Team:UCSF/Project/Background

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Rarely in nature do bacterial strains exist in isolation; they form complex microbial communities that interact with various organisms. In these communities, there are few ways to target specific strains effectively. The way people control bacteria is through antibiotics, which for the most part act indiscriminately. But often, problems stem from a single species that has invaded the microbial community; normal balanced microbiomes are not only harmless, but are often crucial and positively contribute to an environment. Because most therapies disrupt this balance, there is an obvious need for specific targeting of species in microbial communities. </font>
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Rarely in nature do bacterial strains exist in isolation; they form complex microbial communities that interact with various organisms. These complex, balanced communities - called microbiomes - interact with various organisms and exist almost everywhere from the soil to our own digestive system. Microbiomes play an important role in human and environmental health and disruption of the balance in the microbiome is linked to many diseases. Current methods of manipulating microbiomes, such as antibiotic treatment, are nonspecific and indiscriminately kill both good and bad bacteria. Targeting precise bacterial community strains and controlling their growth, activity, and outputs is difficult and requires many new tools.   </font>
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Here we take advantage of conjugation, a natural horizontal gene transfer mechanism to target specific strains of bacteria and affect gene expression, which will have a potential for future applications that require targeted cell death.
 
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Our project incorporates bacterial conjugation and CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats inhibition), a tool repurposed from a natural adaptive immunity system in bacteria, comprised of a catalytically dead Cas9 (dCas9) protein that complexes with guide RNAs (gRNA) complementary to the target bacteria’s DNA sequence. The dCas9 complexes with the gRNA and binds to DNA complimentary to the gRNA and prevents transcription, therefore repressing gene expression.
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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?” 
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The difficulty faced with this situation is in
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<li>Introduce a targeting system into a defined mixture of bacteria such that you can select and introduce manipulations without negatively affecting other bacteria. </li>
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<li>Creating easy to transfer pathways or circuits that can produce a multitude of outcomes (killing, repressing, upregulating) </li>
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The combination of conjugation and CRISPRi allows us to create a system capable of both transferring genetic instructions from one cell to another and targeting unique species in a microbial community through a specific gene. </font></center>
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<b><FONT COLOR="#008000"><u>1. Introducing CRISPRi to a bacterial community:</u></font></b><br><br>
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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. </div>
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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.
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<font face="arial" size = "2"><center>Amended from: http://www.cell.com/abstract/S0092-8674(13)00211-0?script=true
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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><span> -  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. </div>
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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. </font></b></center><br>
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<b><FONT COLOR="#008000"><u>2. Creating scalable CRISPRi circuits that can choose between outcomes based on the input</u></font></b><br><br>
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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. </div>
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Latest revision as of 23:43, 27 October 2013


Operation CRISPR: Deploying precision guided tools to target unique species in a complex microbiome
Rarely in nature do bacterial strains exist in isolation; they form complex microbial communities that interact with various organisms. These complex, balanced communities - called microbiomes - interact with various organisms and exist almost everywhere from the soil to our own digestive system. Microbiomes play an important role in human and environmental health and disruption of the balance in the microbiome is linked to many diseases. Current methods of manipulating microbiomes, such as antibiotic treatment, are nonspecific and indiscriminately kill both good and bad bacteria. Targeting precise bacterial community strains and controlling their growth, activity, and outputs is difficult and requires many new tools.




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
  1. Introduce a targeting system into a defined mixture of bacteria such that you can select and introduce manipulations without negatively affecting other bacteria.
  2. Creating easy to transfer pathways or circuits that can produce a multitude of outcomes (killing, repressing, upregulating)


1. Introducing CRISPRi to a bacterial community:

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.
WHY USE CRISPRi?

1. CRISPRi utilizes gRNAs which are highly specific and customizable.

2. In principle it could be used to take advantage of unique DNA sequences to target specific bacterial species.
Amended from: http://www.cell.com/abstract/S0092-8674(13)00211-0?script=true



As a means to introduce our CRISPRi system into a microbial community we’ve opted to utilize conjugation - 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.

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.


2. Creating scalable CRISPRi circuits that can choose between outcomes based on the input

In addition to our conjugation project, we have developed a CRISPRi circuit, 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.

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