Fig 2.

Use of mCherry as a reporter for streamlined screening of positive CRISPRi clones and exemplifying CRISPRi targeting with radD. (A) Diagram illustrating the construction of CRISPRi plasmid with mCherry as a screening reporter is outlined. (1) Linearization of pZP4C: the pZP4C is cleaved using MscI and NotI enzymes. (2) Replacement of sgRNA segment with the mCherry gene. The mCherry is driven by the PrpsJ_Cd promoter. The resulting plasmid is designated as pZP4C(mCherry). (3) Release of PrpsJ_Cd -mCherry: pZP4C(mCherry) is again cut using MscI/NotI to release PrpsJ_Cd -mCherry for subsequent cloning. (4) Design primers P1 and P2. P1 is a 60-nt primer starting with a 21-nt PrpsJ_FP promoter match, followed by a 20-nt target gene sequence and ending with a 19-nt sgRNA handle region. The sgRNA includes a 20-nt base-pairing region, a 42-nt dCas9-binding RNA structure (dCas9 handle), and a 40-nt transcription termination sequence from S. pyogenes. (5) Amplification of new target sgRNA: use pZP4C plasmid DNA as a template with primers P1 and P2 to generate a 196-bp target sgRNA fragment. (6) Cloning of new sgRNA: insert the new sgRNA fragment into digested pZP4C(mCherry) via Gibson assembly, creating a new CRISPRi plasmid for targeting a new gene. (B) Designing P1 primer with radD gene example. For instance, the radD gene illustrates the P1 primer design. A 20-nt base pair region from the 670th to the 789th base of radD is selected. This sequence, aligned in a 5′ to 3′ orientation on the template strand, is incorporated into the P1 primer [designated P1(radD)]. The PAM (TGG) sequence is indicated through underlining in lowercase. The MscI site in P1 and the NotI site in P2 are shown. (C) Positive clone selection uses a ChemiDoc MP Imaging System (Bio-Rad). Fluorescent cells, considered negative clones, appear pseudocolored in white under the Cy3 setting, whereas non-fluorescent cells (positive clones) are in gray. (D) CRISPRi-mediated repression of the radD gene abolishes coaggregation with A. oris. The wild-type F. nucleatum strain containing pZP4C(radD) was cultured in a TSPC medium under different theophylline concentrations for 22 hours in anaerobic conditions. Subsequently, cells were collected, washed, resuspended in a coaggregation buffer, and assessed for coaggregation with A. oris. A representative result is presented after three experimental repeats. (E and F) Cells subjected to the experimental conditions detailed in panel D undergo SDS-PAGE followed by immunoblotting. Antibodies specific to RadD (α-RadD) and FomA (α-FomA) are used for detection. FomA serves as a loading control. Molecular weight markers (in kilodaltons) are indicated on the left side of the blot.