Fig. 1
Overview of this work. a Proof-of-concept demonstration of the CRISPRi pooled screening for high-throughput functional genomics in E. coli. An sgRNA library targeting genes of interest is synthesized on a DNA microarray. Oligonucleotides are amplified and cloned into expression plasmids, transformed into E. coli expressing dCas9 protein, resulting in cell libraries. The cell libraries are grown under selective and control conditions. NGS libraries are constructed based on the extracted plasmids to determine the log2 change of each sgRNA between the selective and control conditions (sgRNA fitness). The sgRNA fitness distribution (red histogram) of each gene is compared with that of control sgRNAs (no target site in the E. coli genome; gray histogram) to evaluate the extent to which this gene is associated with relevant phenotypes (selective conditions). In the first part of this work, we designed a tiling sgRNA library composed of 2281 members targeting 44 genes with known phenotypes to evaluate the activities of these sgRNAs. b The absolute values of the Z scores for each sgRNA targeting the true positive genes were extracted, and the distribution of each group (categorized via position in the ORFs) against all 468 sgRNAs was quantified by a two-tailed MWU test. Triple asterisks indicate P < 0.01. For clarity, only sgRNAs with absolute Z scores of 0–4 were plotted. c The minimal number of sgRNAs per gene for reliable hit-gene calling. Results are shown for sampling of 3, 5, 10, 15, 20, and 30 sgRNAs for each gene (16 true positive genes in Library I). Two algorithms (position (Supplementary Fig. 4, see Methods) and random) were applied to determine the priority of sgRNA selection during sampling. Results are presented as box plots of the −Log10(P value) (MWU test) for the genes recalculated with the sampled sgRNA subset. Dashed line refers to P = 0.01. d In the second part of this work, we designed a genome-scale sgRNA library for E. coli based on the rules learned from the tiling experiment and performed screening experiments to test our methods
