The application and progression of CRISPR/Cas9 technology in ophthalmological diseases

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is an adaptive immune defence system that has gradually evolved in bacteria and archaea to combat invading viruses and exogenous DNA. Advances in technology have enabled researchers to enhance their understanding of the immune process in vivo and its potential for use in genome editing. Thus far, applications of CRISPR/Cas9 genome editing technology in ophthalmology have included gene therapy for corneal dystrophy, glaucoma, congenital cataract, Leber's congenital amaurosis, retinitis pigmentosa, Usher syndrome, fundus neovascular disease, proliferative vitreoretinopathy, retinoblastoma and other eye diseases. Additionally, the combination of CRISPR/Cas9 genome editing technology with adeno-associated virus vector and inducible pluripotent stem cells provides further therapeutic avenues for the treatment of eye diseases. Nonetheless, many challenges remain in the development of clinically feasible retinal genome editing therapy. This review discusses the development, as well as mechanism of CRISPR/Cas9 and its applications and challenges in gene therapy for eye diseases.

Fig. 1. Schematic representation of the Clustered-regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) 9 system.

Artificial construction of the type II CRISPR/Cas9 system enables direct chimerization of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) to form single guide RNA (sgRNA) by linker loop. sgRNA guides Cas9-mediated cleavage at a specific site in the target gene sequence to generate double-stranded breaks, thus inducing the activation of either error-prone nonhomologous end-joining or generally error-free homology-directed repair to modify DNA in the target cell.

Fig. 2. Schematic representation of a double-stranded break (DSB; blue dotted line), which can be repaired through nonhomologous end-joining (NHEJ), homology-directed repair (HDR) or microhomology-mediated end joining (MMEJ) pathways.

In the absence of DNA repair template, DSBs will cause various mutations via NHEJ (e.g., substitution, deletion, or insertion), which can be used for gene knockout procedures. When exogenous DNA is used as a DNA repair template, accurate base replacement or insertion can be achieved via HDR, which can be used for gene insertion procedures. The ends of the DSB are linked via microhomologous domains when MMEJ occurs, which can cause a sequence deletion at the position.

Fig. 3. The protocol for genome editing in the retina includes in vivo and ex vivo approaches.

In vivo approaches are designed to directly treat mutations in affected cells in situ. Clustered-regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 can be combined with iPSCs to correct gene mutations in vitro; cells with these corrections can then be transplanted into diseased retina.