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

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.