CRISPR/Cas9-mediated tissue-specific knockout and cDNA rescue using sgRNAs that target exon-intron junctions in Drosophila melanogaster

Madison Chilian, Karen Vargas Parra, Abigail Sandoval, Juan Ramirez, Wan Hee Yoon

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

In this protocol, we take CRISPR/Cas9 and Gal4/UAS approaches to achieve tissue-specific knockout in parallel with rescue of the knockout by cDNA expression in Drosophila. We demonstrate that guide RNAs targeting the exon-intron junction of target genes cleave the genomic locus of the genes, but not UAS-cDNA transgenes, in a tissue where Gal4 drives Cas9 expression. The efficiency of this approach enables the determination of pathogenicity of disease-associated variants in human genes in a tissue-specific manner in Drosophila. For complete details on the use and execution of this protocol, please refer to Yap et al. (2021).

Original languageEnglish
Article number101465
JournalSTAR Protocols
Volume3
Issue number3
DOIs
StatePublished - 16 Sep 2022
Externally publishedYes

Keywords

  • CRISPR
  • Genetics
  • Model Organisms

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