Rapid Cell-Free Preparation of Circular Vector DNA for pegRNA Expression in Prime Editing.

The expression of prime editing guide RNA (pegRNA) is crucial for effective CRISPR-Cas9 prime genome editing, traditionally facilitated by DNA plasmids, which require a time-consuming process of bacterial cloning, validation, and purification. The Circular Vector (CV) protocol represents an efficient, fully synthetic, cell-free method that bypasses these traditional steps, allowing for the preparation of pegRNA expression vectors within 3 h. This approach involves circularizing and purifying linear double-stranded DNA (dsDNA) templates containing a promoter with pegRNA elements into compact, bacterial-backbone-free vectors. These CVs offer significant advantages: absence of a bacterial backbone, absence of endotoxins, and no contamination by bacterial nucleic acids, making them a superior choice for transfection in cell culture experiments, where they have shown performance equal to or better than that of traditional plasmids. The CV protocol not only speeds up the preparation process but also reduces costs and complexity, making it an ideal tool for rapid and efficient pegRNA expression in research settings.