Team:UCSF/Project/Background2
From 2013.igem.org
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. 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.
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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