Abstract
Purpose :
Closure of the optic fissure, an opening at the ventral side of the developing vertebrate eye, is required for normal eye development and function. Failure of the two edges of the fissure to fuse together results in a potentially blinding congenital ocular condition known as coloboma, which accounts for ~10% of childhood blindness. Although the embryology of optic fissure closure is well understood, the gene networks underlying this process are largely unknown. By using laser capture microdissection (LCM) of tissues from optic fissure margins in mice embryos (E10.5-E12.5) and microarray, we identified 164 annotated candidate genes that are dynamically expressed during optic fissure closure. In order to investigate the specific roles of the candidate genes with no previous association with coloboma, we are generating knockout (KO) zebrafish lines using CRISPR/Cas9-mediated genome editing technology and screening for ocular coloboma and other eye abnormalities.
Methods :
We used two different approaches to generate CRISPR-KO lines. 1) Target-specific single sgRNAs were designed and co-injected with Cas9 protein to introduce indels. 2) Pairs of sgRNAs were designed to create large exonic deletions (30-1100 bp) in the coding sequence. Efficiency of the designed sgRNAs were assessed and verified using CRISPR Somatic Tissue Activity Test (CRISPR-STAT), a fluorescent PCR-based method. Sanger Sequencing was used to evaluate the nature of the CRISPR-mediated large deletions.
Results :
Genomic DNA isolated from F0 embryos injected with two sgRNAs and Cas9 were found to contain large deletions in the exonic regions of the targeted genes, suggesting that this deletion strategy works in the somatic tissue.
Conclusions :
Injected F0 embryos will be raised to adulthood and backcrossed to identify founders exhibiting germline transmission. F1 embryos will be assessed for coloboma and other eye abnormalities. This screen will expand our understanding of the molecular mechanisms involved in optic fissure closure.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.