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Genomic Tagging of AGO1 Using CRISPR/Cas9-Mediated Homologous Recombination

  • Sanjay GhoshEmail author
  • Ji-Long LiuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1680)

Abstract

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.

Key words

CRISPR/Cas9 Drosophila AGO1 Homologous recombination Epitope tagging Genome engineering Transfection vector 

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Physiology, Anatomy, and Genetics, MRC Functional Genomics UnitUniversity of OxfordOxfordUK
  2. 2.Department of BiochemistryUniversity of CambridgeCambridgeUK
  3. 3.School of Life Science and TechnologyShanghaiTech UniversityShanghaiChina

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