Enhanced prime editing systems by manipulating cellular determinants of editing outcomes
- Cell. 2021 Oct 28;184(22):5635-5652.e29. doi: 10.1016/j.cell.2021.09.018.
- 1. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02141, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
- 2. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
- 3. Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
- 4. Department of Pediatrics, University of Minnesota, Minneapolis, MN 55454, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55108, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA.
- 5. Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA.
- 6. Human Neuron Core, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Boston, MA 02115, USA; Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA.
- 7. Human Neuron Core, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Boston, MA 02115, USA; Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA.
- 8. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
- 9. Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA; Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA. Electronic address: [email protected].
- 10. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02141, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA. Electronic address: [email protected].
While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.