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CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus

View ORCID ProfileSteven M Sanders, Zhiwei Ma, Julia M Hughes, Brooke M Riscoe, Gregory A Gibson, Alan M Watson, Hakima Flici, Uri Frank, Christine E Schnitzler, Andreas D Baxevanis, Matthew L Nicotra
doi: https://doi.org/10.1101/342592
Steven M Sanders
University of Pittsburgh School of Medicine;
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Zhiwei Ma
University of Pittsburgh School of Medicine;
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Julia M Hughes
University of Pittsburgh School of Medicine;
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Brooke M Riscoe
University of Pittsburgh School of Medicine;
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Gregory A Gibson
University of Pittsburgh;
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Alan M Watson
University of Pittsburgh;
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Hakima Flici
National University of Ireland;
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Uri Frank
National University of Ireland;
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Christine E Schnitzler
University of Florida;
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Andreas D Baxevanis
National Human Genome Research Institute
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Matthew L Nicotra
University of Pittsburgh School of Medicine;
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  • For correspondence: matthew.nicotra@pitt.edu
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Abstract

Background: Hydractinia symbiolongicarpus, a colonial cnidarian, is a tractable model system for many cnidarian-specific and general biological questions. Until recently, tests of gene function in Hydractinia have relied on laborious forward genetic approaches, randomly integrated transgenes, or transient knockdown of mRNAs. Results: Here, we report the use of CRISPR/Cas9 genome editing to generate targeted genomic insertions in H. symbiolonigcarpus. We used CRISPR/Cas9 to promote homologous recombination of two fluorescent reporters, eGFP and tdTomato, into the Eukaryotic elongation factor 1 alpha (Eef1a) locus. We demonstrate that the transgenes are expressed ubiquitously and are stable over two generations of breeding. We further demonstrate that CRISPR/Cas9 genome editing can be used to mark endogenous proteins with FLAG or StrepII-FLAG affinity tags to enable in vivo and ex vivo protein studies. Conclusions: This is the first account of CRISPR/Cas9 mediated knockins in Hydractinia and the first example of the germline transmission of a CRISPR/Cas9 inserted transgene in a cnidarian.The ability to precisely insert exogenous DNA into the Hydractinia genome will enable sophisticated genetic studies and further development of functional genomics tools in this understudied cnidarian model.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 08, 2018.
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CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus
Steven M Sanders, Zhiwei Ma, Julia M Hughes, Brooke M Riscoe, Gregory A Gibson, Alan M Watson, Hakima Flici, Uri Frank, Christine E Schnitzler, Andreas D Baxevanis, Matthew L Nicotra
bioRxiv 342592; doi: https://doi.org/10.1101/342592
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CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus
Steven M Sanders, Zhiwei Ma, Julia M Hughes, Brooke M Riscoe, Gregory A Gibson, Alan M Watson, Hakima Flici, Uri Frank, Christine E Schnitzler, Andreas D Baxevanis, Matthew L Nicotra
bioRxiv 342592; doi: https://doi.org/10.1101/342592

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