High-density CRISPRi screens reveal diverse routes to improved acclimation in cyanobacteria

Abstract

Cyanobacteria are the oldest form of photosynthetic life on Earth and contribute to primary production in nearly every habitat, from permafrost to hot springs. Despite longstanding interest in the acclimation of these microbes, it remains poorly understood and challenging to rewire. This study uses a high-density, genome-wide CRISPR interference screen to examine the influence of gene-specific transcriptional variation on the growth of Synechococcus sp. PCC 7002 under environmental extremes. Surprisingly, many partial knockdowns enhanced fitness under cold monochromatic conditions. Transcriptional repression of genes for core subunits of the NDH-1 complex, which are important for photosynthesis and carbon uptake, improved growth rates under both red and blue light but at distinct, color-specific optima. Most genes with fitness-improving knockdowns were distinct to each light color, and dual-target transcriptional repression produced nonadditive effects. Findings reveal diverse routes to improved acclimation in cyanobacteria (e.g., attenuation of genes involved in CO₂ uptake, light harvesting, translation, and purine metabolism) and provide an approach for using gradients in sgRNA activity to pinpoint biochemically influential transcriptional changes in cells.

Keywords: energy transduction; environmental acclimation; genome-wide screens; photosynthesis; spectral tuning.

Fig. 1.

Genome-wide CRISPRi screens reveal condition-specific enrichment of sgRNAs in PCC 7002. (A) For each screen, we transformed a strain of PCC 7002 with dCas9 under the control of an anhydrotetracycline-inducible promoter (ΔacsA:dCas9) with a plasmid-borne sgRNA library, which placed each sgRNA downstream of glpK under the control of an IPTG-inducible promoter. (B) We grew transformed cells under seven growth conditions that differed in temperature (i.e., 22 or 37 °C) or light regime (i.e., color or exposure duration) and harvested cells at an OD₇₃₀ of at least 0.3 (~9 to 10 doublings). (C) We used next-generation sequencing (NGS) to compare the abundance of sgRNAs before and after growth. The plot enumerates genes with or without significant sgRNA depletion in at least one condition (“depleted” or “not depleted”) and compares them with essentiality designations from a prior Tn-Seq analysis of Synechococcus elongatus (10). (D) Genome maps showing the mean sgRNA enrichment for growth under each condition; bar positioning matches the chromosomal location of each gene. Cold, narrow-spectrum conditions reveal numerous growth-enhancing knockdowns (blue bars). Data in panels (B) and (D) represent the mean and SD of n = 2 biological replicates. SI Appendix, Table S1 provides all sgRNA enrichment values.

Fig. 2.

Cold conditions elicit divergent transcriptional sensitivities to blue and red light. (A) The sgRNAs for genes associated with light harvesting, photosynthesis, and cold shock exhibited condition-specific enrichment patterns that diverged most significantly between 22B and 22R. Box plots show the median, upper and lower quartile, and minimum and maximum values of normalized sgRNA enrichment. (B) We analyzed the correlation in sgRNA enrichment between standard growth (37W) and monochromatic conditions. Outlier populations (i.e., sgRNAs with a standardized residual greater than 3) differ between temperatures within each light color. (C) We clustered genes into five groups using enrichment data from 37W, 22R, and 22B as input parameters in k-means clustering. These clusters, which are plotted here with t-SNE, show condition-specific enrichment patterns. (D) We classified clusters by trends in mean sgRNA enrichment. Violin plots show the median, Upper, and Lower quartile, and minimum and maximum values of mean locus Log₂FC. (E) The three major gene ontology (GO) categories for each cluster, which account for 30 to 80% of their constituent genes, reveal functional differences between them. Data depict (A) normalized mean enrichment, (B) DESeq2-calculated sgRNA enrichment, and (D) mean locus enrichment for n = 2 biological replicates. SI Appendix, Table S1 provides all sgRNA enrichment values.

Fig. 3.

Intermediate transcriptional repression can improve fitness under cold, narrow spectrum conditions. (A) The sgRNAs targeting the core subunits of NDH-1 exhibit a broad distribution of enrichments in 22B and 22R. (B) The sensitivity of core subunits to CRISPRi repression under 22B, defined as the interquartile range of gene-specific Log₂FC values normalized by the largest range for any NDH-1 subunit. (C) Under cold, monochromatic conditions, Log₂FC values for guides targeting ndhC and ndhG show similar correlations between cold monochromatic conditions and 37W. Lines show fits to linear and quadratic equations. (D) For three guides targeting ndhG, the extent of sgRNA depletion under 37W correlates with transcriptional repression (i.e., mRNA levels) in PCC 7002. (E) We examined the influence of ndhG on growth under 22B by using (Left) sgRNAs with different strengths (i.e., guides from C) or (Right) a single sgRNA under different levels of induction. In both experiments, intermediate transcriptional repression of ndhG improved fitness (i.e., increased cell density). (F) In a focused library of sgRNAs with different strengths, frequencies at 22 °C changed with blue light intensity relative to the initial distribution. (G) In 22B, mismatches in sgRNAs targeting ndhG and ndhB attenuate repression strength, revealing transcriptional optima. Data in (A), (C), and (G) depict the Log₂FC of individual sgRNAs. Data in D–G depict (D) the mean and SD for n ≥3 technical replicates of n = 2 biological replicates, (E and F) the mean and SE for n = 2 biological replicates, and (G) the mean of n = 3 biological replicates. Statistics: two-tailed t tests. (*P < 0.05 and **P < 0.01).

Fig. 4.

Cold monochromatic conditions induce stress responses that can be mitigated or enhanced by different levels of ndhG repression. (A and B) Low-temperature (77 K) fluorescence emission spectra of PCC 7002 cultures exposed to blue (460 nm), red (660 nm), or white light at 22 °C (22B, 22R, and 22W, respectively). (A) Excitation of chlorophyll a at 440 nm yields fluorescence emission peaks for PSI (λ_em = 720 nm) and PSII (λ_em = 685/695 nm) (34, 35); spectra are normalized to 720 nm. The 22B conditions decrease the PSI:PSII ratio, relative to white and red light. (B) Excitation of phycocyanin at 580 nm yields fluorescence emission peaks for free PBS (660 nm), PBS-PSII (685 nm), and PBS-PSI (712 nm). Spectra are normalized to PBS-PSI (712 nm). The 22B condition increases the proportion of free PBS and PBS-PSII complex. (C) A schematic of photosystem imbalance. At 22 °C, blue light overexcites PSI (28) and enhances PQ oxidation (38), which is mitigated by NDH-1 through CET. Red light overexcites PSII and enhances PQ reduction through LET. (D) Absorbance spectra of cells grown in 22B. Minor repression of ndhG enhances chlorophyll a and phycocyanin abundance; higher levels of repression decrease this effect. (E) Many proteins exhibit significant differences in abundance (red, P < 0.05, |Log₂FC| > 2) between 22B and 22-W in the nontargeting strain. (F–I), Analysis of changes in proteins under 22B. Increasing levels of ndhG repression decrease the relative abundance of both (F) core phycobilisome proteins and (G) iron-handling proteins, (H) leave ribosomal proteins at similar levels of depletion, and (I) increase the relative abundance of DNA repair and uncharacterized proteins. Data in E–I represent the normalized, mean-centered Log₂FC of 4 biological replicates. Full protein names appear in SI Appendix, Table S2.

Fig. 5.

Dual knockdowns display nonlinear effects. (A) Schematic of a single-transcript dual-sgRNA system. Native RNase III cleaves the aceEF RNA hairpin to generate two sgRNAs which complex with dCas9 (46). (B) The Log₂FC of sgRNAs for genes that exhibited prominent sgRNA enrichment under 22R or 22B. Highlight: maximally enriched guides (black). (C–F), Growth curves and absorbance scans of single and double constructs grown in (C and D) red or (E and F) blue light at 300 µmol photons m⁻² s⁻¹ at different inducer concentrations: full (5 mM IPTG, 0.5 µg/mL aTc; C and E) or 1/500 (10 µM IPTG, 1 ng/mL aTc; D and F). Absorbance spectra are normalized to 730 nm. Growth curves evidence sensitivity to guide order and inducer concentration. Data represent the mean and SD of n = 2 biological replicates.