Abstract
Purpose :
Nme2Cas9, known for its compact size, high precision, and broad PAM selection, is a versatile genome editing platform. This study investigates the in vivo efficacy of Nme2Ca9-mediated CRISPR knockdown in mouse retinal ganglion cells (RGCs) by incorporating Nme2Cas9 and a pair of gRNA into one single AAV vector. Given the prevalence of glaucomatous RGC degeneration as a leading cause of irreversible blindness globally, the potential of this system to promote neural repair is explored.
Methods :
We generated an AAV vector that drives Nme2-Cas9 by a 199bp NEFH-hA promoter and two U6-gRNA cassettes. AAV intravitreal injection and retina wholemount and section were used to determine the efficacy of NEFH-hA promoter in RGCs. Paired gRNAs targeting Pten gene were selected in Neuro2A cells and the knockdown efficacy was determined by deep sequencing in N2A cells and in in vivo optic nerve crush model.
Results :
The NEFH-hA-driven gene expression was confirmed in mouse RGCs. The indel rate of targeted region by Pten gRNA pairs was 63.8% in Neuro2A cells. The single AAV-Nme2Cas9/gRNA mediated Pten knockdown in RGCs leads to robust axon regeneration after optic nerve crush injury in mouse model.
Conclusions :
This proof-of-concept study establishes the feasibility of employing Nme2Cas9 for potential gene therapy in RGC-related diseases, such as glaucoma. The findings underscore the promise of single AAV-mediated CRISPR as an efficient research tool for gene manipulation in retinal neurons.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.