Related Experiment Video
Updated: Sep 10, 2025
Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica
Published on: October 20, 2020
Adapting and Testing ReMOT Control for Expanded CRISPR-Era Genome Functions in Non-model Insects
Shivani Dave1,2,3, Chahat Patel1,2,3, Chan Heu4
1Department of Biological Sciences, University of North Texas, Denton, TX, USA.
Abstract:
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.
Insights
Researchers developed ReMOT Control, a method using a Drosophila yolk protein fragment to deliver CRISPR/Cas9 gene editing tools into insect oocytes for heritable mutations. This technique enables precise germline DNA targeting across various arthropod species.
Area of Science:
- Molecular Biology
- Genetics
- Entomology
Background:
- Oocytes are generally protected from external molecules, lacking specific receptors for yolk proteins found in other species.
- The Drosophila yolk protein (DmYP) fragment's ability to facilitate Cas9/guide RNA complex transport into mosquito oocytes was unexpected.
- CRISPR/Cas9 gene editing requires efficient delivery into germlines for heritable trait modification.
Purpose of the Study:
- To provide protocols for adapting the ReMOT Control method for CRISPR/Cas9-mediated gene knockdown in diverse insect species.
- To enable entomologists to identify suitable peptides for oocyte uptake in various insects.
- To facilitate the widespread application of heritable gene editing in insects.
Main Methods:
- Identifying insect vitellogenin receptor-binding regions to find suitable oocyte uptake peptides.
- Generating a fusion protein by linking the identified peptide to Cas9 using the Addgene plasmid pET28a/Cas9-cys.
- Performing adult insect injections with the fusion protein and guide RNA, targeting hemolymph for ovary translocation and gene editing.
Main Results:
- A small DmYP fragment successfully transported Cas9 and guide RNA into mosquito oocytes, enabling germline DNA targeting.
- The ReMOT Control procedure has been successfully adapted for insects, ticks, and crustaceans.
- Protocols are provided for peptide identification, fusion protein generation, and insect injection for heritable gene editing.
Conclusions:
- ReMOT Control is a robust and adaptable method for CRISPR/Cas9-mediated gene knockdown and heritable mutation induction in arthropods.
- The provided protocols support the broad adoption of this gene editing technique by entomologists.
- This method offers a powerful tool for genetic research and manipulation in a wide range of insect species.
Related Concept Videos
01:28CRISPR/Cas9 Genome Editing
01:59CRISPR