Executive Summary : | CRISPR-Cas9 system is widely used for genome editing and for various applications including but not limited to therapeutics, crop production enhancement, pathogen detection, virus resistance etc. The another application of CRISPR-Cas9 apart from genome editing is in genome imaging to visualize in real time the organization and dynamics of cellular components such as visualization of chromatin remodelling, telomere dynamics etc. Although CRISPR has been used extensively, it still suffers from few limitations. Several efforts has been done in last few years to engineer CRISPR system for better cleavage efficiency and increase its cellular stability for both genome editing and genome imaging applications. Both Cas9 protein and sgRNA has been engineered by incorporating various modifications. Although the single guide RNA (sgRNA) modifications are more simple and straightforward. The aim of this proposal is to engineer sgRNA by incorporating a secondary structure element named RNA three-way Junction (3WJ) that is known in literature to be exceptionally thermostable and even stable in serum. The outcome of this proposal is two fold. The addition of 3WJ to sgRNA will make sgRNA more thermally stable to fold it into proper conformation to bind to Cas9 protein and will help in enhanced genome editing. It will also provide cellular stability to sgRNA to enhance residence time with on-target for better cleavage. On the other hand, 3WJ provides extra arms to sgRNA to attach various RNA modules such as RNA aptamers for enhanced imaging. RNA 3WJ has been used in RNA nanotechnology for constructing various 2D and 3D RNA nanoparticles including RNA dendrimers having several arms to attach functionalities. This proposal anticipates that the addition of RNA nanotechnology to CRISPR-Cas9 system would be advantageous not only for genome editing but also for genome imaging applications. |