@article{618360ee18204b7093c8cfe7d6150114,
title = "CRISPR/Cas9-mediated tissue-specific knockout and cDNA rescue using sgRNAs that target exon-intron junctions in Drosophila melanogaster",
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).",
keywords = "CRISPR, Genetics, Model Organisms",
author = "Madison Chilian and {Vargas Parra}, Karen and Abigail Sandoval and Juan Ramirez and Yoon, {Wan Hee}",
note = "Funding Information: This work was supported by the National Institute of Neurological Disorders and Stroke (1R01 NS121298-01) and the National Institute of General Medical Sciences (5 P20 GM103636-09) of the National Institutes of Health. We thank Professor Hugo Bellen for his advice and generous gift of Drosophila stocks. We thank Professor Simon Bullock for a gift of pCFD3.1-w-dU6:3gRNA. We thank Dr. Nam Chul Kim, Dr. SeYeon Chung, and Dr. Ji-Hoon Kim for helpful discussion. We thank Yohan Park and Jae Sun Kang for the Drosophila stock care and maintenance. M.C. and K.V.P. carried out the experiments and wrote the manuscript. A.S. and J.R. prepared tables and provided technical help. W.H.Y. conceived of the approach and experimental design, secured funding, and wrote and edited the manuscript. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2022 The Author(s)",
year = "2022",
month = sep,
day = "16",
doi = "10.1016/j.xpro.2022.101465",
language = "English",
volume = "3",
journal = "STAR Protocols",
issn = "2666-1667",
publisher = "Cell Press",
number = "3",
}