Protocol for generating in-frame seamless knockins in Drosophila using the SEED/Harvest technology

The generation of knockins is fundamental to dissect biological systems. SEED/Harvest, a technology based on CRISPR-Cas9, offers a powerful approach for seamless genome editing in Drosophila. Here, we present a protocol to tag any gene in the Drosophila genome using SEED/Harvest technology. We describe knockin design, plasmid preparation, injection, and insertion screening. We then detail procedures for germline harvesting. The technique combines straightforward cloning and robust screening of insertions, while still resulting in scarless gene editing. For complete details on the use and execution of this protocol, please refer to Aguilar et al.1 [Display omitted] •Guide for in-frame knockin design, protein tag selection, and locus analysis•Step-by-step protocol for the cloning of gRNAs and SEED donors•Instructions for injecting Drosophila embryos•Guidance on screening, harvesting, and confirmation of knockins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The generation of knockins is fundamental to dissect biological systems. SEED/Harvest, a technology based on CRISPR-Cas9, offers a powerful approach for seamless genome editing in Drosophila. Here, we present a protocol to tag any gene in the Drosophila genome using SEED/Harvest technology. We describe knockin design, plasmid preparation, injection, and insertion screening. We then detail procedures for germline harvesting. The technique combines straightforward cloning and robust screening of insertions, while still resulting in scarless gene editing.