Figure 3

SSDR/Cas9 genome engineering. (i) A bacterial strain is transformed with a plasmid containing a Cas9 endonuclease along with a tracrRNA. RecT activity can be provided either in trans on another plasmid or if the bacterium has a RecT protein in the native chromosome endogenous recombinase activity may be sufficient if recombineering activity is high enough. The strain is made electrocompetent and an oligonucleotide containing the desired mutation to be incorporated (denoted by red circle). (ii) Incorporation of the oligonucleotide (green) ultimately results in the alteration of G-G for T-T, which will disrupt the PAM site (iii). Note that the PAM in this example (NGG) is variable depending on the source of Cas9 utilized in the experiment. (iv) Electroporation of a plasmid containing a crRNA targeting the chromosome into the population of recombineered cells. The crRNA will direct Cas9 to cleave the chromosome of the wild-type cells, while mutant chromosomes will not be cut. By selecting for cells that contain the crRNA plasmid by antibiotic selection, only cells that have acquired the mutation via recombineering and avoid Cas9 cleavage will survive.