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.
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