Knocking in Multifunctional Gene Tags into SMC Complex Subunits Using Gene Editing
Condensin, a highly conserved pentameric chromosome complex, is required for the correct organization and folding of the genome. Here, we highlight how to knock protein tags into endogenous loci to faithfully study the condensin complex in vertebrates and dissect its multiple functions. These include using the streptavidin binding peptide (SBP) to create the first genome-wide map of condensin and perform varied applications in proteomics and enzymology of the complex. The revolution in gene editing using CRISPR/Cas9 has made it possible to insert tags into endogenous loci with relative ease, allowing physiological and fully functional tagged protein to be analyzed biochemically (affinity tags), microscopically (fluorescent tags) or both purified and localized (multifunctional tags). In this chapter, we detail how to engineer vertebrate cells using CRISPR/Cas9 to provide researchers powerful tools to obtain greater precision than ever to understand how the complex interacts and behaves in cells.
Key wordsCondensin Streptavidin binding peptide Gene editing CRISPR/Cas9 Chromosomes Mitosis Southern blot hybridization
The work presented in this chapter was supported by National Health and Medical Research Council (Australia) project Grants GNT1127209 (PK and DH) and GNT1145188 (PK and DH) and by the Victorian Government’s Operational Infrastructure Support Program.