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. 2017 Mar 21;7:44929. doi: 10.1038/srep44929

Figure 1. A schematic map of programmable and integrative CRISPR/Cas9 system.

Figure 1

(A) To generate a programmable CRISPR/Cas9 system, synthetic oligos containing a promoter, leader sequence and direct repeat (DR) sequence flanked with the BbsI restriction sites (Supplemental Figure S1) were cloned into the pMK4 shuttle vector, resulting pKS1. Synthetic oligos specific to the target gene (spacer sequence) were cloned into the BbsI site, resulting pKS2. (B) To generate integrative CRISPR/Cas9 system, genes encoding tracr-RNA and Cas9 were amplified from chromosomal DNA of Streptococcus pyogenes SF370 using PCR and cloned into the modified pMAD-secY temperature sensitive shuttle vector, resulting pKS3. To program CRISPR/Cas9 system specific to the target gene, the pre-crRNA (the promoter, leader sequence, DR, and spacer sequence) was amplified from pKS2 and cloned into pKS3, resulting pKS4. (C) The programmed CRISPR/Cas9 system was integrated into the non-coding region of ϕSaBov genome by homologous recombinations. (D) The CRISPR/Cas9 system programmed to target S. aureus is induced and delivered by ϕSaBov. The CRISPR/Cas9 system (tracrRNA, crRNA, and Cas9) is expressed and scanned the PAM sequence and recognize the target sequence in the chromosomal DNA, leading to chromosomal DNA cleavage and bacterial death.