(68f) Long-Term Live Cell Imaging of Endogenous Loci by CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat

Tasan, I., University of Illinois at Urbana-Champaign
Sustackova, G., University of Illinois at Urbana-Champaign
Zhang, L., University of Illinois at Urbana-Champaign
Kim, J., University of Illinois at Urbana-Champaign
Sivaguru, M., University of Illinois at Urbana-Champaign
HamediRad, M., University of Illinois at Urbana-Champaign
Wang, Y., Carnegie Mellon University
Genova, J., University of Illinois at Urbana-Champaign
Ma, J., Carnegie Mellon University
Belmont, A., University of Illinois at Urbana-Champaign
Zhao, H., University of Illinois at Urbana-Champaign
Spatiotemporal organization of the mammalian genome within the nucleus is highly regulated; however, the link between subnuclear localization and gene function remains elusive. To relate genome function to higher order nuclear organization, a direct, microscopy-based method for live cell tracking of the dynamics of any specific endogenous locus of interest is necessary.

Live cell imaging of DNA was previously carried out by using a fluorescent repressor-operator system to enrich fluorescent proteins (FPs) at a specific site on the DNA. In the two commonly used systems, repeating sequences of Lac operators (LacO) or Tet operators (TetO) are used as a DNA tag and FP-fused Lac repressor (LacI) or Tet repressor (TetR), respectively, are used for visualization of the tag. Most previous examples focused on plasmid or BAC integrated transgenes, although their behavior may not fully recapitulate the behavior of the endogenous locus. In a more recent example, these operator arrays were targeted to endogenous loci using homologous recombination (HR). However, a low targeting efficiency was observed, indicating the need for better targeting strategies with higher efficiency.

The efficiency of homology-directed genome editing can be increased by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). CRISPR/Cas9 system includes an sgRNA that recognizes a 20 nt DNA sequence and recruits the Cas9 endonuclease to the target DNA. Upon binding to the target, Cas9 induces a double strand break (DSB). In mammalian cells, non-homologous end joining (NHEJ) is the predominant mechanism for repairing DSBs; however, in the presence of a DNA homologous to the sequences flanking the DSB site, such as an exogenous donor DNA with suitable homology arms (HAs), the DSB can be repaired by HR as well. Depending on the design of the donor, gene knock-in, deletion or replacement can be achieved. One unexplored application of the CRISPR-mediated knock-in is the tagging of the DNA itself to overcome the need for a pre-existing repeat for visualization of chromosome loci.

In this work we developed an easy, efficient and scalable method named SHACKTeR (short homology arm and CRISPR/Cas9 mediated knock-in of a TetO repeat) for tagging and live cell imaging of non-repetitive, endogenous chromosome regions. We created an optimized, irregular TetO repeat, which allowed PCR-based creation of donors with short HAs and simplified the knock-in protocol. Irregular TetO repeats also enabled easy genotyping of positive TetO-labeled clones by PCR. SHACKTeR achieved targeted insertion at 10 different loci and we demonstrated the general applicability of SHACKTeR by showing, for the first time, the feasibility of knock-in into heterochromatin sites. We also show direct evidence that heterochromatin sites prefer NHEJ to repair DSBs and chromatin state can affect knock-in efficiency. SHACKTeR was highly specific given the lack of additional off-target spots. Moreover, integration of TetO repeats did not affect sub-nuclear localization, chromatin status and DNA replication dynamics of the integration site. By using a 96-mer TetO repeat, we were able to perform long-term live cell imaging of an endogenous locus to capture its replication during S-phase of the cell cycle. The ease-of-use and the scalability of SHACKTeR will allow labeling large numbers of endogenous chromosome loci for both fixed and live-cell imaging, which could lead to an improved understanding of the relationship between gene compartmentalization and function.