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Functional study of the AMD-associated gene TMEM97 in retinal pigmented epithelium using CRISPR interference

Jiang-Hui Wang, Daniel Urrutia-Cabrera, Santiago Mesa Mora, Tu Nguyen, Sandy Hung, Alex W. Hewitt, Thomas L. Edwards, View ORCID ProfileRaymond C.B. Wong
doi: https://doi.org/10.1101/2020.07.10.198143
Jiang-Hui Wang
1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital. Australia
2Ophthalmology, Department of Surgery, University of Melbourne, Australia
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Daniel Urrutia-Cabrera
1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital. Australia
2Ophthalmology, Department of Surgery, University of Melbourne, Australia
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Santiago Mesa Mora
1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital. Australia
2Ophthalmology, Department of Surgery, University of Melbourne, Australia
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Tu Nguyen
1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital. Australia
2Ophthalmology, Department of Surgery, University of Melbourne, Australia
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Sandy Hung
1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital. Australia
2Ophthalmology, Department of Surgery, University of Melbourne, Australia
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Alex W. Hewitt
1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital. Australia
2Ophthalmology, Department of Surgery, University of Melbourne, Australia
3Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Australia
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Thomas L. Edwards
1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital. Australia
2Ophthalmology, Department of Surgery, University of Melbourne, Australia
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Raymond C.B. Wong
1Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital. Australia
2Ophthalmology, Department of Surgery, University of Melbourne, Australia
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  • ORCID record for Raymond C.B. Wong
  • For correspondence: wongcb@unimelb.edu.au
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Abstract

Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >65 genetic risk factors for AMD, including the TMEM97 locus. TMEM97 encodes the Sigma-2 receptor which is involved in apoptosis and cytotoxicity across a range of neurodegenerative diseases. However, the expression pattern of TMEM97 in the human retina and its functional role in retinal cells has remained elusive. Here we utilised CRISPR interference (CRISPRi) to investigate the functional role of TMEM97 in the retina. Transcriptome analysis of all major cell types within the human retina showed that TMEM97 is expressed in the RPE, retinal ganglion cells (RGCs) and amacrine cells. Using CRISPRi, we performed loss-of-function study of TMEM97 in the human RPE cell line, ARPE19. We generated a stable ARPE19 cell line expressing dCas9-KRAB which facilitated knockdown of TMEM97 using specific sgRNAs. Our results show that knockdown of TMEM97 in ARPE19 exerts a protective effect against oxidative stress-induced cell death. This work provides the first functional study of TMEM97 in RPE and supports the role of TMEM97 in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi cell line generated here provided an useful in vitro tool for functional studies of other AMD-associated genes.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted July 11, 2020.
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Functional study of the AMD-associated gene TMEM97 in retinal pigmented epithelium using CRISPR interference
Jiang-Hui Wang, Daniel Urrutia-Cabrera, Santiago Mesa Mora, Tu Nguyen, Sandy Hung, Alex W. Hewitt, Thomas L. Edwards, Raymond C.B. Wong
bioRxiv 2020.07.10.198143; doi: https://doi.org/10.1101/2020.07.10.198143
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Functional study of the AMD-associated gene TMEM97 in retinal pigmented epithelium using CRISPR interference
Jiang-Hui Wang, Daniel Urrutia-Cabrera, Santiago Mesa Mora, Tu Nguyen, Sandy Hung, Alex W. Hewitt, Thomas L. Edwards, Raymond C.B. Wong
bioRxiv 2020.07.10.198143; doi: https://doi.org/10.1101/2020.07.10.198143

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