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

Abstract

Prime 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.

Emerging Perspectives on Prime Editor Delivery to the Brain

“…Dirkx et al [ 306 ], achieved increased PE gene modification rates in KCNQ2 and SCN1A genes using single-nicking all-in-one plasmids, simplifying the delivery of PE components. Huang et al [ 307 ] developed a refined uni-vector prime editing system that improves editing outcomes in mammalian cells by incorporating all the necessary components into a single vector, streamlining the delivery process. Petri et al [ 308 ] demonstrated the feasibility of CRISPR prime editing using RNP complexes in zebrafish and primary human cells, providing an alternative delivery method that avoids the challenges associated with vector-based delivery.…”