Evaluation of CRISPR/CasRx-Mediated VEGF mRNA Knockdown in Mouse Retina.

Neovascular eye diseases (NEDs) are a group of diseases caused by the abnormal overgrowth of blood vessels in the eye. Normal vasculature is maintained through a dynamic balance of the vascular endothelial growth factor (VEGF). In diseases such as advanced diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD), an overexpression of VEGF leads to the formation of structurally weak and leaky blood vessels, resulting in vision impairment and, without intervention, legal blindness. In the clinic, NEDs are presently managed through anti-VEGF agents that specifically bind and neutralize VEGF signaling. While effective, this approach requires invasive intravitreal injections monthly and places a heavy burden on patients and healthcare providers. A flexible, long-lasting therapeutic that can reduce or eliminate frequent anti-VEGF treatment will significantly advance NED management.RNA editing with clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) is an emerging strategy to achieve reversible gene editing. The CRISPR-Cas13 system exclusively targets single-stranded RNA and allows gene silencing in a safe manner through RNA knockdown, as the genome is left intact. In addition, the compact CasRx enzyme (930aa) allows RNA silencing to be achieved through the delivery of a single adeno-associated virus (AAV), ideal for gene therapy applications. Herein, we outline methods to target VEGFA mRNA using CRISPR/CasRx in a mammalian cell line and humanized VEGFA transgenic mice.