Part:BBa_K1150000

dCas9

dCas9
Function	Binding protein
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]

Cas9 is the main protein of the CRISPR-Cas system of Streptococcus pyogenes, which is categorized as CRISPR system type II. Like all other CRISPR systems it protects bacteria (and archaea) from phages by recognizing and cleaving of the invading phage DNA. This recognition is based on Watson Crick base pairing between a short RNA (called crRNA), which is in complex with Cas9, and the target DNA [1].
Because of the ability to recognize almost every DNA sequenz, Cas9 became of interest for research concerning DNA targeting. At first it was used in combination with the crRNA and a tracrRNA, which is required to form the protein-RNA-complex, to introduce mutations within the genome of several organisms by causing a double strand break [2][3]. After the exchange of an aminoacid Cas9 was converted from a nuclease to a nickase, introducing only single strand breaks [4]; and very recently converted to a enzymaticly inactive form, called dCas9, by another aminoacid exchange [5].

The here available dCas9 is codon optimized for human cell lines and standardized (RFC 25). It can be used as a DNA binding protein, that can be fused with different effectors in order to regulate gene expression.

References

[1] Westra E.R., Swarts D.C., Staals R.H., Jore M.M., Brouns S.J., van der Oost J. (2012). The CRISPRs, they are a-changin': how prokaryotes generate adaptive immunity. Annu Rev Genet. 46, 311-39
[2] Mali P., Yang L., Esvelt K.M., Aach J., Guell M., DiCarlo J.E., Norville J.E., Church G.M. (2013). RNA-guided human genome engineering via Cas9. Science 339(6121), 823-6
[3] Jiang W., Bikard D., Cox D., Zhang F., Marraffini L.A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 31(3), 233-9
[4] 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
[5] Qi L.S., Larson M.H., Gilbert L.A., Doudna J.A., Weissman J.S., Arkin A.P., Lim W.A. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5), 1173-83

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 248
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]