@article {1272329,
	title = {Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes},
	journal = {Curr Protoc Mol Biol},
	volume = {130},
	year = {2020},
	month = {2020 Mar},
	pages = {e112},
	abstract = {The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or {\textquoteright}knock-in{\textquoteright} of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with \~{}500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. {\textcopyright} 2019 by John Wiley \&amp; Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.},
	issn = {1934-3647},
	doi = {10.1002/cpmb.112},
	author = {Bosch, Justin A and Knight, Shannon and Kanca, Oguz and Zirin, Jonathan and Yang-Zhou, Donghui and Hu, Yanhui and Rodiger, Jonathan and Amador, Gabriel and Bellen, Hugo J and Perrimon, Norbert and Mohr, Stephanie E}
}
