Plasmid2MC: Efficient cell-free recombination of plasmids into high-purity minicircle DNA for use in genome editing applications

Abstract

AbstractDNA plasmids are commonly used for delivering proteins and RNA in genome editing. However, their bacterial components can lead to inactivation, cell toxicity, and reduced efficiency compared to minicircle DNA (mcDNA) lacking such bacterial sequences. Existing commercial kits that recombine plasmids into mcDNA within proprietary bacterial strains are labor-intensive, yield inconsistent results, and often produce low-quality mcDNA. To address this, we developed Plasmid2MC, a cell-free method using ΦC31 recombination to efficiently excise the bacterial backbone from conventionally prepared plasmids, while mcDNA purification steps digest all DNA impurities and reduce endotoxin levels. We demonstrated application of mcDNA to express CRISPR-dCas9 for base editing in HEK293T cells and mouse embryonic stem cells, as well as for homology-independent targeted insertion (HITI) genome editing. The method’s ease of preparation, efficiency and mcDNA purity make it a valuable alternative for applications requiring bacterial backbone-free circular DNA.