Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands
Figure 1
An engineered CRISPR plasmid with spacers targeting the chromosome displays Cas–dependent toxicity.
(A) Strategy for chromosomal targeting (see Materials and Methods). Protospacers were PCR-amplified from target DNA with full or partial repeats and BbsI sites on the primers. PCR products were digested with BbsI and cloned into BbsI-digested plasmids containing a leader sequence and one repeat. This process was repeated to generate multiple spacer inserts. These arrays generate crRNAs that target the template DNA strand but not the mRNA. (B) Transformation plates of P. atrosepticum WT or a Δcas mutant (PCF80) after growth for 36 h on LBA containing Ap. Plasmids transformed contained no spacers (pC1-780), 3 scrambled spacers (pS3-780) and 3 anti-expI spacers (pE3-780). Representatives are shown from experiments performed at least in triplicate.