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.
