s cerevisiae inducible crispri library  (Addgene inc)

Journal: mBio

Article Title: Genome-wide CRISPRi screen and proteomic profiling identify key genes related to ferulic acid’s antifungal activity

doi: 10.1128/mbio.01909-25

Figure Lengend Snippet: Comprehensive genome-wide CRISPRi analysis of S. cerevisiae response to ferulic acid. ( A ) Schematic representation of the CRISPRi library screening and competitive growth. S. cerevisiae cells expressing a genome-wide CRISPRi library were subjected to competitive growth in the presence of FA or DMSO (control). The dCas9-Mxi1 system, guided by gRNAs, repressed target gene transcription upon induction with ATc. Pooled cultures underwent serial dilutions (1:100), followed by plasmid extraction and sequencing to assess gRNA abundance. Enrichment analysis identified genes whose repression either increased sensitivity (depleted gRNAs) or conferred resistance (enriched gRNAs) to FA, as visualized in the volcano plot. ( B ) Correlation matrix showing Pearson correlation coefficients between biological replicates of FA-treated (FA), DMSO control (DM), and initial library samples (Li). The color scale (blue to red) indicates the strength of correlation. ( C ) Volcano plot illustrates differential gRNA abundance between FA-treated and DMSO control samples. Genes significantly depleted (blue) or enriched (orange) are highlighted, with thresholds of log2 fold change >2 or <−2 and P value <0.05. ( D ) Top-ranked genes sensitive to FA treatment. The top 10 genes with the highest RRA scores in negative selection. ( E ) Top-ranked genes contributing to FA resistance. The top 10 genes with the highest RRA scores in positive selection. ( F ) STRING network analysis of 194 genes depleted upon FA treatment. The network is divided into three distinct clusters: blue, green, and red. The blue cluster primarily contains genes involved in ribosomal biogenesis and RNA metabolism. The green cluster includes genes related to mitochondrial function, protein degradation, and stress responses. The red cluster comprises genes associated with mRNA processing, splicing, and transport.

Article Snippet: S. cerevisiae inducible CRISPRi Library (Addgene #161829) was transformed into DH5α Escherichia coli, generating more than 4 M colonies.

Techniques: Genome Wide, Library Screening, Expressing, Control, Plasmid Preparation, Extraction, Sequencing, Selection

Journal: mBio

Article Title: Genome-wide CRISPRi screen and proteomic profiling identify key genes related to ferulic acid’s antifungal activity

doi: 10.1128/mbio.01909-25

Figure Lengend Snippet: Effect of ERG9 silencing on FA resistance and upstream enzyme expression. ( A ) Lollipop plot of ergosterol biosynthesis genes. Log2 fold change (FA-DMSO) obtained from MAGeCK test analysis. Each gene response is colored based on the direction of fold change, with red indicating a negative log-fold change and blue indicating a positive log-fold change. Asterisks denote statistical significance levels: * <0.05, ** <0.01, and *** <0.001. ( B ) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) confirmation of ERG9 silencing. mRNAs were quantified by RT-qPCR analysis from three independent biological repeats, each with three technical repeats, normalized to ACT1 mRNA levels. ( C ) Yeast growth assay comparing the FA resistance of the ERG9 CRISPRi strain to the empty vector (E.V.) control. Growth was monitored in the presence of DMSO and 1.5 mM FA over 48 h. ( D ) Quantitative analysis of Yeast drop assay. Log2 fold-change (FA-DMSO) in the ERG9 CRISPRi strain compared to the empty vector. Error bars represent the standard error of the mean (SEM) from three independent biological repeats, and the P value was calculated by the dependent samples one-tailed t -test. ( E ) Expression levels of nine upstream enzymes in the ergosterol biosynthesis pathway following ERG9 silencing. mRNAs were quantified by RT-qPCR analysis and normalized to ACT1 mRNA levels and empty vector expression levels. The histogram presents the quantification of three independent biological repeats, each with three technical repeats. P values were calculated by the dependent samples one-tailed t -test.

Article Snippet: S. cerevisiae inducible CRISPRi Library (Addgene #161829) was transformed into DH5α Escherichia coli, generating more than 4 M colonies.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Growth Assay, Plasmid Preparation, Control, One-tailed Test

Journal: mBio

Article Title: Genome-wide CRISPRi screen and proteomic profiling identify key genes related to ferulic acid’s antifungal activity

doi: 10.1128/mbio.01909-25

Figure Lengend Snippet: Comprehensive genome-wide CRISPRi analysis of S. cerevisiae response to ferulic acid. ( A ) Schematic representation of the CRISPRi library screening and competitive growth. S. cerevisiae cells expressing a genome-wide CRISPRi library were subjected to competitive growth in the presence of FA or DMSO (control). The dCas9-Mxi1 system, guided by gRNAs, repressed target gene transcription upon induction with ATc. Pooled cultures underwent serial dilutions (1:100), followed by plasmid extraction and sequencing to assess gRNA abundance. Enrichment analysis identified genes whose repression either increased sensitivity (depleted gRNAs) or conferred resistance (enriched gRNAs) to FA, as visualized in the volcano plot. ( B ) Correlation matrix showing Pearson correlation coefficients between biological replicates of FA-treated (FA), DMSO control (DM), and initial library samples (Li). The color scale (blue to red) indicates the strength of correlation. ( C ) Volcano plot illustrates differential gRNA abundance between FA-treated and DMSO control samples. Genes significantly depleted (blue) or enriched (orange) are highlighted, with thresholds of log2 fold change >2 or <−2 and P value <0.05. ( D ) Top-ranked genes sensitive to FA treatment. The top 10 genes with the highest RRA scores in negative selection. ( E ) Top-ranked genes contributing to FA resistance. The top 10 genes with the highest RRA scores in positive selection. ( F ) STRING network analysis of 194 genes depleted upon FA treatment. The network is divided into three distinct clusters: blue, green, and red. The blue cluster primarily contains genes involved in ribosomal biogenesis and RNA metabolism. The green cluster includes genes related to mitochondrial function, protein degradation, and stress responses. The red cluster comprises genes associated with mRNA processing, splicing, and transport.

Article Snippet: To comprehensively identify and characterize molecular pathways and resistance mechanisms associated with FA treatment, we utilized a previously established genome-wide CRISPRi library (Addgene #161829) ( ).

Techniques: Genome Wide, Library Screening, Expressing, Control, Plasmid Preparation, Extraction, Sequencing, Selection