Difference between revisions of "Team:Northwestern/Description"

 
(210 intermediate revisions by 2 users not shown)
Line 1: Line 1:
{{Northwestern}}
+
{{Northwestern_Page_Head}}
 +
{{Navbar}}
 
<html>
 
<html>
<div class="column full_size" >
+
<head>
<h3> PROJECT DESCRIPTION </h3>
+
<meta charset="UTF-8">
<h4> What it the problem we are trying to solve? </h4>
+
<link rel="stylesheet" href="https://www.w3schools.com/w3css/4/w3.css">  
<p>
+
<link rel="stylesheet" href="https://fonts.googleapis.com/css?family=Lora">
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.
+
<meta ="viewport" content="width=device-width, initial-scale=1">
</p>
+
<script src="//use.edgefonts.net/josefin-sans:n1,i1,n3,i3,n4,i4,n6,i6,n7,i7.js"></script>
<h4> Our Proposed Solution </h4>
+
<script src="//use.edgefonts.net/aladin.js"></script>
<p> Cas9, when combined with CRISPR, acts as a gene editing system. The Cas9-CRISPR complex has shown the ability to delete and add genes in almost every organism. Scientists hope to use this system to aid in the pathogenesis of harmful, antibiotic-resistant bacteria.<a href="http://onlinelibrary.wiley.com/doi/10.1111/cmi.12693/full"><sup>(1)</sup></a> However, despite the promise behind Cas9-CRISPR, researchers are having a difficult time determining how to get the complex to infection sites effectively. We hypothesize that if we were able to successfully package and deliver Cas9 and a specific guide RNA that defines the genomic target, the complex could be used in a therapeutic setting to combat antibiotic resistance.</p>
+
<div class="column full_size" >
+
<h4>Cas9 Delivery </h4>
+
<p>
+
The Cas9 endonuclease, when co-localized with a single guide RNA, can generate a double strand break (DSB) at a specified location. 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 excursively 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.
+
</p>
+
<p>
+
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 to be a viable delivery methods for humans.  Bacteriophages as vectors, on the other hand, are efficient in their delivery and expression of genes, but fall short in their size limitations and their potential to harm the immune system of the host, or the potential of the host’s immune system to eradicate the phages.<a href="http://online.liebertpub.com/doi/abs/10.1089/hum.2015.069"><sup>(2)</sup></a>
+
+
Therefore, there is a growing potential for the use of non-viral vectors for the in vivo delivery of Cas9.  Thus, we decided that <b>OMVs</b> are the simplest, most viable option for non-viral lipid-based vectors.
+
  
</p>
+
</head>  
  
  
</div>
+
<!-- Container (Project) -->
 +
  <span style="font-family:Lora" class="w3-center"
 +
<div class="w3-margin">
 +
<div class=" w3-white" id="project"  >
  
 +
<style> 
 +
.border { border: 3px solid black; }
 +
h1 {font-family: aladin, sans-serif;}
 +
 +
</style>
 +
</head>
 +
 +
<h1 style= "font-family:aladin, sans-serif; color: #551A8B; font-size: 30 px"><b> Our Project</b> </h1>
 +
 +
<h1 style= "font-family:aladin, sans-serif; color: #551A8B; font-size: 28px"> What's the Problem? </h1>
 +
<p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px">
 +
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. Here are some factors contributing to antibiotic resistance...</p>
 +
 +
 +
<h1 style= "font-family:aladin, sans-serif; color: #551A8B; font-size: 28px"> Our Proposed Solution:</h1>
 +
<p class="body-cont w4-center"  style ="font-family: josefin-sans, sans-serif; font-size:22px">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>
 
<div class="column full_size" >
 
<div class="column full_size" >
 +
<h1 style= "font-family:aladin, sans-serif; color: #551A8B; font-size: 24px">Cas9 Delivery</h1>
 +
<p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px">
 +
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.
 +
<br />
 +
<br />
 +
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>
 +
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.
  
<h4>What are OMVs?</h4>
+
</p>
  
<p>
+
<h1 style= "font-family:aladin, sans-serif; color: #551A8B; font-size: 24px">What are OMVs?</h1>
Outer Membrane Vesicles, or OMVs, are spheroid structures whose lipid barrier resembles that of the outer membrane of Gram-negative bacteria and internal composition has 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 removing toxic compounds and regulating 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.
+
 
 +
<p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px">
 +
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.
 
</p>
 
</p>
 
<figure>
 
<figure>
  <img src="https://static.igem.org/mediawiki/2017/1/1d/T--Northwestern--OMV.jpg" style="width:300px;height:400px;">
+
<img class="w3-hide-small border" src="https://static.igem.org/mediawiki/2017/1/1d/T--Northwestern--OMV.jpg" >
  <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>
+
<img class="w3-hide-large w3-hide-medium border" src="https://static.igem.org/mediawiki/2017/1/1d/T--Northwestern--OMV.jpg" style="width:100%;height:400px;">
 +
<figcaption><a href="http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12048/full"> <br> Fig1: OMVs forming and pinching off from the outer membrane.<sup>(4)</sup></a></figcaption>
 
</figure>
 
</figure>
</div>
 
 
<div class="column full_size" >
 
  
<h4>Our Project</h4>
+
<h1 style= "font-family:aladin, sans-serif; color: #551A8B; font-size: 24px">Our Project</h1>
<p>This summer, our team will tackle three key features of this project: the incorporation of Cas9 into OMVs, the inclusion of guide RNAs into separate OMVs and finally the fusion of these vesicles with target cells and distribution of their contents.</p>
+
<p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px">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>
<h4><i>Delivery of Cas9 to target cell</i></h4>
+
<h1 style= "font-family:aladin, sans-serif; color: #551A8B; font-size: 24px">Delivery of Cas9 to target cell</h1>
<p>
+
<p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px">
To deliver Cas9 using an OMV, our team is going to start with a particular strain of <i>E. coli</i> (JC8031) that is genetically engineered to be hyper vesiculating (constantly creating OMVs from its outer membrane). These bacteria will be introduced to a plasmid that will allow the cell to metabolically create the Cas9 protein and, using signalling peptides, will encourage the secretion of Cas9 through the inner membrane and into the periplasm. Here we will take a sample and test for the existence of Cas9 in the periplasm using fractionation– lysing only the outer membrane of the cell. Once we have confirmed that the Cas9 has been introduced into the periplasm, the Cas9 protein will exit the cell via an OMV.  
+
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.  
 
</p>
 
</p>
 
<figure>
 
<figure>
   <img src="https://static.igem.org/mediawiki/2017/e/e5/T--Northwestern--BreakingOff.jpeg" style="width:575px;height:400px;">
+
   <img class="w3-hide-small w3-hide-medium border" src="https://static.igem.org/mediawiki/2017/e/e5/T--Northwestern--BreakingOff.jpeg">
   <figcaption><a href="https://www.nature.com/articles/ncomms10515">Fig2: OMV forming from periplasmic content.<sup>(5)</sup></a></figcaption>
+
  <img class="w3-hide-large border" src="https://static.igem.org/mediawiki/2017/e/e5/T--Northwestern--BreakingOff.jpeg" style="width:100%;height:400px;"> <br>
 +
   <figcaption><a href="https://www.nature.com/articles/ncomms10515"> <br> Fig2: OMV forming from periplasmic content.<sup>(5)</sup></a></figcaption>
 
</figure>
 
</figure>
<p>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.
+
<p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px">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.
 
</p>
 
</p>
<h4><i>Delivery of gRNA to target cell</i></h4>
+
<h1 style= "font-family:aladin, sans-serif; color: #551A8B; font-size: 24px">Delivery of gRNA to target cell</h1>
<p>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, 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 chose electroporation as our method to get gRNA into the periplasm of the cell. We will be using the same hypervesicular strain of <i>E. coli</i> to maximize our chances of gRNA ending up in OMVs. (<b> NOT FINISHED, NEED TO READ UP ABOUT THIS </b>).
+
<p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px" ><p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px">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.
</p>
+
</p></p>
<h4><i>Fusion of OMVs to target cell</i></h4>
+
<h1 style= "font-family:aladin, sans-serif; color: #551A8B; font-size: 24px">Fusion of OMVs to target cell</h1>
<p>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 fuse and how they interact with different types of cells, we will gain a better understanding of the concepts behind our experiment.</p>
+
<p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px"><p class="body-cont w4-center" style="font-family: josefin-sans, sans-serif; font-size:22px">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>
  
 +
 +
<h4 style="color: #551A8B">References</h4>
 +
<p style = "font-family: josefin-sans, sans-serif" 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</p>
 +
<p style = "font-family: Lora"; 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</p>
 +
<p style = "font-family: Lora"; 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</p>
 +
<p style = "font-family: Lora"; 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</p>
 +
<p style = "font-family: Lora"; 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>
 +
<p style = "font-family: Lora"; 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>
 
</div>
 
</div>
<div class="column half_size" >
+
</span>
 
+
    <!-- Hide this text on small devices -->
<h4>References</h4>
+
<p><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
+
</p>
+
<p><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>
+
<p><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
+
</p>
+
<p><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
+
</p>
+
<p><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>
+
<p><a href="https://www.nature.com/articles/ncomms10515"><sup>(6)</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>
+
 
</div>
 
</div>
 
</html>
 
</html>
 +
{{Northwestern_Page_Foot}}

Latest revision as of 02:23, 23 September 2017

Northwestern Template Northwestern Template

Our Project

What's the Problem?

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. Here are some factors contributing to antibiotic resistance...

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. (2) 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.


Fig1: OMVs forming and pinching off from the outer membrane.(4)

Our Project

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.

Delivery of Cas9 to target cell

Our team will make use of an E.coli 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.



Fig2: OMV forming from periplasmic content.(5)

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 E. coli (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.

Delivery of gRNA to target cell

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.(6) 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 E. coli 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.

Fusion of OMVs to target cell

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.

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

(4)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

(5)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

(6)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