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.
Exogenous reporter gene activation is useful for quickly screening tCas9 created. However, it is a poor model for regulation of endogenous genes on chromosomes. Unlike on the reporter plasmid, chromosomal genes are controlled by promoters with varying levels of activity. These promoters are often already regulated by transcription factors, adding a level of variability of how well the gene may be upregulated. Lastly, the epigenetic landscape of endogenous genes may also play a role in impeding dCas9-VPR mediated gene regulation. Thus, there is a need to test our truncated dCas9 in the context of endogenous genes.
In this experiment, the reporter plasmid is no longer required. The sgRNA plasmid is retargeted to endogenous housekeeping genes such as ASCL1, TTN and MIAT. These genes were selected as parameters for upregulatory effects on these genes by dCas9-VPR has been previously established in literature. Cells are transfected and incubated for 48H, then sorted by flow-cytometry assisted cells sorting (FACS) for mCherry positive transfected cells. Quantitative assessment of tCas9 activity is then acquired by quantitative PCR (qPCR) for mRNA level of the housekeeping genes.
In continuation from last year’s project, better performing truncated dCas9s were tested on ASCL1 and MIAT. While data generally correlate with exogenous gene activation results, gene activation is by truncated dCas9-VPR is generally poorer than WT. This is even the case for ∆HNH, which had been shown to perform on par with WT in exogenous tests. ∆3ple and further truncations are unable to activate gene expression.
Extended incubation time
As the poor performance of truncated dCas9-VPR is likely due to poor affinity to target DNA, we posited that a longer incubation period may allow more time for the poorer binding truncated dCas9-VPR to interact with the promoters of genes, and better upregulate the genes. Indeed, prior experiments with longer incubation time in reporter experiments consistently demonstrated increased MFI.
Thus, a 72H incubation experiment for ASCL1 is performed. As seen in the results, gene activation by WT and well-performing truncated dCas9-VPR is only mildly affected by the longer incubation. However, poor performing truncated dCas9 such as ∆3ple, ∆3ple ∆REC1-3 and ∆3ple ∆REC1-1 ∆REC 1-3 appears to perform better.
Incubation time may thus be a consideration for therapeutic application – for example, repeated dosages may be required when utilizing extensively truncated dCas9-VPR.
Affinity enhanced truncated dCas9-VPR
Endogenous tests is 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 endogenous context, mirroring exogenous results. However, preliminary results for enhanced ∆3ple appears to only result in improvements for MIAT gene. Further replicates and tests with multiple mutations would be required to confirm results.
ConclusionIn 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 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 has been replicated in endogenous gene activation – a better model for therapeutic applications.
While results suggests that multiply truncated dCas9-VPR perform much more poorly in endogenous tests, this is to be expected. Other than aforementioned differences in endogenous gene environment, regulatory feedback loops are likely to dampen the upregulatory effects of dCas9-VPR. In addition, the genes tested are housekeeping genes, which already has a high basal expression level. In the context of therapeautic applications such as treatment of lost-of-function diseases, the upregulation effect would be more pronounced. Indeed, even a modest increase in gene expression could be helpful in some diseases.
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 does not necessarily improves specificity. The mutations performed improves non-specific affinity to DNA backbone, and thus may even further decrease specificity by improving binding to off-target sequences. Future work would include characterization of off-target effects, and if necessary, improvements to specificity.
