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Resource and Technical AdvanceIn-Press PreviewGeneticsOphthalmology Open Access | 10.1172/jci.insight.189615

CRISPR/Cas-mediated activation of genes associated with inherited retinal dystrophies in human cells for diagnostic purposes

Valentin J. Weber,1 Alice Reschigna,2 Maximilian J. Gerhardt,3 Thomas Heigl,1 Klara S. Hinrichsmeyer,1 Sander van den Engel,1 Dina Y. Otify,4 Zoran Gavrilov,1 Frank Blaser,5 Isabelle Meneau,5 Christian Betz,6 Hanno J. Bolz,6 Martin Biel,4 Stylianos Michalakis,3 and Elvir Becirovic1

1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

Find articles by Weber, V. in: PubMed | Google Scholar

1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

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1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

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1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

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1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

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1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

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1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

Find articles by Otify, D. in: PubMed | Google Scholar

1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

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1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

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1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

Find articles by Meneau, I. in: PubMed | Google Scholar

1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

Find articles by Betz, C. in: PubMed | Google Scholar

1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

Find articles by Bolz, H. in: PubMed | Google Scholar

1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

Find articles by Biel, M. in: PubMed | Google Scholar

1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

Find articles by Michalakis, S. in: PubMed | Google Scholar |

1Department of Ophthalmology, University of Zurich, Zurich, Switzerland

2Department of Ophthalmology, Ludwig-Maximilan-University, Munich, Germany

3Department of Ophthalmology, Ludwig-Maximilian University, Munich, Germany

4Department of Pharmacy, Ludwig-Maximilian University, Munich, Germany

5Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland

6Bioscientia Institute of Medical Diagnostics, Ingelheim, Germany

Find articles by Becirovic, E. in: PubMed | Google Scholar

Published October 7, 2025 - More info

JCI Insight. https://doi.org/10.1172/jci.insight.189615.
Copyright © 2025, Weber et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Published October 7, 2025 - Version history
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Abstract

Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.

Graphical Abstract
graphical abstract
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Version history
  • Version 1 (October 7, 2025): In-Press Preview
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