Difference between revisions of "Part:BBa K3286008"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3286008 short</partinfo>
 
<partinfo>BBa_K3286008 short</partinfo>
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==Usage and Biology==
  
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The expression dCas9 is under control of the constitutive TET promoter. Expression regulation is achieved by the lac operator located between dCas9 and pTET. In an un-induced state, the lacI protein is bound to the lac operator inhibiting expression of the dCas9. Upon addition of the inducer IPTG (or Lactose) the repressor lacI binds to the inducer and dCas9 is being expressed.  
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<p>CRISPR interference (CRISPRi) makes use of catalytically inactive variants of Cas9 proteins to suppress gene expression [1]. Identical to their active counterparts, the co-expression of guide RNAs directs the ribonuclease protein (RNP) to its specific DNA target sequence. However, introduction of mutations in the RuvC1 and HNH nuclease domains of Cas9 cause the Cas9 to lose endonuclease activity, without impeding the DNA binding [2; 3]. This enables the reversible transcriptional inhibition by tightly DNA-bound dCas proteins, contrary to irreversible cleavage by active Cas9. One way to reverse the effect of dCas-mediated gene repression is through their natural inhibitors, known as Anti-CRISPR (Acr) proteins. Acrs are small, phage-derived proteins blocking the natural CRISPR immune system of bacteria [4]. In most cases, they directly interfere with Cas nucleases, blocking binding or cleavage of the target DNA [5]. Therefore, Acrs may represent a powerful tool for the optimization of CRISPR/Cas-based genome editing approaches or the construction of synthetic circuits [6].</p>
  
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<p>bi-directional terminator: https://parts.igem.org/Part:BBa_K3286006</p>
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==Sequence and Features==
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<p>dCas9: https://parts.igem.org/Part:BBa_K3286013</p>
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<p>lac operator: https://parts.igem.org/Part:BBa_K3286004</p>
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CRISPR interference (CRISPRi) is a powerful tool for reversible gene repression The expression dCas9 is under control of the constitutive TET promoter. Expression regulation is achieved by the lac operator located between dCas9 and pTET. In the un-induced state, the <em>lac</em>I protein is bound to the lac operator inhibiting expression of the dCas9. Upon addition of the inducer IPTG (or Lactose) the repressor <em>lac</em>I binds to the inducer and dCas9 is being expressed. Design of CRISPR interference (CRISPRi) spacers is and experiment execution are described extensively by Larson et al., 2013 [1].  
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<p>pTET: https://parts.igem.org/Part:BBa_K3286005</p>
+
−
<p>lacI promoter: https://parts.igem.org/Part:BBa_K2572009</p>
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<p>pTET: [[Part:BBa_K3286005]]</p>
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<p>lacI: https://parts.igem.org/Part:BBa_K143033</p>
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<p>lac operator: [[Part:BBa_K3286004]]</p>
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<p>Prarthana terminator: https://parts.igem.org/Part:BBa_K3286007</p>
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<p>dCas9: [[Part:BBa_K3286012]]</p>
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<p>bi-directional terminator: [[Part:BBa_K3286006]]</p>
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<p>lacI promoter: [[Part:BBa_K2572009]]</p>
 +
<p>lacI: [[Part:BBa_K143033]]</p>
 +
<p>Prarthana terminator: [[Part:BBa_K3286007]]</p>
  
 
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<partinfo>BBa_K3286008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3286008 SequenceAndFeatures</partinfo>
  
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==Fluorescence Loss Assays== 
 +
 +
=== dCas9 - monoplex===
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 +
<p>The strength of dCas9 inhibition was measured in an Fluorescence Loss Assay. GFP, under the control of the lacUV5 promoter, is genomically inserted in an <em>E. coli</em> DH10ß strain. Three spacer were designed (Sp1 - 3) targeting either the -10 element of the lacUV5 promoter (Sp1) or the first 100 nucleotides of the GFP cds. A non-targeting Spacer (SpNT) was included as a control. All spacers were expressed via a single guide (sg)RNA expression cassette. Expression of dCas9 is IPTG inducible via the <em>lac</em>I/<em>lac</em> operator system. </p>
 +
<p>Sp1 - 3 show clear reduction of GFP levels. Leakiness of <em>lac</em>I/<em>lac</em> operator system results in GFP repression even under un-induced conditions. The choice of spacers shows an effect on the efficiency of repression. Targeting the promoter region is more promising for repression of gene expression (Sp1) compared to targeting the beginning of the cds (Sp2&3). For recommendations regarding spacer design see Larson et al., 2013. </p>
 +
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<p> - sgRNA expression cassette (single target): [[Part:BBa_K3286003]] </p>
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<p> - dCas9 can further be used in a gene circuit under the control of an Anti-CRISPR. For more information see: [[Part:BBa_K3286010]]</p>
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<html>
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<figure>
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<img style="width:60%" src="https://static.igem.org/mediawiki/parts/1/12/DCas9_Bronze_monoplex.png">
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<figcaption>
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<b>Figure1: Fluorescence Loss Assay. Amount of GFP fluorescence under the induction of dCas9 targeting GFP.</b> <p> The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 represent three different spacers targeting GFP or the associated promoter. SpNT represents a non-targeting spacer serving as a control. </p>
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</figcaption>
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</figure>
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</html>
 +
 +
===dCas9 - multiplex===
 +
 +
<p>In a similar manner the strength of dCas9 inhibition on two targets, GFP & RFP, was measured simultaneously. GFP & RFP were under the control of the constitutive phage lambda PR & PL promoter, respectively . Sp1 - 3 refer to three different combinations of two spacers targeting each either GFP or RFP. A non-targeting Spacer (SpNT) was included as a control. Sp1 - 3 were expressed via a double sgRNA expression cassette with each sgRNA being linked to either a GFP or RFP targeting spacer. Expression of dCas9 is IPTG inducible via the lacI/lac operator system.</p>
 +
 +
<p> - sgRNA expression cassette (double target): [[Part:BBa_K3286011]]</p>
 +
 +
 +
 +
<html>
 +
<figure>
 +
<img style="width:70%" src="https://static.igem.org/mediawiki/parts/4/4d/DCas9_Bronze_multiplex.png">
 +
<figcaption>
 +
<b>Figure2: Fluorescence Loss Assay. Amount of GFP fluorescence under the induction of dCas9 targeting GFP.</b> <p> The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 refer to three different combinations of two spacers targeting each either GFP or RFP. SpNT represents a non-targeting spacer serving as a control. </p>
 +
</figcaption>
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</figure>
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</html>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 21:18, 21 October 2019


IPTG inducible dCas9 expression module

Usage and Biology

CRISPR interference (CRISPRi) makes use of catalytically inactive variants of Cas9 proteins to suppress gene expression [1]. Identical to their active counterparts, the co-expression of guide RNAs directs the ribonuclease protein (RNP) to its specific DNA target sequence. However, introduction of mutations in the RuvC1 and HNH nuclease domains of Cas9 cause the Cas9 to lose endonuclease activity, without impeding the DNA binding [2; 3]. This enables the reversible transcriptional inhibition by tightly DNA-bound dCas proteins, contrary to irreversible cleavage by active Cas9. One way to reverse the effect of dCas-mediated gene repression is through their natural inhibitors, known as Anti-CRISPR (Acr) proteins. Acrs are small, phage-derived proteins blocking the natural CRISPR immune system of bacteria [4]. In most cases, they directly interfere with Cas nucleases, blocking binding or cleavage of the target DNA [5]. Therefore, Acrs may represent a powerful tool for the optimization of CRISPR/Cas-based genome editing approaches or the construction of synthetic circuits [6].

Sequence and Features

CRISPR interference (CRISPRi) is a powerful tool for reversible gene repression The expression dCas9 is under control of the constitutive TET promoter. Expression regulation is achieved by the lac operator located between dCas9 and pTET. In the un-induced state, the lacI protein is bound to the lac operator inhibiting expression of the dCas9. Upon addition of the inducer IPTG (or Lactose) the repressor lacI binds to the inducer and dCas9 is being expressed. Design of CRISPR interference (CRISPRi) spacers is and experiment execution are described extensively by Larson et al., 2013 [1].

pTET: Part:BBa_K3286005

lac operator: Part:BBa_K3286004

dCas9: Part:BBa_K3286012

bi-directional terminator: Part:BBa_K3286006

lacI promoter: Part:BBa_K2572009

lacI: Part:BBa_K143033

Prarthana terminator: Part:BBa_K3286007

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1200
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3479
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Fluorescence Loss Assays

dCas9 - monoplex

The strength of dCas9 inhibition was measured in an Fluorescence Loss Assay. GFP, under the control of the lacUV5 promoter, is genomically inserted in an E. coli DH10ß strain. Three spacer were designed (Sp1 - 3) targeting either the -10 element of the lacUV5 promoter (Sp1) or the first 100 nucleotides of the GFP cds. A non-targeting Spacer (SpNT) was included as a control. All spacers were expressed via a single guide (sg)RNA expression cassette. Expression of dCas9 is IPTG inducible via the lacI/lac operator system.

Sp1 - 3 show clear reduction of GFP levels. Leakiness of lacI/lac operator system results in GFP repression even under un-induced conditions. The choice of spacers shows an effect on the efficiency of repression. Targeting the promoter region is more promising for repression of gene expression (Sp1) compared to targeting the beginning of the cds (Sp2&3). For recommendations regarding spacer design see Larson et al., 2013.

- sgRNA expression cassette (single target): Part:BBa_K3286003

- dCas9 can further be used in a gene circuit under the control of an Anti-CRISPR. For more information see: Part:BBa_K3286010

Figure1: Fluorescence Loss Assay. Amount of GFP fluorescence under the induction of dCas9 targeting GFP.

The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 represent three different spacers targeting GFP or the associated promoter. SpNT represents a non-targeting spacer serving as a control.

dCas9 - multiplex

In a similar manner the strength of dCas9 inhibition on two targets, GFP & RFP, was measured simultaneously. GFP & RFP were under the control of the constitutive phage lambda PR & PL promoter, respectively . Sp1 - 3 refer to three different combinations of two spacers targeting each either GFP or RFP. A non-targeting Spacer (SpNT) was included as a control. Sp1 - 3 were expressed via a double sgRNA expression cassette with each sgRNA being linked to either a GFP or RFP targeting spacer. Expression of dCas9 is IPTG inducible via the lacI/lac operator system.

- sgRNA expression cassette (double target): Part:BBa_K3286011


Figure2: Fluorescence Loss Assay. Amount of GFP fluorescence under the induction of dCas9 targeting GFP.

The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 refer to three different combinations of two spacers targeting each either GFP or RFP. SpNT represents a non-targeting spacer serving as a control.