Fig 5.

Efficient depletion of M protein and in vivo CRISPRi in GAS. (A) Strain NV25 (NV6 + pDC-sgRNA-emm) was grown in THB containing 2 µg/mL of chloramphenicol at 37°C in microtiter plates in the presence of various concentrations of doxycycline. (B) Strains NV25 (sgRNA-emm), NV26 (sgRNA-control), and M protein knockout GAS M1T1 5448 were grown in THB to mid-logarithmic growth in the presence or absence of 20 ng/mL doxycycline. Bacteria were immunostained using M protein antisera and analyzed by flow cytometry as described in Materials and Methods. (C) Strains NV1 (wild-type GAS 5448) and NV6 (cas9::tetM, tetR, Ptet-dcas9, hsdR::ery) were grown in THB at 37°C, and 1–3 × 10⁸ colony-forming units (CFU) were used to infect CD-1 mice IP (10 mice per group). Disease score and survival were followed for 14 days. There was no statistically significant (n.s.) difference in virulence between both strains (Mantel-Cox Log Rank test). (D) Strains NV26 (NV6 + pDC-sgRNA-control) and NV19 (NV6 + pDC-sgRNA-ftsZ) were grown in THB containing 2 µg/mL of chloramphenicol at 37°C, and 1–3 × 10⁸ CFU were used to infect CD-1 mice IP (10 mice per group). Disease score and survival were followed for 14 days. Doxy-induced mice infected with NV19 showed a statistically significant increased survival (**P = 0.0027, Mantel-Cox Log Rank test).