SYNCAS-Mediated CRISPR-Cas9 Genome Editing in the Jewel Wasp, Nasonia vitripennis

Abstract

Genetic engineering is a formidable approach to study biology. The development of CRISPR-Cas9 has allowed the genetic engineering of insect species from several orders, and in some species, this tool is used routinely for genetic research. However, insect gene editing often relies on the delivery of CRISPR-Cas9 components via embryo injection. This technique has a limitation: some species lay their eggs inside hard substrates or living hosts, making embryo collection impossible or labour-intensive. Recently, a variety of techniques that exploit maternal injection of nucleases have been developed to circumvent embryo injection. Yet, despite this variety of maternal delivery techniques, some insects remain refractory to gene editing. One of these is the parasitoid wasp, Nasonia vitripennis, an important hymenopteran model species. In this study, a recently developed method termed SYNCAS was used to perform knock-out (KO) of the cinnabar gene in this wasp, obtaining KO efficiencies up to ten times higher than reported for other maternal injection approaches. We found up to 2.73% of all offspring to display a KO phenotype, and we obtained up to 68 KO offspring per 100 injected mothers. The optimal timing of injection and provision of hosts for egg laying was determined. With this protocol, routine applications of CRISPR-Cas9 become feasible in this species, allowing reverse genetics studies of genes with unknown associated phenotypes and paving the way for more advanced editing techniques. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/643426v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@170bdd6org.highwire.dtl.DTLVardef@bf3cbaorg.highwire.dtl.DTLVardef@12be928org.highwire.dtl.DTLVardef@1d85e6a_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIMaternal injection of Cas9 RNP together with BAPC and saponins (SYNCAS) results in efficient gene editing of Nasonia vitripennis offspring C_LIO_LITiming of injection and egg collection is important for maximising efficiency and reducing screening effort C_LIO_LIThe efficient genetic engineering of Nasonia vitripennis suggests the applicability of SYNCAS to other parasitoid wasps, a category of insects particularly difficult to genetically engineer with embryonic Cas9 injections. C_LI