20.500.12556/RUL-170446 Analiza učinkovitosti razcepljenih baznih preurejevalcev povezanih z ovitimi vijačnicami v človeški celični liniji HEK293 Genome editing efficiency determination using coiled-coils tethered split base editors in HEK293 human cell line Bazni preurejevalci so biotehnološko orodje za preurejanje genoma in omogočajo natančno spreminjanje baznih parov na tarčnem zaporedju brez ustvarjanja dvojnoverižnih prelomov v DNA. Citozinski (CBE) oziroma adeninski bazni preurejevalci (ABE) so sestavljeni iz fuzije nikaze Cas9 (nCas9) ter citozinske ali adeninske deaminaze. V magistrskem delu predstavljamo sistem baznega preurejanja, ki temelji na razcepljenih baznih preurejevalcih (ccBE). nCas9 in deaminaza sta v tem primeru nekovalentno povezana preko dveh peptidov, ki tvorita ovito vijačnico. V človeško celično linijo HEK293 smo s transfekcijo vnesli plazmide z zapisom za izražanje ccCBE ali osnovnega fuzijskega baznega preurejevalca TadCBEa ter ugotovili, da se njihova učinkovitost na nobenem lokusu ne razlikuje. S titracijo plazmidov z geni za nCas9 in deaminazno podenoto razcepljenega adeninskega baznega preurejevalca smo na poročevalski celični liniji na osnovi HEK293 določili optimalno razmerje plazmidov za nCas9 in deaminazno podenoto 20:1. Na štirih izbranih genomskih lokusih smo primerjali delovanje ccCBE pri razmerjih deaminaze in nCas9 1:1 in 1:20. Pokazali smo, da je pri obeh razmerjih učinkovitost preurejanja tarčne baze ter preurejanja sosednjih netarčnih baz primerljiva. Čeprav izbrano razmerje ni pokazalo večje učinkovitosti pri preurejanju tarčne baze s ccCBE v primerjavi s TadCBE, smo ugotovili, da je sistem enako aktiven, če uporabimo 20-krat nižjo koncentracijo plazmida z deaminazo, kot plazmida z nCas9. Na koncu smo za vse na novo pripravljene preurejevalce določili še okno preurejanja in ugotovili, da imajo najvišjo učinkovitost preurejanja med položaji od tri do sedem baz od začetka sekvence tarčnega zaporedja (5' do 3'). Razliko med preurejevalci smo opazili pri ccCBE 1:1, ki ima na položaju šest in sedem manjše delovanje v primerjavi s ccCBE 20:1 in fuzijskim CBE. Base editors are biotechnological tools used for genome editing that enable precise modification of base pairs at the target sequences, without creating double strand breaks in DNA. Cytosine (CBE) and adenine base editors (ABE) consist of a fusion between Cas9 nickase (nCas9) and a cytosine or adenine deaminase. In this master's thesis, we present a base editing system based on split base editors (ccBE), in which nCas9 and the deaminase are non-covalently linked via two peptides that form a coiled-coil. We transfected human HEK293 cells with plasmids encoding either the ccCBE or the standard fusion base editor TadCBEa and found no significant difference in their editing efficiency across any of the tested loci. Using a reporter HEK293 based cell line, we titrated plasmids carrying genes for nCas9 and the deaminase subunit of the split adenine base editor, and determined that the optimal plasmid ratio for nCas9 to deaminase subunit is 20:1. We then compared the activity of ccCBE at two different nCas9:deaminase ratios 1:1 and 20:1, across four selected genomic loci. We showed that both ratios resulted in comparable editing efficiency of the target base, as well as similar levels of bystander editing at nearby positions. Although the 20:1 ratio did not show increased target base editing efficiency with ccCBE compared to TadCBE, we demonstrated that the system remains equally active when using a 20-fold lower concentration of the deaminase-encoding plasmid than that of nCas9. Finally, we determined the editing windows of all newly developed editors and found that the highest editing efficiency occurred between positions three and seven from the beginning of the target sequence (5' to 3'). A difference was observed with ccCBE 1:1, which showed reduced activity at positions six and seven compared to ccCBE 20:1 and the fusion CBE. CRISPR preurejanje genoma razcepljeni bazni preurejevalci ovite vijačnice CRISPR genome editing split base editors coiled coils true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-07-06 07:16:05 2025-07-06 07:16:13 2025-07-08 14:36:31 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2026-07-15 257738 241816835 49431.pdf 49431.pdf 1 0087187C5D21D7ADE1DA2B3909B51D88 c0a642240aa24409f0e324361756b1d3c02c76c596712228ac8c1ded418324a5 6c9113df-5a26-11f0-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=212450 Biotehniška fakulteta 0 0 0