Abstract
Purpose :
Uveal coloboma is a developmental eye disorder that occurs due to improper closure of the optic fissure in the second month of embryonic development. This results in optic tissue missing at birth, appearing as gaps in different parts of the eye including the iris, the retina, the choroid, or the optic nerve. Coloboma is a congenital condition that accounts for up to 10% of childhood blindness, however, the gene networks regulating optic fissure closure are not well understood. We have previously identified 164 annotated candidate genes by microarray that are expressed in tissues from the optic fissure region of mouse embryos at embryonic day (E) 10.5-E12.5 isolated using laser capture microdissection. We have generated knockout zebrafish lines using CRISPR/Cas9-mediated genome editing technology to investigate the roles of the candidate genes in the ontogenesis of coloboma.
Methods :
To generate CRISPR knockout lines, sgRNAs were designed and injected with Cas9 protein at the one cell stage of the zebrafish embryo to introduce insertions and/or deletions (indels). Large exonic indels (30-1100 bp) were introduced in the coding sequence by pairs of sgRNAs. Multiplexing, or the injection of multiple CRISPRs, was also performed to introduce indels in multiple genes at once in the attempt to be efficient. The CRISPR-mediated insertions and deletions were evaluated by Sanger Sequencing. CRISPR Somatic Tissue Activity Test (CRISPR-STAT) analysis, a fluorescent PCR-based method, was also used to assess the efficiency of the designed sgRNAs.
Results :
Multiplexing results in the introduction of indels in multiple genes at once. However, none of the multiplexed pairs of sgRNAs exhibited coloboma. We found that the genomic DNA from the embryos injected with single gRNAs and Cas9 contained large deletions in the exonic regions of the targeted genes, which suggests that the genes were successfully eliminated. Out of the 164 candidate genes, we have generated 69 CRISPR F0 lines, screened 56 of these lines, and found that 14 lines produce coloboma in F1 zebrafish.
Conclusions :
Injected F0 embryos were raised to adulthood and backcrossed to identify founders exhibiting germline transmission. Currently, F1 and F2 embryos are being assessed for coloboma. This screen will expand our understanding of the molecular mechanisms involved in optic fissure closure and the genes that are involved in uveal coloboma.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.