Periodic Reporting for period 1 - MsgRNA (Modification of single guide (sg) RNA in CRISPR-Cas9 gene editing Tool)
Reporting period: 2019-11-01 to 2021-10-31
DNA stores the genetic information of almost all known organisms. The ability to precisely target DNA and alter its function has been a long-term goal of scientists. Until recently, methods to edit the genome were complex and unintuitive, but the discovery of RNA-guided CRISPR-Cas systems has transformed the situation. However, hurdles such as non-specific nucleobase editing, special information of gene editing programs, and controlling the cleavage of the desired gene are in front of the research in this field. Chemical manipulation of the system could prove vital in addressing these issues. This work is to expand the function of the system in order to ask better questions and simplify its use. The project is important for society because CRISPR is now in a stage of the human clinical trial as CRISPR-Cas9 gene-editing techniques are used in a variety of complex diseases, including cancer, neurodegenerative disorders, and genetic and metabolic diseases. So, modulation of the CRISPR gene-editing technique is the main challenge for society. In the project, we had used a simple technique to control the gene-editing mechanism. Using a multi-disciplinary approach combining innovative state-of-the-art probes and techniques, we characterized in depth the molecular mechanisms of gene editing CRISPR-Cas9 techniques.
The fundamental principle of CRISPR genome editing is the cleavage of double-strand breaks (DSBs) in a DNA region of interest. The desirable efficiency and specificity of CRISPR-based genome editing systems are still the main challenge. Here, we have performed a structure-based approach to sequence-specific cleaving of the genome of interest. For this purpose, our strategy was to target the genomic DNA using Cerium metal-binding ligand, N,N,N′,N′- ethylenediaminetetramethylenephosphonic acid (EDTP) was conjugated to 5’-end of sgRNA of CRISPR. Combining with Cas9 nuclease enzyme results in efficient cleavage site specific of two sides of the target DNA genome. We also explored a novel approach to reducing fluorescent sgRNA signals outside the nucleus using fluorophore-quencher pairs. Upon hybridization of the modified crRNA and tracrRNA, a stable RNA-DNA heteroduplex was formed which is sensitive to native cellular RNase H cleavage that released the quencher. Optimization of the exact distance between the dCas9 protein and the RNA-DNA duplex was done in vitro by monitoring of fluorescence enhancement in real-time upon mixing purified crRNA/tracrRNA, RNase H and dCas9. Controlling gene expression with light and chemical cross-linking was approached in this project. Light-sensitive molecules like azobenzene that are covalently linked to crRNA of CRISPR, blocking gene editing activity and again starting the editing processes using a photocleavable linker. Modulation of the CRISPR gene editing system was performed by illuminating with a specific wavelength of light causing the DNA cleavage and another specific wavelength of light to stop the cleavage of the genome. Interestingly, when one or two azobenzenes were introduced in the seed and non-seed region of crRNA, genomic DNA cleavage was hardly suppressed in the cis-form as compared to the natural trans form of azobenzene. To allow general use of these technologies an efficient ‘ON’ and ‘OFF’ state is a smart approach for this technology. Using the same strategies, we applied chemical cross-linking between the internal loop of sgRNA using click chemistry to stop the gene editing and restore gene editing activity by illumination of the photocleavable linker. The first attempt was internal crosslinking of tetraloop and stem loop 2 and then stem loop 1 and stem loop 3. This internal crosslinking makes sgRNA more rigid and results in loss of its activity. The cleavage activity was restored again by using a coumarin photocleavable linker which absorbs visible light and a nitrobenzene-like photocleavable linker for UV illumination.
The project (MsgRNA-836039) has been deeply affected by the pandemic (Covid-19). Due to department closures of more than four months, all experiments were stopped completely. The research results were highlighted on the internet and the webpage, the host lab. The researcher has attended the virtual conferences and presented virtual posters in ‘OLIGO 2020’ and ‘OLIGO 21’ organized by LibPubMedia conference meetings where he has presented his CRISPR work. Also, he has been invited for a scientific talk at ‘Future of Chemistry’ conference series organized by the Tata Institute of Fundamental Research (TIFR), India. In addition, two papers are being prepared for submission and peer review.
The project (MsgRNA-836039) has been deeply affected by the pandemic (Covid-19). Due to department closures of more than four months, all experiments were stopped completely. The research results were highlighted on the internet and the webpage, the host lab. The researcher has attended the virtual conferences and presented virtual posters in ‘OLIGO 2020’ and ‘OLIGO 21’ organized by LibPubMedia conference meetings where he has presented his CRISPR work. Also, he has been invited for a scientific talk at ‘Future of Chemistry’ conference series organized by the Tata Institute of Fundamental Research (TIFR), India. In addition, two papers are being prepared for submission and peer review.
The MsgRNA project was highly novel in terms of the design of chemically modified CRISPR RNA constructs as new tools for gene-editing techniques. Metal-induced artificial cutters offer site-specific multiple cleavages of the target gene. This serves as a unique tool for the design of more efficient genome sequence-specific cleaving constructs. The novel approach had been developed for live cell imaging via CRISPR-Cas9 systems by fluorophore quencher pairs. This has provided definitive evidence for the ease and simplicity of fsgRNAs for live-cell imaging compared to other methods. Controlling off-target effects of CRISPR-Cas9 systems using light-induced DNA damage was also a novel approach for gene editing technology which was studied in this project. This project has established successful collaborations across Europe for the host laboratory. Introducing photoswitches into the sgRNA provides excellent opportunities to control CRISPR gene editing in biological applications. These photoresponsive promoters could be powerful tools for gene editing application. By using the azobenzene attachment, CRISPR gene editing could be efficiently and reversibly photoregulated. The cis-trans isomerization of azobenzene by UV or visible light provides two different states of the crRNA, which allows on-off switching of DNA cleavage. An efficient ‘ON’ and ‘OFF’ state of gene editing was developed with the help of click chemistry, producing crosslinking between the loops of sgRNA. Site-specific sgRNA crosslinking approaches have been developed for two internal stem loops within sgRNA. By incorporating a photo-cleavable linker, the click crosslinking sgRNA can be released by irradiation with a predetermined wavelength of light. These approaches will accelerate the future development of photo-activated gene editing technologies as well as lead to CRISPR-based gene therapies.