Figure 3. Production of cryaba and cryabb frameshift mutants.

Single gRNAs were designed to target the first exons of the zebrafish cryaba (A) and cryabb (D) genes. After injection into wild-type zygotes, founders were outcrossed to wild-type fish, and resulting heterozygotes with deletions that caused frameshift mutations leading to early stop codons were detected (B and E). Mass spectrometry analysis of adult lenses from mutant lines confirmed the loss of protein coded by each gene. Parallel reaction monitoring of the +3 charge state of αBa-crystallin tryptic peptide 80–89 from the digest of wild-type lenses detected coelution of its three major fragment ions (left), and these were not detectable in cryaba mutant adult lenses (C). Similarly, the +2 charge state of αBb-crystallin tryptic peptide 12–20 from the digest of wild-type lenses detected coelution of its three major fragment ions (left), and these were missing in digests from cryabb mutant adult lenses on the right (F).