Difference between revisions of "Part:BBa K2130000"
 
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__NOTOC__
 
__NOTOC__
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<partinfo>BBa_K2130011 short</partinfo>
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<partinfo>BBa_K2130000 short</partinfo>
  
 
dCas9 is a catalytically inactive Cas9, which still retains it's ability to bind to DNA. For epigenetic regulation, the dCas9 is highly dependent upon the function of it's fusion partner.
 
dCas9 is a catalytically inactive Cas9, which still retains it's ability to bind to DNA. For epigenetic regulation, the dCas9 is highly dependent upon the function of it's fusion partner.
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For this part, we have deleted the HNH domain of the SP-dCas9 and tested it's binding capability by assessing the strength in transcriptional activation via a VP64-p65-Rta.
 
  
  
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<html><center><img src="https://static.igem.org/mediawiki/2016/0/0b/T--NTU-Singapore--ruvc3.jpg" height="60%"width="75%"></center></html>
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===Usage and Biology===
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For this part, we have deleted the HNH domain as well as the RuvCIII-2 region of the SP-dCas9 and tested it's binding capability by assessing the strength in transcriptional activation via by tagging it with a VP64-p65-Rta tripartite activator. A GFP reporter, <html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2130003"> BBa_K2130003</a></html> with two dCas9 binding site was used to measure the strength of the truncated dCss9.
  
  
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https://static.igem.org/mediawiki/2016/a/a2/T--NTU-Singapore--ruvc3delresult.png
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<html><center><img src="https://static.igem.org/mediawiki/2016/0/0b/T--NTU-Singapore--ruvc3.jpg" height="60%"width="75%"><p>Conformation of dCas9 before and after non-targeted strand cleavage. In state B, there are several regions (highlighted purple in state A), that are unresolved which points out that these regions are very dynamic and is not important for DNA interaction. We named this dynamic region as RuvCIII-2. On the other hand, RuvCIII-1(colored in cyan) also has minimal contact with DNA.</p>
  
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<img src="https://static.igem.org/mediawiki/2016/a/a2/T--NTU-Singapore--ruvc3delresult.png" height="60%"width="75%">
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<p>Our results showed that RuvCIII-2 which is highly dynamic can be deleted while RuvCIII-1 couldn't. RuvCIII-2 deletion can be combined with the HNH deletion producing a truncated version of more than 60% of activity.</p>
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<img src="https://static.igem.org/mediawiki/2016/e/e2/T--NTU-Singapore--conclusion.png" height="60%"width="75%">
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<p>We then compared our ∆HNH∆RuvCIII-2 dCas9 with other truncation. This part still has stronger activation strength than the ∆REC2∆HNH mutant although with an extra of ~200bp.</p></center></html>
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===Improvement===
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Team NTU_SINGAPORE 2017 made an improvement to this part by exploring possibilities to further shrink the size of dCas9 into part <html><a href="https://parts.igem.org/Part:BBa_K2316000">BBa_K2316000</a></html>. Furthermore, they even demonstrated that the improved version can be used in CRISPRi system.
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===References===
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<html>
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VPR tripartite activator:
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<p><a href="http://www.nature.com/nmeth/journal/v12/n4/full/nmeth.3312.html">Highly efficient Cas9-mediated transcriptional programming.</a> Chavez A. et. al.
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(2015) Nature Methods 12, 326–328 </p>
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State A:
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<p><a href="http://science.sciencemag.org/content/351/6275/867.long">Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.</a> <br>Jiang, F., Taylor, D.W., Chen, J.S., Kornfeld, J.E., Zhou, K., Thompson, A.J., Nogales, E., Doudna, J.A.(2016) Science 351: 867-871 </p>
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State B:
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<p><a href="http://www.cell.com/cell/abstract/S0092-8674(14)00156-1?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867414001561%3Fshowall%3Dtrue">Crystal structure of Cas9 in complex with guide RNA and target DNA.</a> Nishimasu, H., Ran, F.A., Hsu, P.D., Konermann, S., Shehata, S.I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. (2014) Cell(Cambridge,Mass.) 156: 935-949</p>
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</center></html>
  
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2130011 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2130000 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2130011 parameters</partinfo>
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<partinfo>BBa_K2130000 parameters</partinfo>
 
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Latest revision as of 03:26, 2 November 2017


∆RuvCIII-2 ∆HNH Sp-dCas9

dCas9 is a catalytically inactive Cas9, which still retains it's ability to bind to DNA. For epigenetic regulation, the dCas9 is highly dependent upon the function of it's fusion partner.


Usage and Biology

For this part, we have deleted the HNH domain as well as the RuvCIII-2 region of the SP-dCas9 and tested it's binding capability by assessing the strength in transcriptional activation via by tagging it with a VP64-p65-Rta tripartite activator. A GFP reporter, BBa_K2130003 with two dCas9 binding site was used to measure the strength of the truncated dCss9.


Conformation of dCas9 before and after non-targeted strand cleavage. In state B, there are several regions (highlighted purple in state A), that are unresolved which points out that these regions are very dynamic and is not important for DNA interaction. We named this dynamic region as RuvCIII-2. On the other hand, RuvCIII-1(colored in cyan) also has minimal contact with DNA.

Our results showed that RuvCIII-2 which is highly dynamic can be deleted while RuvCIII-1 couldn't. RuvCIII-2 deletion can be combined with the HNH deletion producing a truncated version of more than 60% of activity.

We then compared our ∆HNH∆RuvCIII-2 dCas9 with other truncation. This part still has stronger activation strength than the ∆REC2∆HNH mutant although with an extra of ~200bp.

Improvement

Team NTU_SINGAPORE 2017 made an improvement to this part by exploring possibilities to further shrink the size of dCas9 into part BBa_K2316000. Furthermore, they even demonstrated that the improved version can be used in CRISPRi system.


References

VPR tripartite activator:

Highly efficient Cas9-mediated transcriptional programming. Chavez A. et. al. (2015) Nature Methods 12, 326–328

State A:

Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.
Jiang, F., Taylor, D.W., Chen, J.S., Kornfeld, J.E., Zhou, K., Thompson, A.J., Nogales, E., Doudna, J.A.(2016) Science 351: 867-871

State B:

Crystal structure of Cas9 in complex with guide RNA and target DNA. Nishimasu, H., Ran, F.A., Hsu, P.D., Konermann, S., Shehata, S.I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. (2014) Cell(Cambridge,Mass.) 156: 935-949


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 251
    Illegal BglII site found at 1188
    Illegal BamHI site found at 2006
    Illegal XhoI site found at 2962
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2350
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2496
    Illegal BsaI site found at 3158
    Illegal BsaI.rc site found at 1411