Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9
Li M, Cook Au LY, Douglah D, Chong A, White BJ, Ferree PM, Akbari OS.
10.1101/096578Abstract
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.
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Generation of heritable germline mutations in the jewel wasp Nasonia vitripennis using CRISPR/Cas9. · DOI 10.1038/s41598-017-00990-3