The idea behind this project is to create an easy and accessible way to insert the CRISPR/Cas9 system into cells using an atoxic C. difficile Toxin. The toxin used is an atoxic C. difficile B (atcdB), more specifically the W102A D288N line, which is glucosyltransferase deficient. Glucosyltransferase causes apoptosis in cells, but the atoxic toxin B does not have any detrimental health effects.
The Cas9 gene from S. pyogenes is used, but also contains an N-terminal SV40 NLS tag and a C-terminal nucleoplasmin NLS tag, both of which direct the protein to the nucleus. The construct is attached to the aTcdB via a glycine-serine linker, and ends with an 6xHis tag in order to aid in the purification process. It is assembled in the pHis1522 plasmid and was transformed in DH5a E. coli. Since E. coli cannot reliably translate such large proteins, the plasmid will ultimately be transformed into Bacillus megaterium which is capable of synthesizing massive proteins. The Cas9-aTcdB protein will be expressed with a xylose inducible promoter. The C-terminal His tag on the construct will be able to bind to a nickel column during purification. The gRNA will be transcribed in vitro, and incubated with the Cas9-aTcdB.
A crucial step in this project is to create a biological template for the Cas9 gRNA that can easily be customized to anneal to any sequence. To this end, our Biobrick was adapted from a sequence used for CRISPR-Cas9 editing in flies. In the original sequence, created by the O'Connor-Giles, Wildonger and Harrison labs, a U6 promoter is used, followed by two Bbs1 cut sites facing opposite directions, the rest of the guide RNA template, and a terminator. The cut sites allow easy integration of a new 20nt long guide sequence.
In our created part, we used a T7 promoter to allow transcription in E. coli or with in vitro transcription kits. This will allow other iGEM teams in the future to have access to an easily customizable construct, one which can be modified by only using one restriction enzyme.