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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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“In general, precise donor integration remains a challenge in the field and in spite of the fact that CRISPR efficiently induces double stranded breaks, CRISPR mediated donor integration importantly comes at the expense of: (i) 3–20-fold higher indel formation rates at the non-targeted allele (56,57) and (ii) significa…”
BackgroundCRISPR/Cas9-mediated multiple single guide RNAs potently abrogate pseudorabies virus replication
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“knock-in can be also executed by homology-independent DNA repair [5, 6, 246].”
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From DNA break repair pathways to CRISPR/Cas-mediated gene knock-in methods
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“However, targeting IRES-GFP to the 30UTR of OCT4 through NHEJ yielded only 0.6% GFPþ cells (He et al., 2016), which is in the range of efficiency observed by Zhu and colleagues using selection-free HDR-mediated targeting of a 2A-mOrange fluorescent reporter targeted to the same locus in hPSCs (Zhu…”
BackgroundMethodInfluentialGenome and Epigenome Editing in Mechanistic Studies of Human Aging and Aging-Related Disease
Lau, C., & Suh, Y. (2016) · Gerontology · doi:10.1159/000452972 · Cited by 22
Applications of CRISPR/Cas9 in retinal degenerative diseases
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“GLVs were introduced into the Gt(ROSA26)Sor locus via NHEJ-dependent knock-in using the CRISPR / Cas9 vectors TetFE CR1 and CR2, which cleaved Gt(ROSA26)Sor intron 1 and the backbone sequence of GLV1, respectively (34).”
Meca‐Cortés, Ó., Guerra‐Rebollo, M., Garrido, C., Borrós, S., Rubio, N., & Blanco, J. (2017) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2017.07.012 · Cited by 22
“Even though solid evidence is not provided for the number of transgene copies per cell in these experiments, CRISPR/Cas9mediated knockin is expected to result in the insertion of a single copy per genome.(17) Nevertheless, Cas9 nuclease activity may produce off-target strand excision/repair events at predictable sites…”
BackgroundCRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in vivo cancer modeling
Mou, H., Özata, D. M., Smith, J. L., Sheel, A., Kwan, S., Hough, S. H., Küçükural, A., Kennedy, Z., Cao, Y., & Xue, W. (2019) · Genome Medicine · doi:10.1186/s13073-019-0627-9 · Cited by 22
CRISPR/CAS9, the king of genome editing tools
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Synthetic biology for improving cell fate decisions and tissue engineering outcomes
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“When compared to NHEJ and MMEJ donors, more exact and properly oriented chromosomal integration of exogenous DNA is achieved through HMEJ and HR donor designs (15).”
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Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange
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Successful Correction by Prime Editing of a Mutation in the RYR1 Gene Responsible for a Myopathy
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“…for generating loss-of-function mutations or utilize homology-directed repair (HDR) using an introduced DNA repair template, such as a double-stranded DNA donor plasmid or a single-stranded oligo DNA nucleotide (ssODN), leading to the precise knock-in of mutations or heterologous genes 14,15 .”
What rheumatologists need to know about CRISPR/Cas9
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Next-generation mammalian genetics toward organism-level systems biology
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“insertion of large multi-kilobase (83) genetic cassettes capable of bearing several reporters at a genetic safe harbor site.”
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Biederstädt, A., Manzar, G. S., & Daher, M. (2022) · Frontiers in Immunology · doi:10.3389/fimmu.2022.1063303 · Cited by 19
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“Hence, the high-specificity SpCas9 nucleases identified here as efficient tools for DSB-dependent genome editing are expected to be particularly suited for gene knock-ins entailing HMEJ and, possibly, other types of ‘double-cut’ donors, such as those prone to NHEJ or MMEJ.”
BackgroundInfluentialCRISPR/CAS9 - КОРОЛЬ ГЕНОМНОГО РЕДАКТИРОВАНИЯ, "Молекулярная биология"
Банников, А. В., & Лавров, А. В. (2017) · Molecular Biology · doi:10.7868/s0026898417040036 · Cited by 19
Enhanced Biosynthesis Performance of Heterologous Proteins in CHO-K1 Cells Using CRISPR-Cas9
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A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells
Brookhouser, N., Nguyen, T., Tekel, S. J., Standage-Beier, K., Wang, X., & Brafman, D. A. (2020) · BMC Biology · doi:10.1186/s12915-020-00929-7 · Cited by 19
Engineering the Drosophila Genome for Developmental Biology
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“7 kb have been achieved by providing linear DNA during CRISPR/Cas9 induced NHEJ in Zebrafish [90], and up to 34 kb in human cell lines [91] using piggyBac or adenoviral vectors that were cleaved by Cas9, targeted via a vector specific gRNA.”
MethodFast and cloning‐free CRISPR/Cas9‐mediated genomic editing in mammalian cells
Manna, P. T., Davis, L. J., & Robinson, M. S. (2019) · Traffic · doi:10.1111/tra.12696 · Cited by 18
“The observed fidelity of the CHoP-In approach thus compares well with that of other NHEJ-mediated knock-in approaches (Lackner et al., 2015, He et al., 2016).”
BackgroundResultInfluentialA homology independent sequence replacement strategy in human cells using a CRISPR nuclease
Danner, E. W., Lebedin, M. Y., de la Rosa, K., & Kühn, R. (2021) · Open Biology · doi:10.1098/rsob.200283 · Cited by 22
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“Genome editing via the HR pathway, an auxiliary cellular repair pathway (4), usually requires an HR donor harboring left and right homology arms (HAs) (5–7).”
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“We found that knock-in via CRISPR/Cas9-induced NHEJ is superior to the commonly used HDR-based method in all human cell lines examined (He et al., 2016).”
BackgroundMethodInfluentialCRISPR/Cas-Based Gene Editing Tools for Large DNA Fragment Integration
Yang, S., Hu, G., Wang, J., & Song, J. (2024) · ACS Synthetic Biology · doi:10.1021/acssynbio.4c00632 · Cited by 17
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Non-viral ex vivo genome-editing in mouse bona fide hematopoietic stem cells with CRISPR/Cas9
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Genome editing in pests: basic science to applications
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“It has demonstrated that using Cas9 nickase or coupling microbial single-strand annealing proteins (SSAPs) with catalytically inactivate dCas9 promotes the knock-in of long sequences and improves knock-in in human cells (He et al. 2016; Wang et al. 2022a).”
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“The double-cut donor might induce insertion of the plasmid BB via the NHEJ pathway [34].”
BackgroundMethodKararoudi, M. N., Likhite, S. B., Elmas, E., Yamamoto, K., Schwartz, M., Sorathia, K., Pereira, M. D. S. F., Devin, R. D., Lyberger, J. M., Behbehani, G. K., et al. (2021) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2021.03.17.435886 · Cited by 15
High-efficiency targeted transgene integration via primed micro-homologues
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Targeted integration into pseudo attP sites of CHO cells using CRISPR/Cas9
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“The sgA sequence referred from the published data (He et al., 2016) was cloned into px459-v2 (Addgene, Watertown, USA, #118632) in a similar manner.”
MethodGene therapy using genome‐edited iPS cells for targeting malignant glioma
Tamura, R., Miyoshi, H., Imaizumi, K., Yo, M., Kase, Y., Sato, T., Sato, M., Morimoto, Y., Sampetrean, O., Kohyama, J., et al. (2022) · Bioengineering & Translational Medicine · doi:10.1002/btm2.10406 · Cited by 13
CRISPR-engineered genome editing for the next generation neurological disease modeling
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“Recently, several studies convincingly demonstrated the NHEJ process can be repurposed for knock-in in mammalian cells [56-60].”
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Efficient Homology-directed Repair with Circular ssDNA DonorsPreprint
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Safe Harbor Targeted CRISPR-Cas9 Tools for Molecular-Genetic Imaging of Cells in Living Subjects
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Calderón, K. I., Rojas-Neyra, A., Carbajal-Lévano, B., Luján-Valenzuela, L., Ticona, J., Isasi-Rivas, G., Montalván-Ávalos, Á., Criollo-Orozco, M., Huaccachi-Gonzalez, E., Tataje‐Lavanda, L., et al. (2022) · Viruses · doi:10.3390/v14040793 · Cited by 13
“The CRISPR-associated 9 (CRISPR/Cas9) type II system, consisting of RNA-guided Cas9 endonuclease from Streptococcus pyogenes, a single guide RNA (sgRNA), and the transactivating crRNA (tracrRNA) [32], was developed for the editing of eukaryotic cells by introducing precise and efficient double-stranded breaks (DSBs) i…”
BackgroundMethodInfluentialSurveying the Delivery Methods of CRISPR/Cas9 for ex vivo Mammalian Cell Engineering
Kelton, W., Pesch, T., Matile, S., & Reddy, S. T. (2016) · CHIMIA International Journal for Chemistry · doi:10.2533/chimia.2016.439 · Cited by 13
Applications of CRISPR technology in cellular immunotherapy
Zhou, X., Renauer, P. A., Zhou, L., Fang, S., & Chen, S. (2023) · Immunological Reviews · doi:10.1111/imr.13241 · Cited by 14
“69 Earlier approaches employ Cas9 RNP electroporation and AAV-HDR template introduction to generate TCR-T cells with tumor antigen-specific TCR that was stably integrated into TRAC locus.”
MethodAntibiotic Combinations That Enable One-Step, Targeted Mutagenesis of Chromosomal Genes
Lee, W., Do, T., Zhang, G., Kahne, D. E., Meredith, T. C., & Walker, S. (2018) · ACS Infectious Diseases · doi:10.1021/acsinfecdis.8b00017 · Cited by 12
Zhang, C., He, X., Kwok, Y. K. Y., Wang, F., Xue, J., Zhao, H., Suen, K. W., Wang, C. C., Ren, J., Chen, G. G., et al. (2018) · BMC Biology · doi:10.1186/s12915-018-0616-2 · Cited by 13
“This is apparently biased because DNA cleavage at the 3rd nucleotide is known to be dominant and prevalent [3, 16].”
InfluentialGenerating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing
Tang, N., Zhang, Y., Pedrera, M., Chang, P., Baigent, S. J., Moffat, K., Shen, Z., Nair, V., & Yao, Y. (2019) · Journal of Visualized Experiments · doi:10.3791/58193 · Cited by 13
Optimization and Validation of CAR Transduction into Human Primary NK Cells Using CRISPR and AAVPreprint
Kararoudi, M. N., Likhite, S. B., Elmas, E., Yamamoto, K., Schwartz, M., Sorathia, K., Pereira, M. D. S. F., Devin, R. D., Lyberger, J. M., Behbehani, G. K., et al. (2021) · SSRN Electronic Journal · doi:10.2139/ssrn.3869896 · Cited by 13
Wang, F., Calvo-Roitberg, E., Rembetsy-Brown, J. M., Fang, M., Sousa, J., Kartje, Z. J., Krishnamurthy, P. M., Lee, J., Green, M. R., Pai, A. A., et al. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac1108 · Cited by 12
“Single clones were seeded and genotyped as previously described (24).”
MethodWu, Y., Xu, K., Ren, C., Li, X., Lv, H., Han, F., Wei, Z., Wang, X., & Zhang, Z. (2017) · FEBS Letters · doi:10.1002/1873-3468.12599 · Cited by 12
CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture
Spiegel, A., Bachmann, M., Jiménez, G. J., & Sarov, M. (2019) · Methods · doi:10.1016/j.ymeth.2019.04.016 · Cited by 12
Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9
Liu, X., Wang, M., Qin, Y., Shi, X., Cong, P., Chen, Y., & He, Z. (2018) · BMC Biotechnology · doi:10.1186/s12896-018-0474-6 · Cited by 12
A Practical Guide to Genome Editing Using Targeted Nuclease Technologies
Porter, S. N., Levine, R. M., & Pruett‐Miller, S. M. (2019) · Comprehensive physiology · doi:10.1002/cphy.c180022 · Cited by 18
Beer, N. L., & Gloyn, A. L. (2016) · F1000Research · doi:10.12688/f1000research.8682.1 · Cited by 11
New Turns for High Efficiency Knock-In of Large DNA in Human Pluripotent Stem Cells
He, X., Li, Y., & Feng, B. (2018) · Stem Cells International · doi:10.1155/2018/9465028 · Cited by 11
“On the other hand, consistent with previous studies [16, 47, 48], He et al. also showed that the knock-in efficiency in human ESC, via either pathway, is much lower than that observed in somatic cell lines [45], which suggest that human ESCs possess unique properties in repairing DNA damage.”
BackgroundInfluentialAdvancing the predictivity of skin sensitization by applying a novel HMOX1 reporter system
Zhong, G., Li, H., Bai, J., Pang, S., He, C., Du, X., Wang, H., Zhang, Q., Xie, S., Du, H., et al. (2018) · Archives of Toxicology · doi:10.1007/s00204-018-2287-8 · Cited by 11
qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging
Philip, R., Sharma, A., Matellan, L., Erpf, A. C., Hsu, W.-H., Tkach, J. M., Wyatt, H. D. M., & Pelletier, L. (2024) · The EMBO Journal · doi:10.1038/s44318-024-00337-5 · Cited by 10
“Collectively, when attempting to insert large transgenes, these limitations often result in extremely low integration rates, making the isolation of homozygous cell lines both challenging and time-consuming (Artegiani et al, 2020; Fueller et al, 2020; He et al, 2016; Roberts et al, 2017).”
Research and Therapeutic Approaches in Stem Cell Genome Editing by CRISPR Toolkit
Mollashahi, B., Latifi‐Navid, H., Owliaee, I., Shamdani, S., Uzan, G., Jamehdor, S., & Naserian, S. (2023) · Molecules · doi:10.3390/molecules28041982 · Cited by 11
“We can use longer homology arms on either side of the insert site to insert chunks longer than the donor DNA [140,141].”
MethodCRISPR/Cas9-mediated generic protein tagging in mammalian cells
Thöne, F. M. B., Kurrle, N., von Melchner, H., & Schnütgen, F. (2019) · Methods · doi:10.1016/j.ymeth.2019.02.018 · Cited by 9
Emergent challenges for CRISPR: biosafety, biosecurity, patenting, and regulatory issues
Braddick, D., & Ramarohetra, R. F. (2020) · Elsevier eBooks · doi:10.1016/b978-0-12-818140-9.00021-0 · Cited by 10
Lu, Y., Wong, K. Y., Tan, C., Ma, J., Feng, B., & Lin, G. (2020) · Journal of Environmental Science and Health Part C · doi:10.1080/26896583.2020.1769409 · Cited by 10
A Simple and Efficient CRISPR Technique for Protein Tagging
Zeng, F., Beck, V., Schuierer, S., Garnier, I., Manneville, C., Agarinis, C., Morelli, L., Quinn, L., Knehr, J., Roma, G., et al. (2020) · Cells · doi:10.3390/cells9122618 · Cited by 11
“The second benefit of our protocol lies in its ease and simplicity: while other NHEJ-based methods rely on the vector expression of gRNA and the Cas9 protein [5,6,25,26], which requires intensive and expensive laboratory work, all reagents used in HiTag methods are readily available from commercial suppliers.”
MethodLu, H., Liu, J., Feng, T., Guo, Z., Yin, Y., Gao, F., Cao, G., Du, X., & Wu, S. (2021) · Genome Research · doi:10.1101/gr.271312.120 · Cited by 10
“Using a two-cut donor flanking the insertion elements with nuclease cleavage sites is a viable option to address this problem for NHEJ-based gene tagging (Lackner et al. 2015; He et al. 2016).”
MethodCRISPR/Cas9 knockin methodology for the sea urchin embryo
Oulhen, N., Morita, S., Warner, J. F., & Wessel, G. M. (2023) · Molecular Reproduction and Development · doi:10.1002/mrd.23672 · Cited by 9
“Some protocols rely instead of nonhomologous end joining (He et al., 2016).”
BackgroundInteractome Analysis Reveals a Link of the Novel ALMS1-CEP70 Complex to Centrosomal Clusters
Woerz, F., Hoffmann, F., Antony, S., Bolz, S., Jarboui, M. A., Junger, K., Klose, F., Stehle, I. F., Boldt, K., Ueffing, M., et al. (2023) · Molecular & Cellular Proteomics · doi:10.1016/j.mcpro.2023.100701 · Cited by 12
“In addition, the homology arms were fl anked by sgRNA-targeted sequences for linearization of the repair construct in vivo which is described to increase repair ef fi ciency (30).”
MethodZhang, J., Park, J.-Y., Kim, S.-W., Choi, Y., Cha, S.-Y., Jang, H., Wei, B., & Kang, M. (2024) · Vaccines · doi:10.3390/vaccines12030226 · Cited by 9
“NHEJ can occur at any phase of the cell cycle, whereas HDR can only occur during the S and G2 phases; the NHEJ pathway is able to repair DNA within 30 min, whereas HDR may require a 7 h delay to complete the process [24–26].”
BackgroundPrecise Gene Knock‐In Tools with Minimized Risk of DSBs: A Trend for Gene Manipulation
Liu, Y. H., Kong, J., Liu, G., Li, Z., & Xiao, Y. (2024) · Advanced Science · doi:10.1002/advs.202401797 · Cited by 10
Knock-in of Labeled Proteins into 5’UTR Enables Highly Efficient Generation of Stable Cell Lines
Ijaz, F., & Ikegami, K. (2021) · Cell Structure and Function · doi:10.1247/csf.21002 · Cited by 9
“…gene loci, efficiency of positive clone acquirement as high as 80% can be achieved in antibiotic selected cells and is incomparable to other CRISPR/Cas9-mediated NHEJ KI strategies (Suzuki et al. , 2016; He et al. , 2016; Schmid-Burgk et al. , 2016; Tálas et al. , 2017; Manna et al. , 2019).”
MethodGerace, D., Boulanger, K. R., Kenty, J. H.-R., & Melton, D. A. (2021) · Stem Cell Research · doi:10.1016/j.scr.2021.102371 · Cited by 9
Roy, P., Moffat, K., Nair, V., & Yao, Y. (2021) · Microorganisms · doi:10.3390/microorganisms9081681 · Cited by 9
“The relative expression of the genes was calculated with the calibration of the genes in the un-transfected controls and determined using the arithmetic comparative 2−∆∆Ct method [24].”
MethodA versatile toolbox for knock-in gene targeting based on the Multisite Gateway technology
Yoshimatsu, S., Sone, T., Nakajima, M., Sato, T., Okochi, R., Ishikawa, M., Nakamura, M., Sasaki, E., Shiozawa, S., & Okano, H. (2019) · PLoS ONE · doi:10.1371/journal.pone.0221164 · Cited by 9
Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing
Tang, N., Zhang, Y., Pedrera, M., Chang, P., Baigent, S. J., Moffat, K., Shen, Z., Nair, V., & Yao, Y. (2019) · Journal of Visualized Experiments · doi:10.3791/58193-v · Cited by 9
Targeted Gene Insertion: The Cutting Edge of CRISPR Drug Development with Hemophilia as a Highlight
Zhang, Z., Zhang, S., Wong, H. T., Li, D., & Feng, B. (2024) · BioDrugs · doi:10.1007/s40259-024-00654-5 · Cited by 9
“Remarkably, NHEJ insertion exhibited much higher efficiency than HDR-based strategy in cellular assays [49] due to the rapid and dominant nature of NHEJ-based DSB repair in mammalian species [51].”
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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