebrafish embryos devoid of wild-type melanocytes (C) that completely mimic the gol mutant phenotype. (D-G) Whereas control siblings develop characteristic pigment patterns as adults (D), fully penetrant gol crispants maintain their mutant phenotype to adulthood (E) and completely transmit mutant gol alleles to the F1 generation when incrossed with other gol crispants (F,G). Adult zebrafish are shown as composite images. F1 phenotypes were assessed from three crispant incrosses resulting in normally sized (75-100) to big (250-300) clutches. (H,I) Crispants for tbx16 can form the characteristic spadetail (spt) phenotype of tbx16 mutants (arrowhead in I, see also Fig. S5K,L). (J-L) Crispants for tbx5a recapitulate the pectoral fin and heart phenotypes of tbx5a mutants and morphants, showing a range of phenotype expressivity including cardiac edema (asterisk, K) and unilateral pectoral fin defects (arrowhead, K), or complete heart tube stretching (asterisk, L) and pectoral fin loss (arrowheads, L). (M-O) Phenotype/genotype correlation of tbx5a and hand2 F2 embryos featuring two different alleles. Red box, target sequence; PAM is in yellow. (P) Phenotype penetrance and expressivity for tested crispants. All embryos summarized in the graph were quality controlled for injection by fluorescence microscopy for the tagged RNP directly after injection; non-fluorescent embryos were discarded; dead embryos were counted at 1dpf and included unfertilized embryos.