Utility of self-destructing CRISPR/Cas constructs for targeted gene editing in the retina
Li, F and Hung, SSC and Mohd Khalid, MKN and Wang, J-H and Chrysostomou, V and Wong, VHY and Singh, V and Wing, K and Tu, L and Bender, JA and Pebay, A and King, AE and Cook, AL and Wong, RCB and Bui, BV and Hewitt, AW and Liu, G-S, Utility of self-destructing CRISPR/Cas constructs for targeted gene editing in the retina, Human Gene Therapy, 30, (11) pp. 1349-1360. ISSN 1043-0342 (2019) [Refereed Article]
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.