Protocol for Prime Editing with Engineered PAM-Flexible Cas9 Variants

Prime editing (PE) is a precision genome-editing technology that allows all types of editing, such as substitutions, insertions, and deletions, at the target site in the genome of a cell without double-strand breaks (DSBs) or donor DNA. However, the targeting range of existing prime editing systems is limited due to their dependence on the NGG protospacer adjacent motif (PAM) recognized by Streptococcus pyogenes Cas9 (spCas9). To address this limitation, we have engineered prime editor variants incorporating PAM-flexible Cas9 proteins. These variants significantly expand the range of targetable genomic sites, allowing for the introduction of mutations previously inaccessible to standard editing methods. Here, we provide detailed methods for applying these PAM-flexible PE variants in human cell lines.