|Use in||Mammalian cells|
|RFC standard||RFC 25|
|Source||Feng Zhang, Addgene|
|Submitted by||Freiburg 2013|
dCas9 is a codon optimized and standardized (RFC 25) protein for human cell lines. Interacting with a DNA-binding RNA and fused with different effector domains it can be used for specific gene regulation.
Cas9 is the main protein of the CRISPR/Cas system II of Streptococcus pyogenes. CRISPR systems protect bacteria and archaea from phages by recognizing and cleaving of invading phage DNA. This recognition is based on Watson Crick base pairing between a short RNA, called crRNA, and the complementary DNA strand. A second RNA, called tracrRNA, connects crRNA and Cas9. These three parts together form a protein-RNA-DNA complex with the targeted DNA strand .
Cas9 became of great interest for research concerning DNA targeting, because of its ability to recognize site specific DNA strands by a crRNA.
At first the functionality of Cas9 was modified by exchanging aminoacids. As a result, Cas9 was able to introduce mutations within the genome of several organisms by causing double strand breaks . Then, it was converted from a nuclease to a nickase introducing single strand breaks  and lately it was converted to an enzymatically inactive form, called dCas9 .
This 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.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 248
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
|Protein data table for BioBrick BBa_K1150000 automatically created by the BioBrick-AutoAnnotator version 1.0|
|Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)|
ATGGCCGGCGACAAGAAG ... GGAGGCGACACCGGT
ORF from nucleotide position -8 to 4107 (excluding stop-codon)
|Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)|
|Sequence features: (with their position in the amino acid sequence, see the list of supported features)|
|Amino acid composition:|
|Amino acid counting|
|Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges|
|Alignments (obtained from PredictProtein.org)|
There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
|Predictions (obtained from PredictProtein.org)|
|There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.|
| The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.|
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 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
 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
 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
 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
 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