Adapting and Testing ReMOT Control for Expanded CRISPR-Era Genome Functions in Non-model Insects.

The movement of the Drosophila yolk protein (DmYP) across the mosquito oocyte membrane was both fortuitous and puzzling; the cells that become future offspring--oocytes--are closed off to molecules that are not specifically recognized by a receptor, but there is no obvious ortholog of the yolk protein/receptor for DmYP in mosquitoes. Nonetheless, a small fragment of DmYP was sufficient to move the massive ribonucleoprotein complex of Cas9 and a guide RNA from the open circulatory system of a female mosquito into the mosquito oocyte for targeting of the germline DNA and heritable mutation. This procedure, known as ReMOT Control, is a robust method for CRISPR/Cas9-mediated gene knockdown that has been adapted for many orders of insects, for ticks, and even for several species of crustacean by first identifying a suitable peptide for oocyte uptake, then expressing Cas9 as a fusion protein with the peptide and finally performing adult injections with expressed, purified protein and guide RNA against a gene with a visible marker phenotype. In order to support the adaptation of this procedure widely among entomologists, herein, we provide the protocols to: (a) Identify a suitable peptide for any insect by identifying the receptor-binding region of vitellogenin. (b) Clone a nucleotide coding the peptide and a fluorescent protein into the commercially available Addgene plasmid pET28a/Cas9-cys to generate a fusion-protein encoding gene. (c) Express a fusion protein for specific delivery of ReMOT Cas9 to the ovaries of an insect of interest. (d) Adapt a generalized procedure for adult injection of insects targeting the hemolymph and detecting ovary translocation and heritable gene editing.