Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair
Open accessAbstract
CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.
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HITI strategy exhibits much higher knock-in efficiency than HDR both in vitro and in vivo.
“The recently developed HITI strategy exhibits much higher knock-in efficiency than HDR both in vitro and in vivo [86, 87].”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [86]
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Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors
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The Problem of the Low Rates of CRISPR/Cas9-Mediated Knock-ins in Plants: Approaches and Solutions
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Site-Specific Fat-1 Knock-In Enables Significant Decrease of n-6PUFAs/n-3PUFAs Ratio in Pigs
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Progress and Perspective of CRISPR‐Cas9 Technology in Translational Medicine
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Current trends in gene recovery mediated by the CRISPR-Cas system
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Development of delivery strategies for CRISPR‐Cas9 genome editing
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Universal toxin-based selection for precise genome engineering in human cells
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A New Era of Genome Integration—Simply Cut and Paste!
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Nano-vectors for CRISPR/Cas9-mediated genome editing
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- Authors
- He, X., Tan, C., Wang, F., Wang, Y., Zhou, R., Cui, D., You, W., Zhao, H., Ren, J., & Feng, B.
- Affiliations
- Chinese University of Hong Kong (HK)
- Corresponding author
- Feng, Bo
- ORCID
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2016-02-04
- Volume
- 44
- Issue
- 9
- Pages
- e85-e85
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC4872082
- OpenAlex
- W2267596434
- Cited by
- 338
- References
- 56
- Citations by year
- 2026: 4 · 2025: 18 · 2024: 29 · 2023: 25 · 2022: 36 · 2021: 42 · 2020: 56 · 2019: 35 · 2018: 43 · 2017: 35 · 2016: 15
- FWCI
- 22.17
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- HighWire Press Open Archive (submittedVersion)
- Funders
- National Natural Science Foundation of China (T13-607/12R, 2015CB964700, NSFC 31171433, JCYJ20140425184428469)
- Chinese University of Hong Kong
- Hong Kong University of Science and Technology
- National Key Research and Development Program of China (2015CB964700)
- 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, non-homologous end joining, homology-directed repair, knock-in, DNA double-strand breaks, human embryonic stem cells, somatic cells, genome editing efficiency
- MeSH terms
- Human Embryonic Stem Cells, Gene Editing, RNA, Guide, CRISPR-Cas Systems, DNA, Humans, Genes, Reporter, Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating), HCT116 Cells, Cell Line, Tumor, Green Fluorescent Proteins, DNA Breaks, Double-Stranded, Gene Knock-In Techniques, HEK293 Cells, DNA End-Joining Repair, Recombinational DNA Repair, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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