期刊
BMC GENOMICS
卷 19, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s12864-018-5032-z
关键词
Genome editing; Immunohistochemistry; Cnidaria; Invertebrate; Model organism; Transgenic; FLAG; eGFP; tdTomato; P2A
资金
- NSF [IOS1557339]
- Thomas E. Starzl Transplantation Institute
- Science Foundation Ireland Principal Investigator award [11/PI/1020]
- NIH [ZIA-HG000140, T32AI074490]
- NATIONAL HUMAN GENOME RESEARCH INSTITUTE [ZIAHG000140] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [T32AI074490] Funding Source: NIH RePORTER
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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