Table 3.

Factors affecting the use of CRISPR/Cas for gene editing in RDs.

Factor	Description
Ethical Issue [98]	While using CRISPR/Cas9, the ethical issues will be there, since CRISPR/Cas based gene editing could result in serious off-target gene manipulations.
Selection of gene [99]	For example, in LCA there are 14 genes that have mutations. Therefore, one should be clear about the gene that needed to be edited for improvement of the complications associated with the RD.
Knock out [100]	Knock out is not always beneficial, until and unless the role of the gene has been vastly understood. For instance, knockout of Vegfa gene is shown in wAMD to stop angiogenesis. But it cannot be applicable for every gene because one gene can be involved in various cellular functions and knockout could cause the loss of some important cellular functions.
Knock in [101]	Some of the RDs require HDR. Since NHEJ is more prominent w.r.t HDR, therefore, one should consider this factor while utilizing the CRISPR/Cas technique.
Vitreal barrier [102]	The presence of vitreous fluid may retard the diffusion of the CRISPR/Cas components toward the posterior segment of the eye. Additionally, the nucleases/proteases present in the vitreous fluid could degrade the CRISPR components.
Targeted Delivery	RDs requires editing of the gene in retinal cells only, therefore, delivering the CRISPR/Cas component specifically to retinal cells could be challenging.
Off-target effect	While designing sgRNA for a specific gene, one should ensure the specificity of the sgRNA toward the gene of interest. Else, it could lead to undesired gene editing.
PAM sequence [100]	As it is known that the CRISPR/Cas perform DSB near the PAM sequences (NGG, GGG), and however, it is not always possible to have a PAM sequence at the desired gene editing site.
Limited delivery route	Blood retinal barrier limits the distribution of therapeutic agents to the eye tissue, therefore localized injection is the only potential approach left. Localized injection such as intravitreal (IVT) injection has the risk of eye damage and requires trained healthcare personnel.