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

STAR Protoc. 2022 Jun 14;3(3):101465. doi: 10.1016/j.xpro.2022.101465. eCollection 2022 Sep 16.


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).

Keywords: CRISPR; Genetics; Model Organisms.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CRISPR-Cas Systems* / genetics
  • DNA, Complementary / genetics
  • Drosophila / genetics
  • Drosophila melanogaster* / genetics
  • Exons / genetics
  • Introns


  • DNA, Complementary