PT - JOURNAL ARTICLE AU - Steven M. Sanders AU - Zhiwei Ma AU - Julia M. Hughes AU - Brooke M. Riscoe AU - Gregory A. Gibson AU - Alan M. Watson AU - Hakima Flici AU - Uri Frank AU - Christine E. Schnitzler AU - Andreas D. Baxevanis AU - Matthew L. Nicotra TI - CRISPR/Cas9-mediated gene knockin in the hydroid <em>Hydractinia symbiolongicarpus</em> AID - 10.1101/342592 DP - 2018 Jan 01 TA - bioRxiv PG - 342592 4099 - http://biorxiv.org/content/early/2018/06/08/342592.short 4100 - http://biorxiv.org/content/early/2018/06/08/342592.full AB - 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.Eef1aEukaryotic elongation factor 1 alphasgRNAsingle guide RNASF-TAPStrepII-FLAG tandem affinity purificationPCRpolymerase chain reaction; IPs - immunoprecipitationsONovernightRTroom temperatureUTRuntranslated region