Fig. 4

Prospectively designed PreMA reagent against tdgf1 can be used to reproduce gross developmental defect in 1 dpf, injected F0 larvae.

A. Top–Wildtype tdgf1 sequence with sgRNA target site annotated in orange. 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. 72% of the mutant allele recovered were of the predicted MH allele. B. Previously reported tdgf1 loss-of-function phenotype was successfully recapitulated using this CRISPR-Cas9. Phenotype severity was graded by the “pinhead” morphology and cyclopia. Pinhead morphology alone was classified as Weak, whereas Moderate and Severe phenotypes also presented with varying degrees of cyclopia judged by the distance of forebrain protrusion. In the Severe class, the forebrain does not separate the eyes, and they are fused together. Box plot demonstrating phenotypic penetrance is provided with each experiment denoted by a unique marker shape. N = 4 with 3 biological and 4 technical replicates. At least 42 injected animals were scored in each experiment.