PT - JOURNAL ARTICLE AU - Ming Li AU - Lauren Yun Cook Au AU - Deema Douglah AU - Abigail Chong AU - Bradley J. White AU - Patrick M. Ferree AU - Omar S. Akbari TI - Generation of heritable germline mutations in the jewel wasp <em>Nasonia vitripennis</em> using CRISPR/Cas9 AID - 10.1101/096578 DP - 2016 Jan 01 TA - bioRxiv PG - 096578 4099 - http://biorxiv.org/content/early/2016/12/23/096578.short 4100 - http://biorxiv.org/content/early/2016/12/23/096578.full AB - 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 a number of important biological phenomena in this organism.