Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing.

Li, F.; Wing, K.; Wang, J.-H.; Luu, C. D.; Bender, J. A.; Chen, J.; Wang, Q.; Lu, Q.; Tran, M. T. N. T.; Young, K. M.; Wong, R. C.; Pebay, A.; Cook, A. L.; Hung, S. S.; Liu, G.-S.; Hewitt, A. W.

doi:10.1101/2020.06.09.141705

bioRxiv · 10.1101/2020.06.09.141705

Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing.

Li, F.·Wing, K.·Wang, J.-H.·Luu, C. D.·Bender, J. A.·Chen, J.·Wang, Q.·Lu, Q.·Tran, M. T. N. T.·Young, K. M.·Wong, R. C.·Pebay, A.·Cook, A. L.·Hung, S. S.·Liu, G.-S.·Hewitt, A. W.

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Abstract

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 sgRNA targeting YFP and a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and 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 vitro and in vivo.

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Li, F., Wing, K., Wang, J.-H., Luu, C. D., Bender, J. A., Chen, J., Wang, Q., Lu, Q., Tran, M. T. N. T., Young, K. M., Wong, R. C., Pebay, A., Cook, A. L., Hung, S. S., Liu, G.-S., Hewitt, A. W.. 2020-06-09. Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing.. https://doi.org/10.1101/2020.06.09.141705

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