Complexity of Detecting CRISPR/Cas9-Mediated Homologous Recombination in Zebrafish


Homology-directed (HD) genome modification offers an opportunity to precisely modify the genome. Despite reported successful cases, for many loci, precise genome editing remains challenging and inefficient in vivo. Here we report an effort to precisely knock-in a GFP reporter into gad locus mediated by CRISPR/Cas9 system in the zebrafish Danio rerio. PCR artifact was detected in testing for homologous recombination (HR), but was mitigated by optimizing PCR condition and decreasing the injected targeting plasmid concentration. Under this optimized condition, time course analysis revealed a decline of the HR-positive embryos at embryogenesis progressed. GFP signals also diminished at later developmental stages. The GFP signals were consistent with PCR detection, both of which suggested the loss of targeted insertion events at later stages. Such loss of insertion might be one underlying reason for the inability to obtain germ-line transgenic lines with GFP knocked into the gad locus. Our results suggest that the low HR efficiency associated with CRISPR-mediated knock-in is in part due to loss of insertion after targeted integration into the gad locus.

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  1. 1

    Asada H., Kawamura Y., Maruyama K., Kume H., Ding R.G., Kanbara N., Kuzume H., Sanbo M., Yagi T., Obata K. 1997. Cleft palate and decreased brain gamma-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase. Proc. Natl. Acad. Sci. U. S. A.94, 6496‒6499.

    CAS  Article  Google Scholar 

  2. 2

    Auer T.O., Del Bene F. 2014. CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. Methods.69, 142‒150.

    CAS  Article  Google Scholar 

  3. 3

    Auer T.O., Duroure K., De Cian A., Concordet J.P., Del Bene F. 2014. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res.24, 142‒153.

    CAS  Article  Google Scholar 

  4. 4

    Bedell V.M., Wang Y., Campbell J.M., Poshusta T.L., Starker C.G., Krug R.G., Tan W.F., Penheiter S.G., Ma A.C., Leung A.Y.H., Fahrenkrug S.C., Carlson D.F., Voytas D.F., Clark K.J., Essner J.J., Ekker S.C. 2012. In vivo genome editing using a high-efficiency TALEN system. Nature.491, 114‒118.

    CAS  Article  Google Scholar 

  5. 5

    Bowman T.V., Zon L.I. 2010. Swimming into the future of drug discovery: In vivo chemical screens in zebrafish. Acs Chem. Biol.5, 159‒161.

    CAS  Article  Google Scholar 

  6. 6

    Feng Z., Zhang B., Ding W., Liu X., Yang D.L., Wei P., Cao F., Zhu S., Zhang F., Mao Y., Zhu J.K. 2013. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res.23, 1229‒1232.

    CAS  Article  Google Scholar 

  7. 7

    Filippi A., Mueller T., Driever W. 2014. vglut2 and gad expression reveal distinct patterns of dual GABAergic versus glutamatergic cotransmitter phenotypes of dopaminergic and noradrenergic neurons in the zebrafish brain. J. Comp. Neurol.522, 2019‒2037.

    CAS  Article  Google Scholar 

  8. 8

    Gagnon J.A., Valen E., Thyme S.B., Huang P., Akhmetova L., Pauli A., Montague T.G., Zimmerman S., Richter C., Schier A.F. 2014. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One.9, e98186.

    Article  Google Scholar 

  9. 9

    Hoshijima K., Jurynec M.J., Grunwald D.J. 2016. Precise editing of the zebrafish genome made simple and efficient. Dev. Cell.36, 654‒567.

    CAS  Article  Google Scholar 

  10. 10

    Hwang W.Y., Fu Y., Reyon D., Maeder M.L., Tsai S.Q., Sander J.D., Peterson R.T., Yeh J.R., Joung J.K. 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol.31, 227‒229.

    CAS  Article  Google Scholar 

  11. 11

    Irion U., Krauss J., Nusslein-Volhard C. 2014. Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. Development.141, 4827‒4830.

    CAS  Article  Google Scholar 

  12. 12

    Jao L.E., Wente S.R., Chen W. 2013. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl. Acad. Sci. U. S. A.110, 13904‒13909.

    CAS  Article  Google Scholar 

  13. 13

    Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science.337, 816‒821.

    CAS  Article  Google Scholar 

  14. 14

    Jinek M., Jiang F.G., Taylor D.W., Sternberg S.H., Kaya E., Ma E.B., Anders C., Hauer M., Zhou K.H., Lin S., Kaplan M., Iavarone A.T., Charpentier E., Nogales E., Doudna J.A. 2014. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science. 343, 1247997.

    Article  Google Scholar 

  15. 15

    Kimmel C.B., Ballard W.W., Kimmel S.R., Ullmann B., Schilling T.F. 1995. Stages of embryonic-development of the zebrafish. Dev. Dynam.203, 253‒310.

    CAS  Article  Google Scholar 

  16. 16

    Lieschke G.J., Currie P.D. 2007. Animal models of human disease: zebrafish swim into view. Nat. Rev. Genet.8, 353‒367.

    CAS  Article  Google Scholar 

  17. 17

    Meng X.D., Noyes M.B., Zhu L.H.J., Lawson N.D., Wolfe S.A. 2008. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol.26, 695‒701.

    CAS  Article  Google Scholar 

  18. 18

    Patton E.E., Zon L.I. 2001. The art and design of genetic screens: zebrafish. Nat. Rev. Genet.2, 956‒966.

    CAS  Article  Google Scholar 

  19. 19

    Platt R.J., Chen S.D., Zhou Y., Yim M.J., Swiech L., Kempton H.R., Dahlman J.E., Parnas O., Eisenhaure T.M., Jovanovic M., Graham D.B., Jhunjhunwala S., Heidenreich M., Xavier R.J., Langer R., et al. 2014. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell.159, 440‒455.

    CAS  Article  Google Scholar 

  20. 20

    Ran F.A., Hsu P.D., Wright J., Agarwala V., Scott D.A., Zhang F. 2013. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc.8, 2281‒2308.

    CAS  Article  Google Scholar 

  21. 21

    Sander J.D., Cade L., Khayter C., Reyon D., Peterson R.T., Joung J.K., Yeh J.R. 2011. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol.29, 697‒698.

    CAS  Article  Google Scholar 

  22. 22

    Shin J., Chen J.K., Solnica-Krezel L. 2014. Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases. Development.141, 3807‒3818.

    CAS  Article  Google Scholar 

  23. 23

    Stemmer M., Thumberger T., Keyer M.D., Wittbrodt J., Mateo J.L. 2015. CCTop: An intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS One. 10, e0124633.

    Article  Google Scholar 

  24. 24

    Sung Y.H., Kim J.M., Kim H.T., Lee J., Jeon J., Jin Y., Choi J.H., Ban Y.H., Ha S.J., Kim C.H., Lee H.W., Kim J.S. 2014. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res.24, 125‒131.

    CAS  Article  Google Scholar 

  25. 25

    Won M., Dawid I.B. 2017. PCR artifact in testing for homologous recombination in genomic editing in zebrafish. PLoS One.12, e0172802.

    Article  Google Scholar 

  26. 26

    Woods I.G., Schier A.F. 2008. Targeted mutagenesis in zebrafish. Nat. Biotechnol.26, 650–651.

    CAS  Article  Google Scholar 

  27. 27

    Zu Y., Tong X.J., Wang Z.X., Liu D., Pan R.C., Li Z., Hu Y.Y., Luo Z., Huang P., Wu Q., Zhu Z.Y., Zhang B., Lin S. 2013. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat. Methods.10, 329‒331.

    CAS  Article  Google Scholar 

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We thank Prof. Wenbiao Chen from Vanderbilt University for providing the pTyr-gRNA plasmid. We also thank Prof. Jennifer Doudna for helpful discussion.


This work was supported by the National Natural Science Foundation of China (no. 31310103032)(K.J.), and grants from National Institute of Health (NIH) of USA (DA035680 and NS095734)(S.G.).

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Correspondence to K. J. Jiang or S. Guo.

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Conflict of interest. The authors declare that they have no conflict of interest.

Statement on the welfare of animals. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.


Y.P. and K.H. contributed equally to this work.

Y.P. and S.G. designed the research study; Y.P., K.H., and F.J. contributed reagents and materials; Y.P., K.H., K.J., W.Z., Z.D., and S.G. performed the experiments and analyzed data; Y.P., K.H., W.Z., K.J., and S.G. wrote the manuscript; all authors participated in the discussion of results and commended on the final paper.


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Pi, Y., He, K.Z., Zhang, W.Q. et al. Complexity of Detecting CRISPR/Cas9-Mediated Homologous Recombination in Zebrafish. Mol Biol 54, 382–390 (2020).

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  • genome modification
  • complexity
  • zebrafish