Genetic studies of human embryonic morphogenesis are constrained by ethical and practical challenges, restricting insights into developmental mechanisms and disorders. Human pluripotent stem cell (hPSC)–derived organoids provide a powerful alternative for the study of embryonic morphogenesis. However, screening for genetic drivers of morphogenesis in vitro has been infeasible due to organoid variability and the high costs of performing scaled tissue-wide single-gene perturbations. By overcoming both these limitations, we developed a platform that integrates reproducible organoid morphogenesis with uniform single-gene perturbations, enabling high-throughput arrayed CRISPR interference (CRISPRi) screening in hPSC-derived organoids. To demonstrate the power of this platform, we screened 77 transcription factors in an organoid model of anterior neurulation to identify ZIC2 , SOX11 , and ZNF521 as essential regulators of neural tube closure. We discovered that ZIC2 and SOX11 are required for closure, while ZNF521 prevents ectopic closure points. Single-cell transcriptomic analysis of perturbed organoids revealed co-regulated gene targets of ZIC2 and SOX11 and an opposing role for ZNF521 , suggesting that these transcription factors jointly govern a gene regulatory program driving neural tube closure in the anterior forebrain region. Our single-gene perturbation platform enables high-throughput genetic screening of in vitro models of human embryonic morphogenesis.

Background Idiopathic pulmonary fibrosis is a fatal lung disease of progressive lung parenchymal scarring caused by the aberrant response of an alveolar epithelium repeatedly exposed to injury. Understanding epithelial dysfunction has been hampered by the lack of physiological alveolar type 2 (AT2) cell models and defined disease triggers. Monogenic forms of familial pulmonary fibrosis (FPF) caused by toxic gain-of-function variants provide an opportunity to investigate early pathogenic events. One such variant, surfactant protein C (SFTPC)-I73T, abnormally localises within AT2 cells and causes their dysfunction. Methods We used base editing of fetal lung-derived AT2 (fdAT2) organoids to create a heterozygous disease model of endogenous SFTPC-I73T expression. We also created an inducible overexpression system to interrogate temporal changes associated with SFTPC-I73T expression. We cultured fdAT2 both in 3D culture and at air-liquid interface to understand the importance of polarity cues and air exposure on disease phenotypes. Results In our heterozygous endogenous expression system, we found that fdAT2 expressing SFTPC-I73T grew without a lumen and were unable to correctly polarise. SFTPC-I73T accumulated with time and caused gross enlargement of early endosomes, preventing correct apico-basal trafficking of multiple endosomally trafficked cargoes including polarity markers and cell adhesion proteins. This phenotype was exacerbated by air exposure and led to loss of epithelial monolayer integrity and abnormal wound healing after injury. Conclusion Using endogenous gene editing for the first time in differentiated alveolar organoids, we have demonstrated that the pathogenic effects of SFTPC-I73T are mediated through endosomal dysfunction and abnormal epithelial organisation. This has important implications for AT2 function in vivo .

The establishment of the body plan during gastrulation represents a hallmark of animal life. It emerges from the interplay of gene-regulatory programs and positional cues, yet how these signals are integrated post-transcriptionally remains largely unexplored. Here, we combine the scalability of mouse gastruloids with a single-cell CRISPR screening platform to functionally dissect germ layer specification at single-cell transcriptomic resolution. Focusing on post-transcriptional regulation, we systematically map drivers of mesodermal and endodermal fate and identify the deadenylase Cnot8 . Loss of Cnot8 leads to widespread poly(A) tail elongation and transcript stabilization, shifting mesoderm differentiation toward ectopic notochord fate, thereby profoundly impacting axial patterning. Collectively, our findings identify mRNA deadenylation as a fundamental mechanism linking cellular identity with morphogenetic signaling during mammalian body plan formation.

Streptococcus mutans is recognized as the primary etiological agent of dental caries, one of the most prevalent infectious diseases globally. Its remarkable acid tolerance enables survival and proliferation in the low-pH biofilm microenvironment, establishing S. mutans as the dominant species in dental plaque and a key contributor to cariogenesis. Although numerous studies have identified genes linked to acid tolerance mechanisms, the full set of essential acid tolerance genes within its genome remains incompletely characterized, largely due to the lack of systematic, genome-scale investigations. To address this knowledge gap, we constructed a genome-wide pooled CRISPR interference (CRISPRi) library targeting 95% of the predicted S. mutans genes and employed next-generation sequencing to identify acid tolerance determinants systematically. Our screen revealed 95 acid tolerance-associated genes, a subset of which were functionally validated through gene knockout studies. Functional enrichment analysis demonstrated significant associations with metabolic pathways (including cofactor biosynthesis and amino/nucleotide sugar metabolism), tRNA modification, and transcriptional regulation. Protein-protein interaction (PPI) network analysis identified critical interactors (ComYC, SMU_1979c, DeoC, AcpP, NadD, and SMU_1988c) and two functionally cohesive modules. These findings provide novel mechanistic insights into the acid adaptation strategies of S. mutans and highlight potential therapeutic targets for caries prevention.

CRISPR activation (CRISPRa) enables precise, locus-specific upregulation of gene expression, offering potential for both ex vivo and in vivo applications. However, the lack of scalable, high-coverage tools has limited its use in comprehensive genetic screens, particularly in murine models. Here, we introduce Partita, a next-generation, whole-genome CRISPRa sgRNA platform designed for unparalleled efficiency and depth in gene activation studies. Partita employs a high-density targeting strategy, deploying 10 sgRNAs per transcription start site, structured into five gene family-specific sub-libraries to maximize transcriptional induction. To demonstrate its capabilities, we performed a series of large-scale screens: an in vitro enrichment/depletion screen in iBMDMs, whole-genome CRISPRa screens in a double-hit lymphoma model to uncover genes driving resistance to pro-apoptotic drugs (venetoclax, nutlin-3a, etoposide), and an in vivo whole-genome screen identifying accelerators of Myc-driven lymphomagenesis. Each experiment revealed both expected and novel regulators of cellular phenotypes, with a high validation rate in secondary assays. By enabling robust, high-throughput gain-of-function screening, Partita unlocks new avenues for functional genomics and expands the toolkit for discovering key drivers of biological processes across diverse research fields.

Clustered regularly interspaced short palindromic repeat Cas endonuclease (CRISPR-Cas) systems, such as RNA-editing CRISPR-Cas13d, are poised to advance the gene therapy of various diseases. However, their clinical development has been challenged by 1) the limited biostability of linear guide RNAs (lgRNAs) susceptible to degradation, 2) the immunogenicity of prokaryotic microorganism-derived Cas proteins in human that restrains their long-term therapeutic efficacy, and 3) off-targeting gene editing caused by the prolonged Cas expression from DNA vectors. Here, we report the development of highly stable circular gRNAs (cgRNAs) and transiently-expressing Cas13d-encoding mRNA for efficient CRISPR-Cas13d editing of target mRNA. We first optimized cgRNA for CRISPR-Cas13d editing of adenosine deaminase acting on RNA type I ( Adar1 ) transcript for the combination immunotherapy of triple negative breast cancer (TNBC). cgRNAs were synthesized by enzymatic ligation of lgRNA precursors. cgRNAs enhanced biostability with comparable Cas13d-binding affinity relative to lgRNA. Next, using ionizable lipid nanoparticles (LNPs), we co-delivered the resulting Adar1 -targeting cgRNA with an mRNA encoding RfxCas13d (mRNA-RfxCas13d), a widely used Cas13d variant, to TNBC cells. As a result, relative to lgRNA, cgRNA significantly enhanced the efficiency of Adar1 knockdown with minimal collateral activity, which sensitized the cancer cells for cytokine-mediated cell apoptosis. In a 4T1 murine TNBC tumor model in syngeneic mice, Adar1 -targeting cgRNA outperformed lgRNA for tumor immunotherapy in combination with immune checkpoint blockade (ICB). Collectively, these results demonstrate the great potential of cgRNA and mRNA-RfxCas13d for RNA-targeted gene editing.

Soil salinity varies widely across geographies both due to natural factors and human activities, including agriculture, road salt application, sea level rise, and desertification. Increases in soil salinity may affect organisms widely and particularly impact soil foodwebs. As parasites, entomopathogenic nematodes (EPNs) occupy crucial links in soil foodwebs and are important for agriculture as biological control agents of insect pests. Previous research found that the EPN Steinernema carpocapsae may exhibit higher salt tolerance than several of its congeners. We recently identified that S. carpocapsae uniquely evolved two amino acid substitutions in the first extracellular loop of the sodium pump (Na□/K□-ATPase). Here, we tested if these substitutions explain S. carpocapsae ’s reported lower sensitivity to salt. Our results confirm that S. carpocapsae exhibits higher salt tolerance and show it can more effectively locate and infect insect hosts than its congeners S. feltiae and S. hermaphroditum in highly saline environments. We then retraced the evolution of the two amino acid substitutions in S. carpocapsae by introducing them alone and in combination in Caenorhabditis elegans using CRISPR genome engineering. We found that C. elegans mutants with single substitutions showed improved salt tolerance. However, this improvement disappeared in the double mutant, whose sodium pump mimicked that of S. carpocapsae . This pattern of negative epistasis between the amino acid substitutions suggests they are not responsible for variation in salt tolerance between Steinernema species. Sodium pump evolution in S. carpocapsae might instead be driven by encounters with cardiac glycosides, which are released into soil by several clades of plants including milkweeds, sequestered by some of this EPN’s herbivorous insect hosts, and known to target the first extracellular loop of the sodium pump. Our findings provide valuable insights into EPN adaptation to changes in environmental sodium levels and may have implications for their use in biological control.

ABSTRACT Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FRφ), here we show that FRφ exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FRφ improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.

Background: The emergence and spread of antimicrobial resistance (AMR) genes in environmental microbiomes pose a critical threat to global health security. Current detection methods are time-consuming and often lack the sensitivity required for early environmental surveillance. Methods We developed a novel CRISPR-Cas13a-based biosensor system coupled with isothermal amplification for rapid, field-deployable detection of clinically relevant AMR genes (blaNDM-1, mcr-1, and vanA) in environmental samples. The system integrates microfluidic sample processing with fluorescent readout and smartphone-based detection. Results Our biosensor demonstrated exceptional sensitivity with detection limits of 10 copies/μL for target AMR genes, achieving 100% specificity against non-target sequences. Field testing across 150 environmental samples from wastewater treatment plants, agricultural runoff, and hospital effluents revealed previously undetected AMR hotspots. The system provided results within 45 minutes compared to 72 hours for conventional PCR-based methods. Longitudinal monitoring revealed seasonal fluctuations in AMR gene prevalence, with peak concentrations coinciding with agricultural antibiotic usage patterns. Conclusions This innovative biosensor platform enables rapid, sensitive detection of AMR genes in environmental settings, providing a powerful tool for real-time surveillance and early warning systems. The technology addresses critical gaps in current AMR monitoring capabilities and offers significant potential for global implementation in resource-limited settings.

Abstract Autosomal dominant tubulointerstitial kidney disease -UMOD (ADTKD-UMOD) is characterized by progressive renal interstitial inflammation and fibrosis. However, its underlying mechanisms remain unclear. Here, we identify a large ADTKD pedigree harboring a novel UMOD p.H36Y mutation. Using CRISPR/Cas9 technology, we generated a UmodH36Y/+ mouse model that recapitulates the key phenotypes observed in affected individuals, including renal dysfunction, cyst formation, interstitial inflammation, and fibrosis. Multi-omics analyses revealed marked macrophage pyroptosis in UmodH36Y/+ kidneys. Treatment with disulfiram (DSF), a pyroptosis inhibitor, significantly alleviated interstitial inflammation and improved renal function. Mechanistically, the Umod p.H36Y variant activated the amyloid precursor protein (App)-Cd74 axis which mediated the crosstalk between renal TECs and macrophages. This axis sustains NF-κB pathway activation in macrophages, initiating pyroptosis and pro-inflammatory cytokine release. Disrupting App-Cd74 signaling effectively suppressed macrophage pyroptosis. Notably, pharmacologic inhibition using ARN2966, a small-molecule App inhibitor, markedly attenuated renal injury in UmodH36Y/+ mice. Collectively, these findings uncover a novel, targetable pathway in ADTKD-UMOD.

GPR132 (G2A), a lipid- and pH-sensing GPCR, has been implicated in both pro- and anti-inflammatory signaling, but its in vivo function in wound repair and infection control remains unknown. Here, we investigated the role of GPR132b, a zebrafish homolog of G2A, in regulating innate immune responses. Using CRISPR-Cas9, we generated gpr132b mutants and found that they exhibit enhanced wound healing following sterile injury but increased susceptibility to Listeria monocytogenes infection, indicating that GPR132b modulates a trade-off between wound repair and antimicrobial defense. The enhanced regrowth phenotype was associated with increased macrophage accumulation at the wound site and reduced basal expression of the pro-inflammatory cytokine tnf-α . Macrophage depletion suppressed the enhanced regrowth phenotype, suggesting a functional role for macrophages in GPR132b-mediated repair. Pharmacological inhibition of cyclooxygenase (COX) and 12-lipoxygenase (12-LOX) pathways mimicked the gpr132b mutant phenotype in wild-type larvae, indicating that GPR132b likely responds to lipid-derived signals. Together, our findings reveal that GPR132b acts as a c ontext-dependent regulator of innate immunity, impairing efficient tissue repair in sterile conditions while supporting pathogen resistance during infection. Our results underscore the importance of GPCR-mediated signaling in orchestrating effective responses to tissue injury and infection.

Twenty causative genes have been reported that cause non-syndromic childhood glaucoma associated with anterior segment dysgenesis. FOXC1, PAX6 and PITX2 are the most well-known, but cases linked to SLC4A11, PITX3 and SOX11 have also been reported. As genetic testing becomes increasingly widespread and rates of molecular diagnosis rise, the extent of phenotypic overlap between the different genetic causes of non-syndromic glaucoma associated with anterior segment dysgenesis is becoming more evident. Taking aniridia as an example, whilst PAX6 mutations remain the predominant cause, variants in CYP1B1, FOXC1, PXDN and SOX11 have also been reported in patients with childhood glaucoma and aniridia. Developments in molecular-based therapies for retinal and corneal disease are advancing rapidly, and pre-clinical studies of gene-based treatments for glaucoma and aniridia are showing promising results. Use of adeno-associated viral vectors for gene delivery is most common, with improvements in intraocular pressure and retinal ganglion cell survival in Tg-MYOCY437H mouse models of glaucoma, and successful correction of a germline PAX6G194X nonsense variant in mice using CRISPR/Cas9 gene editing. This review will explore the actions and interactions of the genetic causes of non-syndromic glaucoma associated with anterior segment dysgenesis and discuss current developments in molecular therapies for these patients.

The nuclear pore complex (NPC) forms a large channel that spans the double lipid bilayer of the nuclear envelope and is the central gateway for macromolecular transport between the nucleus and cytoplasm in eukaryotes. NPC biogenesis requires the coordinated assembly of over 500 proteins culminating in the fusion of the inner and outer nuclear membranes. The molecular mechanism of this membrane fusion step that occurs in all eukaryotes is unknown. Here, we elucidate the mechanism by which two paralogous transmembrane proteins, Brl1 and Brr6, mediate membrane fusion in S. cerevisiae. Both proteins form multimeric, ring-shaped complexes with membrane remodeling activity. Brl1 is enriched at NPC assembly sites via a nuclear export sequence and then interacts with Brr6 across the nuclear envelope lumen through conserved hydrophobic loops. Disrupting this interaction blocks fusion and halts NPC assembly. Molecular dynamics simulations suggest that the Brl1-Brr6 complex drives membrane fusion by forming a channel across bilayers that enables lipid exchange. Phylogenetic analyses reveal that Brl1/Brr6 homologues are broadly distributed across eukaryotes, and functional experiments in human cells and D. melanogaster establish CLCC1 as an NPC fusogen in metazoans. Together, our results uncover a novel, conserved mechanism for membrane fusion in eukaryotes.

Trypanosoma cruzi , the causative agent of Chagas disease, relies on complex gene regulatory mechanisms to adapt to its diverse host environments. In recent years, it has been established that epigenetics plays an essential role in these mechanisms via the regulation of chromatin structure. Bromodomain-containing factors (BDFs), known for recognizing acetylated lysines on histones, have emerged as key factors in chromatin remodeling complexes. Among the eight predicted BDFs in T. cruzi , BDF6 is part of a TINTIN-like complex with MRGx and MRGBP, homologous to components of the NuA4/TIP60 chromatin remodeling complex. We generated knockout (KO) parasites for bdf6 gene using CRISPR/Cas9 gene editing. BDF6-deficient epimastigotes exhibit normal morphology but decreased size and growth and the resulting metacyclic trypomastigotes displayed drastically reduced infectivity. Strikingly, once inside host cells, BDF6-deficient parasites differentiated into amastigotes but failed to replicate. This intracellular arrest was reversed by episomal complementation of BDF6. Consistently, BDF6-KO parasites also exhibited impaired infectivity in mice, a defect that was also rescued in the add back parasite strains. Our findings highlight BDF6 as a critical regulator of intracellular parasite development, operating in stages beyond epimastigotes where epigenetic plasticity is essential for host adaptation. This striking stage-specific phenotype of BDF6 KO underscores its functional importance and highlights the relevance of epigenetic regulators along T. cruzi’s life cycle.

ABSTRACT The TTC22 gene encodes a protein containing seven tetratricopeptide repeats (TPRs), which mediate protein‒protein interactions as chaperones. We previously reported that the level of TTC22 transcript variant 1 ( TTC22v1 ) was downregulated in human colon adenocarcinoma (COAD) and that TTC22 upregulated m6A-mediated WTAP and SNAI1 expression via the TTC22‒RPL4 interaction and subsequently promoted COAD metastasis. Thus, a commercially available C57BL/6N mouse model in which the Ttc22 exon 2&3 encoding the TPR4 (equal to the human TTC22 TPR3) domain was knocked out via CRISPR-Cas9 was used to evaluate the contribution of Ttc22 to the development of mice and COAD. Unfortunately, the long-term observation results demonstrated that Ttc22 knockout (KO, including Ttc22 -/+ or Ttc22 -/- ) did not affect the body weight, development, fertility, or spontaneous tumor incidence of male or female mice. No differences in the incidence of AOM/DSS-induced COAD were observed between these mouse groups, although Ttc22 exon 2&3 deletion partially resulted in the upregulation of adaptive response genes in the colon mucosa. Further study revealed that TPR4 deletion did not disrupt the effect of TTC22 on WTAP upregulation. Consistently, TPR4 deletion did not affect the abundance of total RNA m6A in the colon tissues of the mice, suggesting that the TPR4-deleted Ttc22 mutant remains functional. In conclusion, the TPR4 domain is not essential for the Ttc22 protein. Loss of Ttc22 TPR4 caused no observable changes in the development of C57BL mice or their susceptibility to treatment with the chemical carcinogen AOM/DSS. Whether an essential sequence for a target gene is knocked out should be carefully evaluated before a gene knockout model is employed in formal experiments. Highlights Knockout of Ttc22 exons 2 and 3 does not affect the development of C57BL mice. Ttc22 knockout does not affect the induction of mouse colon cancer by AOM/DSS. Loss of the Ttc22 exons 2 and 3 cannot disrupt the functions of TTC22.