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

Abstract

Summary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Genetics
• Model Organisms
• CRISPR