Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, amethod for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into laminaassociated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by longterm live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.
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