Experiment (PMID: 34297925): We designed an M. tuberculosis CRISPRi library (RLC12) to target all annotated Mtb genes with single guide RNAs (sgRNAs) of varying predicted knockdown efficiencies. In parallel, we constructed a M. smegmatis CRISPRi library (RLC11) to target all annotated Msmeg genes, similar to the Mtb sgRNA library. RLC12 was transformed into M. tuberculosis H37Rv; RLC11 into M. smegmatis mc2-155. We passaged both CRISPRi libraries for nearly 30 generations in the presence or absence of the CRISPRi-inducer anhydrotetracycline (ATc). At the indicated timepoints, genomic DNA was harvested and the representation of individual sgRNAs was counted using Illumina sequencing. Here, we show sgRNA log2 fold-change values (L2FC) (plus versus minus ATc) over consecutive generations for sgRNAs targeting the gene you selected. sgRNAs are color-coded based on their predicted strength from blue (strength=0; weak) to red (strength=1; strong).

In order to quantify gene vulnerability, data were subsequently analyzed with a hierarchical model at the sgRNA level (two-line model) and gene level (Hill curve). The vulnerability quantification is shown below. [More information]

	M. smegmatis Reference Fitness Experiment	M. tuberculosis H37Rv Reference Fitness Experiment	M. tuberculosis HN878 Reference Fitness Experiment
	M. smegmatis Gene-Level Logistic Regression	M. tuberculosis H37Rv Gene-Level Logistic Regression (Most similar ortholog)	M. tuberculosis HN878 Gene-Level Logistic Regression (Most similar ortholog)
Vulnerability Index (VI)	-0.1550	1.0040
VI Lower Bound	-0.2240	-1.0150
VI Upper Bound	-0.0830	3.8240