Development And Application Of CRISPR-mediated Live Cell Imaging

Eukaryote chromosomes are tightly arranged in the nucleus,within which they own their territories.The spatio-temporal organization of chromosomes consists of a strict hierarchical structure,including chromosome regions,topologically associating domains and chromatin loops,which play an essential role in gene expression.However,it is still unclear how the structure and dynamics of chromosomes regulate a series of processes such as transcription,replication and DNA repair.Therefore,if the visualization of chromosome dynamics could be realized in living cells,it will play a key role in revealing how chromosomes dynamically control genome functions,but current technology is not enough for us to track any genomic locus of interest.CRISPR imaging technology can visualize a part of genomic loci,but there are still some limitations,including low signal-to-noise ratio,difficult to visualize non-repetitive sequences and impossible to visualize arbitrary genomic sites.Therefore,we have made efforts to solve these problems.Aiming at the low signal-to-noise ratio,we used the brightest fluorescent protein mNeonGreen combined with the dCas9-SunTag system which achieved high signal-to-noise ratio in labeling repetitive sequences and low repetitive sequences,compared with previous studies.We tracked the movement of telomeres for a long time by the dCas9-SunTag system combined with different fluorescent protein,laying the foundation for realizing the visualization of the three-dimensional structure of the genome.Based on the dCas9-SunTag system combined with mNeonGreen,more Cas9 mutants are introduced for live cell imaging,including dCas9,devoCas9,dxCas9,deCas9 and dCas9-HF1.Our results showed that all of them can label repetitive sequences and low repetitive sequences.In addition,by comparing their labeling efficiency,it was found that dCas9 showed the best ability in CRISPR live cell imaging,but dxCas9 came last among five Cas9 mutants.Furthermore,we constructed a system CRISPRTag-Tet-H2B-mCherry-P2A-puro-24XMS2 knocked into ACTB site of MCF-7 cell line to trace the dynamic changes of the Tet promoter.Observing the dynamics of the promoter before and after doxycycline treatment detected by dCas9-SunTag system and transcription dynamics using MS2 aptamer simultaneously,realized the visualization of cis-regulatory elements.The system can be used to how temporal coordination between different events,from DNA replication to transcription to mRNA processing,translation and decay,providing a platform for revealing how the three-dimensional organization of the genome spatio-temporally regulates gene expression.