Application of CRISPR/TET1-dSaCas9 and CRISPR/VPR-dSpCas9 molecular tools for reactivation of HNF1A gene

Modifikacija sustava CRISPR-Cas9 uvođenjem deaktivirane endonukleaze Cas9 (dCas9), te vezanjem za nju katalitičkih domena epigenetičkih modifikatora i/ili transkripcijskih represora i aktivatora, omogućila je ciljanu manipulaciju epigenoma i ekspresije gena. Upotreba ortologa dCas9 te više sgRNA omogućava istovremenu manipulaciju više različitih gena i/ili epigenetičkih oznaka. Fuzija katalitičke domene enzima TET1 (od eng. Ten-eleven translocation) s dCas9 omogućava ciljanu demetilaciju DNA što rezultira povećanjem u ekspresiji gena dok fuzija transkripcijskog aktivatora VPR s dCas9 direktno utječe na aktivaciju gena. Cilj ovog istraživanja bio je izraditi testove za analizu metilacije DNA metodom bisulfitnog pirosekvenciranja kojima će se analizirati metilacija u CpG mjestima smještenim uzvodno i nizvodno od regulatorne regije gena HNF1A ciljane s fuzioniranim proteinima TET1-dSaCas9 i VPR-dSpCas9 u stanicama HEK293. Pokazala sam da dolazi do smanjenja stupnja metilacije u CpG mjestima smještenim uzvodno i nizvodno od ciljane regije nakon transfekcije stanica fuzijom TET1-dSaCas9 te dvostruke transfekcije stanica fuzijama TET1-dSaCas9 i VPR-dSpCas9. Metodom kvantitativnog PCR-a utvdila sam da su TET1-dSaCas9 i VPR-dSpCas9 uzrokovali povećanje transkripcijske aktivnosti gena HNF1A, a njihovo zajedničko djelovanje induciralo je dugotrajniji efekt na transkripcijsku aktivnost.Modification of the CRISPR-Cas9 system by introducing deactivated Cas9 endonuclease (dCas9) and binding the catalytic domains of epigenetic modifiers and/or transcriptional repressors and activators to it, enabled targeted manipulation of the epigenome and gene expression. The use of the dCas9 orthologs and multiple sgRNAs allows simultaneous manipulation of multiple different genes and/or epigenetic marks. The fusion of the catalytic domain of the TET1 (Ten-eleven translocation) enzyme with dCas9 allows targeted DNA demethylation resulting in an increase in gene expression, while the fusion of the VPR transcriptional activator with dCas9 directly affects gene activation. The aim of this study was to design assays for DNA methylation analysis by bisulfite pyrosequencing that would analyze methylation at CpG sites located upstream and downstream of the regulatory region of the HNF1A gene targeted by TET1-dSaCas9 and VPR-dSpCas9 fusion proteins in HEK293 cells. I have shown that there is a decrease in the degree of methylation in CpG sites located upstream and downstream of the target region after the transfection of cells with TET1-dSaCas9 fusion and double transfection with TET1-dSaCas9 and VPR-dSpCas9 fusions. By using a quantitative PCR method, I have found that TET1-dSaCas9 and VPR-dSpCas9 caused an increase in the transcriptional activity of the HNF1A gene and their joint action induced a long-lasting effect on transcriptional activity