%0 Journal Article %J Curr Protoc Mol Biol %D 2020 %T Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes %A Bosch, Justin A %A Knight, Shannon %A Kanca, Oguz %A Zirin, Jonathan %A Yang-Zhou, Donghui %A Hu, Yanhui %A Rodiger, Jonathan %A Amador, Gabriel %A Bellen, Hugo J %A Perrimon, Norbert %A Mohr, Stephanie E %X 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 'knock-in' 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. © 2019 by John Wiley & 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. %B Curr Protoc Mol Biol %V 130 %P e112 %8 2020 Mar %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/31869524?dopt=Abstract %R 10.1002/cpmb.112