Visualization of Genomic Loci in Living Cells with a Fluorescent CRISPR/Cas9 System
The discovery that the RNA guided bacterial endonuclease Cas9 can be harnessed to target and manipulate user-defined genomic sequences has greatly influenced the field of genome engineering. Interestingly, a catalytically dead Cas9 (dCas9) can be employed as a targeted DNA-binding platform to alter gene expression. By fusing this dCas9 to eGFP, we and others could show that the CRISPR/Cas9 system can be further expanded to label and trace genomic loci in living cells. We demonstrated that by exchanging the sgRNA, dCas9-eGFP could be specifically directed to various heterochromatic sequences within the nucleus. Here, we provide a basic protocol for this versatile tool and describe how to verify new dCas9-eGFP targets.
Key wordsCRISPR/Cas9 sgRNA In vivo labeling Repetitive sequences
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, SFB 1064 and Nanosystems Initiative Munich, NIM), and T.A. thankfully acknowledges the Graduiertenkolleg GRK1721.
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