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Tagging of genes at the endogenous loci is a powerful strategy for the analysis of protein function. We have developed a homologous recombination-based approach for inserting epitope tag into Drosophila AGO1 locus by employing the CRISPR/Cas9 technology. The methodology involves co-expression of sgRNA (containing 20-nucleotide AGO1 targeting sequence) and Cas9 protein, together with a donor template that has HA-AGO1 cassette flanked by sequences homologous to the AGO1 locus. The integration is efficient and readily monitored by immunostaining of the transgenic cell line. This method facilitates rapid generation of stable cell lines and allows insertion of any tag sequence into endogenous loci, thus accelerating characterization of the tagged proteins.

Original publication

DOI

10.1007/978-1-4939-7339-2_15

Type

Chapter

Publication Date

2018

Volume

1680

Pages

217 - 235

Keywords

AGO1, CRISPR/Cas9, Drosophila, Epitope tagging, Genome engineering, Homologous recombination, Transfection vector, Animals, Animals, Genetically Modified, Argonaute Proteins, CRISPR-Cas Systems, Cloning, Molecular, Drosophila Proteins, Gene Expression, Gene Order, Gene Targeting, Genetic Engineering, Genetic Vectors, Homologous Recombination, RNA, Guide, Transfection