Part:BBa_K1150023

SV40:HA-NLS-dCas9-Linker-G9a-NLS:BGH
Function	DNA binding protein fused to a methyl histone transferase
Use in	Mammalian cells
RFC standard	RFC 25
Backbone	pSB1C3
Organism	Streptococcus pyogenes, Mus musculus
Source	Feng Zhang, Addgene Albert Jeltsch, University of Stuttgart
Submitted by	[http://2013.igem.org/Team:Freiburg Freiburg 2013]

uniCAS Histone Modifier (SV40 promoter)

This device is combining the dCas9 protein, that enables multiple gene targeting with the set-domain of the murine G9a. dCas9 is working as a carrier for this histone methyltransferase and enables specific methylation of histone 3 lysine 9 (H3K9me2/3) when targeted to a histone locus that is accessible for DNA binding proteins. Literature indicates that targeting the G9a set-domain to an open locus leads to a transcriptionally inactive state [1]. The usage of the medium strong SV40 promoter optimizes this device for means were a strong expression is suboptimal. If a strong expression is desired check our uniCAS Histone Modificator device with CMV promoter. For more information [http://2013.igem.org/Team:Freiburg/Project/effector#repression klick here].

Figure 1: Complete overview on CMV:dCas9-G9a with all features.

Usage and Biology

H3K9 methylation is a hallmark of repressed transcriptional states. [2] Upon close contact the murine G9a-set domain transfers methyl groups to H3K9 leading to transcriptional repression. [3] The dCas9 protein can simultaneously be targeted to several DNA loci as it interacts with small RNAs thereby forming a complex that will interact with complementary DNA strands. It origins from the bacterial adaptive immune system of Streptococcus pyogenes called CRISPR. Hijacking this system leads to a whole new approach for multiple gene targeting. The team [http://2013.igem.org/Team:Freiburg Freiburg 2013] combined these two elements to create a transcriptional repressor that can be targeted to any desired locus of interest.
This approach offers new possibilities for applied as well as for fundamental research, such as tissue engineering, epigenetics and cancer research.

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 664
  Illegal BglII site found at 5139
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1]Wolffe, A., et al. (1999). Epigenetics: Regulation Through Repression. Science 286169, 481.
[2]Snowden, A., et al. (2002). Gene-Specific Targeting of H3K9 Methylation Is Sufficient for Initiating Repression In Vivo. Current Biology 12, 2159-2166.
[3] Lee, D., et al. (2006). Histone 3 Lysine 9 Methyltransferase G9a Is a Transcriptional Coactivator for Nuclear Receptors. Journal of Biological Chemistry 281, 8476-8485.