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| | Cas9 is the main protein of the CRISPR/Cas system II of Streptococcus pyogenes. CRISPR systems protect bacteria and archaea from phages by recognizing and cleaving of invading phage DNA. In practice, a short gRNA is used as a guide and bind the Cas9 protein to its complementary DNA strand. Cas9 protein would cleave the DNA strand at the binding site. dCas9 is a mutant of Cas9 that lacks DNase activity. dCas9 preserves the ability to bind gRNA and then to gRNA’s complementary DNA strand, but does not cut DNA double strands. Therefore dCas9/gRNA complex is able to inhibit expression without damaging DNA. | | Cas9 is the main protein of the CRISPR/Cas system II of Streptococcus pyogenes. CRISPR systems protect bacteria and archaea from phages by recognizing and cleaving of invading phage DNA. In practice, a short gRNA is used as a guide and bind the Cas9 protein to its complementary DNA strand. Cas9 protein would cleave the DNA strand at the binding site. dCas9 is a mutant of Cas9 that lacks DNase activity. dCas9 preserves the ability to bind gRNA and then to gRNA’s complementary DNA strand, but does not cut DNA double strands. Therefore dCas9/gRNA complex is able to inhibit expression without damaging DNA. |
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| − | ==Fluorescence Loss Assays==
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| − | <p>characterised by iGEM19_Wageningen</p>
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| − | === 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>
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| − | <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> - Complete dCas9 expression cassette: https://parts.igem.org/Part:BBa_K3286008 </p>
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| − | <p> - sgRNA expression cassette (single target): https://parts.igem.org/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: https://parts.igem.org/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>
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| − | ===dCas9 - multiplex===
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| − | <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>
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| − | <p> - Complete dCas9 expression cassette: https://parts.igem.org/Part:BBa_K3286008</p>
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| − | <p> - sgRNA expression cassette (double target): https://parts.igem.org/Part:BBa_K3286011</p>
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| − | <html>
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| − | <figure>
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| − | <img style="width:70%" src="https://static.igem.org/mediawiki/parts/4/4d/DCas9_Bronze_multiplex.png">
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| − | <figcaption>
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| − | <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>
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| − | </figcaption>
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| − | </figure>
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| − | </html>
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Cas9 is the main protein of the CRISPR/Cas system II of Streptococcus pyogenes. CRISPR systems protect bacteria and archaea from phages by recognizing and cleaving of invading phage DNA. In practice, a short gRNA is used as a guide and bind the Cas9 protein to its complementary DNA strand. Cas9 protein would cleave the DNA strand at the binding site. dCas9 is a mutant of Cas9 that lacks DNase activity. dCas9 preserves the ability to bind gRNA and then to gRNA’s complementary DNA strand, but does not cut DNA double strands. Therefore dCas9/gRNA complex is able to inhibit expression without damaging DNA.
