Fig 2.

Development of a doxycycline-inducible CRISPRi system for use in group A Streptococcus. (A) Schematic representation of CRISPRi. An sgRNA encoding a 20 nt spacer region targets dCas9 to the non-template strand containing a protospacer adjacent motif (PAM) on the GAS ftsZ locus. Transcription elongation by RNA polymerase (RNAP) is consequently hampered leading to reduced levels of FtsZ in the cell when dCas9 is induced. (B) Schematic representation of the genomic organization of the cas9 locus in wild-type GAS strain 5448 (NV1), in strain NV4 (cas9::kan) and strain NV6 (cas9::tetM, tetR, Ptet-dcas9). (C) Schematic representation of sgRNA cloning vector pDC-sgRNA. (D) Strain NV9 (NV1, pDC-sgRNA) was grown in Todd Hewitt broth containing 2 µg/mL of chloramphenicol at 37°C, and exponentially growing cells were imaged by differential interference contrast (DIC) and fluorescence microscopy. Scale bar: 2 µm. (E) Oligo-based sgRNA cloning in pDC-sgRNA. Plasmid pDC-sgRNA is cut with Esp3I (or its isoschizomer BsmBI), and its resulting sticky ends are shown. Two complementary oligos of each 24 nt long that include a 20-bp spacer sequence are annealed, phosphorylated, ligated, and electroporated to competent GAS (see Materials and Methods). As an example, the two oligos used to clone the sgRNA targeting GAS ftsZ are shown (for oligo design, see Table S1). Successful clones lost the mCherry cassette and will be white on the plate instead of pink.