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<u>Affinity enhanced truncated dCas9-VPR</u> | <u>Affinity enhanced truncated dCas9-VPR</u> | ||
− | <p>Endogenous tests | + | <p>Endogenous tests are also performed for truncated dCas9-VPR that has improved target DNA affinity by mutation of key residues close to target DNA.</p> |
<img src="https://static.igem.org/mediawiki/2017/0/06/POC_TP_3.jpg" > | <img src="https://static.igem.org/mediawiki/2017/0/06/POC_TP_3.jpg" > | ||
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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.</p> | 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.</p> | ||
<p> | <p> | ||
− | 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 | + | 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 have been replicated in endogenous gene activation – a better model for therapeutic applications. |
</p> | </p> | ||
− | <p>While results | + | <p>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 therapeautic 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.</p> |
<p>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. | <p>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. | 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. | ||
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<p> | <p> | ||
− | For this experiment, we fused Rad52, | + | For this experiment, we fused Rad52, an HDR protein. Rad52 helps to repair DSB by promoting ssDNA annealing between homologous sequence, then assist in the homologous pairing process. Then, the plasmid was transfected into HEK293FT cells. |
</p> | </p> | ||
<p> | <p> | ||
− | We used plasmid constructed by Dr Yuan Ming. The plasmid expresses SpCas9 fused with Rad52 | + | 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 a human U6 promoter. |
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2017/a/a9/Plasmid.png"> | <img src="https://static.igem.org/mediawiki/2017/a/a9/Plasmid.png"> | ||
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<p> | <p> | ||
− | 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 | + | HDR needs the presence of a donor template which contains the 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 action, 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. |
</p> | </p> | ||
<p> | <p> | ||
− | We used flow cytometry to | + | We used flow cytometry to analyze 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 the two-dimensional plot with respect to signal detected by FITC and PE channel of the flow cytometer to capture GFP and mCherry fluorescence, respectively. The HDR efficiency was determined from the GFP knock-in percentage of successfully transfected cells (subsequently referred as knock-in). |
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2017/1/16/Facs.png"> | <img src="https://static.igem.org/mediawiki/2017/1/16/Facs.png"> | ||
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<p> | <p> | ||
− | 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. | + | At first, we tested for GAPDH gene. We were happy because the result showed that there are more cells expressing GFP if transfected with the 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. |
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2017/6/6d/GAPDH.png"> | <img src="https://static.igem.org/mediawiki/2017/6/6d/GAPDH.png"> | ||
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<p style="text-align:center"> | <p style="text-align:center"> | ||
− | Percentage of knock-in event for GAPDH gene. The data were normalized to empty construct. Error bar is +- s.d. | + | Percentage of the knock-in event for GAPDH gene. The data were normalized to empty construct. Error bar is +- s.d. |
</p> | </p> | ||
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<p style="text-align:center"> | <p style="text-align:center"> | ||
− | Percentage of knock-in event for CLTA gene. The data were normalized to empty construct. Error bar is +- s.d. | + | Percentage of the knock-in event for CLTA gene. The data were normalized to empty construct. Error bar is +- s.d. |
</p> | </p> | ||
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<p style="text-align:center"> | <p style="text-align:center"> | ||
− | Percentage of knock-in event for GLUL gene. The data were normalized to empty construct. Error bar is +- s.d. | + | Percentage of the knock-in event for GLUL gene. The data were normalized to empty construct. Error bar is +- s.d. |
</p> | </p> | ||
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<p> | <p> | ||
− | 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 | + | 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 occurred, and hence outcompete NHEJ from repairing the DSB. |
</p> | </p> | ||
<div class="separator" id="EGFR" style="border-bottom: 0px;"></div> | <div class="separator" id="EGFR" style="border-bottom: 0px;"></div> | ||
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− | After cloning, the plasmids were transfected into PC9 cells. Since the plasmids contain the mCherry reporter gene which codes for red fluorescence, successfully transfected cells were sorted via FACS (RFP+) after 24h and 48h. We then extracted the genomic DNA from these cells and PCR | + | After cloning, the plasmids were transfected into PC9 cells. Since the plasmids contain the mCherry reporter gene which codes for red fluorescence, successfully transfected cells were sorted via FACS (RFP+) after 24h and 48h. We then extracted the genomic DNA from these cells and PCR amplified the targeted genomic locus. The amplified products were tested for detection and quantification of indels using T7 endonuclease I (T7E1). |
</p> | </p> | ||
<p> | <p> | ||
− | In T7E assay, the PCR products are denatured and reannealed to allow heteroduplex formation between wild-type DNA and CRISPR/Cas9-mutated DNA. T7E1, which recognizes and cleaves mismatched DNA, is used to digest heteroduplexes. The resulting cleaved and full-length PCR products are visualized by agarose gel electrophoresis. The image of the gel is shown under the proof of concept. The diagram below shows the general workflow for T7E assay. | + | In a T7E assay, the PCR products are denatured and reannealed to allow heteroduplex formation between wild-type DNA and CRISPR/Cas9-mutated DNA. T7E1, which recognizes and cleaves mismatched DNA, is used to digest heteroduplexes. The resulting cleaved and full-length PCR products are visualized by agarose gel electrophoresis. The image of the gel is shown under the proof of concept. The diagram below shows the general workflow for T7E assay. |
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2017/1/1b/Egfr_method_2.jpg" > | <img src="https://static.igem.org/mediawiki/2017/1/1b/Egfr_method_2.jpg" > |
Revision as of 19:12, 1 November 2017