Journal of Cancer Immunology & Therapy

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Short Communication - Journal of Cancer Immunology & Therapy (2021) Volume 4, Issue 2

The Two to control cjcas9 expression in the deletion of pathogenic GAA repeat in Frataxin gene

 Most Friedrich Ataxia cases are caused by an insertion of a GAA repeat sequence (GAAr) in the first intron

of the frataxin gene leading to a decrease in protein expression. Deletion of this GAAr by CRISPR-Cas9
technology leads to an increase in frataxin ex¬pression. Due to the limited size of AAV packaging, GAAr
remov¬al by SpCas9 required two Adeno-associated viruses (AAVs). Us¬ing 2 AAVs reduces the efficiency of
a potential treatment since each cell must be infected by both viruses. We have therefore used CjCas9 because
it is small enough to be delivered with two sgR¬NAs by a single AAV. However, a constitutive expression of the
CjCas9 gene delivered by an AAV in vivo may increase off-target mutations and induce an immune response
against the Cas9 pro¬tein. Temporal expression is important for the CRISPR system. We investigated two
approaches to limit the nuclease expression.
First, molecular Hara Kiri, we simultaneously transfected in HeLa cells plasmids encoding CjCas9, two
guides (pre and post GAAr) targeting the frataxin gene and two guides targeting the CjCas9 gene. Our results
showed that despite the self-destruction of the CjCas9 gene, an effective genome editing of the FXN gene was
obtained in vitro. Second, CRISPR-SCReT (Stop Codon Read Through), we insert¬ed a stop codon (TGA) at
the beginning of the CjCas9 gene to repress its expression. Subsequently, we induced the expression of Cas9
by molecules capable to allow translation despite the pres¬ence of the premature stop codon. This plasmid
was transfected into 293T cells with two sgRNAs (pre and post GAAr). We noted deletion of the GAAr only in
cells treated with G418. molecular Hara Kiri, we simultaneously transfected in HeLa cells plasmids encoding
CjCas9, two guides (pre and post GAAr) targeting the frataxin gene and two guides targeting the CjCas9
gene. Our results showed that despite the self-destruction of the CjCas9 gene, an effective genome editing
of the FXN gene was ob¬tained in vitro. CRISPR-SCReT (Stop Codon Read Through), we inserted a stop
codon (TGA) at the beginning of the CjCas9 gene to repress its expression. Subsequently, we induced the
expression of Cas9 by molecules capable to allow translation despite the pres¬ence of the premature stop
codon. This plasmid was transfected into 293T cells with two sgRNAs (pre and post GAAr). We noted deletion
of the GAAr only in cells treated with G418. These different methods permitted to obtain efficient editing of
the frataxin gene while preventing a sustained Cas9 expression. This could reduce the chances of off-target
mutations and im¬mune reaction against Cas9 protein. These different methods permitted to obtain efficient
editing of the frataxin gene while preventing a sustained Cas9 expression. This could reduce the chances of
off-target mutations and im¬mune reaction against Cas9 protein
Author(s): Pouiré Yaméogo

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