The DOI matches its Crossref record; 75 of the 75 references it cites were found in a registry or an index.
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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Citations
Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016) · Nature · doi:10.1038/nature17946 · Cited by 5,437
Self-citationSearch-and-replace genome editing without double-strand breaks or donor DNA
Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., et al. (2019) · Nature · doi:10.1038/s41586-019-1711-4 · Cited by 4,767
Self-citationOptimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9
Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., et al. (2016) · Nature Biotechnology · doi:10.1038/nbt.3437 · Cited by 4,637
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage
Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R. (2017) · Nature · doi:10.1038/nature24644 · Cited by 4,184
Self-citationGenome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors
Anzalone, A. V., Koblan, L. W., & Liu, D. R. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0561-9 · Cited by 2,237
Self-citationDynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System
Chen, B., Gilbert, L. A., Cimini, B. A., Schnitzbauer, J., Zhang, W., Li, G., Park, J. S., Blackburn, E. H., Weissman, J. S., Qi, L. S., et al. (2013) · Cell · doi:10.1016/j.cell.2013.12.001 · Cited by 1,988
CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia
Frangoul, H., Altshuler, D., Cappellini, M. D., Chen, Y.-S., Domm, J., Eustace, B. K., Foell, J., de la Fuente, J., Grupp, S., Handgretinger, R., et al. (2021) · New England Journal of Medicine · doi:10.1056/nejmoa2031054 · Cited by 1,962
Kosicki, M., Tomberg, K., & Bradley, A. (2018) · Nature Biotechnology · doi:10.1038/nbt.4192 · Cited by 1,883
CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis
Gillmore, J. D., Gane, E., Taubel, J., Kao, J., Fontana, M., Maitland, M. L., Seitzer, J., O’Connell, D., Walsh, K. R., Wood, K., et al. (2021) · New England Journal of Medicine · doi:10.1056/nejmoa2107454 · Cited by 1,801
Cited by
Sorted by citations
Prime editing for precise and highly versatile genome manipulation
Chen, P. J., & Liu, D. R. (2022) · Nature Reviews Genetics · doi:10.1038/s41576-022-00541-1 · Cited by 644
Self-citationPhage-assisted evolution and protein engineering yield compact, efficient prime editors
Doman, J. L., Pandey, S., Neugebauer, M. E., An, M., Davis, J. R., Randolph, P. B., McElroy, A. N., Gao, X. D., Raguram, A., Richter, M. F., et al. (2023) · Cell · doi:10.1016/j.cell.2023.07.039 · Cited by 327
Self-citationAn, M., Raguram, A., Du, S. W., Banskota, S., Davis, J. R., Newby, G. A., Chen, P., Palczewski, K., & Liu, D. R. (2024) · Nature Biotechnology · doi:10.1038/s41587-023-02078-y · Cited by 275
Self-citationImproving prime editing with an endogenous small RNA-binding protein
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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