Abstract
Purpose :
Congenital hereditary endothelial dystrophy (CHED) is an autosomal recessive bilateral disorder of the cornea endothelium that is associated with homozygous or compound heterozygous mutations in the SLC4A11 gene. To investigate the role of SLC4A11 on corneal endothelial cell (CEnC) function, we performed CRISPR-Cas9-mediated gene editing to stably knockout SLC4A11 expression in human CEnC.
Methods :
A human CEnC line, HCEnC-21T, was transfected with SLC4A11 CRISPR-Cas9 guide RNA constructs and subsequently sub-cloned with puromycin selection and limiting dilution. Sanger sequencing and CRISP-ID analysis were performed to genotype and identify sub-clones containing either homozygous or compound heterozygous mutations that are predicted to truncate the encoded SLC4A11 protein at the n-terminal. Western blot was performed to evaluate SLC4A11 protein production of each clone, and only clones with truncating SLC4A11 mutations on both alleles and no expression of SLC4A11 protein were considered as validated SLC4A11-/- CEnC candidates.
Results :
Three unique SLC4A11-/- CEnC clones were generated by CRISPR-Cas9 gene editing and validated by Western blot and Sanger sequencing. The genotype of each of the three SLC4A11-/- CEnC clones are as follows: homozygous c.125dupT (p.Glu43Argfs*9); heterozygous c.123delC (p.Phe42Serfs*73) and heterozygous c.124delT (p.Phe42Serfs*73); and homozygous c.123_135delTTCGAGGATTC (p.Phe42Lysfs*6).
Conclusions :
Human CEnC models of CHED were successfully generated using CRISPR-Cas9 gene editing. The generated SLC4A11-/- CEnC clones will be utilized to study the role of SLC4A11 in CEnC function and in the development of CHED.
This is a 2020 ARVO Annual Meeting abstract.