Team:Hong Kong HKUST/Application
Artificial Control of Cell Cycle Arrest Using Combined CRISPRi and Antisense RNA
Our CRISPRi and asRNA toggle switch can accomplish everything a traditional two-state toggle switch can do, but it’s FASTER, MORE FLEXIBLE AND MORE SPECIFIC! We hope the CRISPRi and asRNA toggle switch can replace traditional switches and be applied widely to biological systems to attain bistability or even multistability.
Below, we have introduced one such preliminary application idea that, we believe, would benefit from the use of our circuit. However, our project is aimed to tackle not just one case, but a myriad of purposes that scientists can apply this tool to.
For instance, a popular topic in synthetic biology is to create circuits that resemble electronic ones. Bistable toggle switches can serve as an RS latch, which is a de-synchronized flip-flop device that can either induce 2 states or reset the switch. The circuit of an RS latch and its truth table is shown in the figure below.
Nevertheless, there are two problems that prevent scientists from successfully making a reliable RS latch with existing switch designs. Firstly, existing switches change states very slowly, making the addition of feedback loops not applicable as they don’t synchronize. The drastic difference between the fast-paced response of the feedback loop and the slow response of the state switching results in a mismatch in the circuit. Secondly, existing toggle switches often “glitch”, meaning that they oscillate between the on and off state slightly.
Our new toggle switch design may have the potential to solve the aforementioned problems given its unique characteristics. As the RNA in our circuit does not need to be translated, our toggle switch responds much faster than existing factor-based switches. This also alleviates the burden of producing more proteins from the cell. Moreover, the affinity between sgRNA and dCas9 is highly specific and effective, given that no competing RNA factors are involved, which may result in less noise.
We can further expand our circuit into a gated-RS latch, a combination of multiple RS latches. Another possibility is to assemble the switch with other components to make it more versatile. In short, we provided a genetic circuit element of superior quality to the synthetic biology community!
A proposed application of a biological switch is to study cell development. The cell cycle can be divided into 4 different states: G1, S, G2, and M. The procession of different states is regulated by the concentration of different types of cyclin, which activates cyclin-dependent kinases (CDK). There are three main ways to perform cell cycle arrest: adding inhibitors that modify CDKs or related proteins, inducing with metallic nanoparticles or activating repressor proteins that will inhibit CDK. We propose to use a multiple lane circuit to directly suppress the level of different cyclins, fixing the cell in a particular state, as illustrated in the figure below. By reversing the switch, we can resume the cyclin concentration and progress to the next cell cycle state. This is a convenient way to study individual cell cycles in cells where apoptosis-related genes, such as HeLa, have been knocked out.
References:
[1] N. *, “Latches: Types, Advantages, Disadvantages, and Their Applications,” ElProCus, 04-Mar-2019. [Online]. Available: https://www.elprocus.com/basics-of-latches-in-digital-electronics/. [Accessed: 21-Oct-2019].
[2] “BOM Tool,” All About Circuits. [Online]. Available: https://www.allaboutcircuits.com/textbook/digital/chpt-10/s-r-latch/. [Accessed: 21-Oct-2019].
[3] D. A. Ferenbach and J. V. Bonventre, “The Molecular Response to Renal Injury,” Kidney Development, Disease, Repair and Regeneration, pp. 367–379, 2016.
[4] D. A. Ferenbach and J. V. Bonventre, “The Molecular Response to Renal Injury,” Kidney Development, Disease, Repair and Regeneration, pp. 367–379, 2016.
[5] P. Hillenbrand, G. Fritz, and U. Gerland, “Biological Signal Processing with a Genetic Toggle Switch,” PLoS ONE, vol. 8, no. 7, 2013.
[6] Y. Li, J. Fan, and D. Ju, “Neurotoxicity concern about the brain targeting delivery systems,” Brain Targeted Drug Delivery System, pp. 377–408, 2019.
[7] S. O’Grady and M. W. Lawless, “Liver Cancer (Hepatocellular Carcinoma),” Epigenetic Cancer Therapy, pp. 269–288, 2015.