When cells enter mitosis with under-replicated DNA, sister chromosome segregation is compromised, which can lead to massive genome instability. The replisome-associated E3 ubiquitin ligase TRAIP mitigates this threat by ubiquitylating the CMG helicase in mitosis, leading to disassembly of stalled replisomes, fork cleavage, and restoration of chromosome structure by alternative end-joining. Here, we show that replisome disassembly requires TRAIP phosphorylation by the mitotic Cyclin B-CDK1 kinase, as well as TTF2, a SWI/SNF ATPase previously implicated in the eviction of RNA polymerase from mitotic chromosomes. We find that TTF2 tethers TRAIP to replisomes using an N-terminal Zinc finger that binds to phosphorylated TRAIP and an adjacent TTF2 peptide that contacts the CMG-associated leading strand DNA polymerase ε. This TRAIP-TTF2-pol ε bridge, which forms independently of the TTF2 ATPase domain, is essential to promote CMG unloading and stalled fork breakage. Conversely, RNAPII eviction from mitotic chromosomes requires the ATPase activity of TTF2. We conclude that in mitosis, replisomes undergo a CDK- and TTF2-dependent structural reorganization that underlies the cellular response to incompletely replicated DNA.

Abstract The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of transgenic lines, as an isogenic and stable transgenic line can in principle be achieved already in the first generation. However, genetic tools for the manipulation of their genes had not been developed until now. Here, we successfully implement CRISPR/Cas9 as a technique to modify the genome of the stick insect Medauroidea extradentata . As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation ( cinnabar and white , second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24h after oviposition, to focus on the monocellular (and haploid) stage of development. The KOs generated resulted in distinct eye and cuticle colour phenotypes for cinnabar and white . Homozygous cinnabar mutants showed pale pigmentation of eyes and cuticle, while homozygous white KO resulted in a completely unpigmented phenotype in developing embryos. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable animals. This genetic toolbox can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism.

Malaria is still a major health issue in many parts of the world, particularly in tropical and subtropical regions of Africa, Asia, and Latin America. Despite significant efforts to control and eliminate the disease, malaria remains a leading cause of illness and death, mainly due to the occurrence of drug-resistant parasites to the frontline antimalarials such as dihydroartemisinin-piperaquine (DHA-PPQ). Artemisinin resistance has been linked to kelch13 mutations, while decreased PPQ sensitivity has been associated with higher plasmepsin II and III gene copies and mutations in the chloroquine resistance transporter. In this study, we demonstrate the effective use of CRISPR/Cas9 technology to generate single knockouts (KO) of plasmepsin II and plasmepsin III , as well as a double KOs of both genes, in two isogenic lines of Cambodian parasites with varying numbers of plasmepsin gene copies. The deletion of plasmepsin II and/or III increased the parasites’ sensitivity to PPQ, evaluated by the area under the curve. We explored several hypotheses to understand how an increased plasmepsin gene copy number might influence parasite survival under high PPQ pressure. Our findings indicate that protease inhibitors have a minimal impact on parasite susceptibility to PPQ. Additionally, parasites with higher plasmepsin gene copy numbers did not exhibit significantly increased hemoglobin digestion, nor did they produce different amounts of free heme following PPQ treatment compared to wildtype parasites. Interestingly, hemoglobin digestion was slowed in parasites with plasmepsin II deletions. By treating parasites with digestive vacuole (DV) function modulators, we found that changes in DV pH potentially affect their response to PPQ. Our research highlights the crucial role of increased plasmepsin II and III gene copy numbers in modulating response to PPQ and begins to uncover the molecular and physiological mechanisms underlying PPQ resistance in Cambodian parasites. Author Summary Global malaria control has plateaued, with drug-resistant Plasmodium falciparum posing a significant challenge. Artemisinin-based combination therapies (ACTs) are becoming less effective, especially in South-East Asia, where resistance to dihydroartemisinin-piperaquine (DHA-PPQ) is leading to treatment failures, notably in Cambodia. Genome-wide studies link artemisinin resistance to kelch13 mutations, while decreased PPQ sensitivity is tied to higher plasmepsin II and III gene copies and mutations in chloroquine resistance transporter. We previously showed a connection between increased plasmepsin gene copies and reduced PPQ sensitivity. In this study we try to understand the biological role of the plasmepsins in PPQ sensitivity. Therefore, we knocked out plasmepsin II and III genes in Cambodian strains using CRISPR/Cas9, and found increased PPQ sensitivity, confirming these genes’ roles in resistance. Plasmepsins are proteases that participate in the hemoglobin degradation cascade in the digestive vacuole of the parasites. Protease inhibitor experiments and hemoglobin digestion studies indicate that digestive vacuole pH fluctuations affect PPQ response, highlighting the need for further research into PPQ resistance mechanisms.

The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through C RISPR- A ssociated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral “spacer” sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus . TON_0321 exhibits 5′ to 3′ exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5′ direction, demonstrating structure-specific endonuclease activity.

ABSTRACT The genetic perturbations caused by spaceflight on biological systems tend to have a system-wide effect which is often difficult to deconvolute into individual signals with specific points of origin. Single cell multi-omic data can provide a profile of the perturbational effects but does not necessarily indicate the initial point of interference within a network. The objective of this project is to take advantage of large scale and genome-wide perturbational or Perturb-Seq datasets by using them to pre-train a generalist machine learning model that is capable of predicting the effects of unseen perturbations in new data. Perturb-Seq datasets are large libraries of single cell RNA sequencing data collected from CRISPR knock out screens in cell culture. The advent of generative machine learning algorithms, particularly transformers, make it an ideal time to re-assess large scale data libraries in order to grasp cell and even organism-wide genomic expression motifs. By tailoring an algorithm to learn the downstream effects of the genetic perturbations, we present a pre-trained generalist model capable of predicting the effects of multiple perturbations in combination, locating points of origin for perturbation in new datasets, predicting the effects of known perturbations in new datasets, and annotation of large-scale network motifs. We demonstrate the utility of this model by identifying key perturbational signatures in RNA sequencing data from spaceflown biological samples from the NASA Open Science Data Repository.

Clavibacter is a phytopathogenic genus that causes severe diseases in economically important crops, yet the role of prophages in their evolution, pathogenicity and adaptation remains poorly understood. In this study, we used PHASTER, Prophage Hunter, and VirSorter2 to identify prophage-like sequences in publicly available Clavibacter genomes. Prophage predictions were checked by hand to make them more accurate. We identified 353 prophages, predominantly in chromosomes, with some detected prophages in plasmids. Most prophages exhibited traits of advanced domestication, such as an unimodal genome length distribution, reduced numbers of integrases, and minimal transposable elements, suggesting long-term interactions with their bacterial hosts. Comparative genomic analyses uncovered high genetic diversity, with distinct prophage clusters showing species-specific and interspecies conservation patterns. Functional annotation revealed prophage-encoded genes were involved in sugar metabolism, heavy metal resistance, virulence factors, and antibiotic resistance, highlighting their contribution to host fitness and environmental adaptation. Defense system analyses revealed that, despite lacking CRISPR-Cas, Clavibacter genomes harbor diverse antiviral systems, including PD-Lambda-1, AbiE and MMB_gp29_gp30, some encoded within prophages. These findings underscore the pervasive presence of prophages in Clavibacter and their role in shaping bacterial adaptability and evolution.

Klebsiella pneumoniae is a prominent opportunistic pathogen associated with multidrug resistance (MDR) and high morbidity and mortality rates in healthcare settings. The emergence of strains resistant to last-resort antibiotics, such as colistin and carbapenems, poses significant therapeutic challenges. This study presents the complete genome analysis of the MDR strain K. pneumoniae BCSIR-JUMIID to elucidate its genetic architecture, resistance mechanisms, and virulence factors. The genome of K. pneumoniae BCSIR-JUMIID, isolated from a pharmaceutical wastewater in Dhaka, Bangladesh, was sequenced using next-generation sequencing technologies. Bioinformatics tools were employed for genome assembly, annotation, and functional analysis. Phylogenetic relationships were established through whole-genome comparisons. Antibiotic resistance genes, virulence factors, and mobile genetic elements were identified using the Comprehensive Antibiotic Resistance Database (CARD), ResFinder-4.5.0, Virulence Factors Database (VFDB), and various phage identification tools. The genome of K. pneumoniae BCSIR-JUMIID consists of 5,769,218 bp with a G+C content of 56.79%, assembled into 343 contigs. A total of 6,062 coding sequences (CDS), including 1,087 hypothetical proteins, 49 tRNA genes, and 4 rRNA genes, were identified. Key loci involved in capsular polysaccharide and O-antigen biosynthesis (KL150, KL107-D1, O3b) were detected. A diverse array of antibiotic resistance genes was uncovered, including those conferring resistance to beta-lactams, quinolones, and colistin. Phage analysis revealed the presence of multiple dsDNA bacteriophages, and CRISPR-Cas systems indicated robust phage defense mechanisms. The genomic analysis of K. pneumoniae BCSIR-JUMIID provides a detailed understanding of its resistance and virulence mechanisms, highlighting its potential for horizontal gene transfer and rapid adaptation. These findings underscore the necessity for continued surveillance and novel therapeutic strategies to combat MDR K. pneumoniae infections effectively.

Yeast mitochondria receive the majority of their lipids from the endoplasmic reticulum (ER) via the heterotetrameric ERMES lipid transport complex. This complex is thought to establish a lipid transporting tube of fixed composition spanning the space between both organelles. Intriguingly, however, some of the lipid-transporting components of the complex can be replaced by an artificial ER-mitochondria tether without lipid transport activity, indicating that ERMES subunits are not all of equal importance for lipid transport. Here, we propose a model whereby lipid transfer by the ERMES complex can occur with various sub-ensembles of ERMES, and minimally with only one of the four members, namely Mmm1. Our results imply flexibility in the composition of the ERMES complex, which might help it accommodate various interorganelle distances.

The PIWI-interacting RNA (piRNA) pathway is crucial for maintaining genomic integrity by suppressing transposable elements in animal germlines. Despite its well-established function in the animal germline, piRNAs and PIWI proteins are expressed in somatic tissues across arthropod species, raising questions about piRNA functions outside the gonads. For example, Aedes albopictus mosquitoes express four PIWI genes, Piwi4 , Piwi5 , Piwi6 and Ago3, in both germline and somatic tissues. We generated Piwi6 knockout Ae. albopictus cell lines and observed a substantial upregulation of Long Terminal Repeat (LTR)-retrotransposons, among which a full-length endogenous retrovirus that we named AalERV1 . We found that Piwi6 silences AalERV1 at the transcriptional level by guiding the deposition of the repressive H3K9me3 histone mark. Importantly, the control of AalERV1 is recapitulated in vivo as Piwi6 knockdown increased AalERV1 expression in both somatic and germline tissues of Ae. albopictus mosquitoes. These results establish Aedes mosquitoes as a model to study nuclear PIWI functions and suggest that somatic piRNA-mediated transposon silencing is evolutionarily conserved across arthropod species.

We report the successful creation of multi-kilobase knockin mice and cell lines using three rAAV donors and CRISPR/Cas9 via consecutive homology-directed repair and reliable generation of over 120 KI mice with rAAV donors. For comprehensive analysis of the post-edit genome, we combined target capture and long-read sequencing for a multiplexable and high-throughput, all-in-one method to detect on-target insertion and as importantly, various undesired editing outcomes in the genome. Together, the two novel methods greatly improve the efficiency and accuracy of challenging large KI mouse and cell models.

Maintaining efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse-transcriptase ACTivity-crispR (REACTR) assay for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR’s sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.

Population-scale resources of genetic, molecular, and cellular information form the basis for understanding human genomes, charting the heritable basis of disease, and tracing the effects of mutations. Pooled perturbation assays applied to cellular models, probing the effect of many perturbations coupled with an scRNA-seq readout (Perturb-seq), are especially potent references for interpreting disease-linked mutations or gene expression changes. However, the utility of existing maps has been limited by the comprehensiveness of perturbations possible, and the relevance of their cell line context. Here, we present the first genome-scale CRISPR interference (CRISPRi) perturbation map with single-cell RNA sequencing readout across many human genetic backgrounds in human pluripotent cells. To do so, we establish large-scale CRISPRi screening in human induced pluripotent stem cells from healthy donors, using over 20,000 guide RNAs to target 7,226 genes across 34 cell lines from 26 genetic backgrounds, and gather expression data from nearly 2 million cells. We comprehensively map trans expression changes induced by the target knockdowns, which complement co-expression patterns in unperturbed cells and facilitate the functional annotation of target genes to biological processes and complexes. Consistency of targeting protein complex members point to protein complexes as a nexus for aggregating transcriptional variation, revealing novel interaction partners. We characterise variation in perturbation effects across donors, with expression quantitative trait loci linked to higher genetic modulation of perturbation effects but overall low replication of trans effects due to knockdown of their corresponding cis regulators. This study pioneers population-scale CRISPR perturbations with single cell readouts that will fuel foundation models for the future of effective modulation of cellular disease phenotypes.

The organization of the cell’s cytoskeletal filaments is coordinated through a complex symphony of signaling cascades originating from internal and external cues. Two major actin regulatory pathways are signal transduction through Rho family GTPases and growth and proliferation signaling through the Hippo pathway. These two pathways act to define the actin cytoskeleton, controlling foundational cellular attributes such as morphology and polarity. In this study, we use human epithelial cells to investigate the interplay between the Hippo and Rho Family signaling pathways, which have predominantly been characterized as independent actin regulatory mechanisms. We identify that the RhoA effector, ARHGAP18, forms a complex with the Hippo pathway transcription factor YAP to address a long-standing enigma in the field. Using super resolution STORM microscopy, we characterize the changes in the actin cytoskeleton, on the single filament level, that arise from CRISPR/Cas9 knockout of ARHGAP18. We report that the loss of ARHGAP18 results in alterations of the cell that derive from both aberrant RhoA signaling and inappropriate nuclear localization of YAP. These findings indicate that the Hippo and Rho family GTPase signaling cascades are coordinated in their temporal and spatial control of the actin cytoskeleton.

Cell competition is a quality control acting from development to the adult that eliminates cells that are less-fit than their neighbours. How winner cells induce the elimination of losers during this process is poorly understood. Here, we address this question by studying the onset of differentiation in mouse, where cell competition eliminates 35% of embryonic cells. These loser cells have mitochondrial dysfunction, and we find that this causes amino-acid deprivation and activation of the integrated stress response (ISR), a pathway essential for their survival. We show that L-Proline is a key amino-acid sensed by the ISR and that in a competitive environment, winner cells induce increased L-Proline uptake in loser cells. This causes ISR repression and their elimination. Our results imply that cell competition is acting as a nutrient sensor, eliminating dysfunctional cells when amino acids are plentiful but sparing them in nutrient poor environments.

Summary CRISPR-Cas9 (clustered, regularly interspaced, short palindromic repeats with CRISPR-associated protein 9) is a powerful, versatile, and cost-effective molecular tool that can be used for genetic engineering purposes and beyond 1 and is especially suited for non-model organisms 2 . Effective delivery of this system, however, remains a challenge for in vivo genetic manipulation of specific tissues 3 , particularly the brain 4 , and in adult indivuduals 5,6 . We designed a new CRISPR-Cas9 plasmid that was inserted into a baculovirus vector to knockdown the octopamine beta subtype 2 receptor ( AmOctβ2R ), a transmembrane protein found in the mushroom body neurons of the honey bee ( Apis mellifera ) brain, to determine if octopamine plays a role in appetite regulation. We first confirmed that gene editing of AmOctβ2R is possible with Sanger sequencing. We then demonstrated expression of the CRISPR-Cas9 system with the baculovirus vector in vitro using live cell imaging, flow cytometry analysis, and in vivo using confocal imaging, showing widespread expression in the cells and throughout the honey bee brain, three days post treatment. There was also in vitro and in vivo knockdown of AmOctβ2R three days post-infection, that corresponded with appetite suppression in starved forager bees. Our findings suggest that we successfully delivered the CRISPR-Cas9 system and knocked down AmOctB2R in neuronal cells of the honey bee brain that were previously inaccessible due to the blood brain barrier and lack of infectivity of lentivirus vectors 7 . The newly characterized AmOctβ2R 8 can now be assigned a functional role and other targets for gene editing are now possible using this CRISPR-Cas9 system. Graphical Abstract

Familial dysautonomia (FD) is a fatal autosomal recessive congenital neuropathy caused by a T-to-C mutation in intron 20 of the Elongator acetyltransferase complex subunit 1 ( ELP1 ) gene, which causes tissue-specific skipping of exon 20 and reduction of ELP1 protein. Here, we developed a base editor (BE) approach to precisely correct this mutation. By optimizing Cas9 variants and screening multiple gRNAs, we identified a combination that was able to promote up to 70% on-target editing in HEK293T cells harboring the ELP1 T-to-C mutation. These editing levels were sufficient to restore exon 20 inclusion in the ELP1 transcript. Moreover, we optimized an engineered dual intein-split system to deliver these constructs in vivo . Mediated by adeno-associated virus (AAV) delivery, this BE strategy effectively corrected the liver and brain ELP1 splicing defects in a humanized FD mouse model carrying the ELP1 T-to-C mutation and rescued the FD phenotype in iPSC-derived sympathetic neurons. Importantly, we observed minimal off-target editing demonstrating high levels of specificity with these optimized base editors. These findings establish a novel and highly precise BE-based therapeutic approach to correct the FD mutation and associated splicing defects and provide the foundation for the development of a transformative, permanent treatment for this devastating disease.

The obligate intracellular parasite Toxoplasma gondii replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV, Toxoplasma endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host ESCRT machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the Plant Vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if Toxoplasma uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide CRISPR screen in wild-type (WT) parasites and Δ gra14 , Δ cpl , and Δ crt mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that Δ cpl and Δ crt mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, TCA cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathway’s potential role in host resource scavenging. Interestingly, ingestion mutants showed an exacerbated growth defect when grown in amino acid-depleted media, suggesting a role for the Toxoplasma ingestion pathway during nutrient scarcity. Importance Toxoplasma gondii is an obligate intracellular pathogen that infects virtually any nucleated cell in most warm-blooded animals. Infections are asymptomatic in most cases but people with weakened immunity can experience severe disease. For the parasite to replicate within the host, it must efficiently acquire essential nutrients, especially as it is unable to make several key metabolites. Understanding the mechanisms by which Toxoplasma scavenges nutrients from the host is crucial for identifying potential therapeutic targets. Our study highlights the function of the ingestion pathway in sustaining parasite metabolites and contributes to parasite replication under amino acid limiting conditions. This work advances our understanding of the metabolic adaptability of Toxoplasma .

Summary Recent massively-parallel approaches to decipher gene regulatory circuits have focused on the discovery of either cis -regulatory elements (CREs) or trans -acting factors. Here, we develop a scalable approach that pairs cis - and trans -regulatory CRISPR screens to systematically dissect how the key immune checkpoint PD-L1 is regulated. In human pancreatic ductal adenocarcinoma (PDAC) cells, we tile the PD-L1 locus using ∼25,000 CRISPR perturbations in constitutive and IFNγ-stimulated conditions. We discover 67 enhancer- or repressor-like CREs and show that distal CREs tend to contact the promoter of PD-L1 and related genes. Next, we measure how loss of all ∼2,000 transcription factors (TFs) in the human genome impacts PD-L1 expression and, using this, we link specific TFs to individual CREs and reveal novel PD-L1 regulatory circuits. For one of these regulatory circuits, we confirm the binding of predicted trans -factors (SRF and BPTF) using CUT&RUN and show that loss of either the CRE or TFs potentiates the anti-cancer activity of primary T cells engineered with a chimeric antigen receptor. Finally, we show that expression of these TFs correlates with PD-L1 expression in vivo in primary PDAC tumors and that somatic mutations in TFs can alter response and overall survival in immune checkpoint blockade-treated patients. Taken together, our approach establishes a generalizable toolkit for decoding the regulatory landscape of any gene or locus in the human genome, yielding insights into gene regulation and clinical impact.

CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and silent mutations. Here, we present a Cas12a-based system that targets each gene with four sgRNAs to overcome these limitations, using Drosophila as a tractable in vivo model. We show that multiplexed sgRNAs act synergistically to create deletions between target sites, substantially increasing the fraction of loss-of-function mutations. To systematically assess off-target effects, we developed a novel screening assay that visualizes CRISPR-induced chromosomal alterations in living animals. This enabled comprehensive screening of more than 2000 sgRNAs clustered in 525 quadruple arrays across 21 megabases of genomic DNA, revealing remarkably high on-target activity (100%, 82/82) and undetectable off-target cutting (0%, 0/443). Quantitative side-by-side comparisons with a current Cas9-based system targeting over 100 genes demonstrates that multiplexed Cas12a-mediated gene targeting achieves superior performance and reveals phenotypes missed by established methods. This highly efficient and specific system provides a framework for reliable functional genomics studies across diverse organisms.

ABSTRACT Common dCas9-based CRISPR interference (CRISPRi) system for gene regulation requires antibiotic selection and exogenous inducer molecules, posing significant challenges when applied in in vivo bacterial infection models. Using Staphylococcus aureus as a model organism, we have developed a programmable, plasmid-based, but selection-free (ppsf)-CRISPRi system that is based on the pCM29- plasmid which is stable without antibiotic selection. In this ppsf-CRISPRi system, dCas9 expression is regulated by an endogenous virulence gene promoter, and sgRNA expression is driven by a constitutive promoter eliminating the need for exogenous inducer molecules. The system was programmed to silence the expression of genes encoding the virulence factor coagulase or peptidoglycan hydrolase autolysin, whenever their respective endogenous promoter was activated. The selection-free functionality was confirmed over at least 27 generations and verified by qPCR and phenotypic assays depending on the protein target, including coagulation of rabbit plasma and THP-1 macrophage cell infection in vitro as well as in vivo infection of Galleria mellonella larvae, in each case phenocopying the observations made using transposon mutant strains. The system is suitable for long-term studies of S. aureus pathogenesis in vitr o or in vivo and represents a blueprint for the development of similar ppsf-CRISPRi systems in other bacterial species.

ABSTRACT Double-stranded RNAs (dsRNAs) produced during viral infections are recognized by the innate immune sensor protein kinase R (PKR), triggering a host translation shutoff that inhibits viral replication and propagation. Given the harmful effects of uncontrolled PKR activation, cells must tightly regulate PKR to ensure that its activation occurs only in response to viral infections, not endogenous dsRNAs. Here, we use CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that exploits translation levels as a readout and identifies PACT as a key inhibitor of PKR during viral infection. We find that cells deficient for PACT hyperactivate PKR in response to several different RNA viruses, raising the question of why cells need to limit PKR activity. Our results demonstrate that PACT cooperates with ADAR1 to suppress PKR activation from self-dsRNAs in uninfected cells. The simultaneous deletion of PACT and ADAR1 results in synthetic lethality, which can be fully rescued in PKR-deficient cells. We propose that both PACT and ADAR1 act as essential barriers against PKR, creating a threshold of tolerable levels to endogenous dsRNA in cells without activating PKR-mediated translation shutdown and cell death.

Programmable epigenome editors modify gene expression in mammalian cells by altering the local chromatin environment at target loci without inducing DNA breaks. However, the large size of CRISPR-based epigenome editors poses a challenge to their broad use in biomedical research and as future therapies. Here, we present Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins (RENDER) for transiently delivering programmable epigenetic repressors (CRISPRi, DNMT3A-3L-dCas9, CRISPRoff) and activator (TET1-dCas9) as ribonucleoprotein complexes into human cells to modulate gene expression. After rational engineering, we show that RENDER induces durable epigenetic silencing of endogenous genes across various human cell types, including primary T cells. Additionally, we apply RENDER to epigenetically repress endogenous genes in human stem cell-derived neurons, including the reduction of the neurodegenerative disease associated V337M-mutated Tau protein. Together, our RENDER platform advances the delivery of CRISPR-based epigenome editors into human cells, broadening the use of epigenome editing in fundamental research and therapeutic applications.

While the most widely used CRISPR-Cas enzyme is the S. pyogenes Cas9 endonuclease (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Although Cas9 can eventually be forcibly removed by extrinsic factors (translocating polymerases, helicases, chromatin modifying complexes, etc), the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Interestingly, it’s been shown that Cas9 can be more easily dislodged when complexes collide with the PAM-distal region of the Cas9 complex or when the strength of Cas9 interactions in this region are weakened. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and still support Cas9 activity. We find that guide truncation promotes much faster Cas9 turnover and used this to capture previously uncharacterized Cas9 reaction states. Kinetics-guided cryo-EM enabled us to enrich for rare, transient states that are often difficult to capture in standard workflows. From a single dataset, we examine the entire conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.

The activity of signaling pathways is required for coordinated cellular and physiological processes leading to normal development of brain structure and function. Mutations in OCRL , a phosphatidylinositol 4,5 bisphosphate [PIP 2 ] 5-phosphatase leads to the neurodevelopmental disorder, Lowe Syndrome (LS). However, the mechanism by which mutations in OCRL leads to the brain phenotypes of LS is not understood. We find that on differentiation of LS patient derived iPSC, developing neural cultures show reduced excitability along with enhanced P levels of Glial Fibrillary Acidic Protein. Multiomic single-nucleus RNA and ATAC seq analysis of neural stem cells generated from LS patient iPSC revealed an enhanced number of cells with a gliogenic cell state. RNA seq analysis also revealed increased levels of DLK1 , a non-canonical Notch ligand in LS patient NSC associated increased levels of cleaved Notch protein and elevation of its transcriptional target HES5 , indicating upregulated Notch signaling. Treatment of iPSC derived brain organoids with an inhibitor of PIP5K, the lipid kinase that synthesizes PIP 2 , was able to restore neuronal excitability and rescue Notch signaling defects in LS patient derived organoid cultures. Overall, our results demonstrate a role for PIP 2 dependent regulation of Notch signaling, cell fate specification and development of neuronal excitability regulated by OCRL activity.

ABSTRACT Macrophages hold tremendous promise as effectors of cancer immunotherapy, but the best strategies to provoke these cells to attack tumors remain unknown. Here, we evaluated the therapeutic potential of targeting two distinct macrophage immune checkpoints: CD47 and CD24. We found that antibodies targeting these antigens could elicit maximal levels of phagocytosis when combined together in vitro. However, to our surprise, via unbiased genome-wide CRISPR screens, we found that CD24 primarily acts as a target of opsonization rather than an immune checkpoint. In a series of in vitro and in vivo genetic validation studies, we found that CD24 was neither necessary nor sufficient to protect cancer cells from macrophage phagocytosis in most mouse and human tumor models. Instead, anti-CD24 antibodies exhibit robust Fc-dependent activity, and as a consequence, they cause significant on-target hematologic toxicity in mice. To overcome these challenges and leverage our findings for therapeutic purposes, we engineered a collection of 77 novel bispecific antibodies that bind to a tumor antigen with one arm and engage macrophages with the second arm. We discovered multiple novel bispecifics that maximally activate macrophage-mediated cytotoxicity and reduce binding to healthy blood cells, including bispecifics targeting macrophage immune checkpoint molecules in combination with EGFR, TROP2, and CD71. Overall, our findings indicate that CD47 predominates over CD24 as a macrophage immune checkpoint in cancer, and that the novel bispecifics we created may be optimal immunotherapies to direct myeloid cells to eradicate solid tumors.