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Figure 1. Effects of different sequences of sgRNAs (sgRNA1-3), including the vector and positive control sgRNA, on gene expression according to RT‒qPCR at 0 and 24 h. (a-e) Fold changes in the dCas9 gene. (f-g) Fold changes in the number of cells positive for the mmpL3 gene. (h-k) Fold changes in the inhA gene. (l) Comparison of the fold changes in inhA gene expression after 24 h of aTc activation. Statistical significance was determined using nonparametric tests (Mann‒Whitney U test) for comparisons between inactivated and activated samples using 100 ng/ml of aTc at each time point for different strains (a-k). The Kruskal‒Wallis test was performed to analyze the significant differences in inhA gene expression from different sgRNAs after induction by aTc for 24 h (l). Error bars represent the standard deviation from the mean of triplicate experiments (*p < 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Journal: Scientific reports

Article Title: The CRISPR-dCas9 interference system suppresses inhA gene expression in Mycobacterium smegmatis.

doi: 10.1038/s41598-024-77442-2

Figure Lengend Snippet: Figure 1. Effects of different sequences of sgRNAs (sgRNA1-3), including the vector and positive control sgRNA, on gene expression according to RT‒qPCR at 0 and 24 h. (a-e) Fold changes in the dCas9 gene. (f-g) Fold changes in the number of cells positive for the mmpL3 gene. (h-k) Fold changes in the inhA gene. (l) Comparison of the fold changes in inhA gene expression after 24 h of aTc activation. Statistical significance was determined using nonparametric tests (Mann‒Whitney U test) for comparisons between inactivated and activated samples using 100 ng/ml of aTc at each time point for different strains (a-k). The Kruskal‒Wallis test was performed to analyze the significant differences in inhA gene expression from different sgRNAs after induction by aTc for 24 h (l). Error bars represent the standard deviation from the mean of triplicate experiments (*p < 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: The PLJR962 CRISPRi vector expressing Streptococcus thermophilus dCas9 (SthdCas9) with a specific site for inserted sgRNAs (Addgene plasmid #115162) was kindly provided by Dr. Sarah Fortune (Department of Immunology and Infectious Diseases, Harvard T.H.

Techniques: Plasmid Preparation, Positive Control, Gene Expression, Comparison, Activation Assay, Standard Deviation

Figure 3. Effects of different aTc concentrations for CRISPRi activation on gene expression, as determined by RT‒qPCR at 0 and 24 h. (a-c) Fold changes in the dCas9 gene. (d-e) Fold changes in the number of positive sgRNAs and mmpL3 gene expression. (f-g) Fold changes in the inhA gene. Statistical significance was determined using nonparametric ANOVA (Kruskal‒Wallis test) to compare Msm inactivated and activated by 50, 100 or 200 ng/ml of aTc at each time point with the vector control, positive sgRNA, or sgRNA2. Error bars represent the standard deviation from the mean of triplicate experiments (*p < 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Journal: Scientific reports

Article Title: The CRISPR-dCas9 interference system suppresses inhA gene expression in Mycobacterium smegmatis.

doi: 10.1038/s41598-024-77442-2

Figure Lengend Snippet: Figure 3. Effects of different aTc concentrations for CRISPRi activation on gene expression, as determined by RT‒qPCR at 0 and 24 h. (a-c) Fold changes in the dCas9 gene. (d-e) Fold changes in the number of positive sgRNAs and mmpL3 gene expression. (f-g) Fold changes in the inhA gene. Statistical significance was determined using nonparametric ANOVA (Kruskal‒Wallis test) to compare Msm inactivated and activated by 50, 100 or 200 ng/ml of aTc at each time point with the vector control, positive sgRNA, or sgRNA2. Error bars represent the standard deviation from the mean of triplicate experiments (*p < 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: The PLJR962 CRISPRi vector expressing Streptococcus thermophilus dCas9 (SthdCas9) with a specific site for inserted sgRNAs (Addgene plasmid #115162) was kindly provided by Dr. Sarah Fortune (Department of Immunology and Infectious Diseases, Harvard T.H.

Techniques: Activation Assay, Gene Expression, Plasmid Preparation, Control, Standard Deviation