Prime editing primarily induces undesired outcomes in mice
Open accessAbstract
Summary: Genome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.
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Non-negligible frequencies of editing-associated large deletions have been reported after the use of the Cas9 D10A nickase in mESCs and prime editing in early mouse embryos.
“However, non-negligible frequencies of editingassociated large deletions have been reported after the use of the Cas9 D10A nickase in mESCs (14) and prime editing in early mouse embryos (43).”
From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [43]
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Cites
Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L., & Church, G. M. (2013) · Nature Biotechnology · doi:10.1038/nbt.2675 · Cited by 1,752 · cited 2× · see citing works
“…requires induction of nicks on both DNA strands has also been utilized to induce DSBs and subsequent indels by zinc finger nucleases (Miller et al., 2007), transcription-activator-like effector nucleases (Miller et al., 2011), and CRISPR Cas9-D10A nickases (Mali et al., 2013; Ran et al., 2013).”
MethodResultTargeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems
Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M., Hara, K. Y., et al. (2016) · Science · doi:10.1126/science.aaf8729 · Cited by 1,349 · see citing works
Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants
Walton, R. T., Christie, K. A., Whittaker, M. N., & Kleinstiver, B. P. (2020) · Science · doi:10.1126/science.aba8853 · Cited by 1,233 · see citing works
Engineered CRISPR-Cas9 nuclease with expanded targeting space
Nishimasu, H., Shi, X., Ishiguro, S., Gao, L., Hirano, S., Okazaki, S., Noda, T., Abudayyeh, O. O., Gootenberg, J. S., Mori, H., et al. (2018) · Science · doi:10.1126/science.aas9129 · Cited by 1,100 · see citing works
Prime genome editing in rice and wheat
Lin, Q., Zong, Y., Xue, C., Wang, S., Jin, S., Zhu, Z., Wang, Y., Anzalone, A. V., Raguram, A., Doman, J. L., et al. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0455-x · Cited by 873 · see citing works
Got mutation? ‘Base editors’ fix genomes one nucleotide at a time.
Ravindran, S. (2019) · Nature · doi:10.1038/d41586-019-03536-x · Cited by 12 · cited 1× · see citing works
“Currently, it is of great interest to understand whether PE broadly works in vivo for animal model production and therapeutic development (Cohen, 2019; Hampton, 2020; Ravindran, 2019; Urnov, 2020).”
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Research works printed but not linked: 1
- Wilde, J.J., Aida, T., Wienisch, M., Zhang, Q., Qi, P., and Feng, G. (2018). Efficient Zygotic Genome Editing via RAD51-Enhanced Interhomolog Repair. BioRxiv 263699.
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Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos
Alanis-Lobato, G., Zohren, J., McCarthy, A., Fogarty, N. M. E., Kubikova, N., Hardman, E., Greco, M., Wells, D., Turner, J. M. A., & Niakan, K. K. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2004832117 · Cited by 234 · see citing works
Chanda, S., Lepikhov, K., Dahlem, C., Schymik, H. S., Hoppstädter, J., Geber, A.-K., Wagner, K., Kessler, S. M., Empting, M., & Kiemer, A. K. (2024) · Frontiers in Bioscience-Landmark · doi:10.31083/j.fbl2901041 · Cited by 8 · see citing works
- Authors
- Aida, T., Wilde, J. J., Yang, L., Hou, Y., Li, M., Xu, D., Lin, J., Qi, P., Lu, Z., & Feng, G.
- Affiliations
- McGovern Institute for Brain Research (US)
- Chinese Academy of Sciences (CN)
- Broad Institute (US)
- Corresponding author
- Aida, Tomomi
- Wilde, Jonathan J.
- Lu, Zhonghua
- Feng, Guoping
- Venue
- bioRxiv (Cold Spring Harbor Laboratory)
- Publisher
- openRxiv
- Published
- 2020-08-06
- OpenAlex
- W3047219457
- Cited by
- 43
- References
- 6
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 8 · 2023: 7 · 2022: 7 · 2021: 14 · 2020: 5
- Type
- posted content
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- Funders
- National Natural Science Foundation of China (81961128019, 2018YFA0801404)
- Chinese Academy of Sciences (2018YFA0801404, 172644KYSB20170004)
- Science, Technology and Innovation Commission of Shenzhen Municipality (JCYJ20180507182505475, JCYJ20170413162938668, JCYJ20180504165804015)
- 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 › Genetics › Animal Genetics and Reproduction
- Keywords
- prime editing, off-target effects, mouse embryo, indels, in vivo gene editing, genome editing efficiency
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