Team:Freiburg/parts/BBa K1150000

From 2013.igem.org


dCas9

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.

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