Fig. 3

PreMA sgRNA against tyr can be used to recapitulate loss-of-melanophore phenotype in 2 dpf, injected F0 larvae.

A. Top–Wildtype tyr sequence with the #2 sgRNA target site annotated in green. The dotted red boxes are MH arms predicted to be used most frequently. Raw sequence alignment of the whole PCR amplicon demonstrates that the majority of reads are the expected 4 bp deletion allele. Bottom–summary data from subcloning analyses. 88% of the mutant allele recovered were of the predicted MH allele. B. Previously reported tyr loss-of-function phenotype was successfully recapitulated using this CRISPR-Cas9. Phenotype severity was graded by the loss of retinal pigmentation. Partial loss of retinal pigmentation was considered a Weak phenotype, whereas complete loss of pigmentation in one or both eyes were considered Moderate and Severe phenotypes, respectively. Box plot demonstrating phenotypic penetrance is provided with each experiment denoted by a unique marker shape. N = 3 biological and technical replicates. At least 12 injected animals were scored in each experiment.