Fig. 1: WG-CRISPRi screening in M. tuberculosis strain mc26206. | Nature Communications

Fig. 1: WG-CRISPRi screening in M. tuberculosis strain mc26206.

From: Whole genome CRISPRi screening identifies druggable vulnerabilities in an isoniazid resistant strain of Mycobacterium tuberculosis

Fig. 1

a Experimental design for WG-CRISPRi essentiality screening. The pooled CRISPRi plasmid library is transformed into appropriate M. tuberculosis strains. WG-CRISPRi screens were performed for 14 days, with varying concentrations of ATc and at day 5 and 10 back diluted (1/20) into fresh media + ATc to maintain log phase growth. gDNA was extracted, gRNA amplified and sequenced from samples collected on days 5, 10 and 14. The proportion of each gRNA at each ATc concentration within the pooled population was quantified relative to ATc-0 for each sampled time point and plotted on a log2-fold scale as a reduction in gRNA abundance. Increased depletion of gRNA was used to identify genes that are either synthetic lethal or have increased vulnerability in INHR-katG. Created in BioRender. Wang, X. (2023) BioRender.com/q45z115. b–g Summary of gRNA abundance in the M. tuberculosis strain (b) mc26206 DS-parent, (d) INHR-katG, and (f) the depletion difference between the DS-parent and INHR-katG. The gRNA abundance is relative to the ATc-0 control at each time point calculated by the exact test and p-values adjusted by the Benjamini–Hochberg method. Unchanged gRNAs are coloured grey, whilst gRNA with significant > 2-fold depletion (Benjamini–Hochberg adjusted p < 0.01) are coloured green. The red dot within each violin plot denotes the mean gRNA depletion. A total of 3991 unique protein-encoding genes were screened. Of these, the number of essential genes identified in the (c) DS-parent and (e) INHR-katG were illustrated under different time points and ATc concentrations. g Genes identified as being more vulnerable to inhibition in INHR-katG are defined as when (i) they were called essential in INHR-katG and (ii) had no less than 2 gRNAs that were depleted by >1-log2 fold more than observed in the DS-parent. Of these, genes were identified as “synthetic lethal” when had no less than 2 gRNAs that were (1) not depleted in the DS-parent and (2) were depleted by >1-log2 fold in INHR-katG more than observed in the DS-parent. Source data are provided as a Source Data file.

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