Team:UT-Dallas/Project/details

Stage 1

Stage 1 of the project deals with testing the cutting efficiency of the Cas9-gRNA complex. For our project, we constructed a library of gRNAs that target an array of genes found in the pathogen Vibrio cholerae. To test whether our library of gRNAs could cut the desired genes, we first began by constructing reporter vectors that have the gRNA binding sequence in the 5' region of our reporter (YFP). When our Cas9 protein is produced in the presence of a tetracycline derivative (doxycycline), formation of the Cas9-gRNA complex can occur, followed by subsequent binding of the reporter vector and cutting of the vector. Once the vector is cut, the bacterial ENZYME, RecBCD nuclease, degrades the linearized vector. The vector also produces the antibiotic cat gene, which offers the bacteria chloramphenicol resistance,so the cells dies once this vector is cut, as we show in the results page for acfA and ctxB gRNA.