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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.

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


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.


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


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