Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair
Open accessAbstract
Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.
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NHEJ-mediated knock-in of a reporter gene displays 10-fold higher efficiency than homologous recombination.
“Compared to homologous recombination (HR), NHEJ-mediated knock-in of reporter gene has been shown to display 10-fold higher efficiency (Auer, Duroure et al., 2014, Li et al., 2015).”
From Pu.1/Spi1 dosage controls the turnover and maintenance of microglia in zebrafish and mammals (2023) · cites this paper as Auer, Duroure et al., 2014
The Discussion reports higher germline transmission for the authors’ homology-independent knock-ins than for a previously reported HR knock-in. Its “at least 10-fold” figure, however, describes the activity of NHEJ relative to HR during early zebrafish development—not a measured 10-fold difference in reporter-gene knock-in efficiency. (LLM, conf=0.88)
Experimental — accuracy not yet measuredNHEJ-mediated knock-in of reporter gene displays 10-fold higher efficiency compared to homologous recombination.
“Compared to homologous recombination (HR), NHEJ-mediated knock-in of reporter gene has been shown to display 10-fold higher efficiency (Auer et al., 2014; Li et al., 2015).”
From Pu.1/Spi1 dosage controls the turnover and maintenance of microglia in zebrafish and mammals (2025) · cites this paper as Auer et al., 2014
The Discussion reports higher germline-transmission rates for homology-independent knock-in than for HR-mediated knock-in in zebrafish. Its “at least 10-fold” figure, however, describes NHEJ repair activity during early zebrafish development, not a measured 10-fold difference in reporter-gene knock-in efficiency. (LLM, conf=0.87)
Experimental — accuracy not yet measured
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Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116 · see citing works
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339 · see citing works
Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013) · Cell · doi:10.1016/j.cell.2013.04.025 · Cited by 3,495 · see citing works
High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292 · see citing works
Efficient genome editing in zebrafish using a CRISPR-Cas system
Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J., & Joung, J. K. (2013) · Nature Biotechnology · doi:10.1038/nbt.2501 · Cited by 2,937 · see citing works
Kimura, Y., Okamura, Y., & Higashijima, S.-I. (2006) · The Journal of Neuroscience · doi:10.1523/jneurosci.4993-05.2006 · Cited by 302 · see citing works
Flagged first, then by citations
Research works printed but not linked: 41
- Akitake CM, Macurak M, Halpern ME, Goll MG 2011 Transgenerational analysis of transcriptional silencing in zebrafish. Dev Biol 352: 191– 201 21223961 PMC3065955
- Balciunas D, Davidson AE, Sivasubbu S, Hermanson SB, Welle Z, Ekker SC 2004 Enhancer trapping in zebrafish using the Sleeping Beauty transposon. BMC Genomics 5: 62 15347431 PMC520745
- Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG, Tan W, Penheiter SG, Ma AC, Leung AYH, 2012 In vivo genome editing using a high-efficiency TALEN system. Nature 491: 114– 118 23000899 PMC3491146
- Campbell PD, Marlow FL 2013 Temporal and tissue specific gene expression patterns of the zebrafish kinesin-1 heavy chain family, kif5s, during development. Gene Expr Patterns 13: 271– 279 23684767 PMC3754906
- Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, Voytas DF, Long CR, Whitelaw CB, Fahrenkrug SC 2012 Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci 109: 17382– 17387 23027955 PMC3491456
- Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF 2011 Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39: e82 21493687 PMC3130291
- Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ 2013 Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res 23: 465– 472 23528705 PMC3616424
- Cho SW, Kim S, Kim JM, Kim JS 2013 Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31: 230– 232 23360966
- Cristea S, Freyvert Y, Santiago Y, Holmes MC, Urnov FD, Gregory PD, Cost GJ 2013 In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration. Biotechnol Bioeng 110: 871– 880 23042119
- Dai J, Cui X, Zhu Z, Hu W 2010 Non-homologous end joining plays a key role in transgene concatemer formation in transgenic zebrafish embryos. Int J Biol Sci 6: 756– 768 21152116 PMC2999851
- Di Donato V, Auer TO, Duroure K, Del Bene F 2013 Characterization of the calcium binding protein family in zebrafish. PLoS ONE 8: e53299 23341937 PMC3547026
- Distel M, Wullimann MF, Köster RW 2009 Optimized Gal4 genetics for permanent gene expression mapping in zebrafish. Proc Natl Acad Sci 106: 13365– 13370 19628697 PMC2726396
- Ellingsen S, Laplante MA, Konig M, Kikuta H, Furmanek T, Hoivik EA, Becker TS 2005 Large-scale enhancer detection in the zebrafish genome. Development 132: 3799– 3811 16049110
- Gaj T, Gersbach CA, Barbas CF 3rd 2013 ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 7: 397– 405 23664777 PMC3694601
- Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM 2013 Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194: 1029– 1035 23709638 PMC3730909
- Gupta A, Hall VL, Kok FO, Shin M, McNulty JC, Lawson ND, Wolfe SA 2013 Targeted chromosomal deletions and inversions in zebrafish. Genome Res 23: 1008– 1017 23478401 PMC3668355
- Hagmann M, Bruggmann R, Xue L, Georgiev O, Schaffner W, Rungger D, Spaniol P, Gerster T 1998 Homologous recombination and DNA-end joining reactions in zygotes and early embryos of zebrafish (Danio rerio) and Drosophila melanogaster. Biol Chem 379: 673– 681 9687016
- Huang P, Xiao A, Zhou M, Zhu Z, Lin S, Zhang B 2011 Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol 29: 699– 700 21822242
- Hwang WY, Fu Y, Reyon D, Maeder ML, Kaini P, Sander JD, Joung JK, Peterson RT, Yeh JR 2013a Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS ONE 8: e68708 23874735 PMC3706373
- Jasin M 1996 Genetic manipulation of genomes with rare-cutting endonucleases. Trends Genet 12: 224– 228 8928227
- Kawakami K, Shima A, Kawakami N 2000 Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc Natl Acad Sci 97: 11403– 11408 11027340 PMC17212
- Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M 2004 A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell 7: 133– 144 15239961
- Kawasaki T, Kurauchi K, Higashihata A, Deguchi T, Ishikawa Y, Yamauchi M, Sasanuma M, Hori H, Tsutsumi M, Wakamatsu Y, 2012 Transgenic medaka fish which mimic the endogenous expression of neuronal kinesin, KIF5A. Brain Res 1480: 12– 21 22975131
- Koster RW, Fraser SE 2001 Tracing transgene expression in living zebrafish embryos. Dev Biol 233: 329– 346 11336499
- Lim S, Wang Y, Yu X, Huang Y, Featherstone MS, Sampath K 2013 A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases. Genome Biol 14: R69 23815890 PMC4054832
- Liu J, Gong L, Chang C, Liu C, Peng J, Chen J 2012 Development of novel visual-plus quantitative analysis systems for studying DNA double-strand break repairs in zebrafish. J Genet Genomics 39: 489– 502 23021549
- Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM 2013 RNA-guided human genome engineering via Cas9. Science 339: 823– 826 23287722 PMC3712628
- Maresca M, Lin VG, Guo N, Yang Y 2013 Obligate ligation-gated recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res 23: 539– 546 23152450 PMC3589542
- Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, 2011 A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29: 143– 148 21179091
- Obholzer N, Wolfson S, Trapani JG, Mo W, Nechiporuk A, Busch-Nentwich E, Seiler C, Sidi S, Sollner C, Duncan RN, 2008 Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells. J Neurosci 28: 2110– 2118 18305245 PMC6671858
- Parinov S, Kondrichin I, Korzh V, Emelyanov A 2004 Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo. Dev Dyn 231: 449– 459 15366023
- Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK 2012 FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30: 460– 465 22484455 PMC3558947
- Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR 2011 Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29: 697– 698 21822241 PMC3154023
- Scott EK, Mason L, Arrenberg AB, Ziv L, Gosse NJ, Xiao T, Chi NC, Asakawa K, Kawakami K, Baier H 2007 Targeting neural circuitry in zebrafish using GAL4 enhancer trapping. Nat Methods 4: 323– 326 17369834
- Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali DAA 2004 Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 22: 589– 594 15064769
- Tesson L, Usal C, Menoret S, Leung E, Niles BJ, Remy S, Santiago Y, Vincent AI, Meng X, Zhang L, 2011 Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol 29: 695– 696 21822240
- Thermes V, Grabher C, Ristoratore F, Bourrat F, Choulika A, Wittbrodt J, Joly JS 2002 I-SceI meganuclease mediates highly efficient transgenesis in fish. Mech Dev 118: 91– 98 12351173
- Wefers B, Meyer M, Ortiz O, Hrabe de Angelis M, Hansen J, Wurst W, Kuhn R 2013 Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc Natl Acad Sci 110: 3782– 3787 23426636 PMC3593923
- Xiao T, Roeser T, Staub W, Baier H 2005 A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection. Development 132: 2955– 2967 15930106
- Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, Zu Y, Li W, Huang P, Tong X, 2013 Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res 41: e141 23748566 PMC3737551
- Zu Y, Tong X, Wang Z, Liu D, Pan R, Li Z, Hu Y, Luo Z, Huang P, Wu Q, 2013 TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat Methods 10: 329– 331 23435258
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“In contrast, experiments in whole organisms such as mice ((Wang et al., 2013), primates (Niu et al., 2014), Zebrafish (Auer et al., 2014) or C. elegans (Dickinson et al., 2013) reported off-target frequencies that were low or not detectable consistent with high specificity of the CRISPR/Cas9…”
ResultHe, X., Tan, C., Wang, F., Wang, Y., Zhou, R., Cui, D., You, W., Zhao, H., Ren, J., & Feng, B. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw064 · Cited by 338 · see citing works
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Bannister, S., Antonova, O., Polo, A., Lohs, C., Hallay, N., Valinciute, A., Raible, F., & Tessmar-Raible, K. (2014) · Genetics · doi:10.1534/genetics.113.161091 · Cited by 56 · see citing works
“Furthermore, as both systems use very different initial mechanisms of DNA sequence recognition, it is possible that in some cases where one system fails or is inefficient, the other might work better (Hwang et al. 2013; Auer et al. 2014).”
BackgroundPu.1/Spi1 dosage controls the turnover and maintenance of microglia in zebrafish and mammals
Wu, Y., Guo, W., Kuang, H., Wu, X., Trinh, T. H., Wang, Y., Zhao, S., Wen, Z., & Yu, T. (2025) · eLife · doi:10.7554/elife.105788 · Cited by 9 · see citing works
Pu.1/Spi1 dosage controls the turnover and maintenance of microglia in zebrafish and mammals
Wu, Y., Guo, W., Kuang, H., Wu, X., Trinh, T. H., Wang, Y., Zhao, S., Wen, Z., & Yu, T. (2023) · bioRxiv (Preprint) · doi:10.1101/2023.12.14.570333 · Cited by 1 · see citing works
“Compared to homologous recombination (HR), NHEJ-mediated knock-in of reporter gene has been shown to display 10-fold higher efficiency (Auer, Duroure et al., 2014, Li et al., 2015).”
- Authors
- Auer, T. O., Duroure, K., De Cian, A., Concordet, J.-P., & Del Bene, F.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Corresponding author
- Concordet, Jean-Paul
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2013-10-31
- Volume
- 24
- Issue
- 1
- Pages
- 142-153
- ISSN
- 1088-9051 (ISSN-L)
- PMC
- PMC3875856
- OpenAlex
- W2121633690
- Cited by
- 631
- References
- 6
- Citations by year
- 2026: 20 · 2025: 27 · 2024: 36 · 2023: 31 · 2022: 37 · 2021: 33 · 2020: 56 · 2019: 53 · 2018: 68 · 2017: 73 · 2016: 82 · 2015: 56 · 2014: 57
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genome Research (publishedVersion)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- HighWire Press Open Archive (submittedVersion)
- Funders
- Infrastructures en Biologie Santé et Agronomie
- Boehringer Ingelheim Fonds
- Agence Nationale de la Recherche
- Institut National de la Santé et de la Recherche Médicale
- Centre National de la Recherche Scientifique (UMR3215)
- FP7 Ideas: European Research Council (311159)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Zebrafish Biomedical Research Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Keywords
- CRISPR/Cas9, knock-in, zebrafish, DNA double-strand break repair, genome editing, transgene integration, enhanced green fluorescent protein, loss-of-function alleles, co-injection
- MeSH terms
- Animals, DNA Repair, Genetic Engineering, Zebrafish, Mutagenesis, Genome, DNA Breaks, Double-Stranded, Gene Knock-In Techniques, RNA, Small Untranslated, Recombinational DNA Repair, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Associated Proteins
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