Difference between revisions of "Team:Calgary/Gene Integration Diagram"

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                 <div style="padding: 10px">
 
                 <div style="padding: 10px">
 
                   <ol>
 
                   <ol>
−
   <li><p>1. CRISPR/Cas9 wild-type binds targeted DNA location on the genome according to sgRNA homology
+
   <li><p>CRISPR/Cas9 wild-type binds targeted DNA location on the genome according to sgRNA homology
 
                     </p></li>
 
                     </p></li>
−
   <li><p>2. Cas9 wild-type cuts DNA in a double stranded fashion</p></li>
+
   <li><p>Cas9 wild-type cuts DNA in a double stranded fashion</p></li>
−
      <li><p>3. The ssDNA insert brought in along with the RNP CRISPR complex is integrated into the genome</p></li>
+
                     <li> <p>The ssDNA insert brought in along with the RNP CRISPR complex is integrated into the genome</p> </li>
−
                     <li> <p>3. The ssDNA insert brought in along with the RNP CRISPR complex is integrated into the genome</p> </li>
+
                     <li> <p>This integration happens via the non-homologous end joining DNA repair mechanism</p> </li>
−
                     <li> <p>4. This integration happens via the non-homologous end joining DNA repair mechanism</p> </li>
+
                     <li><p>This “landing pad” containing the FRT site is recognized by FlpO, which recombines with the
−
                     <li><p>5. This “landing pad” containing the FRT site is recognized by FlpO, which recombines with the
+
 
                         FRT site present on the plasmid, thus bringing in the entire plasmid into the genome</p>
 
                         FRT site present on the plasmid, thus bringing in the entire plasmid into the genome</p>
 
                     <ul>
 
                     <ul>
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                         </li>
 
                         </li>
 
                       </ul></li>
 
                       </ul></li>
−
                     <li><p>6. Within the genome, two sets of the Six 1, NPS, Six 2 combination are recognized by beta
+
                     <li><p>Within the genome, two sets of the Six 1, NPS, Six 2 combination are recognized by beta
 
                       resolvase, excising the sequences in-between</p></li>
 
                       resolvase, excising the sequences in-between</p></li>
 
                   </ol>  
 
                   </ol>  

Revision as of 22:16, 25 November 2018

Team:Calgary/Gene Integration Diagram - 2018.igem.org

GENE INTEGRATION DIAGRAM


  1. CRISPR/Cas9 wild-type binds targeted DNA location on the genome according to sgRNA homology

  2. Cas9 wild-type cuts DNA in a double stranded fashion

  3. The ssDNA insert brought in along with the RNP CRISPR complex is integrated into the genome

  4. This integration happens via the non-homologous end joining DNA repair mechanism

  5. This “landing pad” containing the FRT site is recognized by FlpO, which recombines with the FRT site present on the plasmid, thus bringing in the entire plasmid into the genome

    • The plasmid contains an inactive puromycin gene as the cargo, this is the case in our assays, for use as a proof of concept

  6. Within the genome, two sets of the Six 1, NPS, Six 2 combination are recognized by beta resolvase, excising the sequences in-between

Result: Desired Product contains the puromycin gene, which is now active. The circularised/excised DNA is discarded, unable to return to the genome.