One-step CRISPR-Cas9 protocol for the generation of plug & play conditional knockouts in Drosophila melanogaster

• Published in: STAR protocols 2021-06, Vol.2 (2), p.100560-100560, Article 100560
• Main Authors: Yu, Joyce J.S., Vincent, Jean-Paul, McGough, Ian J.
• Format: Article
• Language: English
• Subjects: CRISPR; Genetics; Model Organisms; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100560

This one-step method generates, for any locus, a conditional knockout allele in Drosophila. The allele carries a bright fluorescent marker for easy screening and an attP landing site for subsequent genetic manipulations. After removing the selectable marker with Cre, the attP site can be used to insert DNA fragments expressing tagged or mutant alleles to determine protein localization and function in a tissue- and stage-specific manner. Only a single round of CRISPR-Cas9-mediated editing is required. For complete details on the use and execution of this protocol, please refer to the DWnt4[cKO] example in Yu et al. (2020). [Display omitted] •One-step CRISPR-Cas9 protocol to generate conditional knockout alleles in Drosophila•Facile screening with a removeable fluorescent marker•Integrase site included to enable subsequent “plug&play” manipulations This one-step method generates, for any locus, a conditional knockout allele in Drosophila. The allele carries a bright fluorescent marker for easy screening and an attP landing site for subsequent genetic manipulations. After removing the selectable marker with Cre, the attP site can be used to insert DNA fragments expressing tagged or mutant alleles to determine protein localization and function in a tissue- and stage-specific manner. Only a single round of CRISPR-Cas9-mediated editing is required.