20.500.12556/RUL-171781 Učinkovitost urejevalcev 'prime' z obvitimi vijačnicami pri urejanju genoma celic HEK293 in pri popravljanju mutacije v genu CTNNB1 Efficiency of Coiled-Coil-Based Prime Editors in Genome Editing of HEK293 Cells and Correction of a CTNNB1 Gene Mutation Urejanje 'prime' (PE, ang. prime editing) je metoda za natančno urejanje genoma, ki omogoča uvedbo poljubnih točkovnih mutacij ali krajših delecij in insercij brez dvoverižnega preloma. Temelji na fuzijskem proteinu PE z dvema funkcionalnima domenama – katalitično oslabljeno nukleazo Cas9, ki ima v eni izmed endonukleaznih domen mutacijo, kar omogoča prelom le ene verige na tarčnem mestu, in reverzne transkriptaze, ki omogoči nastanek nove verige s prepisom iz dodane usmerjevalne molekule RNA. Potencial uporabe sistema PE v terapevtske namene je velik, vendar pa ozko grlo ostaja učinkovita dostava v tarčne celice, saj velikost fuzijskega proteina presega maksimalne kapacitete nekaterih vektorjev. Na Kemijskem inštitutu so razvili sistem urejanja 'prime' z obvitimi vijačnicami (ccPE), kjer sta omenjeni komponenti ločeni, vsaka od njiju pa je kovalentno povezana s CC-peptidom, ki v paru z ortogonalnim partnerjem spontano tvori motiv obvite vijačnice. Na tak način sta podenoti med seboj nekovalentno povezani v delujočo celoto, obenem pa je sistem razdeljen na dva dela, kar reši problem velikosti vključka, saj vsak del vnesemo v svoj vektor. V sklopu magistrskega dela smo primerjali učinkovitost delovanja sistema ccPE z osnovnim PE na različnih tarčah, tako na endogenih lokusih celic HEK293 kot na klinično relevantni mutaciji gena CTNNB1, preizkusili pa smo tudi učinkovitost sistema ccPE z različnimi CC-peptidi. Ugotovili smo, da je sistem ccPE enako ali bolj učinkovit kot PE. Poleg tega smo želeli sistem ccPE preizkusiti tudi na induciranih pluripotentnih matičnih celicah (iPSC), ki vsebujejo mutacijo v eni kopiji gena CTNNB1. V ta namen smo preizkusili različne metode vnosa v človeške celice iPSC. Za uspešno se je izkazala le transdukcija z lentivirusnimi delci, zato smo pripravili konstrukte z zapisom za sistem ccPE in PE v lentivirusnem vektorju. Prime editing (PE) is a precise genome editing method that enables introduction of short insertions, deletions or substitutions without inducing double-strand breaks. It uses a fusion protein, consisting of a Cas9 nickase, which allows induction of a nick in a single DNA strand at the target site, and a reverse transcriptase, which catalyses reverse transcription from an RNA template encoding the novel sequence. PE has a great potential in therapeutic use, but in vivo delivery remains challenging due to the size of PE machinery, which exceeds the packaging capacities of some vectors. At the National Institute of Chemistry an improved version of the system called coiled-coil prime editing (ccPE) was developed, in which the two components are not covalently linked, but instead interact non-covalently via a coiled-coil (CC) motif interaction. The latter is formed between two CC-peptides that are covalently added to each subunit. This approach allows the two subunits to associate non-covalently into a functional complex, while keeping the system divided into two separate parts. This overcomes the size limitation by allowing each component to be delivered separately in its own vector. We compared the efficiency of ccPE with standard PE on different HEK293 cell endogenous loci and on a clinically relevant CTNNB1 gene mutation. We also tested ccPE with various CC-peptides. We found that ccPE performs equally or more effectively than PE. Additionally, we aimed to evaluate the efficacy of ccPE system in patient-derived induced pluripotent stem cells (iPSC) with a mutation at one allele of the CTNNB1 gene. We tested different delivery methods for introducing nucleic acids into iPSCs and of all methods tested, only lentiviral transduction proved effective, which is why we constructed plasmids for the preparations of lentiviral vectors encoding the PE and ccPE systems. urejanje genoma urejanje 'prime' obvite vijačnice sindrom CTNNB1 iPSC genome editing prime editor coiled coil CTNNB1 syndrome iPSC true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-09-02 13:25:01 2025-09-02 13:25:12 2025-10-10 09:50:55 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 24200 252641027 Junger_Greta_-_Ucinkovitost_urejevalcev__prime__z_obvitimi_vijacnicami_pri_urejanju_genoma_celic.pdf Junger_Greta_-_Ucinkovitost_urejevalcev__prime__z_obvitimi_vijacnicami_pri_urejanju_genoma_celic.pdf 1 39678C13E379CC3E09C33418E02B38DD 79faa05bfe7f70765a5c5b21757214fa033abd7e3bd0ad579ce6bccf40b7380a 5a2064a0-87ef-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=215248 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0