Two-plasmid genome-editing system

Plasmid design was based on a previous study⁴², in which efficient E. coli genome editing was achieved using a two-plasmid approach: plasmid 7 (pCasJZ) and plasmid 8 (pUC57_region1_tet). Plasmid 7 encodes SpCas9 under an arabinose promoter, the red λ phage proteins Gam, Beta and Exo under a T7 promoter, the SacB cassette and the chloramphenicol resistance cassette. Plasmid 8 encodes two 500-bp-long homology regions (1 and 2), flanked by protospacer adjacent motifs (PAMs) (1 and 2) and target sequences (1 and 2), two guiding RNA sequences with target sequences (1 and 2) and the tetracycline resistance locus. PAMs 1 and 2 and the target sequences 1 and 2 were chosen to be upstream and downstream, respectively, of CPS region 1 in the E. coli C43 genome. Homology regions 1 and 2 are 500 bp sequences upstream and downstream of target sequences 1 and 2.

Plasmid 7 was generated using the Gam, Beta, Exo, λ tL3 terminator, SacB, SacB promoter, araBAD promoter and SpCas9 elements from plasmid pCasPA (Addgene 113347), which were cloned by Gibson Assembly (NEB) into MCS-1 of the pACYCDuet plasmid. Next, araBAD and araC were cloned in place of the deleted MCS-II of that plasmid. The homology regions, guiding RNAs, PAMs and target sequences were synthesized by Gene Universal into the pUC57 vector. Then, using Gibson assembly, the ampicillin resistance cassette was replaced with the tetracycline resistance cassette from pBR322 plasmid, giving rise to plasmid 8.