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+ | <title>Project</title> | ||
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+ | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_0" href="https://2016.igem.org/Team:Tsinghua">Home</a></span> | ||
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+ | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_1" href="https://2016.igem.org/Team:Tsinghua/Team">Team</a></span> | ||
+ | <ul class="subs"> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_0" href="https://2016.igem.org/Team:Tsinghua/Team">Team members</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_1" href="https://2016.igem.org/Team:Tsinghua/Attributions">Attributions</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_2" href="https://2016.igem.org/Team:Tsinghua/Acknowledgement">Acknowledgement</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_1_Link_3" href="https://2016.igem.org/Team:Tsinghua/Acknowledgement#tag_sponsorship">Sponsorship</a></li> | ||
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+ | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_2" href="https://2016.igem.org/Team:Tsinghua/Description">Project</a></span> | ||
+ | <ul class="subs"> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_0" href="https://2016.igem.org/Team:Tsinghua/Description">Description</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_1" href="https://2016.igem.org/Team:Tsinghua/Experiments">Design</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_2" href="https://2016.igem.org/Team:Tsinghua/Results">Results</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_3" href="https://2016.igem.org/Team:Tsinghua/Proof">Proof of Concepts</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_4" href="https://2016.igem.org/Team:Tsinghua/Notebook">Notebook</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_5" href="https://2016.igem.org/Team:Tsinghua/Model">Modeling</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_2_Link_5" href="https://2016.igem.org/Team:Tsinghua/Safety">Safety</a></li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="lvl1"> | ||
+ | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_3" href="https://2016.igem.org/Team:Tsinghua/Parts">Parts</a></span> | ||
+ | <ul class="subs"> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_0" href="https://2016.igem.org/Team:Tsinghua/Parts#tag_summary">Parts Summary</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_1" href="https://2016.igem.org/Team:Tsinghua/Basic_Part#tag_basic">Basic Parts</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_2" href="https://2016.igem.org/Team:Tsinghua/Composite_Part">Composite Parts</a></li> | ||
+ | <br> | ||
+ | <br> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="lvl1"> | ||
+ | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_3" href="https://2016.igem.org/Team:Tsinghua/Human_Practices">Human Practices</a></span> | ||
+ | <ul class="subs"> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_0" href="https://2016.igem.org/Team:Tsinghua/Human_Practices">Overview</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_1" href="https://2016.igem.org/Team:Tsinghua/Human_Practices#tag_engagement">Public Engagement</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_0" href="https://2016.igem.org/Team:Tsinghua/Collaborations">Collaboration</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_1" href="https://2016.igem.org/Team:Tsinghua/Community_meetup">Community Meetup</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_3_Link_2" href="https://2016.igem.org/Team:Tsinghua/Integrated_Practices">Integrated Practices</a></li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="lvl1"> | ||
+ | <span class="header"><a id="Galaxy1_Hamburger1_Burgers_Link_4" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist">Medal Checklist</a></span> | ||
+ | <ul class="subs"> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_4_Link_0" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist#bronze">Bronze</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_4_Link_1" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist#silver">Silver</a></li> | ||
+ | <li><a id="Galaxy1_Hamburger1_Burgers_Children_4_Link_2" href="https://2016.igem.org/Team:Tsinghua/MedalChecklist#gold">Gold</a></li> | ||
+ | <br> | ||
+ | <br> | ||
+ | </ul> | ||
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+ | <br> | ||
+ | <a href="https://2016.igem.org/Team:Tsinghua/Description">Description</a><br> | ||
+ | <br> | ||
+ | <a href="https://2016.igem.org/Team:Tsinghua/Experiments">Design</a><br> | ||
+ | <br> | ||
+ | <a href="https://2016.igem.org/Team:Tsinghua/Results">Results</a><br> | ||
+ | <br> | ||
+ | <a href="https://2016.igem.org/Team:Tsinghua/Proof">Proof of concepts</a><br> | ||
+ | <br> | ||
+ | <a href="https://2016.igem.org/Team:Tsinghua/Notebook">Notebook</a><br> | ||
+ | <br> | ||
+ | <a href="https://2016.igem.org/Team:Tsinghua/Model" target="_blank">Modeling</a><br> | ||
+ | <br> | ||
+ | <a href="https://2016.igem.org/Team:Tsinghua/Safety" >Safety</a><br> | ||
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+ | <h1 class="contentPage" id="tag_description">description</h1><br> | ||
+ | <h2 class="contentPage">Background</h2> | ||
+ | The fidelity of genomic sequence is the underpinning aspect of normal cellular activities. Even a minor | ||
+ | <br>disruption to DNA, the storage center of genetic information, can lead to detrimental consequences, | ||
+ | <br>including excessive cell death, abnormal proliferation, untimely cell senescence, etc. For example, | ||
+ | <br>genomic instability and mutation is considered as one of the ten hallmarks of cancer, the occurrence | ||
+ | <br>of which generally leads to over-activation of signaling pathways, in turn triggering malignant | ||
+ | <br> proliferation of cells<sup>[1]</sup>. Recurrent hot-spot DNA mutations and chromosomal rearrangements are | ||
+ | <br>commonly observed in cancer samples. Genomic mutation is therefore a primary concern in terms of both | ||
+ | <br>basic research and clinical treatment, demanding multi-disciplinary efforts. | ||
+ | <br> | ||
+ | <br>In order to address this imperative issue, a gene mutation surveillance system that can sensitively and | ||
+ | <br>efficiently detect and remove such aberrations to achieve real-time monitoring <i>in vivo</i> is required. | ||
+ | <br>Essentially by designing such a system, a tool that can recognize aberrant mutation on mRNA sequences, | ||
+ | <br> or more generally at the transcriptomics level, is desired, because transcripts are derived from | ||
+ | <br>genomic sequences and hence are representatives of genomic instability and mutations. More importantly, | ||
+ | <br> their availability and abundance in the cytoplasm allow them to be targeted. Nonetheless, few such | ||
+ | <br> satisfying molecular tools have been developed yet. Consequently, to design such a surveillance | ||
+ | <br>system is not only important but also necessary. | ||
+ | <br> | ||
+ | <br>To serve such purpose, an RNA binding factor with high efficiency and sensitivity is a must, which | ||
+ | <br> is able to distinguish RNA transcripts produced by mutated genes and switch on the suicidal gene | ||
+ | <br> expression afterwards. There are three major RNA targeting methods currently: PUF proteins<sup>[2][3][4]</sup>, | ||
+ | <br>molecular beacons<sup>[5]</sup> and RNA aptamers<sup>[6]</sup>. Due to lack of specificity or stability in vivo, those existing | ||
+ | <br> methods are not suitable for the potential application in our gene mutation surveillance system. | ||
+ | <br> However, it is newly reported that dCas9<sup>[7]</sup>, capable of recognizing specific endogenous mRNA | ||
+ | <br>sequences, turns out to be an ideal candidate. Surveillance Cas9 (suvCas9), termed for the first time | ||
+ | <br> by our team, is a variant of Cas9 without DNA cleavage activity and can target specific mRNA sequences | ||
+ | <br> by manually designed sgRNA and PAM sequences. With this remarkable ability to target mRNA, suvCas9 | ||
+ | <br> can hopefully be adopted into our surveillance system as the monitor as well as the executor | ||
+ | <br> | ||
+ | <br>We propose to construct suvCas9 system in <i>Saccharomyces cerevisiae</i> to function as the monitor | ||
+ | <br> of genomic sequence. <i>Saccharomyces cerevisiae</i> is one of the most commonly used model organisms, | ||
+ | <br> representing a simple eukaryote with a highly manipulatable genome. Apart from many physical properties | ||
+ | <br> and technical advantages, such as rapid growth, minimal pathogenicity, the ease of phenotype identification, | ||
+ | <br> a well-defined genetic system, and most importantly, a highly versatile DNA transformation system<sup>[11]</sup>, | ||
+ | <br> the yeast has been reported to share a similar characteristics of <i>in vivo</i> ssDNA generation by reverse | ||
+ | <br> transcriptase with prokaryotes<sup>[9]</sup>, which will guide suvCas9 to the targeted mRNA. | ||
+ | <br><br> | ||
− | < | + | <h2 class="contentPage">Project Overview</h2> |
+ | <br> | ||
− | < | + | <video style="width:768px;height:432px;margin-left:60px" controls> |
− | < | + | <source src="https://static.igem.org/mediawiki/2016/f/f1/T--Tsinghua--video.mp4" type="video/mp4"> |
− | < | + | </video> |
− | < | + | <br> |
− | < | + | <br> |
− | < | + | Genomic stability and DNA sequence fidelity are critical for normal cellular activities. If such storage |
− | </ | + | <br> center of genetic information is disrupted, detrimental consequences will take place, including but not |
+ | <br> limited to excessive cell death, abnormal proliferation, untimely cell senescence, etc. Therefore, a gene | ||
+ | <br> mutation surveillance system that can sensitively and efficiently detect and remove such aberrations | ||
+ | <br> <i>in vivo</i>is required. | ||
+ | <br> | ||
+ | <br><i>Saccharomyces cerevisiae</i> is chosen as the model organism for designing such a gene surveillance system. | ||
+ | <br> As one of the most commonly used tools, it not only has a highly manipulatable genome, rapid growth | ||
+ | <br> speed, minimal pathogenicity, an ensemble of selectable markers, a well-defined genetic system, and most | ||
+ | <br> importantly, a highly versatile DNA transformation system. | ||
+ | <br> | ||
+ | <br>Essentially by designing such a system, a tool that can recognize aberrant mutation on mRNA sequences, or | ||
+ | <br> more generally at the transcriptomics level, is desired, because transcripts are derived from genomic | ||
+ | <br> sequences and hence are representatives of genomic instability and mutations. Surveillance Cas9 (suvCas9), | ||
+ | <br> termed for the first time by our team, is a variant of Cas9 without DNA cleavage activity and can target | ||
+ | <br> specific mRNA sequences by manually designed sgRNA and PAM sequences. | ||
+ | <br> | ||
+ | <br> That being said, after mutation is detected on mRNAs, how can our system respond? In a nutshell, when | ||
+ | <br> mutations occur, the surveillance system will detect and trigger the suicidal system. The surveillance | ||
+ | <br> system can be further quantitatively optimized to maximize its sensitivity and minimize potential side | ||
+ | <br> effects. | ||
+ | <br> | ||
+ | <br> | ||
+ | <h2 class="contentPage">Contribution</h2> | ||
+ | This year we've also characterized and improved several parts, including the following three: | ||
+ | <br> BBa_K1323002, BBa_K1493504, BBa_K1470002. Please refer to their Registry pages for detailed | ||
+ | <br> documentation. | ||
+ | </div> | ||
+ | </div> | ||
− | </div> | + | <div class="contentPage2"> |
+ | <div class="contentPage" style="margin-left:200px;margin-top:20px;width:1130px;"> | ||
+ | <h2 class="contentPage"><br>Reference<img id="referenceButton" onclick="hideRef()" src="https://static.igem.org/mediawiki/2016/0/06/T--Tsinghua--details_open.png" style="align:center;"></h2> | ||
+ | <br> | ||
+ | <div id="reference" style="display:none;"> | ||
+ | [1] Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: the next generation. Cell, 144(5), 646-674. | ||
+ | <br>[2] Filipovska, A., Razif, M. F., Nygård, K. K., & Rackham, O. (2011). A universal code for RNA recognition | ||
+ | <br> by PUF proteins. Nature chemical biology, 7(7), 425-427. | ||
+ | <br>[3] Ozawa, T., Natori, Y., Sato, M., and Umezawa, Y. (2007). Imaging dynamics of endogenous mitochondrial | ||
+ | <br> RNA in single living cells. Nat. Methods 4, 413–419. | ||
+ | <br>[4] Wang, Y., Cheong, C.G., Hall, T.M., and Wang, Z. (2009). Engineering splicing factors with designed | ||
+ | <br> specificities. Nat. Methods 6, 825–830. | ||
+ | <br>[5] Sokol, D.L., Zhang, X., Lu, P., and Gewirtz, A.M. (1998). Real time detection of DNA.RNA hybridization | ||
+ | <br> in living cells. Proc. Natl. Acad. Sci. USA 95, 11538–11543. | ||
+ | <br>[6] Fouts, D.E., True, H.L., and Celander, D.W. (1997). Functional recognition of fragmented operator sites | ||
+ | <br> by R17/MS2 coat protein, a translational repressor. Nucleic Acids Res. 25, 4464–4473.<br> | ||
+ | <br>[7] Nelles, D. A., Fang, M. Y., O’Connell, M. R., Xu, J. L., Markmiller, S. J., Doudna, J. A., & Yeo, | ||
+ | <br> G. W. (2016). Programmable RNA Tracking in Live Cells with CRISPR/Cas9. Cell, 165(2), 488-496.<br> | ||
+ | <br>[8] Farzadfard, F., & Lu, T. K. (2014). Genomically encoded analog memory with precise in vivo DNA writing | ||
+ | <br> in living cell populations. Science, 346(6211), 1256272. | ||
+ | <br>[9] Miyata, S., Ohshima, A., Inouye, S., & Inouye, M. (1992). In vivo production of a stable single-stranded | ||
+ | <br> cDNA in Saccharomyces cerevisiae by means of a bacterial retron. Proceedings of the National Academy | ||
+ | <br> of Sciences, 89(13), 5735-5739. | ||
+ | <br>[10] Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., ... & Koonin, E. V. | ||
+ | <br> (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature, 520(7546), 186-191. | ||
+ | <br>[11] Sherman, F. (1991). [1] Getting started with yeast. Methods in enzymology, 194, 3-21.<br> | ||
+ | <br>[12] O’Connell, M.R., Oakes, B.L., Sternberg, S.H., East-Seletsky, A., Kaplan, M., and Doudna, J.A. (2014). | ||
+ | <br> Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266. <br> | ||
+ | <br>[13] Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E., & Boeke, J. D. (1996). Reverse two-hybrid and | ||
+ | <br> one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proceedings | ||
+ | <br> of the National Academy of Sciences, 93(19), 10315-10320. | ||
+ | <br>[14] Alexander, W. G., Doering, D. T., & Hittinger, C. T. (2014). High-efficiency genome editing and allele | ||
+ | <br> replacement in prototrophic and wild strains of Saccharomyces. Genetics, 198(3), 859-866. | ||
+ | <br>[15] Boeke, J. D., La Croute, F., & Fink, G. R. (1984). A positive selection for mutants lacking | ||
+ | <br> orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Molecular | ||
+ | <br> and General Genetics MGG, 197(2), 345-346. | ||
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Latest revision as of 02:58, 20 October 2016