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

Abstract

BackgroundHydractinia 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.ResultsHere, 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5032-z) contains supplementary material, which is available to authorized users.

Genetic examination of the marine bacterium Pseudoalteromonas luteoviolacea and effects of its metamorphosis‐inducing factors

“…Although P . luteoviolacea HI1 and Hydractinia were not isolated from the same environment, both partners of this model interaction are genetically tractable (Huang et al ., 2012; Sanders et al ., 2018) and could serve as a strong platform for determining mechanisms underlying bacteria‐induced metamorphosis in cnidarians.…”

The colonial cnidarian Hydractinia

“…A more targeted way to genetically manipulate the animals is provided by CRISPR-Cas9 technology. In Hydractinia, this is performed by microinjecting site-specific short guide [34] and licensed under CC BY 4.0 (link: https ://creat iveco mmons .org/licen ses/by/4.0/)) RNAs (sgRNA) together with recombinant Cas9 to generate loss-of-function mutations [16,20]. Adding to the injecting cocktail a plasmid including a fragment of DNA, flanked by two homology arms, can be used for targeted knock-in of fragments [34].…”

Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

“…For these trials, we chose to target a gene with obvious phenotypic characteristics upon knockdown. We thus targeted the eGFP gene in the offspring of a cross between eggs produced by a wildtype female line (295–8) and sperm from a transgenic Eef1alpha > eGFP male line (354–3) 10 . The transgenic line was created via CRISPR/Cas9-mediated eGFP gene knockin to the endogenous housekeeping gene Eef1alpha (eukaryotic elongation factor 1 alpha) locus.…”

“…In the last decade, a variety of techniques to study gene function have become available for Hydractinia . Most recently, genetic engineering (both gene knockouts 9 and knockins 10 ) has been achieved in Hydractinia by means of CRISPR/Cas9 embryo microinjection. Prior to this, the use of antisense RNA-mediated gene silencing to induce gene-specific knockdown had been frequently employed in Hydractinia , via morpholino microinjection of embryos 11 and double-stranded RNA (dsRNA) soaking of embryos and polyps 7 , 12 – 15 .…”