Comparison of CRISPR/Cas Endonucleases for Retinal Gene Editing

Overview

Journal Front Cell Neurosci

Specialty Cell Biology

Date 2020 Nov 2

PMID 33132845

Citations 21

Authors

Fan Li

Kristof Wing

Jiang-Hui Wang

Chi D Luu

James A Bender

Jinying Chen

Qi Wang

Qinyi Lu

Minh Thuan Nguyen Tran

Kaylene M Young

Raymond C B Wong

Alice Pebay

Anthony L Cook

Sandy S C Hung

Guei-Sheung Liu

Alex W Hewitt

Affiliations

Soon will be listed here.

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 ; 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 .

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References

Maeder M, Stefanidakis M, Wilson C, Baral R, Barrera L, Bounoutas G . Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med. 2019; 25(2):229-233. DOI: 10.1038/s41591-018-0327-9. View

Ran F, Cong L, Yan W, Scott D, Gootenberg J, Kriz A . In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015; 520(7546):186-91. PMC: 4393360. DOI: 10.1038/nature14299. View

Li F, Hung S, Khalid M, Wang J, Chrysostomou V, Wong V . Utility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina. Hum Gene Ther. 2019; 30(11):1349-1360. DOI: 10.1089/hum.2019.021. View

Huang X, Zhou G, Wu W, Duan Y, Ma G, Song J . Genome editing abrogates angiogenesis in vivo. Nat Commun. 2017; 8(1):112. PMC: 5524639. DOI: 10.1038/s41467-017-00140-3. View

Khabou H, Desrosiers M, Winckler C, Fouquet S, Auregan G, Bemelmans A . Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8. Biotechnol Bioeng. 2016; 113(12):2712-2724. DOI: 10.1002/bit.26031. View

Pan X, Philippen L, Lahiri S, Lee C, Park S, Word T . In Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia. Circ Res. 2018; 123(8):953-963. PMC: 6206886. DOI: 10.1161/CIRCRESAHA.118.313369. View

Hung S, Li F, Wang J, King A, Bui B, Liu G . Methods for In Vivo CRISPR/Cas Editing of the Adult Murine Retina. Methods Mol Biol. 2017; 1715:113-133. DOI: 10.1007/978-1-4939-7522-8_9. View

Dalkara D, Byrne L, Klimczak R, Visel M, Yin L, Merigan W . In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci Transl Med. 2013; 5(189):189ra76. DOI: 10.1126/scitranslmed.3005708. View

Jo D, Koo T, Cho C, Kim J, Kim J, Kim J . Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus. Mol Ther. 2018; 27(1):130-136. PMC: 6318782. DOI: 10.1016/j.ymthe.2018.10.009. View

10.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337(6096):816-21. PMC: 6286148. DOI: 10.1126/science.1225829. View

11.

Yu W, Mookherjee S, Chaitankar V, Hiriyanna S, Kim J, Brooks M . Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun. 2017; 8:14716. PMC: 5355895. DOI: 10.1038/ncomms14716. View

12.

Ruan G, Barry E, Yu D, Lukason M, Cheng S, Scaria A . CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10. Mol Ther. 2017; 25(2):331-341. PMC: 5368591. DOI: 10.1016/j.ymthe.2016.12.006. View

13.

Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J . In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2014; 33(1):102-6. PMC: 4492112. DOI: 10.1038/nbt.3055. View

14.

Bakondi B, Lv W, Lu B, Jones M, Tsai Y, Kim K . In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa. Mol Ther. 2015; 24(3):556-63. PMC: 4786918. DOI: 10.1038/mt.2015.220. View

15.

Latella M, Di Salvo M, Cocchiarella F, Benati D, Grisendi G, Comitato A . In vivo Editing of the Human Mutant Rhodopsin Gene by Electroporation of Plasmid-based CRISPR/Cas9 in the Mouse Retina. Mol Ther Nucleic Acids. 2016; 5(11):e389. PMC: 5155324. DOI: 10.1038/mtna.2016.92. View

16.

Xu L, Lau Y, Gao Y, Li H, Han R . Life-Long AAV-Mediated CRISPR Genome Editing in Dystrophic Heart Improves Cardiomyopathy without Causing Serious Lesions in mdx Mice. Mol Ther. 2019; 27(8):1407-1414. PMC: 6697345. DOI: 10.1016/j.ymthe.2019.05.001. View

17.

Jain A, Zode G, Kasetti R, Ran F, Yan W, Sharma T . CRISPR-Cas9-based treatment of myocilin-associated glaucoma. Proc Natl Acad Sci U S A. 2017; 114(42):11199-11204. PMC: 5651749. DOI: 10.1073/pnas.1706193114. View

18.

Hung S, Chrysostomou V, Li F, Lim J, Wang J, Powell J . AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo. Invest Ophthalmol Vis Sci. 2016; 57(7):3470-6. DOI: 10.1167/iovs.16-19316. View

19.

Mali P, Yang L, Esvelt K, Aach J, Guell M, DiCarlo J . RNA-guided human genome engineering via Cas9. Science. 2013; 339(6121):823-6. PMC: 3712628. DOI: 10.1126/science.1232033. View

20.

Kim E, Koo T, Park S, Kim D, Kim K, Cho H . In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun. 2017; 8:14500. PMC: 5473640. DOI: 10.1038/ncomms14500. View