CRISPR interference screens reveal growth–robustness tradeoffs inSynechocystissp. PCC 6803 across growth conditions

Abstract

Barcoded mutant libraries are a powerful tool for elucidating gene function in microbes, particularly when screened in multiple growth conditions. Here, we screened a pooled CRISPR interference library of the model cyanobacterium Synechocystis sp. PCC 6803 in 11 bioreactor-controlled conditions, spanning multiple light regimes and carbon sources. This gene repression library contained 21,705 individual mutants with high redundancy over all open reading frames and non-coding RNAs. Comparison of the derived gene fitness scores revealed multiple instances of gene repression being beneficial in one condition while generally detrimental in others, particularly for genes within light harvesting and conversion, such as antennae components at high light and PSII subunits during photoheterotrophy. Suboptimal regulation of such genes likely represents a tradeoff of reduced growth speed for enhanced robustness to perturbation. The extensive dataset assigns condition-specific importance to many previously unannotated genes, and suggests additional functions for central metabolic enzymes. Phosphoribulosekinase, glyceraldehyde-3-phosphate dehydrogenase, and the small protein CP12 were critical for mixotrophy and photoheterotrophy, which implicates the ternary complex as important for redirecting metabolic flux in these conditions in addition to inactivation of the Calvin cycle in the dark. To predict the potency of sgRNA sequences, we applied machine learning on sgRNA sequences and gene repression data, which showed the importance of C enrichment and T depletion proximal to the PAM site. Fitness data for all genes in all conditions is compiled in an interactive web application.

The energy metabolism ofCupriavidus necatorin different trophic conditions

“…Mutants for the SH genes hoxFUYHI showed moderately reduced fitness scores, similar to the fitness from hoxKGZ , while fitness scores for the hoxA knockout, which disrupts all hox gene expression, were around twice as low. This partial reduction in fitness again suggests that the two iso-enzymes carry roughly equal flux (Miao et al, 2023). The C. necator hydrogenases have been of interest for their oxygen tolerance, and have been structurally and functionally well characterized (Saggu et al, 2009, Fritsch et al, 2013).…”

“…All fds mutants had strongly reduced fitness while all other FDH mutants did not. In library screenings, we often see a partial decrease in fitness when multiple iso-enzymes with similar activity share the metabolic load (Miao et al, 2023), but the FDH genes did not show such a ‘division of labor’. This also means that the overwhelming majority of electron flux from formate oxidation is channeled towards NADH, and only indirectly towards ubiquinone/ubiquinol in the electron transport chain.…”

Trade‐offs between the instantaneous growth rate and long‐term fitness: Consequences for microbial physiology and predictive computational models

“…A fifth challenge is to gather quantitative experimental evidence related to long‐term fitness also for non model organisms. In addition to meticulous experimental studies concerning individual trade‐offs and constraints, novel methods, such as CRISPR interference screens, [ 112 ] allow us to uncover (hypotheses) about trade offs between the instantaneous growth rate and other properties on a genome‐scale, and are increasingly applicable to non model organisms. Likewise, adaptive laboratory evolution (ALEs) is a promising experimental method to use in combination with resource allocation models.…”

Simulations Predict Stronger CRISPRi Transcriptional Repression in Plants for Identical than Heterogeneous gRNA Target Sites

“…In these systems, the catalytically inactivated Cas9 protein (dCas9), either alone or fused to a transcriptional activation or repression domain, binds to a specific promoter region determined by the sequence of a customized guide RNA (gRNA) [8], thereby activating (CRISPRa) or inhibiting (CRISPRi) transcription. Both have previously been used in plants, but have often resulted in a low dynamic range in both plants [9,10,11] and other systems [12,13,14]. However, the efficacy of regulation can be increased by using multiple gRNA target sites to control a single promoter.…”