crispri sgrna libraries set 2  (Addgene inc)

Journal: Nature Communications

Article Title: CRISPRi screen identifies FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa

doi: 10.1038/s41467-025-61208-z

Figure Lengend Snippet: a Evaluation of the tetracycline- and arabinose-inducible reporter system in P. aeruginosa . The efficacy was assessed by measuring luminescence from luxABCDE expression induced by varying concentrations of doxycycline (Dox) or L-arabinose. b Schematic representation of the tet-inducible CRISPRi system. The system employs a SpydCas9-sgRNA complex, that is directed to DNA target region by a 20-base pair sequence in the sgRNA inhibiting transcription via steric hindrance. dcas9 expression is driven by a P tet promoter, and the sgRNA is expressed from a constitutive promoter. Created in BioRender. Liu, X. (2025) https://BioRender.com/kcvjnbt . c Pyocyanin measurements of the strain with CRISPRi targeting phzM . The up panel shows photographs of culture tubes and lower panel for pyocyanin quantification. d , e Repression of swarming motility by CRISPRi. d presents the swarming colonies of indicated strains on medium with indicated concentration of Dox, and e presents the measurements the surface diameter of the colony. f The reversibility of tet-inducible CRISPRi system in P. aeruginosa . Repression of luxABCDE was induced by 25 or 100 ng/mL Dox at OD600 = 0.001. Following Dox removal and dilution at 7.5 h, luminescence was measured in recovering cultures. LB medium served as negative control. g Growth of CRISPRi knockdown strains targeting ftsZ in various P. aeruginosa backgrounds cultured in LB medium with increasing concentrations of Dox. h CcdB-based sgRNA cloning strategy. The ccdB gene is flanked by BsaI sites for Golden Gate assembly; replacement with sgRNA allows colony formation on LB agar in CcdB sensitive WM3064 strain. i Evaluation of the sgRNA cloning efficiency with the CcdB counter selection system. Three sgRNAs were cloned, and ten colonies from each cloning were randomly picked and sequenced. Created in BioRender. Liu, X. (2025) https://BioRender.com/kcvjnbt . Data from all growth and measurement assays are presented as mean ± SD from three biological replicates. Statistical analysis for panel c and e was performed using Two-way ANOVA and Tukey’s multiple-comparison test. Source data are provided as a Source Data file.

Article Snippet: The pooled CRISPRi sgRNA libraries (set 1 and set 2) for P. aeruginosa PA14 were deposited at Addgene ( https://www.addgene.org/pooled-library/liu-crispri-p-aeruginosa/ ).

Techniques: Expressing, Sequencing, Concentration Assay, Negative Control, Knockdown, Cell Culture, Cloning, Selection, Clone Assay, Comparison

Journal: Nature Communications

Article Title: CRISPRi screen identifies FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa

doi: 10.1038/s41467-025-61208-z

Figure Lengend Snippet: a Workflow of the CRISPRi library construction and fitness evaluation. Oligos (set 1 and 2) were synthesized to create sgRNA libraries targeting 5981 and 5971 genes, respectively. These oligos were then amplified by PCR to form double-strand DNA, which was subsequently cloned into pCRISPRi- ccdB . The resulting plasmids were transformed into E. coli WM3064 to generate sgRNA libraries, which were integrated in to the PA14 chromosome via triparental mating. Effect of CRISPRi on strain fitness in pool was evaluated after approx. 7 (G7, ~5 h incubation in exponential phase), 14 (G14, ~10 h), and 21 (G21, ~15 h) using Illumina sequencing and DEseq2 comparison of spacer abundance in libraries with and without CRISPRi activation (Dox +/−). Created in BioRender. Liu, X. (2025) https://BioRender.com/kcvjnbt . b Genome localization of essential genes identified by CRISPRi-seq and Tn-seq. The outer blue, red and cyan tracks represent genes annotated as essential in previous Tn-seq screens reported by refs. , , , respectively, whereas purple track represents genes identified in this study using CRISPRi-seq. c Comparative analysis of candidate essential gene sets from CRISPRi-seq and Tn-seq screens. d , e Line plots showing the log 2 FC trends in CRISPRi data for the non-essential ( d ) and essential ( e ) genes. Black dots represent sgRNA log 2 FC values. The solid black line represents the locally estimated scatterplot smoothing fit of the individual mean linear regression at different generations (0, 7, 14, and 21). Bar chart displays Functional classification (top 5) of genes with fitness changes for each cluster, based on available COG terms and manual classifications defined in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: The pooled CRISPRi sgRNA libraries (set 1 and set 2) for P. aeruginosa PA14 were deposited at Addgene ( https://www.addgene.org/pooled-library/liu-crispri-p-aeruginosa/ ).

Techniques: Synthesized, Amplification, Clone Assay, Transformation Assay, Incubation, Illumina Sequencing, Comparison, Activation Assay, Functional Assay

Journal: Nature Communications

Article Title: CRISPRi screen identifies FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa

doi: 10.1038/s41467-025-61208-z

Figure Lengend Snippet: a Workflow for screening genes involved in sensitivity to Ga(NO 3 ) 3 treatment. The CRISPRi library was induced with Dox (25 ng/ml) for 14 generations, followed by culture with or without 200 μM Ga(NO 3 ) 3 . Created in BioRender. Liu, X. (2025) https://BioRender.com/kcvjnbt . b Growth curves of PA14 in LB broth with various concentrations of Ga(NO 3 ) 3 . c Volcano plots showing the identified genes involved in Ga(NO 3 ) 3 tolerance. d Growth of control strains PA14, and PA14 harboring CRISPRi system with non-targeting sgRNA ( lucff) . e Validation of targets associated with increased fitness: hitA , hitB , PA14_67550 , PA14_56670 , and thiE using CRISPRi with individual sgRNAs. f Schematic depicting HitAB involved in Ga 3+ and Fe 3+ uptake in P. aeruginosa . g Validation of screening hits with decreased fitness. Knockdown strains targeting PA14_22320 , fdx2 , fprB were constructed and their growth was tested in medium containing the indicated concentrations of gallium and Dox. Representative phenotypes under selected concentrations of Ga(NO₃)₃, chosen to best illustrate differential sensitivity, are shown. h Schematic illustrating the role of FprB in NADP(H)-dependent electron transfer. Created in BioRender. Liu, X. (2025) https://BioRender.com/kcvjnbt . i Plate spot assays showing sensitivity of PAO1 mutants to gallium. All growth data are presented as mean ± SD from three biological replicates. Source data are provided as a Source Data file.

Article Snippet: The pooled CRISPRi sgRNA libraries (set 1 and set 2) for P. aeruginosa PA14 were deposited at Addgene ( https://www.addgene.org/pooled-library/liu-crispri-p-aeruginosa/ ).

Techniques: Control, Biomarker Discovery, Knockdown, Construct

Journal: Nucleic Acids Research

Article Title: Mechanism of Cas9 inhibition by AcrIIA11

doi: 10.1093/nar/gkaf318

Figure Lengend Snippet: AcrIIA11 inhibition mechanism. ( A ) Cas9 initially binds off-target DNA and samples PAMs (gray) in search of the PAM (black square) that is adjacent to the target site. Next, Cas9 forms an R-loop and cleaves the target DNA. ( B ) AcrIIA11 forms a 1:1 complex with Cas9. The complex binds PAM-rich off-target sites and prevents SaCas9 diffusion and target search. ( C ) AcrIIA11 also inhibits nuclease activity for those Cas9s that found the target.

Article Snippet: The TS–SUMO– Sa Cas9–sgRNA cell lysate was applied to a 5 ml of Strep-Tactin Superflow 50% suspension (IBA Life Sciences, 2-1206-025) gravity column equilibrated in Lysis Buffer containing 200 mM NaCl, 25 mM HEPES, pH 7.5, and 2 mM DTT supplemented with protease inhibitors and DNase.

Techniques: Inhibition, Diffusion-based Assay, Activity Assay