The Application of CRISPR/Cas9 for the Treatment of Retinal Diseases

Abstract

The CRISPR/Cas9 system of genome editing has revolutionized molecular biology, offering a simple, and relatively inexpensive method of creating precise DNA edits. It has potential application in gene therapy treatment of retinal diseases providing targeted disruption, alteration, or transcriptional regulation of pathogenic genes. In vivo studies have demonstrated therapeutic benefit for a variety of diseases. Despite this, there are many challenges to clinical use of CRISPR/Cas9, including editing efficiency, off-target effects, and disease heterogeneity. This review details the mechanisms of the CRISPR/Cas9 system and the treatment strategies that can be applied to retinal diseases. It gives an overview of in vivo studies published to date and discusses the challenges and potential solutions to the wide-scale clinical use of CRISPR/Cas9 as a therapeutic intervention.

Keywords: CRISPR; Cas9; HDR; NHEJ; gene editing; gene therapy; retinal disease; sgRNA.

Figure 1

Applications of CRISPR/Cas9 for transcriptional regulation and genomic modification. Following Cas9 binding, cleavage of both DNA strands allows DNA modification. In the absence of any homologous sequences, the cell will undergo non-homologous end joining, resulting in small insertions or deletions around the cut site. If donor DNA is supplied which has homologous arms matching the genomic DNA it will be incorporated into the genome via homology directed repair. Catalytically inactive dCas9 can be targeted to a promoter to alter transcriptional regulation. Fusing a transcriptional activator to dCas9 will upregulate gene expression (termed CRISPR activation) while fusing a transcriptional repressor to dCas9 will downregulate gene expression (termed CRISPR interference).

Figure 2

Allele-specific Cas9 targeting via sgRNA design or novel PAM sites. Cas9 can be targeted to the mutant allele by designing the sgRNA to the region containing the mutation. The discrepancy between the sgRNA sequence and wild type may be sufficient to prevent binding. If the target mutation generates a novel PAM site this will allow Cas9 binding on the mutant but not the wild type strand.