Hey guys! Sorry for putting this out late. It took me some extra time to thoroughly explain everything I’m working on, yet keep it reader friendly and as concise as possible, and include some helpful visual aids. I put a lot of work into this one. Please read and comment!
As I’ve said before, my work involves both the Buchler Lab and the iGEM team. Dr. Buchler does research on ultrasensitivity and molecular titration and iGEM is using both of these concepts in their synthetic bio project this year: programmable cell death. So, I am doing my own research based on Dr. Buchler’s previous research that will help the iGEM team because due to limitations on time and resources, they are unable to do the extensive research on ultrasensitivity and molecular titration that would be applicable to their project.
Ultrasensitivity is a phenomenon in molecular biology where within an input/output system, steadily increasing the input will eventually cause a sudden and sharp increase in output. On a graph with input as X and output as Y, the curve would be S shaped.
Many cells use ultrasensitivity as a way to signal changes in the cell cycle and/or for important cell fate decisions. Ultrasensitivity can be generated via molecular titration, which is a buffering mechanism that earned its name for being similar to the titration of acids and bases in chemistry.
A is the active molecule and B is the titrating molecule. B sequesters A, forming an inactive complex, AB. However the concentration of B is constant, while A is constantly being synthesized. Eventually, A reaches the “threshold point” where it fills up the sink that B was creating. Without anymore B to sequester the active molecule, A quickly begins to accumulate, creating an ultrasensitive response. The strength of the ultrasensitivity is measured by the stoichiometric binding parameter, BT/Kd, the total concentration of the titrating molecule divided by the inactive complex dissociation constant (Buchler, 2008).
iGem works with CRISPR (clustered regularly interspaced short palindromic repeats), which is a prokaryotic immune system in which guide RNA (gRNA) are created that match the DNA sequence of viral DNA. The gRNA then directs the endonuclease protein, Cas9, to the viral DNA, at which point Cas9 destroys it. To learn more about it, check out this animation. The gRNA can be engineered so that instead of targeting viral DNA, it can target DNA within the prokaryote’s own genome. Furthermore, the Cas9 can be decatalyzed into dCas9. Without its endonuclease function, the dCas9 will simply sit on the DNA sequence that the gRNA directs it to. It will not destroy the sequence, but it will block the RNA polymerase from expressing it as mRNA. So, through engineering what was originally an immune system, CRISPR has become a tool to control gene expression.
CRISPR’s repression of gene expression creates a graded response. It is analogous to slowly turning a volume dial on a radio down to zero. But, what if CRISPR could be engineered to be more like an on/off switch, instantly repressing gene expression? If that were the case, then an ultrasensitive response would be created and the CRISPR system could be used as a signaling mechanism for cell fate decisions. iGEM is using an ultrasensitive CRISPR system to trigger the programmable cell death they are developing. My goal is to find ways to make the ultrasensitive response of CRISPR systems more powerful.
I am working on two methods that will increase ultrasensitivity through molecular titration. The first is decoy binding sites, which are DNA sequences that match the gRNA of CRISPR, but are not associated with the gene that is actually targeted for repression, so they cause the gRNA to direct the dCas9 to a false target. The dCas9/gRNA complex is the active molecule, A. The decoy binding sites are the titrating molecule, B. As long as there are decoy sites present, the dCas9/gRNA will bind to those sites instead of the actual target sequences on the gene, forming an inactive dCas9/gRNA/decoy binding site complex. However, once all the decoy sites are occupied, the dCas9/gRNA will actually start to bind to the true target sequences and the gene will be repressed. According to Dr. Buchler’s equation for the stoichiometric binding parameter that I mentioned before, BT/Kd, the repression should be ultrasensitive and the degree to which it is ultrasensitive should be directly proportional to the number of decoy binding sites I use (BT).
Last year, iGEM worked on decoy binding sites, but they were not able to achieve very strong ultrasensitivity, as seen in the graph below.
I am improving the decoy binding sites in two ways. First, I am increasing the number of decoy sites. Second, I am increase the binding affinity of the dCas9/gRNA complex to the decoy sites by making the gRNA and the decoy sites sequence a perfect match, while creating mismatches in the target sequence. Both of these techniques have the potential to increase ultrasensitivity.
The other method I am working on is multiple binding sites. I am creating gRNAs with different target sequences, but they all target the same gene.
Last year, iGEM created a theoretical model for this that showed great potential, but did not have the time nor resources to develop experiments.
I am first performing experiments using the decoy binding sites and the multiple binding sites separately, but my ultimate goal is to combine both into a CRISPR system that maximizes ultrasensitivity.
Woah, that was a lot! Thanks for hanging in there. As a reward, here are this week’s science memes.
