Part:BBa_K1137012

gRNA anti KAN

This rather simple part contains the UG6 promoter that controls the gRNA anti-KAN cassette. This cassette consists of a seed sequence of the kanamycin resistance gene (Aminoglycoside N6’-acetyltransferase) and the gRNA scaffold. This part is ready to use and can generate together with the CAS9 a double strand break in the kanamycin resistance gene. By definition, the gRNA is hybrid RNA out of the crRNA and tracrRNA. The gRNA is necessary to guide the CAS9 to the specific site and to cause there a double strand break. The design of the gRNA is taken from the DiCarlo 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.

CRISPR anti-Kan plasmids target specifically kanamycin resistant E. coli.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]