CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY CRISPR Journal Pub Date : 2023-12-01 DOI:10.1089/crispr.2023.0039
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
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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.

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CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.
Pro23His 等视黄素(RHO)突变是北美显性遗传性视网膜色素变性症的主要病因。与其他显性视网膜营养不良症一样,这些突变导致产生有毒的蛋白质产物,治疗需要敲除突变等位基因。本研究的目的是开发一种 CRISPR-Cas9 介导的转录抑制策略,使用的是与 Krüppel-associated box(KRAB)转录抑制结构域融合的催化活性金黄色葡萄球菌 Cas9(dCas9)。利用克隆在 RHO 启动子片段(核苷酸 -1403 至 +73)下游的携带绿色荧光蛋白(GFP)的报告基因构建物,我们证明 RHOpCRISPRi 处理的 RHO 启动子活性比仅用质粒的对照组降低了 74-84%。在对人类视网膜外植体和转基因 Pro23His 突变体猪进行视网膜下转导后,视网膜上的视紫红质蛋白被显著敲除。体内突变转基因的抑制与内质网(ER)应激和细胞凋亡标记物的减少以及感光细胞层厚度的保持有关。
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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, 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.
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