20.500.12556/RUL-139535 Izboljšava sistema za preurejanje genoma na osnovi CRISPR z uporabo obvitih vijačnic Improvement of the CRISPR Prime Edit System Using Coiled Coils Sistem prime urejanja (PE) je strategija genomskega preurejana, pri kateri je mogoče brez dvojnoverižnih prelomov uvesti poljubno točkovno mutacijo na tarčno mesto v DNA. Temelji na fuziji nikaze Cas9 ter reverzne transkriptaze, povezanih preko peptidnega povezovalca. V tej nalogi predstavimo prilagojen sistem prime urejanja , ki zaobide potrebo po kovalentni povezavi posameznih proteinskih podenot. V nalogi pokažemo, da je posamezne proteine sistema ccPE možno pridobiti z rekombinantnim izražanjem v E. coli in da jih lahko do neke mere očistimo z uporabo gravitacijske Ni-NTA IMAC kromatografije ter gelske izključevalne FPLC kromatografije. S transfekcijo celic HEK293 s plazmidno obliko prilagojenega sistema prime urejanja in poročevalskim plazmidom pokažemo, da je uspešnost preurejanja na izbranem lokusu nekoliko višja od osnovnega sistema PE. Pri transfekciji celic HEK293 z ribonukleoproteinsko obliko prime edit sistema in poročevalskim plazmidom sistem ni kazal uspešnega preurejanja. V prihodnosti bi bilo potrebno optimizirati količine proteinskih in RNA komponent sistema za vnos v celice. Prime editing (PE) is a strategy for genome editing without creating double strand breaks. With prime editing it is possible to introduce any desired point mutation. It is based on the fusion of the Cas9 nickase and a reverse transcriptase. With this work we present a prime editing system based on non-covalent fusion of the protein components. We successfully expressed the N6-MMLVRT, nCas9-N5, nSpRY-N5, nCas9-MMLVRT and nSpRY-MMLVRT proteins in E. coli and were able to purify them to some degree using Ni-NTA gravity IMAC chromatography and using FPLC size exclusion chromatography. By transfecting HEK293 cells with the plasmid version of the non-covalent prime editors and a reporter plasmid we showed that this version achieved editing with greater efficiency than the classic PE system. Using ribonucleoproteins to edit the reporter plasmid showed no efficient editing. For future experiments amounts of proteins and epegRNAs would have to be optimized. CRISPR Preurejanje genoma Urejevalci prime CRISPR Genome editing Prime edit true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2022-09-04 07:15:57 2022-09-04 07:16:03 2022-10-12 09:44:11 0000-00-00 00:00:00 2022 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2023-09-02 208561 125235203 25959.pdf 25959.pdf 1 92D704F40BC5C2B3003433F3203E4F59 901c1d2248fdfa1f38d95b8314f69a76ffbca1a4f78367efee2dac75c700f9f9 a09933b5-2c10-11ed-92af-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=160109 Biotehniška fakulteta 0 0 0