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Curr Protoc Mol Biol. 2020 Mar;130(1):e112. doi: 10.1002/cpmb.112.

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

Author information

1
Department of Genetics, Harvard Medical School, Boston, Massachusetts.
2
Drosophila RNAi Screening Center, Harvard Medical School, Boston, Massachusetts.
3
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas.
4
Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, Texas.
5
Department of Neuroscience, Baylor College of Medicine, Houston, Texas.
6
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas.
7
Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts.

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.

KEYWORDS:

CRISPR; CRISPaint; Drosophila; GFP fusion; cell culture; fluorescent protein tagging; gene tagging; knock-in; ssDNA Drop-In

PMID:
31869524
DOI:
10.1002/cpmb.112

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