Part:BBa_K1150034

dCas RNA plasmid: "uniCAS RNAimer"

pH1:tracrRNA pU6:DR_dummy:DR
Function	two small RNAs giving rise to specific binding of dCas9 to DNA loci
Use in	Mammalian cells
RFC standard	RFC 25
Backbone	pSB1C3
Organism	Streptococcus pyogenes
Source	Feng Zhang, Addgene
Submitted by	[http://2013.igem.org/Team:Freiburg Freiburg 2013]

This device contains loci that can code for small RNAs that are able to target the dCas9 protein [1] to any desired locus containg a so-called PAM seqeunce. It consists of a human H1 promoter driving the expression of the tracrRNA which mediates the interaction between the dCas9 protein and the second RNA, the crRNA. This crRNA gives rise to the specific binding of dCAS9 to DNA and is under control of the human U6 promoter. The 30bp target site can be inserted by simply opening the plasmid with BbsI and ligating it into the plasmid. The flanking direct repeats (DR) will mediate interaction with the tracrRNA. [1]
Multiple target sites can be combined on one plasmid by using the BioBrick standard and enables to target multiple loci at once with a small amount of plasmids.
The very minimalistic pSB1C3 backbone guarantees strong expression of the RNAs, which are suspected to be the limiting factor of dCAS9 interaction.
This plasmid should be used in combination of dCAS9 effector proteins, that can be found in the registry, e.g. [2] or [3].

An efficient tool for designing target sites can be found at Freiburg 2013 teams website. [http://2013.igem.org/Team:Freiburg/Project/crrna]

References

[1]Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., Zhang, F. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339 (6121), 819-23

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 224
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 216