TL;DR
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 how to assess editing efficiency via flow cytometry and next-generation sequencing is provided.
This study demonstrates donor-complementary prime editing (DoPE) as a one-step, DSB-free and library-compatible method for precise insertion of large DNA fragments without requiring recombinases or transposases.
Genome engineering is evolving from a DNA-cutting system to a precise and programmable genome editing system, which may support the future development of safer and more precise therapeutic strategies.
Recent advances in programmable large-DNA writing in the human genome are reviewed across four mechanistic categories: recombinase-, prime editing-, transposase-, and retrotransposon-mediated technologies.
A programmable gene replacement tool, named prime assembly (PA), which adapts prime editors to produce one or two pairs of 3'-flaps on both the genome and donor DNA, allowing megabase-scale genomic excision and/or kilobase-scale donor insertion at the gene of interest.
This chapter provides a practical protocol for base editing by electroporation by electroporation in adherent and suspension stem cells and details essential steps for cell handling and recovery, followed by standard readouts.
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