Scholarlink

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:

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

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

  • 实验方案

  • 发表日期：

    2025年

查看全文
  • Shivani Dave,
  • Chahat Patel,
  • Chan Heu,
  • Vanessa M. Macias

摘要

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: