Team:NTU SINGAPORE/Results
Proof of Concept
Truncation Project
Exogenous Gene ActivationExogenous gene activation of ZSGreen fluorescent reporter gene is first performed. Quantitative assessment of tCas9 activity is acquired by flow cytometry. 3 plasmids are utilized in exogenous gene activation. The reporter plasmid contains ZSGreen under TREtight minimal promoter preceded by 2X protospacers. The sgRNA plasmid expresses sgRNA under U6 promoter. This plasmid also contains a constitutive mCherry expression, to report successfully transfected cells during flow cytometry. The dCas9-VPR plasmid expresses dCas9-VPR under constitutive CMV promoter. dCas9 can then utilized the sgRNA to bind to protospacer, while the fused VPR greatly upregulates expression of ZSGreen. Cells are transfected and incubated for 48H. Flow cytometry is then perfomed by gating for mcherry positive cells indicated successful transfection. The ZSGreen mean fluorescence is then acquired for these cells, reporting the gene actvation activity of the truncated dCas9-VPR.
Improvements to truncated dCas9-VPR
We have demonstrated in our Improve section that ∆3ple is the best set of truncations we have achieved with our current approach. While we demonstrated a new application of ∆3ple for bacterial CRISPRi, we are still interested in using it for mammalian gene activation. Thus, to expand on our truncation approach, we are interested in improving our truncated dCas9 protein for gene activation, especially for ∆3ple.
Truncation of dCas9 led to poorer gene activation activity as expected. The folding of protein is essentially to achieve the lowest free energy state possible with the amino acids in a polypeptide chain – that is the protein’s native, functional state. Natural selection in evolution would select for the best amino acids capable of folding into secondary and tertiary structures properly. Thus, truncations to domains and subdomains would likely disrupt an otherwise finely tuned molecular machine.
Consider the crystal structure of dCas9. In the following images, the 3 key truncations were highlighted. Note the space left behind when the domains are deleted. While the truncations in this project are designed to minimally impact secondary structures, the loss of structural support by these domains and subdomains is bound to have led to disruptions in tertiary structure.
Images on the left indicates before truncation and the right images, after truncation. Note that the domains/subdomains are only removed from the structure; no predictions on alteration to structure were performed.
In addition, consider the hydrophobicity of protein surfaces. Soluble proteins such as Cas9 has a largely hydrophilic outer surface, coupled with a hydrophobic core. Such an organization greatly decreases free energy at native state – the hydrophilic surface interacts with the aqueous environment in the cytoplasm, while the hydrophobic sidechains are sequestered within the protein. The truncation of domains and subdomains, especially those near the surface of the protein, is likely to reveal the hydrophobic core. Such an example could be seen in the following image of ∆RuvCIII-2 truncation, which reveals the dark red hydrophobic core (indicated by a black arrow). To achieve a lower free energy state, the hydrophilic sidechains in the vicinity would likely move in to cover the hydrophobic core, thus disrupting tertiary structure.
Surface hydrophobicity of dCas9 before and after RuvCIII truncation, as calculated by color_h plugin in Pymol. Red indicates hydrophobic, while white indicates hydrophilic.
Indeed, very poor gene activation activity by some truncations and when excessive truncations were combined is likely a result in a completely misfolded or even insoluble protein.
Removal of domains and subdomains is likely to result in a disturbed tertiary structure, with DNA binding residues shifted to suboptimal positions. The resulting drop in target DNA binding activity is likely the biggest contributor to poor gene activation activity, especially for smaller dCas9s with multiple truncations.
Examples of mutations performed as indicated on the crystal structure of WT-Cas9 (above). Note its proximity to nucleic acid backbone (brown).
To test the hypothesis, as well as to improve the gene activation ability of our dCas9, mutation is done to improve target DNA binding affinity again. Crystal structure information of the wild type Cas9 were once again utilized to identify key residues close to DNA backbone, which were then mutated to positive charged side chains such as Arg or Lys. A total of 21 residues were identified and rapidly screened on ∆HNH ∆REC1-3 (indicated as ∆HNH ∆REC1-c in graph). Subsequent mentions of mutated residues are referred to as QC1-21.
Affinity enhanced truncated dCas9-VPR
Endogenous tests were also performed for truncated dCas9-VPR that has improved target DNA affinity by mutation of key residues close to target DNA.
Enhanced ∆REC1-3 ∆HNH results indicated that mutations to enhance target DNA binding do indeed improve gene activation in the endogenous context, mirroring exogenous results. However, preliminary results for enhanced ∆3ple appear to only result in improvements for MIAT gene. Further replicates and tests with multiple mutations is required to confirm results.
Conclusion
In conclusion, our team has built upon past work on truncated dCas9 for gene activation. We explored the truncation of a new subdomain, REC3. In addition, we also explored new combinations of truncations and its effect on gene activation. Multiple truncations led to a decrease in gene activation, likely due to decreased target DNA affinity. We demonstrated that mutations to DNA backbone proximal residues to improve target DNA affinity can improve gene activation by multiply truncated dCas9-VPR. Results in exogenous gene activation have been replicated in endogenous gene activation – a better model for therapeutic applications.
While results suggest that multiply truncated dCas9-VPR perform much more poorly in endogenous tests, this is to be expected. Other than the aforementioned differences in endogenous gene environment, regulatory feedback loops are likely to dampen the up-regulatory effects of dCas9-VPR. In addition, the genes tested are housekeeping genes, which already have high basal expression levels. In the context of therapeutic applications such as in the treatment of loss-of-function diseases, the upregulatory effect will be more pronounced. Indeed, even a modest increase in gene expression could be helpful in some diseases.
More importantly, we have established that improving DNA binding specificity in our truncated dCas9 is a viable way to improve gene activation, both in exogenous and endogenous gene activation. Other methods to improve DNA binding can be explored. The truncations performed are likely to have negatively impacted specificity, in addition to affinity observed. It is worth noting that the mutations applied to improve DNA backbone affinity in our multiply truncated dCas9-VPR do not necessarily improve specificity. The mutations performed improve non-specific affinity to the DNA backbone, and thus may even further decrease specificity by enhancing binding to off-target sequences. Future work will include the characterization of off-target effects, and if necessary, improvements to specificity.
In our human practices, we held a workshop to educate the public on intricacies of utilizing Cas9 for therapeutic applications. We also introduced the public to novel applications of Cas9 beyond genome editing – for example, fusion to transcription activators for gene regulation. We concluded the workshop with the analogy of Cas9 being a molecular Swiss army knife.
