CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vivo.

CRISPR / Cas为某些遗传性视网膜疾病打开了直接基因校正疗法的前景。先前的工作已经证明了腺相关病毒（AAV）介导的向视网膜细胞传递的作用体内; 然而，随着CRISPR / Cas核酸内切酶种类的增加，尚不清楚其中哪一种对视网膜编辑最有效体内。我们试图比较使用单和双AAV传递策略在视网膜细胞中进行基因编辑的CRISPR / Cas核酸内切酶活性。具有SpCas9，SaCas9，Cas12a，CjCas9和sgRNA靶向的双载体系统的质粒YFP，以及带有SaCas9 / YFP sgRNA的单个载体系统，均已生成，并通过流式细胞仪和T7E1分析在表达YFP的HEK293A细胞中进行了验证。然后将配对的CRISPR / Cas内切核酸酶及其性能最好的sgRNA包装到AAV2衣壳衍生物AAV7m8中，然后玻璃体内注射到CMV-Cre：Rosa26-YFP小鼠中。SpCas9和Cas12a的敲除效率比SaCas9和CjCas9更好。而且，在YFP在单和双CRISPR / SaCas9载体系统之间发现了基因编辑。随着YFP阳性视网膜细胞的明显减少，发现在所有研究的核酸内切酶中，AAV7m8递送的SpCas9具有最高的敲除功效。我们证明，AAV7m8介导的CRISPR / SpCas9构建体传递实现了神经感觉性视网膜细胞中最有效的基因修饰体内。