"
Team:Waterloo/Silence
From 2014.igem.org
| Line 35: | Line 35: | ||
<h2>CRISPRi: Silencing Transcription</h2> | <h2>CRISPRi: Silencing Transcription</h2> | ||
<p> | <p> | ||
| - | Background | + | <h3>Background</h3> |
CRISPR systems (clustered regularly interspaced short palindromic sequences) are involved in the adaptive immune systems of bacteria and archaea. Mature CRISPR RNA (crRNA) binds to trans-activating crRNA (tracrRNA) to form a complex that is recognized by the CRISPR associated protein (Cas9). The Cas9 protein is directed to a target DNA site where it performs a double stranded break (Karginov and Hannon, 2010). An improvement to the original CRISPR system is the use of sgRNA which is a chimera of the crRNA and tracrRNA complex and this simplified version has been shown to be effective more (Jinek et al., 2012). | CRISPR systems (clustered regularly interspaced short palindromic sequences) are involved in the adaptive immune systems of bacteria and archaea. Mature CRISPR RNA (crRNA) binds to trans-activating crRNA (tracrRNA) to form a complex that is recognized by the CRISPR associated protein (Cas9). The Cas9 protein is directed to a target DNA site where it performs a double stranded break (Karginov and Hannon, 2010). An improvement to the original CRISPR system is the use of sgRNA which is a chimera of the crRNA and tracrRNA complex and this simplified version has been shown to be effective more (Jinek et al., 2012). | ||
| - | + | </p><p> | |
The CRISPR system can be modified further to regulate gene expression. The CRISPRi (CRISPR interference) system involves a catalytically dead Cas9 protein (dCas9) paired with a single guide RNA. Together, they are able to interfere with transcription and halt the expression of the target gene (Qi et al., 2013). This process is reversible and nonfatal; since dCas9 is catalytically dead, it does not cleave targeted DNA in the way an endonuclease normally would (Qi et al., 2013). | The CRISPR system can be modified further to regulate gene expression. The CRISPRi (CRISPR interference) system involves a catalytically dead Cas9 protein (dCas9) paired with a single guide RNA. Together, they are able to interfere with transcription and halt the expression of the target gene (Qi et al., 2013). This process is reversible and nonfatal; since dCas9 is catalytically dead, it does not cleave targeted DNA in the way an endonuclease normally would (Qi et al., 2013). | ||
| + | </p> | ||
| - | + | <img src="https://static.igem.org/mediawiki/2014/1/1f/Waterloo_CRISPRi.png" alt="CRISPRi Mechanism"/> | |
| - | + | ||
</p> | </p> | ||
</div> | </div> | ||
Revision as of 01:52, 18 October 2014
Silence
Overview: Silencing Antibiotic Resistance
By silencing the expression of genes responsible for antibiotic resistance, antibiotic-resistant bacteria can be converted to antibiotic-sensitive bacteria. In the case of MRSA, silencing the expression of mecA and its regulatory elements create a population of antibiotic-sensitive MRSA (Meng et al., 2006; Hou et al., 2007; Sakoulas et al., 2001). To model silencing in MRSA, our team aimed to silence YFP expression in S. epidermidis using CRISPRi and RNAi.
CRISPRi: Silencing Transcription
Background
CRISPR systems (clustered regularly interspaced short palindromic sequences) are involved in the adaptive immune systems of bacteria and archaea. Mature CRISPR RNA (crRNA) binds to trans-activating crRNA (tracrRNA) to form a complex that is recognized by the CRISPR associated protein (Cas9). The Cas9 protein is directed to a target DNA site where it performs a double stranded break (Karginov and Hannon, 2010). An improvement to the original CRISPR system is the use of sgRNA which is a chimera of the crRNA and tracrRNA complex and this simplified version has been shown to be effective more (Jinek et al., 2012).The CRISPR system can be modified further to regulate gene expression. The CRISPRi (CRISPR interference) system involves a catalytically dead Cas9 protein (dCas9) paired with a single guide RNA. Together, they are able to interfere with transcription and halt the expression of the target gene (Qi et al., 2013). This process is reversible and nonfatal; since dCas9 is catalytically dead, it does not cleave targeted DNA in the way an endonuclease normally would (Qi et al., 2013).
RNAi: Silencing Translation
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
content 2
Design
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
Results and Future Work
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
content 3
