| Popis: |
CRISPR labeling is a powerful technique to study the chromatin architecture in live cells. In CRISPR labeling, a catalytically dead CRISPR-Cas9 mutant is employed as programmable DNA-binding domain to recruit fluorescent proteins to selected genomic loci. The fluorescently labeled loci can then be identified as fluorescent spots and tracked over time by microscopy. A limitation of this approach is the lack of temporal control of the labeling process itself: Cas9 binds to the g(uide)RNA-complementary target loci as soon as it is expressed. The decoration of the genome with Cas9 molecules will, however, interfere with gene regulation and-possibly-affect the genome architecture itself. The ability to switch on and off Cas9 DNA binding in CRISPR labeling experiments would thus be important to enable more precise interrogations of the chromatin spatial organization and dynamics and could further be used to study Cas9 DNA binding kinetics directly in living human cells.Here, we describe a detailed protocol for light-inducible CRISPR labeling. Our method employs CASANOVA, an engineered, optogenetic anti-CRISPR protein, which efficiently traps the Streptococcus pyogenes (Spy)Cas9 in the dark, but permits Cas9 DNA targeting upon illumination with blue light. Using telomeres as exemplary target loci, we detail the experimental steps required for inducible CRISPR labeling with CASANOVA. We also provide instructions on how to analyze the resulting microscopy data in a fully automated fashion. |
