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Takashi Miyai, Kodai Kitamoto, Yukako Taketani, Yasuo Ouchi, Tomohiko Usui; Genome-editing with CRISPR-Cas9 system targeting intronic CTG repeat of TCF4 gene in corneal endothelium.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2173.
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© ARVO (1962-2015); The Authors (2016-present)
Intronic CTG repeat expansion in intron 2 of TCF4 gene on chromosome 18 is a major mutation of late onset Fuchs endothelial corneal dystrophy (FECD). We investigated the methodology of excising the intronic CTG repeat by genome editing with CRISPR-cas9 system in corneal endothelium.
We have designed two types of pX458 plasmids with guide RNAs sandwiching the 18.1 CTG triplet repeat site. The plasmids were transfected into human corneal endothelial cell line, HCEnC-21T, by Fugene 6 and transfection efficacy was assessed for 6, 24, 48hr after transfection. Single cell sorting of GFP positive cells was conducted with FACS and the sorted cells were cultured in growing media. DNA was extracted from the colony of the cells. DNA including the repeat area was amplified by PCR for agarose gel electrophoresis and DNA sequencing.
Transfection efficiency was 1.39% for 6 hr, 10.25% for 24hr and 22.97% for 48hr. By agarose gel electrophoresis, 500 bp bands were detected in control cells and 300bp bands in the pX458 transfected cells. DNA sequencing showed the CTG repeat in intron 2 of TCF4 was removed in the transfected cells.
We could excise CTG repeat in intron 2 of TCF4 in HCEnC-21T by genome editing with CRISPR-Cas 9 system. This methodology suggested future gene therapy for the type of FECD patients with TCF4 triplet repeat expansion.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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