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The two ways to control CjCas9 expression in the deletion of pathogenic GAA repeat in frataxin gene

Pouiré Yaméogo

 Most Friedrich Ataxia cases are caused by an insertion of aGAA repeat sequence (GAAr) in the first intron of the frataxingene leading to a decrease in protein expression. Deletion ofthis GAAr by CRISPR-Cas9 technology leads to an increase infrataxin expression. Due to the limited size of AAV packaging,GAAr removal by SpCas9 required two Adeno-associated viruses(AAVs). Using 2 AAVs reduces the efficiency of a potentialtreatment since each cell must be infected by both viruses. Wehave therefore used CjCas9 because it is small enough to bedelivered with two sgRNAs by a single AAV. However, a constitutiveexpression of the CjCas9 gene delivered by an AAV invivo may increase off-target mutations and induce an immuneresponse against the Cas9 protein. Temporal expression is importantfor the CRISPR system. We investigated two approachesto limit the nuclease expression. First, molecular Hara Kiri,we simultaneously transfected in HeLa cells plasmids encodingCjCas9, two guides (pre and post GAAr) targeting the frataxingene and two guides targeting the CjCas9 gene. Our resultsshowed that despite the self-destruction of the CjCas9 gene,an effective genome editing of the FXN gene was obtained invitro. Second, CRISPR-SCReT (Stop Codon Read Through),we inserted a stop codon (TGA) at the beginning of the CjCas9gene to repress its expression. Subsequently, we induced the expressionof Cas9 by molecules capable to allow translation despitethe presence of the premature stop codon. This plasmidwas transfected into 293T cells with two sgRNAs (pre and postGAAr). We noted deletion of the GAAr only in cells treatedwith G418. These different methods permitted to obtain efficientediting of the frataxin gene while preventing a sustainedCas9 expression. This could reduce the chances of off-targetmutations and immune reaction against Cas9 protein.