Figure 4: Identification of differential vulnerabilities in $\Delta$whiB7.

(A) Differential vulnerability screen in $\Delta$whiB7 M. smegmatis. This screen identifies genes that become more or less sensitive to CRISPRi inhibition between wild-type and $\Delta$whiB7 M. smegmatis. Please see the Materials and Methods section and Bosch et al.²⁹ for further details on screen analysis.

(B) Expression-fitness relationships for the five indicated M. smegmatis genes. The fitness cost (beta_E) is plotted as a function of predicted sgRNA strength (an estimate of the magnitude of target knockdown). Both aspC, asd, and hisC are more vulnerable in $\Delta$whiB7 M. smegmatis (tourquise).

(C) Growth of the indicated M. smegmatis CRISPRi strains monitored by spotting serial dilutions of each strain on the indicated media. Supplemental alanine and aspartate were added at 1 mM. Note that hypomorphic sgRNAs that are predicted to lead to intermediate levels of knockdown are shown for trpC and mmpL3, as strong sgRNAs leading to high-level knockdown would block growth of both wild-type and $\Delta$whiB7 M. smegmatis and not be relevant controls for differential vulnerability.

(D) Growth of the indicated M. tuberculosis dual-gene knockdown CRISPRi strains in 7H9 + ATc, with or without supplemental alanine (1 mM). Dual NT represents a CRISPRi plasmid encoding two non-targeting sgRNAs; NT + whiB7 KD represents a CRISPRi plasmid encoding a single non-targeting sgRNA and a whiB7 targeting sgRNA; aspC KD + whiB7 KD represents a CRISPRi plasmid encoding one sgRNA targeting aspC and a separate sgRNA targeting whiB7.