Abstract
Purpose :
The CRISPR/Cas-based targeted transgene strategies are generally classified into homology-dependent and homology-independent approaches. In this article, we comprehensively compared the homology-mediated end joining (HMEJ) pathways based on homology-dependent strategy with homology-independent targeted integration (HITI) strategy. By rescuing the popular mutation of BCD, c.802-8_810del17insGC in vitro cells and in vivo retina, we explored the different application scenarios of HMEJ and HITI for inherited eye diseases.
Methods :
The HMEJ method was taken by selected sgRNAs targeting on c.802-8_810del17insGC, following donor containing approximately 0.8 kb homologous arm, total 1.6kb sequences. The second HITI approach was taken by selected sgRNAs targeting intron 6 before the mutation and following inserted donor containing exon 7-11, approximately 1.5kb sequences. The comparison was taken in HEK 293T cells, BCD-iPSC-RPE cells using plasmid transfection in vitro. A humanized Cyp4v3 mouse containing mutation was also subretinal injected with two rAAV2/8 AAV vectors (1E9), one encoding CRISPR components and the other containing corresponding donor. Apart from editing efficiency, we also compared the morphology in rescued BCD-iPSC-RPE cells, ERG, fundus imaging, OCT and histologic analysis in mouse.
Results :
The HMEJ strategy showed high editing efficient (29.14%±1.31%) in HEK 293T cells and produced normal morphology in edited BCD-iPSC-RPE cells. We also found no significant difference between one and two cutting sites (p=0.96). In mouse model at 9 months, however, we cannot detect the editing efficiency using Hi-TOM. ERG, fundus imaging and HE staining also performed as poor as untreated disease model. For HITI approach, it showed normal morphology in treated BCD-iPSC-RPE cells, high editing efficiency in both HEK 293T cells (33.5%±4.1%) and in vivo retina (25.3%±15.5%). In addition, the HITI-based editing restores visual function and retinal degeneration in mouse.
Conclusions :
The results showed that HMEJ achieves efficient knock-in in HEK 293T cells and BCD-iPSC-RPE cells, however undetectable editing in BCD mouse retina. In contrast, HITI based method achieved precise and effective editing both in vitro and in vivo, suggesting it is considered to be a more promising therapeutic approach to manage inherited eye disorders.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.