PRJNA817825
Designing prime editing experiments in mammalian cells
Prime editing (PE) is a precision gene editing technology that enables the programmable installation of substitutions, insertions, and deletions in cells and animals without requiring double-stranded DNA breaks (DSBs). The mechanism of prime editing makes it less dependent on cellular replication and endogenous DNA repair than HDR-based approaches, and its ability to precisely install edits without creating DSBs minimizes indels and other undesired outcomes. The capabilities of prime editing have also expanded since its original publication. Enhanced prime editing systems, PE4 and PE5, manipulate DNA repair pathways to increase prime editing efficiency and reduce indels. Other advances that improve prime editing efficiency include engineered pegRNAs (epegRNAs), which include a structured RNA motif to stabilize and protect pegRNA 3' ends, and the PEmax architecture, which improves editor expression and nuclear localization. New applications such as twin prime editing (twinPE) can precisely insert or delete hundreds of base pairs of DNA and can be used in tandem with recombinases to achieve gene-sized insertions and inversions. Achieving optimal prime editing requires careful experimental design, and the large number of parameters that influence prime editing outcomes can be daunting. This protocol describes current best practices for conducting prime editing and twinPE experiments and describes the design and optimization of pegRNAs. We also offer guidelines for how to select the proper PE system (PE1 to PE5, and twinPE) for a given application. Finally, we provide detailed instructions on how to perform prime editing in mammalian cells. Relative to other procedures for editing human cells, this protocol for prime editing provides greater precision and versatility and can be completed within 2-4 weeks.