Background

The Retina

The retina is a 250𝜇m thick light-sensitive multilayered neural tissue that lines the interior of the eye. It has the ability to convert electromagnetic waves into intelligible electrical signals. As the light passes through the lens, it reaches the surface layer of the retina known as the photoreceptor. In glaucoma or age related blindness, the ganglion neurons are degenerated causing vision impairment. This process are irreversible. 

Figure 1: Diagram of the retinal layers and associated retinal cells.

The Problem

There are currently no autologous treatments that can regenerate the retina to successfully restore vision. The human retina's complexity highlights the need for efficient methods of generating retinal neurons to study retina development and treat irreversible vision loss. Though some transcription factors and genes have been identified for retinal development, it is difficult to identify other genes implicated in proper retinal cell fate, differentiation, and phenotype. Current approaches in ophthalmology research are limited by the lack of high-throughput screening to study the retina and its formation. CRISPR technology, which can perform high-throughput screening, still has not been widely applied to the regenerative ophthalmology field. By focusing on integrating CRISPRa with regenerative medicine approaches, important transcription factors can be identified and overexpressed to regenerate retinal neurons that can potentially restore vision and improve patient quality of life. 

Current methods of screening and overexpressing transcription factors involve constructing individual gene cassettes for a library and transfecting them into stem cells, which is laborious and tedious. The CRISPRa system must be versatile enough that different genes and gRNA sequences for each gene can easily be cloned in and tested. Additionally, to make accurate comparisons between sequences and genes, the system must be consistent and effective at gene activation and morphological changes of stem cells when testing transcription factors already known to be crucial for retinal development. 

Figure 2: Global causes of blindness as a percentage of total blindness.

Edited by: Thawinee Thongprajiad