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Antibiotics are among the most frequently administered drugs in human medicine. However, incorrect dosing and failure to complete the prescribed course have contributed to microbes becoming resistant.
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<h3> Our Proposed Solution:</h3>
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<p class="body-cont w4-center"  style="font-family:Lora; font-size:18px">Cas9, when bound to guide RNA, results in a versatile gene editing tool that gives rise to a wide range of potential applications. Through non-homologous end joining of the double stranded break, bases are added and subtracted knocking out the gene of interest. Scientists hope to use this system to battle infectious diseases and treat multi-drug resistant microorganisms.<a href="http://onlinelibrary.wiley.com/doi/10.1111/cmi.12693/full"><sup>(1)</sup></a> Despite the promise behind CRISPR-Cas9, the shift from its use as a research tool to a therapeutic device poses many challenges such as undesirable host immune responses and cleavage in unwanted locations due to low system specificity.  Our team is researching the use of Outer Membrane Vesicles (OMVs) as a Cas9 delivery system. We hypothesize that the successful packaging and delivery of Cas9, when combined with a specific guide RNA that defines the genomic target, could be used in a therapeutic setting to combat antibiotic resistance.</p>
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<h3>Cas9 Delivery</h3>
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Typical methods for delivery of Cas9 into cell cultures include electroporation, nucleofection, and lipofectamine-mediated transfection.<a href="http://online.liebertpub.com/doi/abs/10.1089/hum.2015.069"><sup>(2)</sup></a> However, these methods work exclusively for <i>in vitro</i> delivery of Cas9, and not <i>in vivo</i>.  Thus, an alternative delivery system to transport this protein to target cells is needed.
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Two common in vivo methods for Cas9 delivery are viral vectors and hydrodynamic injection. The latter of these two approaches resulted in both liver and cardiovascular damage to mice, and therefore does not seem viable for humans. Alternatively, bacteriophages have been effectively used for the delivery and expression of genes, but fall short in their size limitation and their potential to harm the host’s immune system.<a href="http://online.liebertpub.com/doi/abs/10.1089/hum.2015.069"> <sup>(2) </sup></a>
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Thus, we believe that a non-viral approach is optimal and have selected <b>OMVs</b> as the simplest, most viable option for delivery of Cas9 as well as the guide RNA.
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<h3>What are OMVs?</h3>
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Outer Membrane Vesicles, or OMVs, are spheroid structures whose lipid barrier resembles that of the outer membrane of Gram-negative bacteria and internal composition shows a high degree of similarity with the bacterial periplasm. OMVs pinch off from the outer cell membrane and move independently from the host cell, allowing them to serve numerous functions such as the removal of toxic compounds and regulation of bacterial colonies by facilitating cell-cell communication.<a href="https://www.ncbi.nlm.nih.gov/pubmed/26373371"><sup>(3)</sup></a>Because OMVs are innately produced by bacteria, they are excellent candidates for non-viral lipid-based delivery systems.
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<figcaption><a href="http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12048/full">Fig1: OMVs forming and pinching off from the outer membrane.<sup>(4)</sup></a></figcaption>
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<p class="body-cont w4-center" style="font-family:Lora; font-size:18px">This summer, our team will tackle three key features of this project: the incorporation of Cas9 into OMVs, the packaging of guide RNAs into separate OMVs and finally the fusion of these vesicles with target cells and distribution of their contents.</p>
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<h3><i>Delivery of Cas9 to target cell</i></h3>
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Our team will make use of an <i>E.coli</i> strain that is genetically engineered to hypervesiculate (JC8031). These bacteria will be transformed with a plasmid that allows the metabolic production of the Cas9 protein attached to amino acid sequences which have been found to be good candidates for periplasmic localization. To test for the presence of Cas9 in different cell compartments we will make use of fractionation - lysing only the outer membrane of the cell. Following the confirmation of Cas9 production, we will proceed by analyzing the contents of OMVs purified by the transformed strain.
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  <figcaption><a href="https://www.nature.com/articles/ncomms10515">Fig2: OMV forming from periplasmic content.<sup>(5)</sup></a></figcaption>
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<p class="body-cont w4-center" style="font-family:Lora; font-size:18px">The team will then separate the OMVs from the original cells using ultracentrifugation. Then, we will take a sample, lyse the cells, and test for Cas9 using a western blot. Once the presence of Cas9 in the OMVs has been confirmed, the OMVs will be introduced to a strain of <i>E. coli</i> (DH5-Alpha) with a gene for antibiotic resistance-- the same gene that the Cas9-CRISPR complex will have been programmed to cut.  When introduced, the OMVs will fuse with the target cell and the Cas9 will be introduced to the periplasm of the antibiotic resistant bacteria.
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<h3><i>Delivery of gRNA to target cell</i></h3>
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<p class="body-cont w4-center" style="font-family:Lora; font-size:18px" ><p class="body-cont w4-center" style="font-family:Lora; font-size:18px">To introduce the gRNA to OMVs, the team looked at a few different methods: Electroporation, chemical induction, and cholesterol-binding. Electroporation has proven to be a viable method for introducing synthetic RNA into periplasm in other contexts, but cell death is a possibility.<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612299/"><sup>(6)</sup></a> However, due to very few protocols on chemically inducing gRNA uptake and introducing gRNA through cholesterol-binding, we still chose electroporation as our method to get gRNA into the periplasm of the cell. Using the same hypervesicular strain of <i>E. coli</i> to maximize our chances of gRNA ending up in OMVs, we will electroporate the cells in a gRNA rich medium to introduce the gRNA into the periplasm of the cell. Then, when OMVs form, we expect to see gRNA in the vesicles as well (which can be tested by adding a fluorescent dye to the guide RNA). These OMVs will then fuse with the same target cell as the Cas9 OMVs.
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<h3><i>Fusion of OMVs to target cell</i></h3>
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<p class="body-cont w4-center" style="font-family:Lora; font-size:18px"><p class="body-cont w4-center" style="font-family:Lora; font-size:18px">Our team wants to determine exactly how OMVs fuse to cells and what types of cells accept OMVs. To do this, we will put CFSE (a hydrophobic dye) inside the vesicles. With this dye and a microscope, we will be able to observe the OMV as it fuses to the cell and confirm that the contents of the OMV end up in the target cell's periplasm. Additionally, we can test the OMV's ability to fuse with different types of bacteria such as Gram-positive and Gram-negative. By observing how OMVs interact with different types of cells, we will gain a better understanding of the concepts behind our experiment.</p></p>
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<h3><center>What is the problem we are trying to solve?</h3></center>
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<p class="body-cont w4-center"><a href="http://onlinelibrary.wiley.com/doi/10.1111/cmi.12693/full"><sup>(1)</sup></a> Doerflinger, M., Forsyth, W., Ebert, G., Pellegrini, M., & Herold, M. (2016). CRISPR/Cas9-The ultimate weapon to battle infectious diseases? Cellular Microbiology, 19(2). doi:10.1111/cmi.12693
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<p class="body-cont w4-center"><a href="http://online.liebertpub.com/doi/abs/10.1089/hum.2015.069"><sup>(2)</sup></a> Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nature Reviews Microbiology,13(10), 605-619. doi:10.1038/nrmicro3525
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<p class="body-cont w4-center"><a href="https://www.ncbi.nlm.nih.gov/pubmed/26373371"><sup>(3)</sup></a> Berleman, J., & Auer, M. (2012). The role of bacterial outer membrane vesicles for intra- and interspecies delivery. Environmental Microbiology, 15(2), 347-354. doi:10.1111/1462-2920.1204
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<p class="body-cont w4-center"><a href="http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12048/full"><sup>(4)</sup></a> Li Ling, He Zhi-Yao, Wei Xia-Wei, Gao Guang-Ping, and Wei Yu-Quan. Human Gene Therapy. June 2015, 26(7): 452-462. https://doi.org/10.1089/hum.2015.069
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<p class="body-cont w4-center"><a href="https://www.nature.com/articles/ncomms10515"><sup>(5)</sup></a> Roier, S., Zingl, F. G., Cakar, F., Durakovic, S., Kohl, P., Eichmann, T. O., . . . Schild, S. (2016). A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria. Nature Communications, 7, 10515. doi:10.1038/ncomms10515 </p>
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<p class="body-cont w4-center"><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612299/"><sup>(6)</sup></a> Sjöström, A. E., Sandblad, L., Uhlin, B. E., & Wai, S. N. (2015). Membrane vesicle-mediated release of bacterial RNA. Scientific Reports, 5, 15329. doi:10.1038/srep15329</p>
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<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big"> <br>Antibiotics are among the most frequently administered drugs in human medicine. However, overprescription and failure to complete the prescribed course have contributed to <b>microbes becoming resistant.</b> In 2014, the World Health Organization classified antibiotic resistance as a global epidemic, highlighting the need for a solution. Inactivating resistance genes via Cas9 nuclease-mediated cleavage has been shown to be an effective means of combating this epidemic; however, methods of in vivo delivery are currently limited.<font></p>
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  <p style= "font-size: 24px"; class="w3-center"><em> Get to know the brains behind the brawn.<br> Click on the images to make them bigger and learn more about 2017 NU iGEM team !</em></p><br>
 
  
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<h3><center>Our proposed solution</h3></center>
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<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big"> Cas9, when bound to guide RNA, results in a versatile gene editing tool that gives rise to a wide range of potential applications. Through non-homologous end joining of the double stranded break, bases are added and subtracted knocking out the gene of interest. Scientists hope to use this system to battle infectious diseases and treat multi-drug resistant microorganisms.<a href="http://onlinelibrary.wiley.com/doi/10.1111/cmi.12693/full"><sup>(1)</sup></a> Despite the promise behind CRISPR-Cas9, the shift from its use as a research tool to a therapeutic device poses many challenges such as undesirable host immune responses and cleavage in unwanted locations due to low system specificity. Our team is researching the use of <b>Outer Membrane Vesicles (OMVs) as a Cas9 delivery system</b>. We hypothesize that the successful packaging and delivery of Cas9, when combined with a specific guide RNA that defines the genomic target, could be used in a therapeutic setting to combat antibiotic resistance.<font></p>
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      <img src="https://static.igem.org/mediawiki/2017/8/8a/T-Northwestern_headshot_chuck.jpeg" style="width:75%" onclick="onClick(this)" class="w3-hover-opacity" alt= "Charley, Major: Mechanical Engineering, Year: Junior || If you need any proof that irony is a fickle mistress, I am a local Evanston townie who grew up making fun of nerdy NU students. As a Wildcat, I spend my days as a mechanical engineering student who loves math and building things. Some of my many eclectic interests include product design, hydroponics, manufacturing, renewable energy, and why people are interested in the Kardashians. When I’m not doing STEM related activities, you can find me at the beach, a Will Ferrel Movie, or doing a cheesy 90’s workout video.">
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      <img src="https://static.igem.org/mediawiki/2017/f/f1/T-Northwesternkaterina.jpg" style="width:100%" style= "height:150%" onclick="onClick(this)" class="w3-hover-opacity" alt="Katerina, Major: Biomedical Engineering, Year: Senior || I am a fourth-year Biomedical Engineering student hoping to pursue medical school following graduation. Having grown up in Athens, Greece scientific terminology does not intimidate me, although I still struggle to pronounce the most obscure names of bacteria. I am currently involved in imaging research for cancer diagnostics, but recently became fascinated by the field of synthetic biology and by how we can engineer bacteria to do the unimaginable. When not in lab, I’m probably reading mystery novels, solving Rubik’s cube puzzles or trying to improve my tennis serve.
 
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<h3><center>Cas9 delivery</h3></center>
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<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big"> Typical methods for delivery of Cas9 into cell cultures include electroporation, nucleofection, and lipofectamine-mediated transfection.<a href="http://online.liebertpub.com/doi/abs/10.1089/hum.2015.069"><sup>(2)</sup></a> However, these methods work exclusively for in vitro delivery of Cas9, and not in vivo. Thus, an alternative delivery system to transport this protein to target cells is needed. Two common in vivo methods for Cas9 delivery are viral vectors and hydrodynamic injection. The latter of these two approaches resulted in both liver and cardiovascular damage to mice, and therefore does not seem viable for humans. Alternatively, bacteriophages have been effectively used for the delivery and expression of genes, but fall short in their size limitation and their potential to harm the host’s immune system. Thus, we believe that a non-viral approach is optimal and have selected OMVs as the <b>simplest, most viable option for delivery of Cas9 as well as the guide RNA.</b><font></p>
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Major: Biomedical Engineering
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Year: Junior ||
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I’m a junior studying Biomedical Engineering. I have had very little wet lab experience coming into iGEM, but I am always up for the challenge and excited to get my feet wet (see what I did there?). Genetic engineering fascinates me and I’m ready to take part in furthering the field. I’m from Chicago, born and bred, something I almost never fail to mention.When I find free time, I like to take pictures, watch Christopher Nolan movies, listen to rap and exploring the city.
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Major: Chemical Engineering
 
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I’m a senior studying chemical engineering and pursuing a minor in biotechnology. My interest in research began after entering a biomedical research lab for the first time at the age of six (possibly illegally), on a trip with my mom to pick up my dad after work. After around 6 summers of derailing graduate students’ research on occasion, I’ve found my place in iGEM. I’m excited to see what research is ‘Really About’ and experience the inner workings of the entire research design process.  
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<h3><center>What are OMVs</h3></center>
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<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big"> Outer Membrane Vesicles, or OMVs, are spheroid structures whose lipid barrier resembles that of the outer membrane of Gram-negative bacteria and internal composition shows a high degree of similarity with the bacterial periplasm. OMVs pinch off from the outer cell membrane and move independently from the host cell, allowing them to serve numerous functions such as the removal of toxic compounds and regulation of bacterial colonies by facilitating cell-cell communication. <a href="https://www.ncbi.nlm.nih.gov/pubmed/26373371"><sup>(3)</sup></a> Because OMVs are innately produced by bacteria, they are excellent candidates for non-viral lipid-based delivery systems.<font></p>
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When I am able to escape tech, I can be found stuffing myself with ramen, taking creeper shots of dogs, and drinking copious amounts of milk tea.
 
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I’m a second-year chemical engineering student that will hopefully end up with a master’s in biotechnology. My first and last exposure to biology was in freshman year AP biology, but that was all I needed to dedicate my life to synthetic biology. I believe the key to healthcare is through genes, and I’ll be darned if I don’t find it. When I’m not struggling to understand things in the lab, I’m producing music for my brother’s friends, watching documentaries, or skyping my 4 pets/kids.
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Major: Electrical Engineering
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I am a second-year electrical engineering student interested in everything STEM related. Although I work in an astrophysics lab on campus, I’m excited to pursue my interest in synthetic biology and its applications through iGEM 2017. When I’m not working, I spend my time building rockets, beating my friends in FIFA, and rewatching the American Pie movies.
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I am a junior majoring in Chemical Engineering and Music Composition, and most of my research experience before iGEM was actually concentrated in ethnomusicology. Even though I grew up in both Sweden and Germany, I went to Northwestern because of my interest in their Dual Degree program. I have always been fascinated with research at the intersection of chemistry and biology, and I hope to one day obtain my PhD in a related field.
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<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big"> <br><a href="http://onlinelibrary.wiley.com/doi/10.1111/cmi.12693/full"><sup>(1)</sup></a>      Doerflinger, M., Forsyth, W., Ebert, G., Pellegrini, M., & Herold, M. (2016). CRISPR/Cas9-The ultimate weapon to battle infectious diseases? Cellular Microbiology, 19(2). doi:10.1111/cmi.12693</font></p>
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<p style="padding-top:2%; padding-right: 15%; padding-left:15%; font-size:14px;" class="big"> <a href="http://online.liebertpub.com/doi/abs/10.1089/hum.2015.069"><sup>(2)</sup></a>      Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nature Reviews Microbiology,13(10), 605-619. doi:10.1038/nrmicro3525</p>
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Latest revision as of 02:39, 2 November 2017

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Overview Silver Gold Collaborations Engagement
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What is the problem we are trying to solve?


Antibiotics are among the most frequently administered drugs in human medicine. However, overprescription and failure to complete the prescribed course have contributed to microbes becoming resistant. In 2014, the World Health Organization classified antibiotic resistance as a global epidemic, highlighting the need for a solution. Inactivating resistance genes via Cas9 nuclease-mediated cleavage has been shown to be an effective means of combating this epidemic; however, methods of in vivo delivery are currently limited.

Our proposed solution

Cas9, when bound to guide RNA, results in a versatile gene editing tool that gives rise to a wide range of potential applications. Through non-homologous end joining of the double stranded break, bases are added and subtracted knocking out the gene of interest. Scientists hope to use this system to battle infectious diseases and treat multi-drug resistant microorganisms.(1) Despite the promise behind CRISPR-Cas9, the shift from its use as a research tool to a therapeutic device poses many challenges such as undesirable host immune responses and cleavage in unwanted locations due to low system specificity. Our team is researching the use of Outer Membrane Vesicles (OMVs) as a Cas9 delivery system. We hypothesize that the successful packaging and delivery of Cas9, when combined with a specific guide RNA that defines the genomic target, could be used in a therapeutic setting to combat antibiotic resistance.

Cas9 delivery

Typical methods for delivery of Cas9 into cell cultures include electroporation, nucleofection, and lipofectamine-mediated transfection.(2) However, these methods work exclusively for in vitro delivery of Cas9, and not in vivo. Thus, an alternative delivery system to transport this protein to target cells is needed. Two common in vivo methods for Cas9 delivery are viral vectors and hydrodynamic injection. The latter of these two approaches resulted in both liver and cardiovascular damage to mice, and therefore does not seem viable for humans. Alternatively, bacteriophages have been effectively used for the delivery and expression of genes, but fall short in their size limitation and their potential to harm the host’s immune system. Thus, we believe that a non-viral approach is optimal and have selected OMVs as the simplest, most viable option for delivery of Cas9 as well as the guide RNA.

What are OMVs

Outer Membrane Vesicles, or OMVs, are spheroid structures whose lipid barrier resembles that of the outer membrane of Gram-negative bacteria and internal composition shows a high degree of similarity with the bacterial periplasm. OMVs pinch off from the outer cell membrane and move independently from the host cell, allowing them to serve numerous functions such as the removal of toxic compounds and regulation of bacterial colonies by facilitating cell-cell communication. (3) Because OMVs are innately produced by bacteria, they are excellent candidates for non-viral lipid-based delivery systems.

References


(1) Doerflinger, M., Forsyth, W., Ebert, G., Pellegrini, M., & Herold, M. (2016). CRISPR/Cas9-The ultimate weapon to battle infectious diseases? Cellular Microbiology, 19(2). doi:10.1111/cmi.12693

(2) Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nature Reviews Microbiology,13(10), 605-619. doi:10.1038/nrmicro3525

(3)Berleman, J., & Auer, M. (2012). The role of bacterial outer membrane vesicles for intra- and interspecies delivery. Environmental Microbiology, 15(2), 347-354. doi:10.1111/1462-2920.1204