Utility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina

Human Gene Therapy
Fan LiGuei-Sheung Liu

Abstract

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 ...Continue Reading

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Citations

Oct 1, 2020·Human Gene Therapy·Joel QuinnKanmin Xue
Nov 3, 2020·Frontiers in Cellular Neuroscience·Fan LiAlex W Hewitt
Dec 29, 2020·Molecular Therapy : the Journal of the American Society of Gene Therapy·Camilo BretonJames M Wilson
Jul 1, 2020·Advanced Drug Delivery Reviews·Wenhan Yu, Zhijian Wu
Oct 19, 2021·Frontiers in Medicine·Jennifer Hernández-JuárezShyamanga Borooah

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Methods Mentioned

BETA
transfection
protein assay
PCR
dissecting
cleavage assay
ICE

Software Mentioned

GraphPad Prism
OFFinder
Synthego
FlowJo
GraphPad
ZEN2
ImageJ
Cas
Prism

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