Difference between revisions of "Part:BBa K3365000"
(→‎Usage and Biology)
 
(17 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K3365000 short</partinfo>
 
<partinfo>BBa_K3365000 short</partinfo>
  
−
An RNAP &#969; subunit, RpoZ is fused to dCas9 C-terminal.
+
An RNA polymerase ω subunit is fused to the C-terminal of dCas9.
  
−
<!-- Add more about the biology of this part here
+
<!-- -->
 
===Usage and Biology===
 
===Usage and Biology===
−
An RNAP ω subunit, RpoZ is fused to dCas9 C-terminal.
+
The dCas9 is a Cas9 nuclease mutant. Mutations D10A and H840A in the RucC and HNH domains, respectively, abolish cleavage but do not impair DNA binding. The dCas9 provides a simple and robust technology for gene repression and activation, and can target almost any DNA sequence aided by the sgRNA. The ω subunit can activate transcription by recruiting the RNAP holoenzyme. The fusion between dCas9 and ω subunit can activate gene transcription downstream of the protospacer. The CRISPR interference inhibits transcription by sterically blocking the RNA polymerase(RNAP).
 +
 
 +
<center>[[File:DCas9_omega.png]]</center>
 +
 
 +
<center><b>Figure1.</b> Gene circuit</center>
 +
 
 +
 
 +
<center>[[File:Mechanism_of_CRISPR-mediated_transcriptional_activation.png]]</center>
 +
 
 +
 
 +
<center><b>Figure2.</b> Mechanism of CRISPR-mediated transcriptional activation</center>
 +
 
  
 
<!-- -->
 
<!-- -->

Latest revision as of 18:32, 27 October 2020


dCas9-ω

An RNA polymerase ω subunit is fused to the C-terminal of dCas9.

Usage and Biology

The dCas9 is a Cas9 nuclease mutant. Mutations D10A and H840A in the RucC and HNH domains, respectively, abolish cleavage but do not impair DNA binding. The dCas9 provides a simple and robust technology for gene repression and activation, and can target almost any DNA sequence aided by the sgRNA. The ω subunit can activate transcription by recruiting the RNAP holoenzyme. The fusion between dCas9 and ω subunit can activate gene transcription downstream of the protospacer. The CRISPR interference inhibits transcription by sterically blocking the RNA polymerase(RNAP).

DCas9 omega.png
Figure1. Gene circuit


Mechanism of CRISPR-mediated transcriptional activation.png


Figure2. Mechanism of CRISPR-mediated transcriptional activation


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1340
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1340
    Illegal NheI site found at 1099
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1340
    Illegal BamHI site found at 3378
    Illegal BamHI site found at 4212
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1340
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1340
  • 1000
    COMPATIBLE WITH RFC[1000]