Abstract
The revolutionary RNA-guided endonuclease CRISPR/Cas9 system has proven to be a powerful tool for gene editing in a plethora of organisms. Here, utilizing this system we developed an efficient protocol for the generation of heritable germline mutations in the parasitoid jewel wasp, Nasonia vitripennis, a rising insect model organism for the study of evolution, development of axis pattern formation, venom production, haplo-diploid sex determination, and host–symbiont interactions. To establish CRISPR-directed gene editing in N. vitripennis, we targeted a conserved eye pigmentation gene cinnabar, generating several independent heritable germline mutations in this gene. Briefly, to generate these mutants, we developed a protocol to efficiently collect N. vitripennis eggs from a parasitized flesh fly pupa, Sarcophaga bullata, inject these eggs with Cas9/guide RNA mixtures, and transfer injected eggs back into the host to continue development. We also describe a flow for screening mutants and establishing stable mutant strains through genetic crosses. Overall, our results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in N. vitripennis, with strong potential for expansion to target critical genes, thus allowing for the investigation of several important biological phenomena in this organism.
Highlights
The last decade has experienced a rapid increase in the genetic toolkit to study the biology of N. vitripennis and its interesting interactions with bacterial symbionts and genetic parasites
Over the past decade or so, several important genetic, genomic, and cell biological studies have been conducted in the jewel wasp N. vitripennis[19, 32,33,34,35]
These studies have been facilitated by the development of several important experimental resources including a high resolution genome sequence[8], several genome wide transcriptional profiling studies[7, 10], procedures for performing embryonic in situ hybridizations to detect spatial patterns of mRNA expression[34], and systemic, parental RNA interference (RNAi) which can be used in certain tissue contexts to study gene function using reverse genetics[12, 13]
Summary
The last decade has experienced a rapid increase in the genetic toolkit to study the biology of N. vitripennis and its interesting interactions with bacterial symbionts and genetic parasites. Methods to functionally disrupt gene expression relying on RNA interference (RNAi) by injecting in vitro transcribed dsRNA into either female pupae[12] or larvae[13] have advanced capabilities of performing reverse genetics on this organism These features have rendered N. vitripennis as a burgeoning model organism[13,14,15,16] for studying complex genetic, cellular and developmental processes including venom production[17, 18], sex determination[19], host symbiont interactions[3, 20], evolution and development of axis pattern formation[21,22,23,24], and development of haplodiploidy[24]. While N. vitripennis has many amenable experimental tools and resources described above, to date there have been no successful methods developed that allow for direct gene mutagenesis in this organism This absence can, in part, be attributed to the difficulty in using previous gene disruption technologies, e.g. TALENs and ZNFs25, in addition to a lack of detailed published protocols for performing embryonic microinjection in N. vitripennis. We demonstrate an efficient, effective, inexpensive, and straightforward CRISPR-Cas[9] heritable gene disruption approach for N. vitripennis, and to our knowledge this study represents one of the first gene disruption-based techniques conducted in a hymenopteran insect
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