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

Abstract

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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ACKNOWLEDGMENTS

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

Funding

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.

AUTHORS CONTRIBUTIONS

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). https://doi.org/10.1134/S0026893320030139

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Keywords:

  • CRISPR
  • genome modification
  • complexity
  • zebrafish