One-step CRISPR-based Strategy for Endogenous Gene Tagging in <em>Drosophila melanogaster</em>

• DOI: 10.3791/64729
• Publication Date: 2024-01-27T00:01:51Z
• Authors: Yangbo Xiao; Ye Yuan; Swathi Yadlapalli
• Keywords: Genetic screen; Model Organism; Marker gene

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent techniques that allow tagging endogenous genes to produce fluorescent fusion proteins. These methods enable researchers visualize behavior real time, providing valuable insights into their functions interactions within cellular environment. Many current gene studies employ a two-step process where visible markers, such as eye color changes, are used identify genetically modified organisms first step, marker is excised second step. Here, we present one-step protocol perform precise rapid Drosophila melanogaster, which enables screening for engineered lines without marker, offering significant advantage over past methods. To screen successful gene-tagging events, PCR-based technique genotype individual flies analyzing small segment from middle leg. Flies pass criteria then stable stocks. detail design construction CRISPR editing plasmids confirmation lines. Together, this improves efficiency significantly processes vivo.