CRISPR-Cas9-mediated functional dissection of the foxc1 genomic region in zebrafish identifies critical conserved cis-regulatory elements.

FOXC1 encodes a forkhead-domain transcription factor associated with several ocular disorders. Correct FOXC1 dosage is critical to normal development, yet the mechanisms controlling its expression remain unknown. Together with FOXQ1 and FOXF2, FOXC1 is part of a cluster of FOX genes conserved in vertebrates. CRISPR-Cas9-mediated dissection of genomic sequences surrounding two zebrafish orthologs of FOXC1 was performed. This included five zebrafish-human conserved regions, three downstream of foxc1a and two remotely upstream of foxf2a/foxc1a or foxf2b/foxc1b clusters, as well as two intergenic regions between foxc1a/b and foxf2a/b lacking sequence conservation but positionally corresponding to the area encompassing a previously reported glaucoma-associated SNP in humans. Removal of downstream sequences altered foxc1a expression; moreover, zebrafish carrying deletions of two or three downstream elements demonstrated abnormal phenotypes including enlargement of the anterior chamber of the eye reminiscent of human congenital glaucoma. Deletions of distant upstream conserved elements influenced the expression of foxf2a/b or foxq1a/b but not foxc1a/b within each cluster. Removal of either intergenic sequence reduced foxc1a or foxc1b expression during late development, suggesting a role in transcriptional regulation despite the lack of conservation at the nucleotide level. Further studies of the identified regions in human patients may explain additional individuals with developmental ocular disorders.