Erin R Burnight, Luke A Wiley, Nathaniel K Mullin, Malavika K Adur, Mallory J Lang, Cathryn M Cranston, Chunhua Jiao, Stephen R Russell, Elliot H Sohn, Ian C Han, Jason W Ross, Edwin M Stone, Robert F Mullins, Budd A Tucker
{"title":"CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.","authors":"Erin R Burnight, Luke A Wiley, Nathaniel K Mullin, Malavika K Adur, Mallory J Lang, Cathryn M Cranston, Chunhua Jiao, Stephen R Russell, Elliot H Sohn, Ian C Han, Jason W Ross, Edwin M Stone, Robert F Mullins, Budd A Tucker","doi":"10.1089/crispr.2023.0039","DOIUrl":null,"url":null,"abstract":"<p><p>Rhodopsin (<i>RHO</i>) 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 <i>Staphylococcus aureus</i> Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the <i>RHO</i> promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in <i>RHO</i> promoter activity in <i>RHOp</i>CRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene <i>in vivo</i> was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"6 6","pages":"502-513"},"PeriodicalIF":3.7000,"publicationDate":"2023-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11304754/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"CRISPR Journal","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1089/crispr.2023.0039","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"GENETICS & HEREDITY","Score":null,"Total":0}
引用次数: 0
Abstract
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 Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (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 versus plasmid-only controls. After 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 endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.
CRISPR JournalBiochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍:
In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR.
Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.