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Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes.

Current protocols in molecular biology (2019-12-24)
Justin A Bosch, Shannon Knight, Oguz Kanca, Jonathan Zirin, Donghui Yang-Zhou, Yanhui Hu, Jonathan Rodiger, Gabriel Amador, Hugo J Bellen, Norbert Perrimon, Stephanie E Mohr
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 '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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
LB Broth (Lennox), Highly-referenced microbial growth powder medium, low salt, suitable for salt-sensitive E.coli culture.
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 5.2, ≥96%
Sigma-Aldrich
Puromycin, Dihydrochloride, Cell Culture-Tested - CAS 58-58-2 - Calbiochem, Puromycin, CAS 58-58-2, is a protein synthesis inhibitor that causes premature release of nascent polypeptide chains.