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. 2023 Aug 30;11(5):e01123-23. doi: 10.1128/spectrum.01123-23

Fig 2.

Fig 2

Development of a CSYi system for gene silencing. (A) Steps to construct plasmids for targeted gene repression (czcR as an example). The csy operon which contains csy1, csy2, csy3, and csy4 genes was obtained from the genome of P. aeruginosa PA14. Ptrc promoter was obtained from the pACRISPR plasmid. The Ptrc promoter and csy operon were assembled between the EcoRI and SacI sites on the plasmid of pBBR1-MCS5, generating pCsy. Plac promoter together with its downstream BamHI and KpnI sites was obtained from pUC57 and inserted at the BamHI site of the plasmid pMS402, generating pEmpty. Oligonucleotides U1 and U2, D1 and D2 were in vitro phosphorylated and annealed to form double-stranded DNA fragments, respectively, and then assembled between the BamHI and KpnI sites of pEmpty, generating pCzcR which contained a mini-CRISPR to encode a crRNA targeting the promoter region of the czcR gene. NNNN indicated the 28-bp repeat sequence (5′-GTTCACTGCCGTATAGGCAGCTAAGAAA-3′) of the CRISPR array. 32-bp spacer sequence was highlighted in red. The Plac promoter and the mini-CRISPR were amplified together and inserted at the XbaI site of the pCsy plasmid, generating pCsy-CzcR. A diagram of the crRNA-guided Csy-crRNA complex binding to the promoter of the czcR gene was shown. (B) Relative expression of the czcR gene in PAO1 strains containing the pCsy-CzcR plasmid (czcR-i) or the control plasmid (Ctrl) which did not carry the mini-CRISPR. (C) Relative expression of the czcR, katE, acrA genes in PA_HN002, P. putida KT2440, E. coli MG1655 strains containing the CSYi plasmids targeting the czcR, katA, acrA genes, respectively, or the control plasmid (Ctrl), which did not carry mini-CRISPR. (D) Diagram of the pA3Csy-CzcR plasmid and its working mechanism to achieve targeted gene silencing in strains containing native type I-F CRISPR-Cas systems. (E) Relative recovery rate of the PA14 strain by electroporation of the control plasmid (Ctrl), the pCsy-CzcR plasmid (czcR-i), the pA3Csy-CzcR plasmid (A3czcR-i), and the pA23Csy-CzcR plasmid (A23czcR-i). (F) Relative expression of the czcR gene in PA14 strains containing the pA3Csy-CzcR plasmid (A3czcR-i) or the control plasmid (A3Ctrl), which did not carry the mini-CRISPR, and PA14 strains containing the pA23Csy-CzcR plasmid (A23czcR-i) or the control plasmid (A23Ctrl), which did not carry the mini-CRISPR. Statistical significance is calculated based on the Student’s t-test (*P < 0.05; ***P < 0.001).