Abstract
Purpose :
Uveal coloboma, a congenital condition that accounts for up to 10% of childhood blindness, results from improper optic fissure closure in the second month of embryonic development. Clinically, coloboma presents as missing tissue (iris, retina/RPE/choroid, and/or optic nerve) at birth, usually in the inferonasal quadrant. Although the genetic contribution to coloboma is widely recognized, the genes and networks regulating optic fissure closure are not well understood. We previously identified 164 annotated genes expressed during optic fissure closure in mouse embryos. Using CRISPR/Cas9-mediated genome editing as a screening method, we generated knockout (KO) zebrafish lines to investigate the roles of these candidate genes in the ontogenesis of coloboma.
Methods :
To generate CRISPR KO lines, sgRNA pairs were designed and injected with Cas9 protein at the zebrafish embryo one-cell stage to introduce insertions and/or deletions (indels). The CRISPR-mediated mutations in founders (F0) and later generations, were evaluated by Sanger Sequencing and fluorescent PCR-based CRISPR Somatic Tissue Activity Test (CRISPR-STAT) analysis. Gene expression was established by in situ hybridization in zebrafish embryos. Morpholinos were also injected into zebrafish embryos to knockdown target genes and check for coloboma phenotype.
Results :
Genomic DNA from embryos injected with sgRNAs and Cas9 contained significant indels in the exonic regions of targeted genes, suggesting successful gene KO. We generated 72 CRISPR F0 lines. From bred F0 incrosses, 19 lines displayed coloboma in F1 zebrafish; 11 of the 19 lines also had other anatomical defects. Of the 19 genes, 14 encoded signaling pathway proteins and 5 encoded transcription factors. Expression patterns for most tested genes showed localization to the eye. We are outcrossing F1 zebrafish with wildtype for all 19 genes to separate alleles in an F2 generation. Netrin 1 (ntn1) is among confirmed coloboma genes in accordance with data in other animal models.
Conclusions :
Ntn1 identification validates our CRISPR-mediated methodology for detecting true novel genes involved in coloboma. Upon allele segregation, F2 zebrafish with the same allele mutation will be incrossed to produce homozygous null F3 zebrafish for use in further developmental and molecular studies. Our screen and validation will expand our understanding of the genetics of uveal coloboma.
This is a 2020 ARVO Annual Meeting abstract.