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Sanders, SM,Ma, ZW,Hughes, JM,Riscoe, BM,Gibson, GA,Watson, AM,Flici, H,Frank, U,Schnitzler, CE,Baxevanis, AD,Nicotra, ML
2018
September
BMC Genomics
CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus
Published
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Genome editing Immunohistochemistry Cnidaria Invertebrate Model organism Transgenic FLAG eGFP tdTomato P2A STEM-CELLS MODEL SYSTEM HOX GENE CNIDARIAN REGENERATION ANIMALS EXPRESSION REVEALS ALLORECOGNITION INVESTIGATE
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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.
10.1186/s12864-018-5032-z
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