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. 2016 Apr 1;44(8):3801–3810. doi: 10.1093/nar/gkw214

Figure 1.

Figure 1.

CRISPR/dCas9 system blocks initiation of replication at the oriC locus. (A) Schematic of initiation of replication by DnaA. Cooperate binding of DnaA proteins to oriC induces unwinding of an adjacent AT-rich region, thus providing single-stranded DNA substrate that is recognized by primosome complexes. Green – helicase DnaB, yellow – helicase loader DnaC. (B) The CRISPR/dCas9 system consists of two plasmids, one coding for dCas9 under the control of an aTc-inducible promoter and the other coding for sgRNA under control of a constitutive promoter. When CRISPR/dCas9 binds to the oriC region, DnaA cannot bind and unwind the DNA, and initiation of replication is blocked. (C) Simultaneous expression of dCas9 and sgRNA has a lethal effect on cells. Serial 10-fold dilutions of liquid bacterial cultures were plated either on the media supplemented (+aTc) or not (−aTc) with 200 ng/ml of aTc. Only in presence of both CRISPR/dCas9 components, cells are not viable.