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Katarina Jovanovic, Amelia Lane, Nele Schwarz, Alison J Hardcastle, Michael E. Cheetham; Probing the function of Arl3 in CRISPR modified cell models. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3061.
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Recently described missense variants in ARL3 have been associated with dominantly inherited retinal dystrophies, but their significance is unclear. ARL3 has an important role in cilia associated lipidated protein traffic and the ARL3 effectors, UNC119 and PrBP, and GTPase activating protein RP2 are essential for retinal function. We therefore examined the function of ARL3 using CRISPR-Cas9 gene editing and induced pluripotent stem (iPSC) cells to model the cell types found in the retina, and assess the role of ARL3 therein.
To probe the function of ARL3 in human retinal cells, CRISPR-Cas9 gene editing targeting ARL3 was used to generate cell lines lacking ARL3 expression. This was performed in hTERT RPE cells as well as control human dermal fibroblasts that were simultaneously reprogrammed into iPSC. The gene edited iPSC lines were differentiated in retinal pigment epithelium (RPE) cells and immunocytochemistry used to assess cellular differences between gene edited and control cells.
CRISPR-Cas9 gene editing was used to generate a homozygous ARL3 knock out in hTERT-RPE cells. These cells had defects in cellular proliferation, and cilia incidence and length were significantly reduced. Simultaneous gene editing and reprogramming of human dermal fibroblast resulted in a large number of successfully edited iPS cell clones. Two clones were selected for further examination, one bearing a homozygous 1bp deletion leading to a complete knock out of ARL3 protein expression, whereas the second line had a 2bp deletion on one allele only, leading to a heterozygous knock-out with significantly reduced ARL3 expression. Both clones were able to successfully differentiate into RPE cells. Cilia incidence and length was also compromised in ARL3 KO iPSC derived RPE, but not in the heterozygous knock-out cells.
Ciliation defects in ARL3 knock out cells confirm an important role for this protein in both cilia formation and elongation in retinal cells. Further studies are warranted to determine the impact of reduced ARL3 function in retinal cells. These studies highlight how recent advances in gene editing and stem cell differentiation can enable modelling of human retinal biology and the in depth analysis of potential disease associated genetic variation.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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