Fig. 2: Identification of essential genes and evaluation of gene vulnerability in P. aeruginosa by CRISPRi-seq.

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. ^40,41,37, 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 log₂FC trends in CRISPRi data for the non-essential (d) and essential (e) genes. Black dots represent sgRNA log₂FC 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. 2c. Source data are provided as a Source Data file.