Abstract
Purpose :
The usefulness of zebrafish for examining molecular mechanisms of lens development could be enhanced by producing consistent terminology for abnormal lens phenotypes. Here we characterize a variety of lens abnormalities stemming from knockout of crystallin, transcription factor and RNA-binding protein genes after CRISPR/Cas9 editing.
Methods :
We used two approaches to disable individual genes with CRISPR/Cas9 editing. First, we produced two gRNAs designed to delete a region spanning a gene’s promoter and first exon to prevent translation of any resulting mRNA. Second, we designed a mix of four gRNAs to produce multiple gene mutations in the coding region of a targeted gene. In each case, we co-injected one nanoliter into one-cell stage zebrafish zygotes containing a total of 500 picograms of gRNA and 250 picograms of recombinant Cas9 protein. For the first method, we raised the injected embryos to adulthood, outcrossed them to wildtype individuals, and genotyped offspring to identify null mutants, which were then used to produce lines of homozygous mutant fish. In the case of the four guide mix, we examined phenotypes directly in injected embryos.
Results :
We generated zebrafish missing ~800 basepairs of the cryaa gene and confirmed by western analysis a decrease in αA-crystallin protein levels. Seventy-eight embryos from a cross of injected fish with this large cryaa deletion showed a decreased proportion of normal lenses compared to wildtype embryos at four dpf (46.2% versus 94.3%; p-value<0.0001). Lenses in these cryaa mutant embryos showed a mix of three different abnormalities (prevalence shown in parentheses): a central roughness (43.6%), disorganization of the concentric fiber cell rings (20.5%), and excessive pitting (6.4%). Mixes of four guides targeting several genes resulted in observable lens phenotypes directly in injected embryos. Targeting of both zebrafish γN-crystallin genes (crygn1 or crygn2) produced lenses with excessive gaps between fiber cells. Similar gaps were seen after targeting the transcription factor gene prox2. A unique lens abnormality affecting the central lens was seen after targeting the ribonuclease endou2.
Conclusions :
These data demonstrate that gene disruption can produce diverse phenotypes in the lens. We suggest that future studies using zebrafish as a model for lens development carefully characterize these abnormalities so that they can be compared between studies.
This is a 2021 ARVO Annual Meeting abstract.