The research team, led by Nana Matoba, Jessica McAfee, Hyejung Won and Jason Stein at the University of North Carolina at Chapel Hill, deployed a massively parallel reporter assay, or MPRA, to interrogate 9,092 genetic variants associated with human cortical structure. The logic of the assay is elegant. Each candidate variant is synthesized as a short 150-base-pair segment of DNA, cloned into a plasmid upstream of a minimal promoter that drives a luciferase reporter gene, and tagged with a random 20-base-pair barcode. When the pooled library is introduced into cells, the amount of barcode RNA produced by each construct reflects the regulatory power of the DNA segment attached to it. By sequencing both the RNA transcripts and the plasmid DNA, researchers can calculate a transcription rate for every element, and by testing both alleles of a variant side by side, they can detect whether one version of the sequence drives expression more strongly than the other.

The cellular context matters enormously here. The team performed the assay in primary human neural progenitor cells, the fetal-derived stem cells that generate the neurons of the developing cortex. Previous work had shown that variants associated with cortical structure are enriched in regulatory regions active in these progenitors and colocalize with genetic effects on chromatin accessibility and gene expression in the same cells. This supports a model in which noncoding variants act during prenatal development, altering transcription factor binding and gene expression in progenitor cells, and thereby influencing decisions about cell proliferation and fate that ultimately determine cortical size in adulthood. The researchers transfected the library into 16 biological replicates, and after 24 hours split the cultures: half received a vehicle control, while half were treated with CHIR99021, a potent inhibitor of GSK3β that activates the Wnt signaling pathway, a key developmental signal known to influence brain size.

The results were striking in their scale and reproducibility. Regulatory activity measurements correlated between replicates with a median Pearson’s coefficient of 0.99, and the positive control promoters behaved exactly as expected. Among 17,837 elements tested under baseline conditions, 988 showed regulatory activity significantly above the negative controls, representing 918 variants drawn from 150 of the 198 cortical-structure-associated loci examined—meaning that 76 percent of the loci contained at least one variant with detectable regulatory function. More than half of the active variants also showed allelic effects, meaning one DNA letter change measurably altered transcription. The team dubbed these expression-modulating variants, or emVars, and they represent the strongest candidates for the true causal variants hiding within blocks of correlated genetic variation that have long frustrated geneticists.

Perhaps the most surprising finding came when the researchers asked what sequence features distinguished the active elements. Active elements were dramatically enriched in short interspersed nuclear elements, or SINEs—a class of retrotransposons, the so-called jumping genes that copy and paste themselves throughout the genome. Within the SINE family, the enrichment was driven almost entirely by Alu elements, primate-specific repeats that have expanded in prevalence alongside increases in brain size over human evolution. A typical Alu element carries two conserved sequences in its left arm, the A box and the B box, which function as promoters for RNA polymerase III. The most active MPRA elements sat precisely in this left arm, and elements containing both boxes showed the highest activity of all. Variants with the strongest allelic effects clustered near these promoter boxes, and even a scrambled negative control that accidentally contained a B-box-like sequence showed strong activity—a striking confirmation of the mechanism.

Skeptics might worry that such activity is an artifact of the episomal plasmid system, in which DNA is not packaged into chromatin as it would be in the native genome. The team addressed this concern with multiple lines of endogenous evidence. Fetal brain tissue shows among the lowest levels of repressive chromatin marks, such as H3K9me3 and H3K27me3, deposited on Alu elements of any tissue in the body, and the MPRA-active Alu elements are rarely repressed in fetal brain specifically. The active elements were also enriched for enhancer RNAs and promoter upstream antisense RNAs, noncoding transcripts previously shown to mediate enhancer-promoter communication through Alu sequences. Finally, using CUT&RUN profiling, the researchers showed that MPRA-active Alu elements are significantly enriched within binding sites of TFIIIC, a transcription factor complex associated with polymerase III and Alu activity. Together, these data suggest that the reporter activity reflects genuine regulatory potential in the developing brain.

The evolutionary implications are tantalizing. Alu elements can be dated by their accumulated mutations, measured as sequence divergence from the ancestral consensus. The younger Alu elements—those with fewer than 100 mismatches per thousand bases—showed significantly higher regulatory activity than older ones, particularly when located in the left arm of the element. Because Alu insertions have proliferated in the primate lineage in step with brain size expansion, the findings suggest that relatively recent retrotransposon insertions created new regulatory elements that influenced neurodevelopmental genes and may have contributed to the expansion of the human cortex. The authors caution that a neutral decay of regulatory potential in older elements over evolutionary time could also explain the pattern, but the convergence with known primate-specific biology makes the adaptive interpretation compelling.

The study also connected individual variants to specific transcription factors and, remarkably, to specific brain regions. Active elements were enriched for binding motifs of three transcription factor clusters—ZNF135/ZNF460, MEF2A-D and Zfx—and the magnitude of a variant’s allelic effect correlated strongly with how severely it disrupted a predicted motif. Disruption of RARA, RARG, ZNF135 and NR2F1 motifs produced the largest impacts. Intriguingly, the consensus sequences of recently evolved Alu subfamilies contain retinoic acid receptor binding sites, linking the transposon story to developmental signaling. Even more strikingly, when the team compared the regional pattern of a variant’s effect on cortical structure with the expression of the disrupted transcription factor across prenatal brain regions from the BrainSpan Atlas, they found significant correlations for seven variants. A variant that disrupts the RARG binding motif, for example, reduced surface area most strongly in regions where RARG expression was highest—suggesting that the regional specificity of genetic effects is governed by where the relevant transcription factors are actually present.

Wnt stimulation added another layer of complexity. Under CHIR treatment, the team detected a similar number of active elements, but 32 elements gained activity specifically under stimulation while seven lost it, and 12 variants showed allelic effects that differed significantly between conditions. One example, rs4670759, associated with the surface area of the inferior temporal cortex, showed enhanced activity and increased chromatin accessibility under Wnt stimulation; it sits in the promoter of CDC42EP3, a gene involved in actin cytoskeleton formation whose expression rises with Wnt activation, and the risk allele is predicted to disrupt a PLAGL2 motif that is itself upregulated by the pathway. Another variant, rs2802295 within an intron of FOXO3, showed an allelic effect only under stimulation near a Wnt-responsive element. The team then used CRISPR interference to silence that element in progenitor cells and confirmed that FOXO3 expression dropped—experimentally tying a cortical-structure-associated variant to its target gene in a condition-specific manner.

The study is not without limitations. The MPRA measures regulatory potential on episomal plasmids, and the overlap with endogenous chromatin accessibility quantitative trait loci was limited, indicating that native chromatin context modifies some effects. The analysis also focused on variants tested primarily in populations of European ancestry and did not address sex-dependent effects. Nevertheless, by experimentally separating correlated variants, identifying the transcription factors they disrupt, and demonstrating condition-dependent function, the work delivers a genome-scale functional framework for one of the most complex traits in human neuroimaging genetics. It transforms a catalog of statistical associations into a mechanistic hypothesis space—one in which ancient jumping genes, developmental signaling pathways and spatially patterned transcription factors jointly sculpt the most distinctive feature of the human brain.

Subject of Research: Functional characterization of noncoding genetic variants associated with human cortical structure using massively parallel reporter assays in neural progenitor cells

Article Title: Massively parallel assessment of gene regulatory activity at human cortical-structure-associated variants

Article References: Matoba, N., McAfee, J. C., Krupa, O., Beltran, A. A., Bell, J. L., Le, B. D., Valone, J. M., Min, H., Crawford, G. E., Raab, J. R., Won, H., & Stein, J. L. (2026). Massively parallel assessment of gene regulatory activity at human cortical-structure-associated variants. Nature Neuroscience. https://doi.org/10.1038/s41593-026-02454-2

Image Credits: AI Generated

DOI: 10.1038/s41593-026-02454-2

Keywords: cortical structure, massively parallel reporter assay, noncoding variants, Alu elements, neural progenitor cells, Wnt signaling, transcription factors, ENIGMA, gene regulation, brain development, retrotransposons, CRISPRi

The new platform belongs to a family of techniques known as CRISPR interference and activation, or CRISPRi/a. Unlike standard CRISPR gene editing, which cuts DNA and permanently alters the genome, CRISPRi/a uses a catalytically inactive version of a Cas protein, often called a dead Cas, that can bind DNA without slicing it. When fused to regulatory proteins and guided to a gene’s promoter region by short RNA molecules, these dead enzymes can dial gene expression up or down. This is especially valuable for studying essential genes, which cannot simply be deleted because doing so kills the organism. By tuning expression levels instead, researchers can probe what a gene does while keeping the parasite alive.

The team’s previous work had established inducible dCas9-based CRISPRi/a systems in P. falciparum, fusing the dead Cas9 to PfGCN5, a histone acetyltransferase that activates genes, or to PfSir2a, a histone deacetylase that represses them. But Cas9 has a fundamental limitation in this parasite: it requires a short DNA motif called NGG, the protospacer adjacent motif or PAM, to bind its targets. The P. falciparum genome is roughly 80 percent adenine and thymine, making NGG sites relatively scarce, particularly in the promoter regions where gene regulation happens. Cas12a, by contrast, recognizes a T-rich PAM called TTTV, and the new study shows this makes an enormous difference.

Systematic scanning of the parasite’s reference genome revealed that Cas12a PAM sites are about 2.5 times more abundant than Cas9 sites overall, and roughly five times more common in annotated promoter regions. After filtering out guide RNAs with predicted off-target effects, the researchers found approximately seven times more usable Cas12a-based guide RNAs than Cas9-based ones in promoters, amounting to about eight clean targets per gene compared with roughly three for Cas9. Using the invasion gene eba-175 as a test case, the 804-base-pair promoter region contained nearly eight times as many Cas12a target sites as Cas9 sites. This expanded targeting space gives researchers far more flexibility in designing experiments.

Building the new system required adapting the inducible architecture the group had previously developed for dCas9. A green fluorescent protein cassette, followed by a transcriptional terminator, is inserted upstream of the dCas12a fusion gene, blocking its expression. This blocking cassette is flanked by loxP sites. When researchers add rapamycin to the culture, a split Cre recombinase reassembles and excises the floxed cassette, switching expression from GFP to the dCas12a fusion protein. The team used a nuclease-inactive LbCas12a variant from the bacterium Lachnospiraceae, tagged for detection, and introduced the constructs into a DiCre-expressing P. falciparum 3D7 line. Within 30 hours of a two-hour rapamycin pulse, GFP fluorescence vanished and the dCas12a fusion proteins became detectable by Western blot, confirming tight, leak-free induction. The fusion proteins did not impair parasite growth on their own.

To benchmark performance, the researchers targeted Pfatg18, an autophagy gene they had previously regulated with dCas9. Three guide RNAs positioned 70, 580, and 1,396 base pairs upstream of the gene’s start codon were tested in both the repression and activation lines. In the dCas12a-Sir2a parasites, Pfatg18 transcripts fell to between 10 and 30 percent of control levels, causing marked growth defects. In the dCas12a-GCN5 line, two of the guides boosted expression roughly 2.5-fold and 1.8-fold, which also retarded growth, echoing earlier findings that Pfatg18 overexpression disrupts autophagy homeostasis. A parallel experiment on eba-175 showed repression to as low as 15 percent of normal and activation up to 3.6-fold, demonstrating that the Cas12a platform matches or exceeds its Cas9 predecessor on both sides of the regulatory dial.

One of Cas12a’s signature features proved especially powerful: the enzyme processes its own guide RNA transcripts. Because Cas12a has intrinsic RNase activity, multiple guide sequences can be strung together in a single array, separated by direct repeat sequences, and the enzyme itself chops the long transcript into individual guides. The researchers assembled three-guide arrays for both Pfatg18 and eba-175. For interference, the arrays outperformed individual guides: the Pfatg18 array pushed repression below 10 percent, and the eba-175 array drove expression down to roughly 4 percent, substantially stronger than any single guide achieved. Activation via arrays was effective but did not exceed the best individual guides, likely because histone acetylation at promoters can become saturated. This multiplexing capacity dramatically simplifies construct design for pooled genetic screens.

The system’s real test came with Pfset7, a lysine methyltransferase gene that previous knockout attempts had failed to disrupt, suggesting it is essential. Three guide RNAs targeting the Pfset7 promoter reduced transcript levels by 50 to 70 percent, with corresponding drops in protein confirmed by antibodies raised against the recombinant PfSET7 protein. The consequences were striking: parasites with strong knockdown showed severe growth inhibition, egress defects in 39 to 64 percent of schizonts, meaning the mature parasites struggled to burst from their host red blood cells, and invasion efficiency of purified merozoites dropped by 35 to 52 percent. Intriguingly, the reverse experiment told the same story from the other direction. Overexpressing Pfset7 by 3.7 to 6.6-fold also caused growth defects, presumably because excessive methylation of the gene’s target proteins disrupts essential cellular processes. This bidirectional evidence, in which both depletion and excess impair the parasite, provides unusually strong confirmation that PfSET7 is essential and likely orchestrates egress and invasion, mirroring the role of its homolog in Toxoplasma gondii.

The researchers acknowledge limitations. Not every guide RNA worked; of nine guides tested, one activation guide and two interference guides had little effect, and one guide reduced mRNA without reducing protein levels, possibly reflecting compensatory translation. Guide performance did not correlate with predicted RNA folding stability, so empirical optimization remains necessary, though the abundance of Cas12a target sites makes it easy to test multiple guides or deploy arrays. Off-target effects, minimized through stringent computational filtering with tools like CRISPOR and CHOPCHOP, cannot be entirely excluded and require validation. Still, the combination of inducibility, bidirectional control, and multiplexing positions the dCas12a platform as a scalable foundation for forward genetic screens under drug pressure, heat shock, or nutrient stress, conditions where conditional gene requirements often emerge.

The implications extend beyond basic biology. Because the system can modulate, rather than abolish, gene function, it opens a window onto the roughly half of the parasite’s genome that remains uncharacterized, including essential genes that knockout approaches cannot touch. The plasmids have been deposited with Addgene, and the parasite lines are available to the community, lowering barriers to adoption. As artemisinin resistance consolidates in Africa and chloroquine resistance long ago became global, tools that rapidly expose new drug targets are urgently needed. A tunable, multiplexable genetic dial for the world’s deadliest malaria parasite may prove to be exactly the kind of instrument that turns genomic data into therapeutic insight.

Subject of Research: Development of an inducible dCas12a-based CRISPR interference and activation system for functional genomics in Plasmodium falciparum

Article Title: Inducible dCas12a system expands CRISPRi/a capabilities for functional genomics in malaria parasite Plasmodium falciparum

Article References: Lucky, A. B., Li, X., Kobakhidze, G., Gonzales, J. L., Wang, C., Cui, L., Dong, G., & Miao, J. (2026). Inducible dCas12a system expands CRISPRi/a capabilities for functional genomics in malaria parasite Plasmodium falciparum. iScience, 29(10), Article 117704. https://doi.org/10.1016/j.isci.2026.117704

Image Credits: AI Generated

DOI: Not provided

Keywords: CRISPRi, CRISPRa, dCas12a, Plasmodium falciparum, malaria, functional genomics, PfSET7, DiCre, gene regulation, Cas12a, epigenetics, drug targets

The commercial stakes are considerable. The global organic acids market is projected to reach 36.86 billion dollars by 2026, and in 2004 the United States Department of Energy identified twelve high-priority building-block chemicals obtainable from biomass, roughly seventy percent of which are organic acids. These include succinic acid, 3-hydroxypropionic acid, itaconic acid, and levulinic acid, compounds that serve as precursors for biodegradable plastics, solvents, pharmaceuticals, and food additives. Traditionally these chemicals come from petrochemical synthesis or heterotrophic fermentation, both of which carry substantial carbon footprints. Cyanobacteria offer a third route: photoautotrophic biosynthesis that consumes the greenhouse gas responsible for climate change as its raw material.

The metabolic logic behind this capability is elegant. Cyanobacteria fix atmospheric carbon dioxide through the Calvin-Benson-Bassham cycle, generating central intermediates such as pyruvate, phosphoenolpyruvate, and acetyl-CoA that feed into the tricarboxylic acid cycle. From these nodes, carbon can be channeled into acetate, lactate, citrate, succinate, fumarate, and malate. Intriguingly, cyanobacteria possess an atypical, incomplete TCA cycle lacking the canonical 2-oxoglutarate dehydrogenase complex. Instead, they bypass this step via 2-oxoglutarate decarboxylase and succinic semialdehyde dehydrogenase, an evolutionary adaptation that appears designed to preserve carbon skeletons for biosynthesis rather than burning them off as carbon dioxide. Some strains also harbor the glyoxylate shunt and a gamma-aminobutyric acid shunt, alternative routes that connect carbon metabolism with nitrogen assimilation and redox regulation.

The organisms also display a striking diurnal rhythm that researchers are learning to exploit. During daylight, cyanobacteria assimilate carbon dioxide into glycogen stores; when darkness falls and oxygen dwindles, they ferment these reserves, secreting organic acids and hydrogen. Under dark, anoxic conditions, Synechocystis sp. PCC 6803 has been shown to release more than 400 milligrams per liter of acetate, along with lactate, succinate, and malate, independent of nitrogen source. This natural overflow metabolism acts as a redox-balancing mechanism, dumping excess reducing equivalents when the cell’s usual sinks, such as glycogen and protein synthesis, are unavailable. Engineers are now working to redirect this flux deliberately toward single target products under continuous illumination.

Case studies demonstrate how far metabolic engineering can push these yields. In Synechocystis PCC 6803, inserting a codon-optimized phosphoketolase from Pseudomonas aeruginosa boosted acetate production fortyfold, and co-expressing phosphotransacetylase raised titers to 2.3 grams per liter, an eightyfold increase over the starting strain. Lactate engineering has been even more dramatic: combining overexpression of malic enzyme and d-lactate dehydrogenase with deletion of the acetate kinase gene produced a record 26.6 grams per liter of d-lactate from high-density cell cultures after seventy-two hours of fermentation. Because lactate is the monomer for polylactic acid, a biodegradable plastic used in packaging and 3D printing, such strains could eventually supply the bioplastics industry with carbon-negative feedstock.

Succinate, a top Department of Energy platform chemical used to make 1,4-butanediol, gamma-butyrolactone, and biodegradable polymers, has seen similar success. In Synechococcus elongatus PCC 7942, researchers used CRISPR interference to repress glycogen synthesis and succinate dehydrogenase while overexpressing key carboxylation and decarboxylation enzymes, achieving the highest cyanobacterial succinate titer reported to date: 4.8 grams per liter in twenty-eight days, rising to 8.9 grams per liter with reinoculation. A separate photomixotrophic strategy engineered the same strain to import glucose at low pH and export succinate at high pH, exploiting opposing proton symporters to reach 5.0 grams per liter in ten days. Even citrate, long considered too tightly regulated to accumulate, has been produced photoautotrophically in PCC 7002 using a theophylline-responsive riboswitch to throttle TCA flux, yielding more than a hundredfold increase over wild type.

Redox engineering has emerged as a central theme in these efforts. Cyanobacteria inherently produce abundant NADPH through photosynthesis, yet many fermentative enzymes prefer NADH, creating a cofactor mismatch that limits yields. Solutions include overexpressing transhydrogenases such as PntAB to interconvert the two pools, and site-directed mutagenesis to swap enzyme cofactor preferences, as demonstrated with an NADH-dependent lactate dehydrogenase from Lactobacillus bulgaricus that was redesigned to accept NADPH. Environmental tuning matters too: raising cultivation temperature from 30 to 37 degrees Celsius enhanced d-lactate and succinate production in PCC 6803, while mildly acidic conditions increase passive secretion of weak acids by boosting membrane permeability. Nitrogen starvation can push glycogen-deficient strains into a photo-catalytic state, continuously excreting alpha-ketoglutarate and pyruvate even without growth.

Formidable obstacles remain before these microbes can compete with petrochemical plants. Cyanobacteria grow slowly, allocate only a minor fraction of fixed carbon to target products, and suffer from genetic instability, low transformation efficiency, and byproduct formation as carbon leaks into competing pathways. Product recovery is another bottleneck: organic acids exist in ionized form at neutral pH, and traditional acidification with mineral acids generates mountains of inorganic salt waste. Photobioreactors struggle with uneven light penetration, limited gas transfer, and biofouling, and techno-economic and life-cycle assessments for engineered cyanobacterial systems remain scarce, making it difficult to judge commercial feasibility. Most studies to date remain at proof-of-concept or laboratory scale.

The road ahead is nonetheless rich with opportunity. Emerging tools include CRISPR-based gene repression and activation, riboswitches and light-inducible promoters that decouple growth from production, and adaptive laboratory evolution to breed acid-tolerant strains. Machine learning-guided flux modeling and multi-omics integration are converting strain design from trial-and-error into predictive optimization, while next-generation photobioreactors with optical waveguides and gas-permeable membranes promise better scalability. Co-cultures with heterotrophs, photomixotrophy using waste-derived carbon, and multi-product biorefineries coupling organic acids with biofuels and bioplastics could further improve economics. Greener recovery technologies, including membrane filtration, electrodialysis, and in situ product removal, are being developed to replace salt-generating acidification. If these threads converge, cyanobacteria could transform sunlight and carbon dioxide into the molecular backbone of a circular, fossil-free chemical economy, turning the planet’s oldest oxygen-makers into its newest industrial workhorses.

Subject of Research: Metabolic engineering of cyanobacteria for sustainable photosynthetic production of organic acids

Article Title: Advances in organic acid production using cyanobacteria: strategies and applications

Article References: Pathania, R., & Srivastava, A. (2025). Advances in organic acid production using cyanobacteria: strategies and applications. Blue Biotechnology, 2(1), Article 22. https://doi.org/10.1186/s44315-025-00045-7

Image Credits: AI Generated

DOI: 10.1186/s44315-025-00045-7

Keywords: cyanobacteria, organic acids, metabolic engineering, synthetic biology, carbon dioxide fixation, succinate, lactate, TCA cycle, bioplastics, photobioreactors, CRISPRi, sustainable biomanufacturing

The centerpiece of the study is an in vivo CRISPR interference, or CRISPRi, screening platform. Unlike traditional knockouts, which permanently delete genes and can be impractical at genome-wide scale, CRISPRi uses a catalytically dead Cas9 protein, dCas9, guided by hundreds of thousands of small RNA molecules to reversibly silence transcription of individual genes. Each bacterial cell in a massive pooled library carries a guide RNA targeting a specific gene, so the abundance of each variant after a period in the host serves as a direct readout of how important the silenced gene was for survival. If a gene is essential in the gut, cells whose guide suppresses it will dwindle in number; if it is dispensable, those cells persist. Gene fitness, measured by deep sequencing of guide RNA abundance with unique molecular identifiers to reduce noise, thus becomes a high-resolution functional reporter of adaptation.

To make the experiment tractable, the researchers colonized mice with OligoMM12, a defined minimal microbial community of twelve mouse gut strains. This gnotobiotic approach, building on earlier genome-guided designs of minimal microbiotas, provides a reproducible yet realistic ecological backdrop in which E. coli must compete with other residents for nutrients and space, without the overwhelming complexity of a conventional microbiome. The team introduced genome-wide CRISPRi libraries into three very different E. coli strains: MG1655, the classic laboratory K-12 derivative; CFT073, a uropathogenic strain; and LF82, an adherent-invasive E. coli, or AIEC, associated with Crohn’s disease. As the libraries circulated through the mice, sequencing revealed which genes each strain depended on under specific conditions.

The first major finding concerns diet. When mice were fed standard chow, a high-fiber diet, or a high-fat Western-style diet, the metabolic landscape facing E. coli changed dramatically, and the essential gene profile shifted with it. Genes required for growth on one diet were dispensable or even deleterious on another, confirming at functional-genomic resolution that what an animal eats directly rewrites the nutritional rules of the gut. The screen also exposed cross-feeding interactions, identifying genes whose loss was tolerated only because other members of the defined community supplied the missing metabolites. In this way the platform does more than list essential genes; it sketches the metabolic web that connects E. coli to its microbial neighbors, showing that the bacterium’s survival strategy is woven into the ecology of the whole community.

Comparing the three strains side by side revealed that there is no single genetic playbook for intestinal colonization. Although the core essential genomes overlapped, reflecting shared housekeeping functions, each strain displayed a distinct signature of requirements. The analysis highlighted divergent strategies in motility, stress response and respiration. MG1655, CFT073 and LF82 each relied on different subsets of genes to cope with bile, acidity, oxygen tension and immune pressures, consistent with the idea that genetic diversity across the E. coli species translates into fundamentally different ecological tactics. The results extend earlier transposon-insertion sequencing studies in enterohemorrhagic E. coli, K1 strains and other pathogens, but with the added precision and tunability that CRISPRi offers, since partial silencing can reveal dose-sensitive phenotypes that complete knockouts miss.

One of the most medically relevant strands of the study concerns AIEC LF82, a strain strongly enriched in the ileal mucosa of Crohn’s disease patients. When the researchers induced intestinal inflammation with dextran sulfate sodium, they found that LF82 preferentially colonized the small intestine in the inflammatory environment, and its genetic dependencies shifted accordingly. Genes tied to iron acquisition became more important, echoing the known biology of lipocalin 2 and neutrophil gelatinase-associated lipocalin, host proteins that sequester iron-bearing siderophores during inflammation. Respiratory genes also rose in prominence, in line with evidence that inflammation disrupts intestinal oxygen balance and creates new terminal electron acceptors that facultative anaerobes can exploit. The inflammatory gut, in other words, is a different nutritional world, and the screen captured that transformation in genetic terms.

Perhaps the most striking discovery involves a mobile genetic element. LF82 carries the Gally prophage, and the CRISPRi data revealed that this integrated virus plays an important role in modulating the strain’s fitness in the inflamed gut. Follow-up analyses quantified phage particle release and showed that the prophage’s influence on fitness differed between inflamed and healthy conditions, adding to growing evidence that prophages are not merely parasitic passengers but functional modulators of host physiology. Previous work has shown that phage integration can rewire the respiratory strategy of E. coli, and the new findings place the Gally element squarely within the colonization toolkit of a Crohn’s-associated pathobiont. The observation that phage production itself appears to be regulated, and blocked under some host-like conditions, underscores how finely tuned these relationships are.

Technically, the study sets a new benchmark for rigor. Gene-level calls were derived using MAGeCK with robust ranking aggregation and Benjamini-Hochberg false-discovery-rate correction across individual mice as biological replicates, and guide activity predictions were used to improve on-target performance, drawing on earlier CRISPR-dCas9 screening work in E. coli. Raw CRISPRi and 16S sequencing data are available through the Sequence Read Archive under accession PRJNA1293909, and processed log2 fold-change values, statistical results, analysis scripts and notebooks are shared through public repositories, allowing other groups to reproduce and extend the work. The authors acknowledge support from the European Research Council, the Agence Nationale de la Recherche, the Fondation pour la Recherche Médicale and the Swiss National Science Foundation’s NCCR AntiResist program.

The broader significance of the work extends well beyond E. coli. By demonstrating that gene fitness can serve as a quantitative sensor of the gut environment, the platform offers a way to probe intestinal conditions directly through the organisms that live there, effectively turning bacteria into living microsensors of diet, inflammation and microbial competition. For microbiome science, where correlational studies abound, this is a shift toward mechanism: every essential gene identified is a hypothesis about a nutrient, a stress or a host defense that can be tested. For medicine, the strain-specific genetic requirements of Crohn’s-associated AIEC and the role of the Gally prophage suggest new angles for interventions that disarm pathobionts without indiscriminately wiping out beneficial residents. As researchers refine gnotobiotic models and CRISPRi delivery, in vivo functional genomics of this kind could become a routine tool, mapping not just which microbes are present in the gut but precisely which molecular functions make them winners or losers in one of the most densely populated ecosystems in the mammalian body.

Subject of Research: In vivo CRISPRi functional genomics of Escherichia coli adaptation to the mouse gut

Article Title: In vivo CRISPRi screens reveal Escherichia coli functional adaptations in the mouse gut

Article References: Maire, A., Ortelli, M., Tkacz, E., Dehio, C., Chassaing, B., Sokol, H., Rolhion, N., & Bikard, D. (2026). In vivo CRISPRi screens reveal Escherichia coli functional adaptations in the mouse gut. Nature Microbiology. https://doi.org/10.1038/s41564-026-02471-8

Image Credits: AI Generated

DOI: 10.1038/s41564-026-02471-8

Keywords: E. coli, CRISPRi, gut microbiome, Nature Microbiology, adherent-invasive E. coli, Crohn's disease, Gally prophage, gene essentiality, mouse model, OligoMM12, functional genomics, intestinal colonization