Abstract
Purpose :
The single copy vertebrate lens crystallin gene for αB-crystallin is duplicated in the zebrafish. We previously showed that the two resulting paralogs, cryaba and cryabb, are differently expressed in developing embryos, and that cryaba becomes lens specific in adults. Here we use CRISPR gene editing to disable each αB-crystallin gene separately to assess their role in lens development and homeostasis.
Methods :
We used qRT-PCR to measure cryaba and cryabb expression levels between 12 hpf and 5 dfp in whole embryos and single-cell RNA sequencing (scRNA-seq) to identify cell types that expressed each gene between 1 and 5 dpf. Guide RNAs targeting exon 1 of cryaba and cryabb were co-injected with Cas9 protein into zebrafish zygotes and resulting fish were outcrossed to wildtypes to identify deletions resulting in early termination of protein translation. Adults lacking each functional αB-crystallin gene were incrossed and lenses from their embryos assessed by DIC microscopy at 4 dpf.
Results :
We produced a mutant cryaba allele with a premature stop codon at position 64 and cryabb allele with a stop at position 26. Embryos from incrosses of homozygous mutants for the cryaba knockout allele showed no lens abnormalities through 4 dpf. Similar analyses are currently underway for the cryabb knockout allele. qRT-PCR suggested very low transcription levels of both cryab paralogs in whole zebrafish embryos through 4 dpf, consistent with publicly available RNA-Seq data. scRNA-seq identified expression of cryaba and cryabb, but not from lens cell clusters identified by expression of known lens marker genes.
Conclusions :
Our data suggest that disabling zebrafish cryaba has no noticeable effect on lens development through 4 dpf. This result may reflect the observation that cryaba is not expressed at detectable levels in lens cells through at least 5 dpf as shown by our scRNA-seq data. Our lack of lens phenotype is similar to past work on a Cryab knockout mouse, which also lacked abnormalities in lens development. However, our findings differ from a recently published zebrafish cryaba and cryabb knockout study that identified lens abnormalities in both mutants. We are in the process of examining our cryabb knockout line for any phenotype. Our cryaba and cryabb knockout lines will be a valuable tool for exploring the role of αB-crystallins in maintaining lens transparency during aging.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.