Team:UCSF/Project/Conjugation/Design1

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<h1><center> Operation CRISPR: Deploying precision guided tools to target unique species in a complex microbiome </center></h1>
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<h2><center> Transmitting CRISPRi Circuits through Cell-to-Cell Conjugation </center></h2>
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<p2>Rarely in nature do bacterial strains exist in isolation; they form complex microbial communities that interact with various organisms. We ourselves contain a major microbial community in our digestive tract that has shown to directly affect our health and well-being.  As shown on the left, to improve and maintain healthly living it would be useful to have the ability to change the microbial community. For example, if a large of amount of a certain sugar was present in your gut ("signal #1") you might want to slow the growth of a certain bacterial populations . In another scenario ("signal #2") it might be useful to increase the growth of other specific bacteria in your gut. But targeting precise bacterial community strains and controlling their growth, activity, and outputs is difficult and requires many new tools.</p2>
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<p1>GOAL: To construct a specific gene repression system using CRISPRi that can be efficiently transmitted between cells by conjugation.</p1>
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<h3>What is conjugation? </h3>
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<p2><br>In nature, bacterial strains rarely exist as distinct populations. Instead, they are almost always found in mixed populations where they compete for resources. Conjugation is a naturally occurring process in bacteria that allows genetic material to be transferred between populations of bacterial cells. This process promotes gene diversity, and in certain situations, provides a competitive advantage for the recipient cell.</p2>
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<p2>At the beginning of this summer, we asked ourselves a question: "What could we introduce to a microbiome which would allow targeting and eventual gene expression changes in a specific bacteria?" The difficulty faced with this situation is in:</p2>
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<p2>By combining CRISPRi and conjugation, we’ve come up with a system that will allow us to specifically target certain populations within a microbiome. To do this, an engineered cell capable of conjugating must be introduced into a microbiome of interest. The engineered cell, or donor cell, is capable of conjugating (proteins necessary for conjugation are contained in the genome) and carries a conjugative plasmid, which codes for a catalytically dead Cas9 (dCas9) protein and guide RNA (gRNA) for a specific gene that is present in the targeted population.</p2>
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<p2>  1. Introduce a targeting system into a defined mixture of bacteria such that you can select and introduce manipulations without negatively affecting other bacteria. </p2>
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<p2>  2. Creating easy to transfer pathways or circuits that can produce a multitude of outcomes (killing, repressing, upregulating). </p2>
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<h3>1. Introducing CRISPRi to a bacterial community</h3>
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<p2>To selectively target and eliminate harmful bacteria, we are utilizing the CRISPRi system, a tool repurposed from a natural adaptive immunity system in bacteria (see diagram below). This tool is comprised of a catalytically dead Cas9 (dCas9) protein that complexes with guide RNAs (gRNA) complementary to the target bacteria’s DNA sequence. This complex binds to DNA complementary to the gRNA and prevents transcription, therefore repressing gene expression.</p2>
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<p2>WHY USE CRISPRi? <br><br>1. CRISPRi utilizes gRNAs which are highly specific and customizable.<br><br>
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2. In principle it could be used to take advantage of unique DNA sequences to target specific bacterial species.</p2>
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<p2>Upon conjugation with the target population, the conjugative plasmid would be transferred. Both dCas9 and gRNA would subsequently be expressed in the recipient cell, and the complex formed will repress the targeted gene specified by the gRNA, shutting down certain cell functions. </p2>
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<p3><center>Amended from: http://www.cell.com/abstract/S0092-8674(13)00211-0?script=true</center></p3>  
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<p2> As a means <a href="https://2013.igem.org/Team:UCSF/Project/Conjugation/Design" > to introduce our CRISPRi system into a microbial community we've opted to utilize  conjugation </a> - a naturally occurring mechanism bacteria use to transfer DNA.  By utilizing this mechanism, we are able to target specific strains of bacteria and affect gene expression. This will have a potential for future applications that require targeting individual strains in a bacterial community. </p2></div>
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<p2>For the summer, we used fluorescent proteins to differentiate between our target cell strains and our unaffected cell strains. Our targeted cells will be marked with red fluorescent protein (RFP) while our unaffected cells with be marked with the fluorescent protein, citrine. Both cell strains will receive the conjugative plasmid from the donor. The gRNA-dCAS9 complex will then form and repress the production of RFP in our target cells. The RFP cell strain will no longer be able to fluoresce, since the gRNA in our conjugative plasmid only recognizes a specific site on RFP, while the citrine cell strain will be left unaffected because there is no gRNA in the conjugative plasmid that recognizes citrine. </p2>
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<p1><center>The combination of conjugation and CRISPRi allows us to create a system capable of both transferring genetic instructions from one cell to another as well as targeting unique species in a microbial community through a specific gene. </center></p1>
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<h3>2. Creating scalable CRISPRi circuits that can choose between outcomes based on the input</h3>
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<p2>In addition to our conjugation project, we have developed a <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Design" >CRISPRi circuit</a><span>, which could be delivered by the same conjugation system, that could apply to future regulatory applications (upregulation of bacterial growth, bacterial activity and behavior, gene expression, and other bacterial processes, etc.).  Our circuit is multi-functional, eliciting different responses with the presence of different inducers and is scalable by incorporating additional designed plasmids or guide RNAs. The circuit relies on the use of CRISPRi gRNAs to provide scalability - several genes can be targeted for silencing, upregulation, or other needs. </p2>
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Revision as of 09:44, 28 October 2013

Transmitting CRISPRi Circuits through Cell-to-Cell Conjugation

GOAL: To construct a specific gene repression system using CRISPRi that can be efficiently transmitted between cells by conjugation.

What is conjugation?


In nature, bacterial strains rarely exist as distinct populations. Instead, they are almost always found in mixed populations where they compete for resources. Conjugation is a naturally occurring process in bacteria that allows genetic material to be transferred between populations of bacterial cells. This process promotes gene diversity, and in certain situations, provides a competitive advantage for the recipient cell.
By combining CRISPRi and conjugation, we’ve come up with a system that will allow us to specifically target certain populations within a microbiome. To do this, an engineered cell capable of conjugating must be introduced into a microbiome of interest. The engineered cell, or donor cell, is capable of conjugating (proteins necessary for conjugation are contained in the genome) and carries a conjugative plasmid, which codes for a catalytically dead Cas9 (dCas9) protein and guide RNA (gRNA) for a specific gene that is present in the targeted population.

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Upon conjugation with the target population, the conjugative plasmid would be transferred. Both dCas9 and gRNA would subsequently be expressed in the recipient cell, and the complex formed will repress the targeted gene specified by the gRNA, shutting down certain cell functions.

For the summer, we used fluorescent proteins to differentiate between our target cell strains and our unaffected cell strains. Our targeted cells will be marked with red fluorescent protein (RFP) while our unaffected cells with be marked with the fluorescent protein, citrine. Both cell strains will receive the conjugative plasmid from the donor. The gRNA-dCAS9 complex will then form and repress the production of RFP in our target cells. The RFP cell strain will no longer be able to fluoresce, since the gRNA in our conjugative plasmid only recognizes a specific site on RFP, while the citrine cell strain will be left unaffected because there is no gRNA in the conjugative plasmid that recognizes citrine.

Retrieved from "http://2013.igem.org/Team:UCSF/Project/Conjugation/Design1"