sgrna library pcr product  (New England Biolabs)

Journal: bioRxiv

Article Title: Genome-wide high-density CRISPR interference screens reveal condition-specific metabolic vulnerabilities in Pseudomonas aeruginosa PAO1

doi: 10.1101/2025.08.12.669819

Figure Lengend Snippet: a, Schematic of construction of pBx-Gm and pBx-Amp, two plasmids for high-efficiency, high-fidelity sgRNA cloning. b , Schematic of the positions of PAMs for which sgRNAs were included in the library in the NT (green triangles) or T (red triangles) strand of coding sequences, or on both strands in intergenic regions (orange triangles). PAMs on the T strand for which no sgRNAs were included are shown by dotted triangles. CDS, coding sequence. c , Schematic of the sgRNA oligo pool design and the PCR-based second-stranding and amplification method using oligos 14919 and 14920 priming in universal flanking M13 and M13R binding regions. RC, reverse complement. d , Distribution of number of sgRNAs per gene or intergenic region. e , Correlation of number of sgRNAs per gene and gene length. f , Distribution of sequencing reads for all designed sgRNAs in the final pBx-Gm-based library. g , Distribution of gene length of genes that are covered by at least 3 sgRNAs, each with at least 10 sequencing reads (orange), and those that do not meet this criterion (blue) shown for the pBx-Gm-based library. Note that characteristics of the pBx-Amp-based library are very similar to the ones shown here for the pBx-Gm-based library.

Article Snippet: The sgRNA library was then cloned in pBx-Gm or pBx-Amp using Golden Gate assembly in a reaction containing 10 μl pBx_Gm (67 ng/μl) or pBx_AmpR (72 ng/μl), 10 μl sgRNA library PCR product (16 ng/μl), 4000 units of T4 DNA ligase (NEB #M0202S) and 120 units of BsaI-HFv2 (NEB #R3733S) in ligase buffer (NEB #B0202) in a total volume of 200 μl.

Techniques: Cloning, Sequencing, Amplification, Binding Assay

Journal: bioRxiv

Article Title: Genome-wide high-density CRISPR interference screens reveal condition-specific metabolic vulnerabilities in Pseudomonas aeruginosa PAO1

doi: 10.1101/2025.08.12.669819

Figure Lengend Snippet: a , Heatmap of log2 fold-changes (LFC) of all genes that showed significant enrichment or depletion (P-val<0.05, |LFC|>1) in at least one condition plotted using the seaborne.clustermap function and average clustering method. b , Venn diagrams depicting pairwise comparisons of Median and Second-best quantification methods after 24h or 48h of growth (panels i-v) and indicated comparisons to the gene essentiality data by Lee et al ., 2015 (panel vi). Note that for panels i-v both enriched and depleted genes were included, whereas for comparisons in panel vi only depleted genes, i.e. genes whose CRISPRi-mediated repression causes fitness defects, were used. The number of genes in each sector of the Venn diagrams is indicated. c , Top: Functionally enriched categories in the current (red) or the Lee et al . dataset (blue) on LB medium as determined using the String database ( Szklarczyk et al , 2023 ). Bottom: LFCs for all sgRNAs of select genes under different growth conditions. Each dot represents the LFC of a single sgRNA calculated from four independent biological replicates, and the median of all sgRNA LFCs for one gene is indicated by a brown line. The grey dashed line marks the -1 LFC threshold. For genes, PA numbers (locus tags) as well as their annotations according to a reference genome annotation file ( Stover et al , 2000 ) are given at the bottom. d , Venn diagrams for genes for which sgRNAs were depleted in at least one of the three tested growth conditions upon induction versus the LB uninduced control are shown along with functionally enriched categories according to the String database. Single select genes are listed in parentheses if the belonged to an enriched category or without if they were not recognized as such by String. e , Similar to d, but for genes whose downregulation improved fitness, i.e. their sgRNAs were enriched. The dotted boxes for glucose and succinate conditions indicate that no functionally enriched categories were identified; instead, we highlight individual genes that deemed interesting to us.

Article Snippet: The sgRNA library was then cloned in pBx-Gm or pBx-Amp using Golden Gate assembly in a reaction containing 10 μl pBx_Gm (67 ng/μl) or pBx_AmpR (72 ng/μl), 10 μl sgRNA library PCR product (16 ng/μl), 4000 units of T4 DNA ligase (NEB #M0202S) and 120 units of BsaI-HFv2 (NEB #R3733S) in ligase buffer (NEB #B0202) in a total volume of 200 μl.

Techniques: Control

Journal: bioRxiv

Article Title: Genome-wide high-density CRISPR interference screens reveal condition-specific metabolic vulnerabilities in Pseudomonas aeruginosa PAO1

doi: 10.1101/2025.08.12.669819

Figure Lengend Snippet: a , Schematic of the central carbon metabolism of P. aeruginosa PAO1. Metabolites are shown in black, enzymes and the reactions they potentially catalyze (arrows) are color-coded according to their CRISPRi scores: blue, no significant change under any conditions; dark orange, essential under all growth conditions; light orange, essential under minimal medium conditions in general; green, essential in glucose conditions only; magenta, essential in succinate conditions only. Parts of the EDEMP cycle that are derived from the EMP, ED or PP pathways are highlighted by different colors, as is the TCA cycle (light blue). Abbreviations for metabolites are defined in the main text or are as follows: DHAP, dihydroxyacetone phosphate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; E4P, erythrose-4-phosphate; S7P, sedoheptulose-7-phosphate; X5P, xylulose-5-phosphate; CIT, citrate; SUC, succinate; FUM, fumarate; MAL, malate. b , LFCs for all sgRNAs of central carbon metabolism genes/proteins mapped in panel a are shown for the three different growth conditions: LB-Miller, MOPS/glucose and MOPS/succinate. Each dot represents the log2 fold-change (LFC) of a single sgRNA calculated from four independent biological replicates, and the median of all sgRNA LFCs for one gene is indicated by a brown line. The grey dashed line marks the -1 LFC threshold. For genes, PA numbers (locus tags) as well as their annotations according to a reference genome annotation file ( Stover et al , 2000 ) are given at the bottom. Asterisks indicate genes that were generally essential as defined by our cutoff, but showed clear quantitative differences between glucose- and succinate-grown cells. GapN (PA2323) and GapB (PA3001) together with Pgk (PA0552), key enzymes in lower glycolysis which are differentially required during growth on glucose and succinate and discussed in the main text, are highlighted in bold and with green and magenta backgrounds, respectively.

Article Snippet: The sgRNA library was then cloned in pBx-Gm or pBx-Amp using Golden Gate assembly in a reaction containing 10 μl pBx_Gm (67 ng/μl) or pBx_AmpR (72 ng/μl), 10 μl sgRNA library PCR product (16 ng/μl), 4000 units of T4 DNA ligase (NEB #M0202S) and 120 units of BsaI-HFv2 (NEB #R3733S) in ligase buffer (NEB #B0202) in a total volume of 200 μl.

Techniques: Derivative Assay