Abstract
Purpose :
With recent enhancements in CRISPR/Cas systems, this technique has gained increased efficacy for precise gene manipulation. To propel fundamental research in retinal studies, we refined the latest CRISPR gene editing systems—leveraging HyperdCas12 for gene activation, CasRx for gene knock-down, and prime editing for tag insertion. Integrating these advancements with the established in vivo plasmid electroporation system, we achieved a robust and cost-effective approach to conduct gene manipulation in the mammalian retina in vivo.
Methods :
We utilized diverse CRISPR systems to edit the Otx2 gene locus, capitalizing on the well-established knowledge of Otx2 in retinal development. For gene activation, we designed 3 sgRNAs targeting the Otx2 promoter region. These sgRNAs were incorporated into plasmids expressing HyperdCas12a. Similarly, for gene knockdown, 3 sgRNAs were designed to target the open reading frame of Otx2 and integrated into plasmids expressing CasRx. Both plasmids were delivered in a Cre-dependent or independent manner. Furthermore, we assessed the efficacy of the CRISPR-based prime editing system in the mouse retina through in vivo electroporation. We generated pegRNAs directing the insertion of an HA (hemagglutinin) tag into either the Otx2 or Lmnb1 gene locus. All plasmids were delivered into the mouse retina via in vivo electroporation at post-natal day 0 (P0) or P1, with the co-electroporation of the CAG-tagBFP plasmid as a control for electroporation efficiency. The outcomes of gene activation, knockdown, or HA insertion were validated through antibody staining and phenotypic examination at P14
Results :
The HyperdCas12a and CasRx plasmids effectively induced overexpression or knockdown of Otx2 in the retina, with the knockdown efficiency notably surpassing that of previously published Otx2 shRNAs. Importantly, the necessity for in vitro prescreening of sgRNAs is eliminated for both systems across most gene loci. Although there is room for improvement in efficiency, we successfully detected HA+ cells expressing Otx2 or Lmnb in the retina in vivo.
Conclusions :
The HyperdCas12a and CasRx systems demonstrate high efficiency in gene manipulation through in vivo electroporation. Additionally, prime editing enables the insertion of HA tags into the gene of interest within the mouse retina in vivo.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.