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BMC Genomics. 2018 Sep 3;19(1):649. doi: 10.1186/s12864-018-5032-z.

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

Author information

1
Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA.
2
Pittsburgh Center for Evolutionary Biology and Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
3
Center for Biological Imaging and Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA.
4
Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
5
Whitney Laboratory for Marine Bioscience, and Department of Biology, University of Florida, St. Augustine, FL, USA.
6
Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
7
Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA. matthew.nicotra@pitt.edu.
8
Pittsburgh Center for Evolutionary Biology and Medicine, University of Pittsburgh, Pittsburgh, PA, USA. matthew.nicotra@pitt.edu.
9
Department of Immunology, University of Pittsburgh, Pittsburgh, PA, USA. matthew.nicotra@pitt.edu.

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.

KEYWORDS:

Cnidaria; FLAG; Genome editing; Immunohistochemistry; Invertebrate; Model organism; P2A; Transgenic; eGFP; tdTomato

PMID:
30176818
PMCID:
PMC6122657
DOI:
10.1186/s12864-018-5032-z
[Indexed for MEDLINE]
Free PMC Article

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