Li Fan, Wing Kristof, Wang Jiang-Hui, Luu Chi D, Bender James A, Chen Jinying, Wang Qi, Lu Qinyi, Nguyen Tran Minh Thuan, Young Kaylene M, Wong Raymond C B, Pébay Alice, Cook Anthony L, Hung Sandy S C, Liu Guei-Sheung, Hewitt Alex W
Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-sen University, Guangzhou, China.
Front Cell Neurosci. 2020 Sep 10;14:570917. doi: 10.3389/fncel.2020.570917. eCollection 2020.
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 ; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing . 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 , 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 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 .
CRISPR/Cas技术为某些遗传性视网膜疾病的直接基因矫正治疗开辟了前景。先前的研究已经证明了腺相关病毒（AAV）介导的视网膜细胞递送的实用性；然而，随着CRISPR/Cas核酸内切酶种类的不断增加，目前尚不清楚哪种酶对视网膜编辑最有效。我们试图通过单AAV和双AAV递送策略比较CRISPR/Cas核酸内切酶在视网膜细胞中的基因编辑活性。构建了含有SpCas9、SaCas9、Cas12a、CjCas9和靶向sgRNA的双载体系统质粒，以及含有SaCas9/YFP sgRNA的单载体系统质粒，并通过流式细胞术和T7E1检测在表达YFP的HEK293A细胞中进行了验证。然后将配对的CRISPR/Cas核酸内切酶及其表现最佳的sgRNA包装到AAV2衣壳衍生物AAV7m8中，并通过玻璃体腔注射到CMV-Cre:Rosa26-YFP小鼠体内。SpCas9和Cas12a的敲除效率优于SaCas9和CjCas9。此外，单CRISPR/SaCas9和双CRISPR/SaCas9载体系统在基因编辑方面没有显著差异。在YFP阳性视网膜细胞显著减少的情况下，发现AAV7m8递送的SpCas9在所有研究的核酸内切酶中具有最高的敲除效率。我们证明，AAV7m8介导的CRISPR/SpCas9构建体递送在神经感觉视网膜细胞中实现了最有效的基因修饰。
