Comparison of CRISPR/Cas endonuclease gene editing efficiency of retinal cells in vivo

Abstract

Purpose : Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has opened the prospect of direct gene correction therapy for some inherited retinal disease. Previous work has demonstrated that adeno-associated virus (AAV) mediated in vivo delivery of retinal cells using Streptococcus pyogenes Cas9 (SpCas9). However, with the expanding repertoire of CRISPR/Cas endonucleases, such as Cas12a, Streptococcus aureus Cas9 (SaCas9) and Campylobacter jejuni (CjCas9), it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare the CRISPR/Cas endonuclease activity in retinal cells of CMV-Cre::Rosa26-YFP transgenic mice.

Methods : Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and sgRNA targeting YFP and a single vector system with SaCas9/YFP sgRNA were generated. For each endonuclease, different sgRNAs targeting YFP were designed and validated by FACS and T7E1 assay in Stable YFP expressing HEK293A cells. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2-based variant, AAV7m8, and injected intravitreally into CMV-Cre::Rosa26-YFP mouse. A total of 150 mice were tested, including dual vector with SpCas9 (n=20), SaCas9 (n=20), Cas12a (n=20), CjCas9 (n=41), single vector SaCas9 (n=20) and mCherry control (n=29) experiment cohort. Five months after injection, viral transduction was identified by retinal cryosection and flat-mount. Gene knockout efficacy was determined by quantifying percentage of YFP knockout among the viral infected retinal cells using FACS. One-way ANOVA was used for statistical analysis.

Results : For in vitro validation, SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 (single and dual vector) and CjCas9 in YFP expressing HEK293A cells (Figure 1). AAV7m8-mediated CRISPR/Cas construct achieved effective transduction into outer retina layer and we found a 21.23% (SD=12.17, n=14) reduction of YFP-positive cells in viral-infected retinal cells with SpCas9, with the highest knockout efficacy among all Cas endonucleases, which is consistent with in vitro validation result. However, variation in knockout efficacy with other Cas endonucleases was observed.

Conclusions : We demonstrate that AAV7m8-mediated delivery of CRISPR/Cas constructs have effective transduction and SpCas9 achieves the most efficient gene modification in retinal cells in vivo.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.