CRISPR Interference–Potential Application in Retinal Disease

Abstract

The treatment of dominantly inherited retinal diseases requires silencing of the pathogenic allele. RNA interference to suppress gene expression suffers from wide-spread off-target effects, while CRISPR-mediated gene disruption creates permanent changes in the genome. CRISPR interference uses a catalytically inactive ‘dead’ Cas9 directed by a guide RNA to block transcription of chosen genes without disrupting the DNA. It is highly specific and potentially reversible, increasing its safety profile as a therapy. Pre-clinical studies have demonstrated the versatility of CRISPR interference for gene silencing both in vivo and in ex vivo modification of iPSCs for transplantation. Applying CRISPR interference techniques for the treatment of autosomal dominant inherited retinal diseases is promising but there are few in vivo studies to date. This review details how CRISPR interference might be used to treat retinal diseases and addresses potential challenges for clinical translation.

Non-targeting control for MISSION® shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

“…The situation is even better for CRISPRi due to the very mechanism of action. The efficient inhibition of transcription requires long-term and high-affinity interaction between 17-20 nucleotide guide sequence and target DNA and therefore mismatch tolerance is low and the number of off-targets – negligible (Gilbert et al, 2014; Peddle, Fry, McClements, & MacLaren, 2020).…”

Genome-Editing Strategies for Treating Human Retinal Degenerations

“…In addition to gene editing, CRISPR-Cas9 can be used for transcriptional regulation, in which catalytically inactivated "dead" Cas9 (dCas9) is fused to transcriptional effectors to repress genes directly (termed CRISPR interference or CRISPRi) or modified to act as a functional transcriptional activator (CRISPRa) 27 (Fig.1D). Multiple groups have shown that the mere binding of dCas9 to promoters and other regulatory regions can repress transcription by sterically hindering the RNA polymerase machinery 27 .…”

“…In addition to gene editing, CRISPR-Cas9 can be used for transcriptional regulation, in which catalytically inactivated "dead" Cas9 (dCas9) is fused to transcriptional effectors to repress genes directly (termed CRISPR interference or CRISPRi) or modified to act as a functional transcriptional activator (CRISPRa) 27 (Fig.1D). Multiple groups have shown that the mere binding of dCas9 to promoters and other regulatory regions can repress transcription by sterically hindering the RNA polymerase machinery 27 . Nevertheless, the repressive capacity of the system is vastly improved when dCas9 is linked to a transcriptional repressor domain 28 For example, CRISPR-mediated epigenome editing may present a novel approach for robust and long-term suppression of vascular endothelial growth factor (VEGF-A) and reversal of the AMD-related phenotype (Table 1) 30 .…”

“…In addition to gene editing, CRISPR-Cas9 can be used for transcriptional regulation, in which catalytically inactivated “dead” Cas9 (dCas9) is fused to transcriptional effectors to repress genes directly (termed CRISPR interference or CRISPRi) or modified to act as a functional transcriptional activator (CRISPRa) 27 ( Fig. 1D ).…”

Differences in the Response to DNA Double-Strand Breaks between Rod Photoreceptors of Rodents, Pigs, and Humans

“…The CRISPR-Cas9 technique relies on the endonuclease Cas9 to generate double-stranded breaks (DSBs) at a specific site of the genome with the subsequent repair of the induced breaks by the endogenous DSB repair machinery. Additionally, Cas9 lacking endonuclease activity can be used to selectively perturb genome expression by blocking transcription [2,3]. Repair of Cas9-induced DSBs takes place either by non-homologous end-joining (NHEJ) which is available in all cell cycle phases or by homologous recombination (HR).…”