June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
CRISPR activation of endogenous CRB1 expression in fibroblasts from retinitis pigmentosa 12 patients
Author Affiliations & Notes
  • Sang Yoon Moon
    Ocular Tissue Engineering Laboratory, Lions Eye Institute, Nedlands, Western Australia, Australia
    Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, Australia
  • Dan Zhang
    Ocular Tissue Engineering Laboratory, Lions Eye Institute, Nedlands, Western Australia, Australia
  • Shang-Chih Chen
    Ocular Tissue Engineering Laboratory, Lions Eye Institute, Nedlands, Western Australia, Australia
  • Tina M Lamey
    Australian Inherited Retinal Disease Registry and DNA Bank, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
    Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, Australia
  • Jennifer A Thompson
    Australian Inherited Retinal Disease Registry and DNA Bank, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
    Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, Australia
  • Terri L McLaren
    Australian Inherited Retinal Disease Registry and DNA Bank, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
  • John N DeRoach
    Australian Inherited Retinal Disease Registry and DNA Bank, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
  • Fred K Chen
    Ocular Tissue Engineering Laboratory, Lions Eye Institute, Nedlands, Western Australia, Australia
    Department of Ophthalmology, Royal Perth Hospital, Perth, Western Australia, Australia
  • Samuel McLenachan
    Ocular Tissue Engineering Laboratory, Lions Eye Institute, Nedlands, Western Australia, Australia
    Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, Australia
  • Footnotes
    Commercial Relationships   Sang Yoon Moon None; Dan Zhang None; Shang-Chih Chen None; Tina Lamey None; Jennifer Thompson None; Terri McLaren None; John DeRoach None; Fred Chen None; Samuel McLenachan None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 753. doi:
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      Sang Yoon Moon, Dan Zhang, Shang-Chih Chen, Tina M Lamey, Jennifer A Thompson, Terri L McLaren, John N DeRoach, Fred K Chen, Samuel McLenachan; CRISPR activation of endogenous CRB1 expression in fibroblasts from retinitis pigmentosa 12 patients. Invest. Ophthalmol. Vis. Sci. 2023;64(8):753.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Mutations in the Crumbs homolog 1 (CRB1) gene are associated with various phenotypes, including retinitis pigmentosa 12 (RP12). Functional evidence for the effects of CRB1 sequence variants on RNA processing and protein expression in the retina is lacking due to inability to access patient retina. To overcome this limitation, we developed a fibroblast assay for determining the effects of CRB1 variants on mRNA expression using CRISPR activation (CRISPRa) of CRB1 gene expression.

Methods : Dermal fibroblasts were cultured from three RP12 patients carrying compound heterozygous mutations in the CRB1 gene and one healthy control subject. The Edit-R CRISPRa system was used to activate CRB1 gene expression in fibroblasts. For comparison with CRISPRa fibroblasts, induced pluripotent stem cells (iPSC) were reprogrammed from RP12 patient fibroblasts and differentiated into retinal organoids (RO). CRB1 mRNA expression and transcript structure was analysed by qPCR, gel electrophoresis and Sanger sequencing.

Results : CRB1 expression was undetectable in human fibroblast cultures, but was induced 1,000-fold after CRISPRa. All three retinal CRB1 isoforms (CRB1-A, -B and -C) were expressed in both CRISPRa fibroblasts and RO. The CRB1 c.4005+1G>A mutation caused skipping of exon 11 in both CRISPRa fibroblasts and RO. Transcripts containing the c.2555T>C, c.2843G>A, c.2850_2855delAAATGC or c.3014A>T sequence variants were expressed in both CRISPRa fibroblasts and patient RO.

Conclusions : CRISPRa of CRB1 provides a robust system for determining the effects of CRB1 sequence variants on mRNA expression and splicing in patient-derived fibroblasts. This platform will enable rapid screening of novel variants and could potentially be applied to assist pathogenicity classifications for other inherited retinal disease genes not expressed in fibroblasts.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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