Kasturi001 (Talk | contribs) |
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+ | <p>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. </p> | ||
+ | <p>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. </p> | ||
+ | <p>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.</p> | ||
− | < | + | <img src="https://static.igem.org/mediawiki/2017/b/ba/POC_TP_1.jpg" > |
− | < | + | <img src="https://static.igem.org/mediawiki/2017/3/33/POC_TP_2.jpg" > |
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+ | <u>Extended incubation time</u> | ||
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− | + | <p>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. </p> | |
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− | < | + | <img src="https://static.igem.org/mediawiki/2017/8/81/72H_NTU.jpg" > |
− | < | + | <p> 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.</p> |
− | </ | + | <p>Incubation time may thus be a consideration for therapeutic application – for example, repeated dosages may be required when utilizing extensively truncated dCas9-VPR. </p> |
− | < | + | <img src="https://static.igem.org/mediawiki/2017/f/f0/ASCL1_48H.jpg" > |
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− | <p> | + | <u>Affinity enhanced truncated dCas9-VPR</u> |
− | <p> | + | <p>Endogenous tests is also performed for truncated dCas9-VPR that has improved target DNA affinity by mutation of key residues close to target DNA.</p> |
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+ | <img src="https://static.igem.org/mediawiki/2017/0/06/POC_TP_3.jpg" > | ||
+ | <img src="https://static.igem.org/mediawiki/2017/a/a9/POC_TP_4.jpg" > | ||
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+ | <p>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. </p> | ||
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+ | <u>Conclusion</u> | ||
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+ | <p>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.</p> | ||
+ | <p>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.</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. | ||
+ | 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. | ||
+ | </p> |
Revision as of 14:36, 1 November 2017