A refined Uni-vector prime editing system improves genome editing outcomes in mammalian cells

Biotechnol J. 2024 Feb;19(2):e2300353. doi: 10.1002/biot.202300353.ABSTRACTPrime editing is an advanced technology in CRISPR/Cas research with increasing numbers of improved methodologies. The original multi-vector method hampers the efficiency and precision of prime editing and also has inherent difficulty in generating homozygous mutations in mammalian cells. To overcome these technical issues, we developed a Uni-vector prime editing system, wherein the major components for prime editing were constructed in all-in-one plasmids, pPE3-pPuro and pePEmax-pPuro. The Uni-vector prime editing plasmids enhance the editing efficiency of prime editing and improved the generation of homozygous mutated mammalian cell lines. The editing efficiency is dependent of the transfection efficiency. Remarkably, the Uni-vector ePE5max system achieved an impressive editing rate approximately 79% in average, even in cell lines that are traditionally difficult to transfect, such as FaDu cell line. Furthermore, it resulted in a high frequency of homozygous knocked-in cells, with a rate of 99% in HeLa and 85% in FaDu cells. Together, our Uni-vector approach simplifies the delivery of editing components and improves the editing efficiency, especially in cells with low transfection efficiency. This approach presents an advancement in the field of prime editing.PMID:38403398 | DOI:10.1002/biot.202300353

Source: Biotechnology Journal - February 25, 2024 Category: Biotechnology Authors: Ching-Hui Huang Szu-Ying Chiu Yu-Chi Chou Kou-Juey Wu Source Type: research