The rockefeller university

A remarkable feature of CRISPR-Cas systems is their ability to acquire short sequences from invading viruses to create a molecular record of infection. These sequences, called spacers, are inserted into the CRISPR locus and mediate sequence-specific immunity in prokaryotes. In type II-A CRISPR systems, Cas1, Cas2 and Csn2 form a supercomplex with Cas9 to integrate viral sequences. While the structure of the integrase complex has been described, a detailed functional analysis of the spacer acquisition machinery is lacking. We developed a genetic system that combines deep mutational scanning (DMS) of Streptococcus pyogenes cas genes with a method to select bacteria that acquire new spacers. Here, we show that this procedure reveals key interactions at the Cas1-Cas2 interface critical for spacer integration, identifies Cas variants with enhanced spacer acquisition and immunity against phage infection, and provides insights into the molecular determinants of spacer acquisition, offering a platform to improve CRISPR-Cas-based applications.

Characterizing genetic essentiality across various conditions is fundamental for understanding gene function. Transposon sequencing (TnSeq) is a powerful technique to generate genome-wide essentiality profiles in bacteria and has been extensively applied to Mycobacterium tuberculosis (Mtb). Dozens of TnSeq screens have yielded valuable insights into the biology of Mtb in vitro, inside macrophages, and in model host organisms. Despite their value, these Mtb TnSeq profiles have not been standardized or collated into a single, easily searchable database. This results in significant challenges when attempting to query and compare these resources, limiting our ability to obtain a comprehensive and consistent understanding of genetic conditional essentiality in Mtb. We address this problem by building a central repository of publicly available Mtb TnSeq screens, the Mtb transposon sequencing database (MtbTnDB). The MtbTnDB is a living resource that encompasses to date approximate to 150 standardized TnSeq screens, enabling open access to data, visualizations, and functional predictions through an interactive web app (). We conduct several statistical analyses on the complete database, such as demonstrating that (i) genes in the same genomic neighborhood have similar TnSeq profiles, and (ii) clusters of genes with similar TnSeq profiles are enriched for genes from similar functional categories. We further analyze the performance of machine learning models trained on TnSeq profiles to predict the functional annotation of orphan genes in Mtb. By facilitating the comparison of TnSeq screens across conditions, the MtbTnDB will accelerate the exploration of conditional genetic essentiality, provide insights into the functional organization of Mtb genes, and help predict gene function in this important human pathogen.

Colibactin, a human microbiome-derived genotoxin, promotes colorectal cancer by damaging the host gut epithelial genomes. While colibactin is synthesized via a hybrid non-ribosomal peptide synthetase (NRPS)polyketide synthase (PKS) pathway, known as pks or clb, the structural details of its biosynthetic enzymes remain limited, hindering our understanding of its biosynthesis and clinical application. In this study, we report the cryo-EM structures of two colibactin-producing PKS enzymes, ClbC and ClbI, captured in different reaction states using a substrate-mimic crosslinker. Our structural analysis revealed the binding sites of carrier protein (CP) domains of the ClbC and ClbI on their ketosynthase (KS) domains. Further, we identified a novel NRPS-PKS docking interaction between ClbI and its upstream enzyme, ClbH, mediated by the C-terminal peptide ClbH and the dimeric interface of ClbI, establishing a 1:2 stoichiometry. These findings advance our understanding of colibactin assembly line and provide broader insights into NRPS-PKS natural product biosynthesis mechanisms.

Lung cancer continues to be the leading cause of cancer-related deaths globally. Unraveling the regulators behind lung cancer growth and its metastatic spread, along with understanding the underlying mechanisms, is crucial for developing novel and effective therapeutic strategies. While much research has focused on identifying potential oncogenes or tumor suppressors, the roles of certain genes can vary depending on the context and may even exhibit contradictory effects. In this study, we demonstrate that acyl-CoA binding domain containing 3 (ACBD3), a Golgi resident protein, promotes primary lung cancer growth by recruiting phosphatidylinositol (PI)-4-kinase III beta (PI4KB) to the Golgi, thereby enhancing oncogenic secretion in chromosome 1q-amplified lung cancer cells. Conversely, in chromosome 1q-diploid lung cancer cells, ACBD3 acts as a suppressor of lung cancer metastasis by inhibiting the NOTCH signaling pathway and reducing cancer cell motility. This highlights the intricacy of cancer progression and cautions against simplistic approaches targeting individual oncogenes for cancer therapy.

Endothelial cells form the inner layer of blood vessels and play key roles in circulatory system development and function. A variety of endothelial cell types have been described through gene expression and transcriptome studies; nonetheless, the transcriptional programs that specify endothelial cell fate and maintenance are not well understood. To uncover such regulatory programs, we studied the C. elegans head mesodermal cell (HMC), a noncontractile mesodermal cell bearing molecular and functional similarities to vertebrate endothelial cells. Here, we demonstrate that a Forkhead transcription factor, LET-381, is required for HMC fate specification and maintenance of HMC gene expression. DMD-4, a DMRT transcription factor, acts downstream of and in conjunction with LET-381 to mediate these functions. Independently of LET-381, DMD-4 also represses the expression of genes associated with a different, non-HMC, mesodermal fate. Our studies uncover essential roles for FoxF transcriptional regulators in endothelial cell development and suggest that FoxF co-functioning target transcription factors promote specific non-contractile mesodermal fates.

Nguni sheep (Ovis aries) are indigenous to the Southern Africa region and common within the smallholder and poor resources farming systems. They are well adapted to different agroecological regions. However, limited genomic resources such as high-quality reference genomes have hindered our understanding of its adaptation and establishment of an effective breeding program. To address this, we assembled a chromosomal-level genome of Nguni sheep using a combination of PacBio HiFi reads and Omni-C reads. The genome size was estimated to be 2.9 Gb with a contig/scaffold N50 74 Mb and 99.6 Mb and a genome completeness of 96.1%, as estimated by the Benchmarking Universal Single-Copy Orthologs (BUSCO) program. The final genome encompassed a total of 25,926 protein-coding genes. The findings of this study provide a valuable genomic resource for understanding the adaptability of the Nguni sheep and the establishment of effective breeding programs.

Hearing hinges upon the ear's ability to enhance its responsiveness by means of an energy-expending active process that amplifies the very mechanical inputs that it detects. This process is defined by four properties that, although seemingly unrelated, consistently occur together: amplification, sharp frequency tuning, compressive nonlinearity, and spontaneous otoacoustic emission. In nonmammal tetrapods, the active process is evident in individual hair cells. The hair bundles of the bullfrog, for example, exhibit all four attributes by operating near a Hopf bifurcation-a critical regime in which these properties naturally coalesce. In mammals, however, the delicate nature of the cochlea has restricted the evidence for an active process to studies in vivo, where it is generally attributed to the collective effort of the outer hair cells that energize the traveling wave along the cochlear spiral. As a result, the cellular mechanisms that underlie the properties of mammalian hearing remain contested, with uncertainty about whether criticality plays a role in the cochlea's active process. Here we show that, when placed in a recording chamber that closely mimics the in vivo physiological environment, a segment of the mammalian cochlea ex vivo displays the features of the active process-amplification, frequency tuning, compressive nonlinearity, and the generation of distortion products. We show that this process operates locally, independently of traveling waves, and that the sensory epithelium achieves active amplification by operating near criticality at a Hopf bifurcation. The results reveal the existence of a unified biophysical principle that underlies auditory processing across species and even phyla.

The rate-limiting isozyme of de novo guanosine biosynthesis, IMPDH2, was identified as an essential gene in Merkel cell carcinoma (MCC) but the consequences of its functional disruption were unclear. Inhibition of IMPDH2 led to reduced MCC cell viability, independent of functional p53 or Merkel cell polyomavirus status, but dependent on depletion of guanylate nucleotides. In contrast to other cancer models, inhibition of IMPDH2 in MCC led to rapid ablation of nascent DNA synthesis and the onset of replication stress without a significant effect on total or ribosomal RNA biosynthesis. Combining IMPDH inhibitors with ataxia telangiectasia mutated and Rad3-related (ATR) inhibitors significantly increased levels of replication stress in vitro and reduced tumor growth in vivo. These findings support replication stress as the dominant consequence of IMPDH2 inhibition in MCC and, when combined with ATR inhibition, indicate a potential therapeutic strategy.

JGP study (Han et al. https://doi.org/10.1085/jgp.202413729) reveals that glycerol storage increases the titin-based stiffness of muscle fibers, suggesting that this commonly used method should be avoided by researchers interested in the passive properties of muscle.

Background: Recent single-cell studies indicated that IL-17-producing T cells (T17) have diverse subsets expressing IL-17A, IL-17F, or a combination in human psoriasis skin. However, it is unknown how T17 subsets are differently regulated by IL-23 versus IL-17A blockade. Objective: We sought to investigate how systemic monoclonal antibody injections blocking IL-23 versus IL-17A differently modify immune cell transcriptomes in human psoriasis skin. Methods: We analyzed a total of 93 human skin single-cell libraries, including 42 psoriasis pretreatment lesional skin, 25 psoriasis pretreatment nonlesional skin, 12 psoriasis posttreatment after IL-23 inhibition, 4 psoriasis posttreatment after IL-17A inhibition, and 10 control skin samples. ClinicalTrials.gov NCT04630652. Results: Of the six T17 subsets identified, an IL17A+IFNG+ subset and an IL17F+IL102 subset expressed the IL-23 receptor along with other inflammatory cytokines, and IL-23 inhibition downregulated these potentially pathogenic T17 subsets. In contrast, T17 cells expressing both IL-17A and IL-17F did not express the IL-23 receptor, and the percentage of this potentially nonpathogenic T17 subset increased after IL-23 inhibition. In addition, the expression of the IL-17-negative regulation genes, such as TNFAIP3, increased in myeloid cells more after IL-23 inhibition than after IL-17A inhibition. Conclusions: This study suggests multiple immune mechanisms of how IL-23 inhibition can modify the complex inflammatory environment present in psoriatic skin, highlighting the roles of specific T17 subsets in psoriasis development and background skin protection. (J Allergy Clin Immunol 2025;155:1898-912.)