Methods for Applying Prime Editing and Inverse Prime Editing in Mammalian Cell Culture

Prime editing (PE) is a powerful method for introducing point mutations into the genomes of living organisms. PE utilizes a Cas9 nickase fused to an engineered Moloney Murine Leukemia Virus reverse transcriptase (MLV-RT), paired with an extended guide RNA known as pegRNA, which contains a primer binding site (PBS) and a reverse transcriptase template (RTT) complementary to the non-target strand DNA. Recently described inverse prime editing (iPE) also employs reverse transcriptase and pegRNAs; however,  it utilizes an RNA template complementary to the target strand, resulting in the polymerization of target-strand DNA in the opposite direction. In this work, we provide a practical protocol for using either PE or iPE to introduce point mutation(s) or small-to-medium sized insertions and deletions in cell culture, and demonstrate how to assess editing efficiency via flow cytometry and next-generation sequencing. We include recommendations for prime editor selection and straightforward guidelines for pegRNA design, intended for researchers unfamiliar with genome editing technologies.