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.
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