BBa_K3218000
The introns are used as docking sequences for the dCas9 mutation mechanism. The introns themselves are the specific gRNA recognition sequence flanked by intron recognition sequences. The intron recognition sequences are those used in S.cerevisiae and the gRNAs used do not have any matches within the yeast genome.
BBa_K3218001
The Ribozyme-gRNA-Ribozyme cassette is a self-cleaving mechanism for proper expression of the gRNA. The gRNA is flanked by two ribozymes, the Hammerhead ribozyme and the hepatitis delta virus ribozyme. This part can be used to express gRNAs in a proper manner.
BBa_K3218002
dCas9 is the targeting mechanism of our system and it is a dead or deactivated version of the Cas9 endonuclease. It binds to the gRNA and forms a complex that then can bind the target DNA and can allows proteins of interest that can be attached to the dCas9 to function on the target DNA.
BBa_K3218003
This part is composite consisting of the dCas9 protein used to recognize the specific gRNA sequence. Puting linkers between the dCas9 and the FokI elements proved problematic to order straight from IDT. To resolve this issue a Type-IIS cleavage site was inserted between dCas9 and FokI in order to insert the linkers between them. Multiple linker lengths would be tested allowing for some variability for optimization. This mechanism has a slight advantage since it requires two dCas9-FokI proteins in order to cleave the DNA, which allows for higher specificity, and causes a double stranded break.
BBa_K3218004
The AID mutation mechanism is slightly different since it does not require any linkers and only requires one dCas9 - AID complex. This mechanism does not cause double stranded breaks but deaminates cytosine into uracil. This version of AID has a limited range and is composed of the dCas9 recognition protein and the AID as the mutation mechanism.
BBa_K3218005
eAID is an enhanced version of the AID which has a broader range than regular AID. eAID is used to enhance the range of the AID.
