Figure 1. cryaa null mutant.

Produced with a single gRNA to generate a five basepair deletion and frame shift mutation in exon 1, with the relative position noted by the red line (A). Injected embryos were outcrossed to wild-type fish to identify heterozygote mutants, with a five-base pair deletion allele identified that leads to an early stop codon after amino acid 28 (B). Incrossed heterozygotes were used to generate a homozygous null line, which was bred to assess the effects of cryaa loss. Mass spectrometry confirmed that embryos generated by incrossing this null line produced no detectable αA crystallin protein (C). Parallel reaction monitoring of the 3 major fragment ions of the +3 charge state of cryaa peptide 52–65 from the digest of wild-type lenses showed coelution of its three major fragment ions on the left and no corresponding peaks in the digest from KO lenses on the right (C). Compared to wild-type lenses at 3 dpf (C), cryaa null embryos showed various defects, such as roughness in the primary fiber cell region (E) and a general disorganization of central fiber cells (F). Lenses are from 3 dpf embryos, imaged using DIC optics and arranged with retina to the left. Scale bars are 20 microns.