Summary
I'm currently focused on enhancing the contractile injection system of the bacterium P. asymbiotica to broaden its potential applications. The contractile injection system is a specialized mechanism to introduce proteins into host cells with precision and efficiency. My project involves enabling the system to deliver guide RNA (gRNA) along with Cas9 as payloads. In previous experiments, it was demonstrated that this system can effectively deliver Cas9. However, in those experiments, the target cell had to contain the necessary gRNA already.
By incorporating both gRNA and Cas9 as payloads in the contractile injection system, I aim to simplify and improve genetic manipulation processes. This improvement eliminates the need for the target cell to independently possess gRNA, making the delivery system more versatile and efficient for precise gene editing and regulation.
State of art
The extracellular contractile injection systems (eCISs) can be reprogrammed to target new cells by engineering the Pvc13 protein, the system's recognition element. By modifying this protein with specific binding domains, such as the adenovirus 5 (Ad5) binding domain, the system can be retargeted to recognize and deliver payloads to human cells . The system's protein delivery capability was demonstrated by loading retargeted PVCs with Pvc13-Ad5-knob fused with Cas9. This resulted in the specific insertion and deletion of genes in human cells, showcasing the system's ability to deliver various payloads, including large proteins like Cas9 (Kreitz et. al, 2023).
Studies have utilized cryo-EM to analyze the structures of PVC particles, revealing key proteins and their interactions. Models have been built and refined based on density maps, allowing for a deeper understanding of the tube and sheath proteins in PVC particles. Additionally, bioinformatics analyses have been conducted to further characterize these components (Jiang et. al, 2019)
Innovative
My project is innovative in enhancing the contractile injection system of P. asymbiotica. The innovation lies in integrating a specialized linker into the polypeptide structure of the tube that contains the payloads. This linker facilitates the binding of guide RNA (gRNA), enabling the system to deliver it. This novel method streamlines genetic manipulation and allows for the simultaneous delivery of guide RNA (gRNA) and Cas9.
Significance
My final project holds significant significance in addressing a pressing problem in genetic engineering and biotechnology. By enhancing the contractile injection system of P. asymbiotica to deliver guide RNA (gRNA), it leverages the system's inherent specificity to improve precision in genetic manipulation.
The importance of this problem lies in its impact on various fields such as medicine, agriculture, and biotechnology. Efficient and targeted gene editing has the potential to revolutionize disease treatments, crop improvements, and biotechnological applications. This project contributes to society by providing a versatile tool for researchers and practitioners to conduct more precise genetic modifications, leading to advancements in healthcare, food security, and environmental sustainability.
Aims