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bioRxiv · 10.1101/162156

CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci

Tasan, I.·Sustackova, G.·Zhang, L.·Kim, J.·Sivaguru, M.·HamediRad, M.·Wang, Y.·Genova, J.·Ma, J.·Belmont, A. S.·Zhao, H.
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Abstract

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, a high-throughput method for tracking any locus of interest is desirable. 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. Compared to alternatives, our method does not require a nearby repetitive sequence and it 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 PCR, was successful at labeling 9 different loci in HCT116 cells with up to 20% efficiency. These loci included both nuclear speckle-associated, euchromatin regions and nuclear lamina-associated, heterochromatin regions. 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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Tasan, I., Sustackova, G., Zhang, L., Kim, J., Sivaguru, M., HamediRad, M., Wang, Y., Genova, J., Ma, J., Belmont, A. S., Zhao, H.. 2017-07-12. CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci. https://doi.org/10.1101/162156

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