Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells
Abstract
The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.
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The desired recombination efficacy of HDR often occurs infrequently, which presents enormous challenges for robust applications.
“However, the desired recombination efficacy often occurs infrequently, which presents enormous challenges for robust applications [61, 82–84].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [82]
Inhibiting the NHEJ pathway with gene silencing or chemical inhibitors can partially increase the efficiency of HDR pathway, but the gene repair efficacy still needs to be further improved for disease therapy.
“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 [82]
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- Authors
- Chu, V. T., Weber, T., Wefers, B., Wurst, W., Sander, S., Rajewsky, K., & Kühn, R.
- Affiliations
- Max Delbrück Center (DE)
- German Center for Neurodegenerative Diseases (DE)
- Corresponding author
- Rajewsky, Klaus
- Kühn, Ralf
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2015-03-24
- Volume
- 33
- Issue
- 5
- Pages
- 543-548
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- OpenAlex
- W2072299899
- Cited by
- 1,210
- References
- 28
- Citations by year
- 2026: 44 · 2025: 59 · 2024: 81 · 2023: 90 · 2022: 115 · 2021: 111 · 2020: 139 · 2019: 127 · 2018: 124 · 2017: 132 · 2016: 137 · 2015: 49 · 2014: 1
- FWCI
- 63.90
- Type
- journal article
- Language
- en
- Update notices
- erratum (2018-02-01)
- Open access
- closed
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (acceptedVersion)
- Site cant be reached (submittedVersion)
- DZNE Pub (submittedVersion)
- PuSH - Publication Server of Helmholtz Zentrum München (submittedVersion)
- 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
- homology-directed repair, CRISPR/Cas9, non-homologous end joining, Ku70, Ku80, DNA ligase IV, gene silencing, mammalian cells
- MeSH terms
- DNA Ligase ATP, Adenoviridae, Animals, Cell Line, Gene Expression Regulation, Genetic Engineering, Humans, DNA Ligases, Viral Proteins, Genome, Human, Adenovirus E4 Proteins, Mice, DNA Breaks, Double-Stranded, Homologous Recombination, DNA End-Joining Repair, CRISPR-Cas Systems
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