(F-231) Simultaneous Genomic Locus Imaging and DNA Repair Monitoring by Elastin-like Proteins in Live Cells

Introduction:: Current Hi-C technologies have enabled us to scrutinize the structural information of genomes. However, these technologies can not provide enough temporal information for the rapidly changing genomes in live cells. To visualize live-cell nucleus behaviors, many tools based on the CRISPR system have been developed, but most of them can be only used to target relatively repetitive loci. Here we developed a novel tool for imaging the non-repetitive loci in the cells based on CRISPR and Elastin-Like Proteins (ELP), namely CRISPR-ELP droplets. We further include the transcription activator VP64 into the CRISPR-ELP droplet so that this system can not only label specific genomic loci but also drives the downstream gene expression. Using this tool, we tracked the double-strand DNA break repair dynamics in the live cell using 53BP1 and found that the DNA repair is faster when transcription is active. Further investigation indicates that the chromatin accessibility at the transcription active site positively affects the DNA repair kinetics.


Materials and Methods:: To target specific gene loci, we used 24xSunTag fused dCas9 and SunTag-specific scFv fused ELP to amplify the signal, where ELP forms droplets at the target loci due to high local concentration (Panel A, Left). For the concurrent labeling and visualization of DNA repair, we developed an orthogonal heat-inducible AsCas12a to trigger the DNA double-strand break, and 53BP1 was used to monitor the DNA repair dynamics (Panel A, Right). To study the transcription activity on DNA repair, we compared the 53BP1 dwelling time before and after CRISPR-ELP droplet targeting (Panel F). Based on the U2-OS ATAC-seq data, we picked the promoter regions of the IL-1B gene and the ACTB gene, which represent closed and open chromatin, respectively, to study the DNA repair difference in closed and open chromatin regions (Panel H).


Results, Conclusions, and Discussions:: We first confirmed the feasibility of simultaneous genome labeling and DNA repair monitoring in the U2-OS cells and observed multiple rounds of DNA repair (Panel B-D). We also tested the gene induction power of the CRISPR-ELP droplet on the inactive gene IL-1B, and we found 10,000-fold gene induction, confirming the gene inducibility of the droplet (Panel E). Using this tool, we targeted the CASP9 gene in the engineered U2OS-SJ cell, which has multiple TetO insertions in the chr1p36 region for TetR labeling. We compared the 53BP1 dwelling time of this gene among three groups, TetR labeling (no occupancy and no induction), no VP64 droplet labeling (occupied but not induced), and VP64 droplet labeling (occupied and induced) (Panel F). The result showed that DNA repair is faster in the CRISPR-ELP droplet labeling group, indicating transcription-coupled DNA repair is faster (Panel G). Furthermore, we quantified the DNA repair dynamics for the promoter region of IL-1B and ACTB, representing open and closed chromatin regions. We found that the average repair time for ACTB is faster than the IL-1B gene (Panel H). Treating the cells with SAHA, a histone deacetylase inhibitor, opens the global chromatin, and results in accelerated DNA repair at the IL-1B gene. What’s more, transfecting the cells with H3.3K27M, which reduces the global level of H3K27 methylation, also showed a similar result. This result suggests that chromatin accessibility can positively affect DNA repair kinetics. Transcription-active genes are usually associated with euchromatin and thus have more open chromatin, which could be one reason why DNA repair is faster at these sites. This novel imaging and manipulation system can be further used to investigate other biological events, such as DNA repair in the context of T cell-mediated killing. Beyond live cell imaging, different modular domains, such as KRAB or p300, can be combined with ELP to form local droplets to control cell behaviors.


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