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| Status |
Public on Mar 28, 2024 |
| Title |
CRISPRi mediated treatment of dominant rhodopsin-associated retinitis pigmentosa. |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Rhodopsin (RHO) mutations such as Pro23His, are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive S. aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying GFP cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ~74%-84% reduction in RHO promoter activity in RHOpCRISPRi treated vs plasmid only controls. Following subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in ER-stress and apoptosis markers and preservation of photoreceptor cell layer thickness.
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| Overall design |
Human donor retinal explants were transduced with AAV5-RHOp-CRISPR-dSaCas9. RNA was isolated from transduced and control explants and used to prepare RNA sequencing libraries to examine gene expression changes between donors and following treatment.
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| Contributor(s) |
Mullin NK, Burnight ER, Tucker BA |
| Citation(s) |
38108516 |
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| Submission date |
Oct 06, 2023 |
| Last update date |
Jun 27, 2024 |
| Contact name |
Nathaniel Kevin Mullin |
| Organization name |
University of Iowa
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| Street address |
375 Newton Road
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| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52246 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA1024894 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE244786_RAW.tar |
13.3 Mb |
(http)(custom) |
TAR (of TAR) |
SRA Run Selector |
| Raw data are available in SRA |
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