Cas13bt3 knockdown of VEGFA mRNA in retinal organoid [scRNA-Seq]

Purpose: We report expression of a new Cas13 enzyme in retinal cells and evaluate its efficacy in targeting the VEGFA mRNA to establish safety and efficacy of the enzyme got anti-VEGFA therapy. Methods: Three retinal organoids were cultured and treated, (1) Untreated, (2) AAV2.7m8.Cas13bt3.VEGFA.sgRNA, (3) AAV2.7m8.Cas13bt3.NTsgRNA. Single-cell RNA sequencing was performed to determine expression of Cas13bt3 across retinal cell types, and the corresponding VEGFA mRNA expression. Results: All retinal cell showed observable transduction and RPE cells were found most susceptible to infection, while bipolar cells had the least infection rate. Significant silencing of VEGFA was observed in RPE cells. Conclusions: AAV2.7m8 can be used for retina-wide transduction, and Cas13bt3 may be a potential new tool for control of VEGFA. Examination of Cas13bt3 transduction and anti-VEGFA potential in mature 3D retinal orgnaoids.