Team:NTU SINGAPORE/Results
Proof of Concept
Truncation Project
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. The graphical form of the results are shown below. 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.
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.
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 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 suggest 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 up-regulatory effects of dCas9-VPR. In addition, the genes tested are housekeeping genes, which already has a high basal expression level. In the context of therapeutic applications such as treatment of loss-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 improve 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.
HDR Project
For this experiment, we fused Rad52, a HDR protein. Rad52 helps to repair DSB by promoting ssDNA annealing between homologous sequence, then assist in homologous pairing process. Then, the plasmid was transfected to HEK293FT cells.
We used plasmid constructed by Dr Yuan Ming. The plasmid expresses SpCas9 fused with Rad52, and then linked with P2A to a mCherry reporter. P2A is a self-cleaving protein. If this chain of protein is expressed, the P2A will self-cleave and release mCherry, hence the cell expressing it will fluoresce red. In other words, cells that are successfully transfected will fluoresce red. In the same plasmid, the sgRNA was cloned in and constitutively expressed under human U6 promoter.
Schematics of the parts of the plasmid
HDR needs the presence of a donor template which contains homologous sequence. In order to detect the presence of HDR, we used a GFP plasmid donor. In this plasmid, there is a GFP gene bearing homology sequence to the gene we targeted. This donor sequence was transfected with the plasmid with fusion protein and guide RNA. If there is a DSB resulting from the cutting activity, the cell can use the GFP donor to repair the DSB, and consequently, GFP will be expressed. Hence, if we could measure HDR percentage indirectly by analyzing the percentage of cells expressing the GFP protein.
We used flow cytometry to analyse the cells expressing the fluorescent proteins (mCherry and GFP) seven days after transfection. We gated for living cells based on the forwards and side scatter, and the plotted in two-dimensional plot with respect to signal detected by FITC and PE channel of flow cytometer to capture GFP and mCherry fluorescence, respectively. The HDR efficiency was determined from the GFP knock-in percentage of successfully transfected cells.
A representative sample of how the knock-in event is calculated
At first we tested for GAPDH gene. We were happy because the result showed that there are more cells expressing GFP if transfected with Rad52 fusion protein. This means that HDR level improved by expressing this protein. Moreover, the percentage of HDR using fusion protein is more than twice compared to the wild-type Cas9.
Percentage of knock-in event for GAPDH gene. The data were normalized to an empty construct. Error bar is ± s.d.
In order to confirm our result, we also tested the construct targeting CLTA and GLUL gene. In these two genes, Rad52-fusion is also shown to improve HDR events.
Our results showed that the Rad52-SpCas9 fusion was able to promote HDR level in HEK293FT cells for all the genes tested. Although it is still unknown how this protein would improve the efficiency, we hypothesized that now Rad52 is available immediately after the DSB occured, and hence outcompete NHEJ from repairing the DSB.
