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

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.

Detection of target DNA with a visual CRISPR-associated hyperbranched rolling circle amplification technique.

This paper presents a novel clustered regularly interspaced short palindromic repeat (CRISPR)-associated HRCA technique (CART). During the entire detection process of CART, the target DNA is first specifically recognized and cleaved by a pair of Cas9/sgRNA complexes; then, the cleaved product is ligated into circular DNA as the template of HRCA, and the circular DNA is efficiently amplified by HRCA. Therefore, CART has the advantages of Cas9/sgRNA (single-base mismatch specificity) and HRCA (isothermal reaction temperature and high sensitivity). This technique has been verified by detecting various human papillomavirus (HPV) genes with numerous subtypes. In summary, this study provides a new and effective method for the detection of nucleic acids.

Evaluation of CRISPR/Cas9 site-specific function and validation of sgRNA sequence by a Cas9/sgRNA-assisted reverse PCR technique.

The effective application of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system in biology, medicine and other fields is hindered by the off-target effects and loci-affinity of Cas9-sgRNA, especially at a genome-wide scale. In order to eliminate the occurrence of off-target effects and evaluate loci-affinity by CRISPR/Cas9 site-specific detection and screening of high-affinity sgRNA sequences, respectively, we develop a CRISPR/Cas9-assisted reverse PCR method for site-specific detection and sgRNA sequence validation. The detection method based on PCR can be used directly in the laboratory with PCR reaction conditions, without the need for an additional detection system, and the whole process of detection can be completed within 2 h. Therefore, it can be easily popularized with a PCR instrument. Finally, this method is fully verified by detecting multiple forms of site mutations and evaluating the affinity of a variety of sgRNA sequences for the CRISPR/Cas9 system. In sum, it provides an effective new analysis tool for CRISPR/Cas9 genome editing-related research. A CRISPR/Cas9-assisted reverse PCR method was developed for Cas9/sgRNA site-specific detection and sgRNA sequence validation. The technique detects target DNA in three steps: (1) target DNA is specifically cut by a pair of Cas9/sgRNA complexes; (2) the cleaved DNA is rapidly linked by T4 DNA ligase; (3) the ligated DNA is efficiently amplified by PCR (PCR or qPCR).

RPE-derived exosomes rescue the photoreceptors during retina degeneration: an intraocular approach to deliver exosomes into the subretinal space.

Retinal degeneration (RD) refers to a group of blinding retinopathies leading to the progressive photoreceptor demise and vision loss. Treatments against this debilitating disease are urgently needed. Intraocular delivery of exosomes represents an innovative therapeutic strategy against RD. In this study, we aimed to determine whether the subretinal delivery of RPE-derived exosomes (RPE-Exos) can prevent the photoreceptor death in RD. RD was induced in C57BL6 mice by MNU administration. These MNU administered mice received a single subretinal injection of RPE-Exos. Two weeks later, the RPE-Exos induced effects were evaluated via functional, morphological, and behavior examinations. Subretinal delivery of RPE-Exos efficiently ameliorates the visual function impairments, and alleviated the structural damages in the retina of MNU administered mice. Moreover, RPE-Exos exert beneficial effects on the electrical response of the inner retinal circuits. Treatment with RPE-Exos suppressed the expression levels of inflammatory factors, and mitigated the oxidative damage, indicating that subretinal delivery of RPE-Exos constructed a cytoprotective microenvironment in the retina of MNU administered mice. Our data suggest that RPE-Exos have therapeutic effects against the visual impairments and photoreceptor death. These findings will enrich our knowledge of RPE-Exos, and highlight the discovery of a promising medication for RD.