University of Tasmania
137539 - Utility of self-destructing CRISPR - Final author version.pdf (32.49 MB)

Utility of self-destructing CRISPR/Cas constructs for targeted gene editing in the retina

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journal contribution
posted on 2023-05-20, 11:06 authored by Li, F, Hung, SSC, Mohd Khalid, MKN, Wang, J-H, Chrysostomou, V, Wong, VHY, Vikrant Singh, Kristof WingKristof Wing, Tu, L, James BenderJames Bender, Pebay, A, Anna KingAnna King, Anthony CookAnthony Cook, Wong, RCB, Bui, BV, Alexander HewittAlexander Hewitt, Guei-Sheung LiuGuei-Sheung Liu
Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing "kamikaze" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were initially designed to target Streptococcus pyogenes Cas9 (SpCas9) and after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein [YFP]), in the presence of mCherry. Both constructs were packaged into AAV2 vectors and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks, the expression of SpCas9 and the efficacy of YFP gene disruption were quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated YFP/SpCas9 targeting CRISPR/Cas compared with those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (∼85.5% reduction compared with LacZ/SpCas9 targeting CRISPR/Cas) in the transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive "kamikaze" CRISPR/Cas system can be used as a robust tool for genome editing in the retina, without compromising on-target efficiency.


Publication title

Human Gene Therapy










Menzies Institute for Medical Research


Mary Ann Liebert Inc Publ

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2 Madison Avenue, Larchmont, USA, Ny, 10538

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Copyright 2019 Mary Ann Liebert, Inc. Final publication is available from Mary Ann Liebert, Inc., publishers

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  • Restricted

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Clinical health not elsewhere classified

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