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Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining
Open accessAbstract
Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator-like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.
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“Even in the presence of donor template DNA, NHEJ is the more frequent repair mechanism observed from CRISPR/Cas9 editing (Maruyama et al., 2015).”
From Delivering CRISPR: a review of the challenges and approaches (2018) · cites this paper as Maruyama et al., 2015
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“or inhibiting the NHEJ pathway [113] have shown to be useful as means of increasing the efficiency of HDR after Cas9 cleavage.”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [113]
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“Although inhibiting the NHEJ pathway with gene silencing or chemical inhibitors can partially increase the efficiency of HDR pathway, the gene repair efficacy still needs to be further improved for disease therapy [82, 85].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [85]
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Metadata
- Authors
- Maruyama, T., Dougan, S. K., Truttmann, M. C., Bilate, A. M., Ingram, J. R., & Ploegh, H. L.
- Affiliations
- Whitehead Institute for Biomedical Research (US)
- Corresponding author
- Ploegh, Hidde L
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2015-03-23
- Volume
- 33
- Issue
- 5
- Pages
- 538-542
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4618510
- OpenAlex
- W2008147758
- Cited by
- 1,137
- References
- 32
- Citations by year
- 2026: 39 · 2025: 58 · 2024: 62 · 2023: 67 · 2022: 108 · 2021: 101 · 2020: 117 · 2019: 122 · 2018: 125 · 2017: 139 · 2016: 143 · 2015: 55
- FWCI
- 66.47
- Type
- journal article
- Language
- en
- Update notices
- erratum (2016-02-05)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- PMC
- Funders
- National Institute of Allergy and Infectious Diseases (R01AI087879)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR/Cas9, genome editing efficiency, non-homologous end joining, homology-directed repair, DNA ligase IV, DNA double-strand break repair, zinc finger nucleases, TALEN
- MeSH terms
- DNA Ligase ATP, Animals, Cell Line, DNA Repair, Genetic Engineering, DNA Ligases, Pyrimidines, Schiff Bases, Zinc Fingers, Genome, Mice, DNA Breaks, Double-Stranded, Homologous Recombination, DNA End-Joining Repair, CRISPR-Cas Systems
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