Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing William Klobasa*,1, Fu-Chyun Chu*,1, Ordom Huot1, Nathaniel Grubbs1, Dorith Rotenberg1, Anna E. Whitfield1, Marcé D. 1 Lorenzen 1 Department of Entomology and Plant Pathology, North Carolina State University * These authors contributed equally Corresponding Author Abstract Marcé D. Lorenzen
[email protected] The corn planthopper, Peregrinus maidis, is a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA Citation interference (RNAi) in P. maidis through microinjection of double-stranded RNAs Klobasa, W., Chu, F.C., (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated Huot, O., Grubbs, N., Rotenberg, D., Whitfield, A.E., via this technique are transient and lack long-term Mendelian inheritance. Therefore, Lorenzen, M.D. Microinjection of the P. maidis toolbox needs to be expanded to include functional genomic tools that Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome would enable the production of stable mutant strains, opening the door for researchers Editing. J. Vis. Exp. (169), e62417, to bring new control methods to bear on this economically important pest. However, doi:10.3791/62417 (2021). unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based Date Published genome editing and germline transformation do not easily cross cell membranes. As March 26, 2021 a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for DOI success. To that end, an agarose-based egg-lay method was developed to allow 10.3791/62417 embryos to be harvested from P.