Scientists have developed a novel method for imaging chromosome, chromatin structure and specific genes in living mammalian cells. By rationally designing and optimizing structures of sgRNAs, inventors have enabled the dynamic imaging, editing and regulation of specific genomic elements via the CRISPR system.
The imaging system consists of an EGFP-tagged, endonuclease-deactivated dCas9 protein and a custom designed sgRNA. The structure of the sgRNA has been intensively modified to enhance its expression level, stability, target specificity and molecular assembly with the dCas9 protein in mammalian cells. The optimized sgRNA sequence and structure has proprietary advantages over original sgRNAs that are used for all genome editing and gene editing and regulation applications. The sgRNA also contains a designable 20-30 nt base pairing region that is complementary to any chromosome site of interest.
The method is highly sensitive with high resolution, and can be used for chromosome or gene copy detection and dynamic analyses in the living cells. This optimized CRISPR system not only enables dynamic tracking of repetitive elements such as telomeres and centromeres, it also enables visualization of non-repetitive genomic sequences in the human genome. Investigators are currently working on the direct delivery of RNAs for cellular imaging, multi-color imaging, and using this method to detect and diagnose genetic disorders.
The best current technology for imaging specific DNA sequences in living cells has relied on fluorescently tagged DNA-binding proteins, an approach that is restricted to imaging artificial repetitive sequences inserted into the genome or specialized genomic elements. While fluorescence in situ hybridization (FISH) enables target sequence flexibility through base pairing of the nucleic acid probes, it is incompatible with live imaging due to sample fixation and DNA denaturation. A genome-imaging technique that combines the flexibility of nucleic acid probes and the live imaging capability of DNA-binding proteins would thus be a major advancement in enabling the study of genomic conformation and dynamics in living cells.
This novel invention provides the following advantages:
• Non-invasive live cell imaging of specific genomic elements (i.e., telomeres, centromeres, single genes) in mammalian cells.
• Detects spatiotemporal organization and dynamics of native chromatin structure.
• Enhanced efficiency and specificity (>50% increase) of genomic imaging, regulation and editing.
• Robust, flexible, highly sensitive and high resolution platform.
• High-throughput detection of multiple genomic events and disease-associated elements (i.e., aberrations in gene copy numbers, deletion, duplication & rearrangement).
• Modified and optimized sgRNA structure with increased stability, improved expression level and Cas9 assembly, and thus enhanced targeting efficiency.
To develop this technology into a molecular kit for genome editing, a reagent for genome imaging or a diagnostics kit for disease specific genetic assays.