This complex part contains the tracrRNA and the CAS9 under the control of constitutive promoters. Note that there is no RBS before the tracRNA. The tracrRNA-CAS9 part is the heart of the CRISPR system. The tracrRNA and the corresponding target crRNA dimerize and are processed and hybridized before binding to the CAS9 protein. The tracrRNA has no specific part that is responsible for binding to the targeted DNA. Therefor this part can be used together with any crRNA. The CAS9 gene encodes for the CRISPR associated protein 9 that is needed to generate a double strand break at a target site. CAS9 binds to the tracRNA-crRNA dimer/gRNA and is then guided by them/it to the target site where it generate a double strand break. This part can be used together with the crRNA anti KAN or the gRNA anti KAN and cause double strand breaks. It can of course also be used together with new generated crRNAs/gRNAs. The tracRNA-CAS9 part is taken from the CRISPER plasmid of the Jiang et al. 2013 paper.
We tested the gRNA anti KAN together with the Cas9 in a killing assay. As there is a constant DNA damage if the system works, in cells that contain a kanamycin resistance and the two plasmids of gRNA and Cas9, this most likely leads to a SOS response and in the dead of the cell. To study that we used two strains, WT and WT+kanR, co-transformed the two plasmids and then plated them on Amp, Chl and Amp/Chl plates. The results can be seen in the figure below.
We introduced our CRISPR-based DNA cleavage system to two strains of E. coli : one WT (blue bars) and one carrying a genomically integrated kanamycin resistance casette (KanR, yellow bars). The strains were co-transformed with two plasmids. One plasmid carried the Cas9 construct and ampicillin resistance. The other plasmid carried the anti-kanamycin guideRNA and chloramphenicol resistance. WT E. coli could be efficiently transformed with either or both plasmids, as determined by selective plating. However, the KanR strain produced few viable transformants when we selected for both plasmids together. We attribute this to Cas9-induced cleavage of the chromosome specifically at the KanR casette. These results are consistent with about 99% efficient targeting from our construct. Sequence and Features
- 10INCOMPATIBLE WITH RFCIllegal EcoRI site found at 1721
- 12INCOMPATIBLE WITH RFCIllegal EcoRI site found at 1721
Illegal NheI site found at 1480
- 21INCOMPATIBLE WITH RFCIllegal EcoRI site found at 1721
Illegal BamHI site found at 3759
- 23INCOMPATIBLE WITH RFCIllegal EcoRI site found at 1721
- 25INCOMPATIBLE WITH RFCIllegal EcoRI site found at 1721
- 1000COMPATIBLE WITH RFC