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Designing and executing prime editing experiments in mammalian cells
Open accessAbstract
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-strand DNA breaks (DSBs). The mechanism of PE makes it less dependent on cellular replication and endogenous DNA repair than homology-directed repair-based approaches, and its ability to precisely install edits without creating DSBs minimizes indels and other undesired outcomes. The capabilities of PE have also expanded since its original publication. Enhanced PE systems, PE4 and PE5, manipulate DNA repair pathways to increase PE efficiency and reduce indels. Other advances that improve PE 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 PE (twinPE) can precisely insert or delete hundreds of base pairs of DNA and can be used in tandem with recombinases to achieve gene-sized (>5 kb) insertions and inversions. Achieving optimal PE requires careful experimental design, and the large number of parameters that influence PE outcomes can be daunting. This protocol describes current best practices for conducting PE 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 PE in mammalian cells. Compared with other procedures for editing human cells, PE offers greater precision and versatility, and can be completed within 2-4 weeks.
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“As there are less variables in base editing versus prime editing, efficiencies of target mutation induction are, in our hands, more predictable without extensive optimizations.18”
From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [18]
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Yan, J., Oyler-Castrillo, P., Ravisankar, P., Ward, C. C., Levesque, S., Jing, Y., Simpson, D., Zhao, A., Li, H. Z., Yan, W., et al. (2024) · Nature · doi:10.1038/s41586-024-07259-6 · Cited by 274
Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing
Tao, J., Bauer, D. E., & Chiarle, R. (2023) · Nature Communications · doi:10.1038/s41467-023-35886-6 · Cited by 232
Metadata
- Authors
- Doman, J. L., Sousa, A. A., Randolph, P. B., Chen, P. J., & Liu, D. R.
- Affiliations
- Broad Institute (US)
- Corresponding author
- Liu, David R.
- Venue
- Nature Protocols
- Published
- 2022-08-08
- Volume
- 17
- Issue
- 11
- Pages
- 2431-2468
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC9799714
- OpenAlex
- W4290673775
- Cited by
- 172
- References
- 75
- Citations by year
- 2026: 45 · 2025: 56 · 2024: 38 · 2023: 27 · 2022: 6
- FWCI
- 3.31
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
- Funders
- Division of Intramural Research, National Institute of Allergy and Infectious Diseases (U01AI142756)
- U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (RM1HG009490)
- U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (R01EB031172)
- U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (R35GM118062)
- Howard Hughes Medical Institute (Liu investigatorship)
- Bill and Melinda Gates Foundation (Liu grant)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- prime editing, pegRNA design, DNA repair pathways, experimental design optimization
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Animals, DNA, DNA Repair, Humans, Mammals, RNA, Guide, Kinetoplastida, DNA Breaks, Double-Stranded, CRISPR-Cas Systems
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