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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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“Researchers have also developed a homology-independent targeted integration (HITI) strategy as an alternative to HDR, a technique that allows DNA integration in both dividing and non-dividing cells in vitro and in vivo [114,115].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [115]
Experimental — accuracy not yet measuredIn the citing paper
“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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“To confirm that ASAP is endogenously expressed, we inserted a Flag-tag at the C 162 terminal of the ORF1 (Flag-KI) using CRISPR/Cas9 system (28) (Figure 1H).”
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“Furthermore, neither lentiviral transduction nor CRISPR/Cas9 knockin are suitable for the insertion of large transgenes: lentiviral vector transgenic payloads are limited to a few kilobases due to decreased titer stemming from viral packaging limits (11) while homology directed repair is inefficient for large inserts…”
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“Recently, it was demonstrated that NHEJ can also be used to facilitate large DNA fragment (IRES-GFP) knock-in with higher efficiencies than HDR.(52)”
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“Recent studies have shown that by designing a CRISPR-Cas9 system that includes the same gRNA cut site in the donor vector as the genomic target site, the NHEJ repair pathway will more efficiently lead to transgene integration in zebrafish (37) and mammalian cells (38, 39).”
BackgroundEvolving AAV-delivered therapeutics towards ultimate cures
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ResultOptimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV
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“It has been shown that the efficiency of recombination increases as the length of HAs increases (Ran et al., 2013; MacLeod et al., 2017; He et al., 2016; Song and Stieger, 2017; Li et al., 2014).”
BackgroundGlycosphingolipids are mediators of cancer plasticity through independent signaling pathways
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Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency
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“untranslated genome regions [14,24,25], and in the case of gene replacement, this mechanism can create serious problems.”
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“6-kb internal ribosome entry site (IRES)-enhanced green fluorescent protein (eGFP) fragment was integrated into the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) site of somatic LO2 cells, 20% of the cells were GFP positive (24).”
MethodSite-Specific Fat-1 Knock-In Enables Significant Decrease of n-6PUFAs/n-3PUFAs Ratio in Pigs
Li, M., Ouyang, H., Yuan, H., Li, J., Xie, Z., Wang, K., Yu, T., Liu, M., Chen, X., Tang, X., et al. (2018) · G3 Genes Genomes Genetics · doi:10.1534/g3.118.200114 · Cited by 49
“As a powerful and widely used tool, CRISPR/Cas9 could efficiently produce flexible genome modifications, including deletion, insertion (Wu et al. 2016b; Chu et al. 2016; He et al. 2016), point mutation (Armstrong et al. 2016; Wang et al. 2016) and replacement (Lin and Potter 2016; Luo et al. 2016).”
MethodProgress 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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Yuan, M., Zhang, J., Gao, Y., Yuan, Z., Zhu, Z., Wei, Y., Wu, T., Han, J., & Zhang, Y. (2021) · Journal of Biological Chemistry · doi:10.1016/j.jbc.2021.100497 · Cited by 43
“By comparing the gene integration efficiency between the HDR-, HMEJ-, NHEJ-, and MMEJ-based methods, interestingly, different results were observed in different cell types or species (16, 20).”
MethodResultMaking ends meet: targeted integration of DNA fragments by genome editing
Yamamoto, Y., & Gerbi, S. A. (2018) · Chromosoma · doi:10.1007/s00412-018-0677-6 · Cited by 49
“A similar approach created > 10 kb (and as large as 34 kb) insertions in human cell lines and embryonic stem cells (He et al. 2016).”
MethodBi, Y., Tu, Z., Zhang, Y., Yang, P., Guo, M., Zhu, X., Zhao, C., Zhou, J., Wang, H., Wang, Y., et al. (2020) · Cell Reports · doi:10.1016/j.celrep.2020.02.090 · Cited by 43
Yang, P., Chou, S., Li, J., Hui, W., Liu, W., Sun, N., Zhang, R. Y., Zhu, Y., Tsai, M., Lai, H. I. A. M., et al. (2020) · Science Advances · doi:10.1126/sciadv.abb7107 · Cited by 42
Universal toxin-based selection for precise genome engineering in human cells
Li, S., Akrap, N., Cerboni, S., Porritt, M. J., Wimberger, S., Lundin, A., Möller, C., Firth, M. A., Gordon, E., Lazovic, B., et al. (2021) · Nature Communications · doi:10.1038/s41467-020-20810-z · Cited by 46
Huang, J. T., Rowe, D., Subedi, P., Zhang, W., Suelter, T., Valent, B. S., & Cook, D. E. (2022) · Nature Communications · doi:10.1038/s41467-022-34736-1 · Cited by 41
CRISPR-based strategies for targeted transgene knock-in and gene correction
Lau, C., Tin, C., & Suh, Y. (2020) · Faculty Reviews · doi:10.12703/r/9-20 · Cited by 46
He, X., Zhang, Z., Xue, J., Wang, Y., Zhang, S., Wei, J., Zhang, C., Wang, J., Urip, B. A., Ngan, C. C., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-34898-y · Cited by 43
Yoshimi, K., Oka, Y., Miyasaka, Y., Kotani, Y., Yasumura, M., Uno, Y., Hattori, K., Tanigawa, A., Sato, M., Oya, M., et al. (2020) · Human Genetics · doi:10.1007/s00439-020-02198-4 · Cited by 40
Paving the way towards precise and safe CRISPR genome editing
Śledziński, P., Dabrowska, M., Nowaczyk, M., & Olejniczak, M. (2021) · Biotechnology Advances · doi:10.1016/j.biotechadv.2021.107737 · Cited by 43
A New Era of Genome Integration—Simply Cut and Paste!
Liu, Z., Liang, Y., Ang, E. L., & Zhao, H. (2017) · ACS Synthetic Biology · doi:10.1021/acssynbio.6b00331 · Cited by 39
Chang, K., Huang, C., Ou‐Yang, C., Ho, C.-H., Lin, H., Hsu, C., Chen, Y., Chou, Y., Chen, Y.-J., Chen, Y., et al. (2021) · Stem Cell Research & Therapy · doi:10.1186/s13287-021-02585-2 · Cited by 39
“Previous reports demonstrated a 2–5% efficiency of HDR after creating DSBs in iPSCs or embryonic stem cells [47, 48].”
BackgroundLi, P., Zhang, L., Li, Z., Xu, C., Du, X., & Wu, S. (2019) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-019-03396-8 · Cited by 38
CRISPR/Cas9‐mediated 2‐sgRNA cleavage facilitates Pseudorabies virus editing
Tang, Y., Guo, J., Wang, T.-Y., Zhao, K., Liu, J., Gao, J., Tian, Z., An, T., & Cai, X. (2018) · The FASEB Journal · doi:10.1096/fj.201701129r · Cited by 37
“First, NHEJ-mediated DNA repair is intrinsically more common than HDR in mammalian cells (33, 34).”
BackgroundEnhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain
Ma, S., Wang, X., Hu, Y., Lv, J., Liu, C., Liao, K., Guo, X., Wang, D., Lin, Y., & Rong, Z. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa779 · Cited by 36
“For commercial re-use, please contact journals.permissions@oup.com ic.oup.com /nar/advance-article/doi/10.1093/nar/gkaa779/5912567 by guest on 30 Septem ber 2020 CRISPR-mediated targeted transgene integration is generally based on homology-directed repair (HDR) (6,7), non-homologous end-joining (NHEJ) (8,9), microhomo…”
InfluentialNano-vectors for CRISPR/Cas9-mediated genome editing
Yang, P., Lee, A. Y.-T., Xue, J., Chou, S., Lee, C., Tseng, P., Zhang, T. X., Zhu, Y., Lee, J., Chiou, S., et al. (2022) · Nano Today · doi:10.1016/j.nantod.2022.101482 · Cited by 41
Xie, K., Starzyk, J., Majumdar, I., Wang, J., Rincones, K., Tran, T. T. D., Lee, D., Niemi, S., Famiglietti, J., Suter, B., et al. (2024) · Nature Biotechnology · doi:10.1038/s41587-024-02504-9 · Cited by 36
Liu, C., Xu, X., Koivisto, O., Zhou, W., Jacquemet, G., Rosenholm, J. M., & Zhang, H. (2021) · Nanoscale · doi:10.1039/d1nr02872c · Cited by 38
Functional interrogation of non-coding DNA through CRISPR genome editing
Canver, M. C., Bauer, D. E., & Orkin, S. H. (2017) · Methods · doi:10.1016/j.ymeth.2017.03.008 · Cited by 36
CRISPR/Cas‐mediated gene editing using purified protein in D rosophila suzukii
Kalajdzic, P., & Schetelig, M. F. (2017) · Entomologia Experimentalis et Applicata · doi:10.1111/eea.12599 · Cited by 34
“Homology-directed repair knock-in experimental data show that different lengths of the left and the right homology arm of the DNA template plasmid can impact knock-in efficiency (Inbar et al., 2000; Lu et al., 2003; He et al., 2016).”
BackgroundDNA, RNA, and Protein Tools for Editing the Genetic Information in Human Cells
Chen, X., & Gonçalves, M. A. F. V. (2018) · iScience · doi:10.1016/j.isci.2018.08.001 · Cited by 36
“7% transgene knock-in at the GAPDH locus in LO2 cells and embryonic stem cells, respectively (He et al., 2016).”
BackgroundZhang, K., Wan, P., Wang, L., Wang, Z., Tan, F., Li, J., Ma, X., Cen, J., Yuan, X., Liu, Y., et al. (2024) · Cell stem cell · doi:10.1016/j.stem.2024.04.022 · Cited by 36
Ways of improving precise knock-in by genome-editing technologies
Smirnikhina, S., Anuchina, A. A., & Лавров, А. В. (2018) · Human Genetics · doi:10.1007/s00439-018-1953-5 · Cited by 32
Xenotransplantation: Progress Along Paths Uncertain from Models to Application
Platt, J. L., Cascalho, M., & Piedrahita, J. A. (2018) · ILAR Journal · doi:10.1093/ilar/ily015 · Cited by 34
“a gene into a preselected genomic region).(56,57) CRISPR-Cas9, in particular, has shown wide applicability and ease of use.”
BackgroundShakirova, A. I., Karpov, T. E., Komarova, Y., & Lepik, K. V. (2023) · Frontiers in Genome Editing · doi:10.3389/fgeed.2023.1068637 · Cited by 33
“…utilize plasmid donor template for targeted knock-in under respectively optimized donor structure (Yoshimi et al., 2021)In the first case, without homology between the donor and the target, the knock-in efficiency usually did not exceed 1% in model human cells (Bachu et al., 2015; He et al., 2016).”
BackgroundChang, P., Yao, Y., Tang, N., Sadeyen, J.-R., Sealy, J. E., Clements, A. L., Bhat, S., Munir, M., Bryant, J. E., & Iqbal, M. (2018) · Viruses · doi:10.3390/v10020081 · Cited by 32
“The universal bait sequence from copGFP was adapted from published data [15].”
BackgroundMethodInfluentialMassey, A. J. (2018) · PLoS ONE · doi:10.1371/journal.pone.0195050 · Cited by 32
Jing, R., Jiao, P., Chen, J., Meng, X., Wu, X., Duan, Y., Shang, K., Qian, L., Huang, Y., Liu, J., et al. (2021) · Small Methods · doi:10.1002/smtd.202100071 · Cited by 32
Overview of advances in CRISPR/deadCas9 technology and its applications in human diseases
Kanafi, M. M., & Tavallaei, M. (2022) · Gene · doi:10.1016/j.gene.2022.146518 · Cited by 37
Gene Therapy for Inherited Retinal Degeneration
Arbabi, A., Liu, A., & Ameri, H. (2019) · Journal of Ocular Pharmacology and Therapeutics · doi:10.1089/jop.2018.0087 · Cited by 30
“172,173 However, the efficiency of CRISPR/Cas9-mediated gene editing via HDR pathway is much lower compared with the efficiency of gene editing mediated by the NHEJ pathway.”
Nanotechnology‐based CRISPR/Cas9 delivery system for genome editing in cancer treatment
Zhou, S., Li, Y., Wu, Q., & Gong, C. (2024) · MedComm – Biomaterials and Applications · doi:10.1002/mba2.70 · Cited by 30
Metadata
- 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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