Mammalian cells can be directed toward specific fates by overexpression of transcription factors (TFs). However, discovering and optimizing which TFs in combination produce a state of interest remains challenging. Here, we develop a scalable screening platform that addresses this challenge by combining high multiplicity of infection (MOI), pooled delivery of barcoded TF open reading frames (ORFs), data augmentation, targeted cell enrichments, and single-cell transcriptomic readouts. As proof of principle, we apply the platform to optimize the generation of hematopoietic stem and progenitor-like cells (HSPCs) from human embryonic stem cells. Our data demonstrate technical performance across a range of key metrics and reveal a richly structured reprogramming fitness landscape over millions of TF combinations. In silico optimization of HSPC-similarity metrics over this landscape revealed two TF combinations that demonstrate superior potency in generating naive multipotent hematopoietic progenitors relative to gold-standard controls. This study demonstrates a powerful approach for data-driven cell fate engineering using complex combinatorial perturbations.

RNA-guided DNA nucleases Cas9 and IscB (insertion sequences Cas9-like OrfB) are components of type II CRISPR–Cas adaptive immune systems and transposon-associated OMEGA (obligate mobile element-guided activity) systems, respectively. Sequence and structural comparisons indicate that IscB (~500 residues) evolved into Cas9 (~700–1,600 residues) through protein expansion coupled with guide RNA miniaturization. However, the specific sequence of events in this evolutionary transition remains unknown. Here, we report cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases—two IscBs and two Cas9s—each in complex with its cognate guide RNA and target DNA. Comparisons of these four complex structures to previously reported IscB and Cas9 structures indicate that evolution from IscB to Cas9 involved the loss of the N-terminal PLMP domain and the acquisition of the zinc-finger-containing REC3 domain, followed by bridge helix extension and REC1 domain acquisition. These structural changes led to expansion of the REC lobe, increasing the target DNA cleavage specificity. Additionally, the structural conservation of the RNA scaffolds indicates that the dual CRISPR RNA (crRNA) and trans-activating crRNA guides of CRISPR–Cas9 evolved from the single ωRNA guides of OMEGA systems. Our findings provide insights into the succession of structural changes involved in the exaptation of transposon-associated RNA-guided nucleases for the role of effector nucleases in adaptive immune systems. Nagahata, Kato and Yamada et al. provide cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases—HfmIscB, TbaIscB, YnpsCas9 and NbaCas9—each in complex with its guide RNA and target DNA, providing insights into CRISPR–Cas9 evolution.

The intracellular delivery of therapeutic macromolecules remains a major challenge in biomedicine. Here we reconstitute the Drosophila melanogaster Arc1 (dArc1) retroelement-derived capsid entirely from purified recombinant protein components and in vitro transcribed RNA, creating a fully defined, cell-free assembled protein nanoparticle system. Through affinity engineering of the dArc1 RNA-binding domain, we enable efficient encapsulation of mRNA payloads and Cas9 ribonucleoproteins. Unexpectedly, we discover that dArc1 capsids bind mammalian cells through a direct interaction with the surface receptor SORCS2. Leveraging this interaction, we show that intramuscular injection of dArc1 capsids carrying Cas9 gene editors achieves up to 18% exon skipping and restores dystrophin expression in muscle fibers of mdx mice, a model of Duchenne muscular dystrophy. Enhanced delivery efficiency in regenerating and dystrophic muscle correlates with upregulated SORCS2 expression, supporting our finding that SORCS2 facilitates cellular uptake of dArc1 capsids. This work demonstrates the potential of in vitro-assembled protein nanoparticles for delivery of molecular cargoes.

Prokaryotes employ diverse defense strategies to detect and halt the progression of phage infection. Multiple defense systems sense phage proteins through direct binding, including antiviral STAND NTPases (Avs), which oligomerize upon target recognition to induce programmed cell death. The widespread Avs2 family was previously shown to detect the large terminase subunit of tailed phages, but the mechanism of terminase sensing was unknown. Here, we determine the structural basis of terminase recognition by Avs2 from Escherichia coli (EcAvs2). A cryo-EM structure at 2.3 Å resolution reveals that EcAvs2 forms a flat, C4-symmetric tetramer in which each protomer is bound to a single terminase monomer. Terminase recognition is mediated by a large, shape complementary binding pocket in the EcAvs2 sensor domain, including specific contacts with an unexpected ATP molecule at the interface of EcAvs2 and terminase. Furthermore, we demonstrate that the defense protein Upx also recognizes diverse phage terminases, despite lacking sequence and structural homology to Avs. AlphaFold 3 models indicate that Upx binds an unfolded state of the core terminase ATPase domain, mediated by β-augmentation. These findings highlight the distinct modes of terminase recognition across structurally diverse defense proteins. Direct recognition of viral proteins is key to bacterial immunity. Here, Evans et al. uncover the mechanisms used by two distinct defense proteins to detect the phage DNA packaging motor mechanisms: bridging ATP ligand or binding an unfolded state.

Human induced pluripotent stem cells (hiPSC) are an invaluable resource for investigating the molecular mechanisms regulating cell fate specification during brain development. However, most directed differentiation methods exhibit significant cell fate heterogeneity and require several months to become functional. To address this challenge, we developed a green fluorescent protein (GFP) reporter system in hiPSC by targeting the genomic locus of Forebrain Enriched Zinc Finger 2 (FEZF2), which encodes a transcription factor essential for the fate specification of sub-cerebral projection neurons (SCPN) during forebrain development. Using this FEZF2-GFP reporter hiPSC line, we optimized a directed differentiation protocol to rapidly and efficiently generate pallial progenitors and glutamatergic neuronal subgroups after 3 weeks. Through fluorescence activated cell sorting for both GFP and CD200, isolated post-mitotic SCPN immediately displayed electrophysiological properties and formed glutamatergic synapses within 4 additional weeks of in vitro cell culture. Co-culture with hiPSC-derived spinal motor neurons further enhanced these electrophysiological characteristics, improved viability, and increased synapse formation in SCPN. This study presents a streamlined and effective strategy to generate, isolate, and characterize human motor neuron circuits, providing insights into the molecular determinants regulating synaptogenesis and functional maturation.

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases, SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis, we identify putative immunogenic epitopes on each nuclease. Using computational modeling, we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components, including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses, yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9, but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection.

R2 elements, a class of non-long terminal repeat (non-LTR) retrotransposons, have the potential to be harnessed for transgene insertion. However, efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here, we structurally and biochemically characterize R2 from Taeniopygia guttata (R2Tg). We show that R2Tg cleaves both strands of its ribosomal DNA target and binds a pseudoknotted RNA element within the R2 3' UTR to initiate target-primed reverse transcription. Guided by these insights, we engineer and characterize an all-RNA system for transgene insertion. We substantially reduce the system's size and insertion scars by eliminating unnecessary R2 sequences on the donor. We further improve the integration efficiency by chemically modifying the 5' end of the donor RNA and optimizing delivery, creating a compact system that achieves over 80% integration efficiency in several human cell lines. This work expands the genome engineering toolbox and provides mechanistic insights that will facilitate future development of R2-mediated gene insertion tools.

The Photorhabdus virulence cassette is a microbial nanosyringe that can be engineered to deliver protein cargos into human cells. Here we further modify this system to incorporate exogenous cargos and targeting moieties in vitro. We show that this method, termed SPEAR, enables loading of different types of cargo (including folded ribonucleoproteins and single-stranded DNA) and targeting of defined cell types both in vitro and in vivo.

During chronic stress, cells must support both tissue function and their own survival. Hepatocytes perform metabolic, synthetic, and detoxification roles, but chronic nutrient imbalances can induce hepatocyte death and precipitate metabolic dysfunction-associated steatohepatitis (MASH, formerly NASH). Despite prior work identifying stress-induced drivers of hepatocyte death, chronic stress’ functional impact on surviving cells remains unclear. Through cross-species longitudinal single-cell multi-omics, we show that ongoing stress drives prognostic developmental and cancer-associated programs in non-transformed hepatocytes while reducing their mature functional identity. Creating integrative computational methods, we identify and then experimentally validate master regulators perturbing hepatocyte functional balance, increasing proliferation under stress, and directly priming future tumorigenesis. Through geographic regression on human tissue microarray spatial transcriptomics, we uncover spatially structured multicellular communities and signaling interactions shaping stress responses. Our work reveals how cells’ early solutions to chronic stress can prime future tumorigenesis and outcomes, unifying diverse modes of cellular dysfunction around core actionable mechanisms.

Naturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifications to the enzymes introduced to improve the desired function; however, it is difficult to engineer variants with enhanced activity while maintaining specificity. Here we engineer the compact Obligate Mobile Element Guided Activity (OMEGA) RNA-guided endonuclease IscB and its guiding RNA (ωRNA) by combining ortholog screening, structure-guided protein domain design and RNA engineering, and deep learning-based structure prediction to generate an improved variant, NovaIscB. We show that the compact NovaIscB achieves up to 40% indel activity (~100-fold improvement over wild-type OgeuIscB) on the human genome with improved specificity relative to existing IscBs. We further show that NovaIscB can be fused with a methyltransferase to create a programmable transcriptional repressor, OMEGAoff, that is compact enough to be packaged in a single adeno-associated virus vector for persistent in vivo gene repression. This study highlights the power of combining natural diversity with protein engineering to design enhanced enzymes for molecular biology applications.

Gratitude practices have been suggested to be helpful in decreasing negative and increasing positive affect. Using large-scale data from a smartphone app (How We Feel) in the United States across three waves of the COVID-19 pandemic before spring 2021 (Wave 1: 61,267 users; 677,246 observations; Wave 2: 16,037 users; 267,938 observations; Wave 3: 9128 users; 265,575 observations), the current study examined associations between a brief gratitude practice and individuals' daily discrete positive and negative affect from Days 1 to 6 after the practice. The results showed that writing down one thing a person feels grateful for was associated with decreased stress, anxiety, tiredness, and loneliness, and most of these effects were sustained one to six days post-practice. However, the brief practice resulted in counterintuitive findings in relation to positive affect; individuals reported feeling less happy, hopeful, and optimistic after writing about what they were grateful for at the beginning of the pandemic. The discussion focuses on the effectiveness of brief, low-cost digital gratitude practices on individuals' affect.

RNA-guided systems provide remarkable versatility, enabling diverse biological functions. Through iterative structural and sequence homology-based mining starting with a guide RNA-interaction domain of Cas9, we identified a family of RNA-guided DNA-targeting proteins in phage and parasitic bacteria. Each system consists of a tandem interspaced guide RNA (TIGR) array and a TIGR-associated (Tas) protein containing a nucleolar protein (Nop) domain, sometimes fused to HNH (TasH)- or RuvC (TasR)-nuclease domains. We show that TIGR arrays are processed into 36-nucleotide RNAs (tigRNAs) that direct sequence-specific DNA binding through a tandem-spacer targeting mechanism. TasR can be reprogrammed for precise DNA cleavage, including in human cells. The structure of TasR reveals striking similarities to box C/D small nucleolar ribonucleoproteins and IS110 RNA-guided transposases, providing insights into the evolution of diverse RNA-guided systems.

Biomolecular condensates organize numerous subcellular processes and have been implicated in diseases, including neurodegeneration and cancer. Protein sequences intrinsically encode their propensity to form condensates, but specific sequence features that regulate this behavior have not been systematically explored at scale. Here, we develop CondenSeq, a high-throughput pooled imaging with in situ sequencing approach to measure propensities of thousands of protein sequences to form nuclear condensates. Leveraging the large scale of these experiments, we evaluated the impacts of dozens of sequence features across a wide range of sequence contexts, identifying several features with highly consistent, context-independent effects and others with less-consistent effects. We also identified multiple classes of condensates and discovered distinct sequence properties that drive their formation. Our results provide a systematic overview of the relationships between protein sequences and nuclear condensate formation and establish a general approach for further dissecting these relationships at scale.

The known diversity of CRISPR-Cas systems continues to expand. To encompass new discoveries, here we present an updated evolutionary classification of CRISPR-Cas systems. The updated CRISPR-Cas classification includes 2 classes, 7 types and 46 subtypes, compared with the 6 types and 33 subtypes in our previous survey 5 years ago. In addition, a classification of the cyclic oligoadenylate-dependent signalling pathway in type III systems is presented. We also discuss recently characterized alternative CRISPR-Cas functionalities, notably, type IV variants that cleave the target DNA and type V variants that inhibit the target replication without cleavage. Analysis of the abundance of CRISPR-Cas variants in genomes and metagenomes shows that the previously defined systems are relatively common, whereas the more recently characterized variants are comparatively rare. These low abundance variants comprise the long tail of the CRISPR-Cas distribution in prokaryotes and their viruses, and remain to be characterized experimentally.

Introduction: Aging impairs immune function and leads to increased vulnerability to infection, malignancy, and vaccine failure.Immunosenescence, marked by reduced thymic output, naive T cell attrition, and progenitor exhaustion, undermines vaccine responses and anti-tumor immunity in aged individuals. While cytokine-based and stem cell-modifying therapies have attempted to reverse this decline, they remain limited by toxicity, durability, or translational feasibility. Here, we present an mRNA approach that transiently reconstitutes thymic signaling pathways in an unrelated organ. By expressing Delta-like ligand 1 (DLL1), Fms-like tyrosine kinase 3 ligand (FLT3-L), and interleukin-7 (IL-7) - collectively termed DFI - in the liver, we observed rejuvenated aged immunity via coordinated modulation of thymopoiesis, lymphoid progenitors, and immune function. Methods: We identified age-related decline of Notch, IL-7, and FLT3-L signaling in thymic epithelial cells and peripheral T cells using spatial transcriptomics (Slide-seq v2) and single-cell RNA-seq across the human and murine lifespan. Modified mRNAs encoding DFI were encapsulated in SM-102 lipid nanoparticles (LNPs) and administered to aged mice (72 weeks). Hepatic expression, pharmacokinetics, and tissue specificity were assessed via RIBOmap and immunofluorescence. Functional impact on thymopoiesis, hematopoiesis, and immune function was assessed via flow cytometry, V(D)J sequencing, TREC analysis, peptide vaccination, tumor challenge (B16-OVA and MC38-OVA), and immune checkpoint inhibitor (ICI) therapy. Autoimmune safety was evaluated in NOD, Act-mOVA, and EAE models. Results: DFI mRNA was selectively translated in hepatocytes, yielding membrane-bound DLL1 and secreted IL-7 and FLT3-L without off-target organ expression. This induced expansion of naïve CD4⁺ and CD8⁺ T cells, increased TRECs, and restored thymic cellularity in aged mice. Mechanistically, DFI did not reprogram hematopoietic stem cell (HSC) fate or reverse myeloid bias but significantly expanded common lymphoid progenitors (CLPs) in the bone marrow. These CLPs exhibited increased CCR9 expression and thymus-homing potential, consistent with peripheral DLL1-mediated Notch signaling and improved thymic seeding. Early thymocyte subsets (DN1-DN3) were selectively enriched, accompanied by rapid induction of Rag2 and Nur77 in thymocytes, supporting enhanced de novo thymopoiesis. Despite HSC aging signatures remaining intact, DFI circumvented upstream deficits by expanding committed lymphoid progenitors and reinvigorating thymic output. Beyond the T cell compartment, DFI restored conventional dendritic cell type 1 (cDC1) populations and boosted co-stimulatory molecule expression (CD40, CD83, CD86). In parallel, it decreased dysfunctional age-associated B cells and increased follicular B cells, resulting in more robust antigen-specific IgG responses and IgM-to-IgG class-switching after peptide vaccination. Functionally, DFI pre-treatment doubled vaccine-specific CD8⁺ T cells in aged mice and improved antigen recall cytokine production (IFN-γ, IL-2). In tumor models, DFI conditioning enhanced spontaneous rejection of MC38-OVA and B16-OVA tumors and improved survival. It restored intratumoral CD8⁺ T cell infiltration and diversity and reversed age-associated T cell exhaustion signatures. When combined with ICI, DFI synergistically enhanced tumor control and survival in aged mice with established tumors. Importantly, DFI did not trigger autoimmune responses. In NOD mice, DFI did not accelerate type 1 diabetes or increase autoreactive TCRs. In Act-mOVA mice, tolerance to endogenous OVA was preserved. In the EAE model, DFI increased peripheral MOG-specific T cells but did not worsen CNS inflammation or clinical scores. DFI's immunostimulatory effects were strictly transient, dissipating 4 weeks post-treatment without long-term adverse effects. Conclusions: Transient hepatic expression of DLL1, IL-7, and FLT3-L safely and effectively reactivates thymopoiesis, expands functional T cell pools, and enhances antigen-specific immune responses in aged hosts. By bypassing stem cell-intrinsic defects and rejuvenating the thymic niche via liver-derived trophic cues, DFI overcomes major roadblocks of immune aging. This mRNA-based, non-integrating platform supports scalable, cyclic immunostimulation without autoimmunity, suggesting a viable strategy to enhance vaccine efficacy and cancer immunotherapy in elderly patients.

Cas13 is an RNA-guided RNA endonuclease derived from the type VI CRISPR-Cas system, which has been used in numerous RNA-targeting technologies, such as RNA knockdown, detection and editing. The catalytically inactive Prevotella sp. Cas13b (dPspCas13b) fused to the human adenosine deaminase acting on RNA 2 (ADAR2) deaminase domain can edit adenosine in target transcripts to inosine, in an RNA-editing technology called REPAIR (RNA editing for programmable A-to-I replacement), which has potential for gene therapy. Here we report the cryo-electron microscopy structures of the PspCas13b-guide RNA binary complex, the PspCas13b-guide RNA-target RNA ternary complex and the dPspCas13b-ADAR2-guide RNA-target RNA complex. These structures provide mechanistic insights into RNA cleavage and editing. We applied our structural insights to engineer a compact and efficient dPspCas13b-ADAR2 complex (REPAIR-mini). Overall, our findings advance the understanding of CRISPR-Cas13 effector nucleases and could enable the development of improved RNA-targeting technologies.

Machado-Joseph disease (MJD) is an autosomal dominantly-inherited neurodegenerative disorder, caused by an over-repetition of the polyglutamine-codifying region in the ATXN3 gene. Strategies based on the suppression of the deleterious gene products have demonstrated promising results in pre-clinical studies. Nonetheless, these strategies do not target the root cause of the disease. In order to prevent the downstream toxic pathways, our goal was to develop gene editing-based strategies to permanently inactivate the human ATXN3 gene. TALENs and CRISPR-Cas9 systems were designed to target exon 2 of this gene and functional characterization was performed in a human cell line. After the demonstration of TALEN and CRISPR-Cas9 efficiency on gene disruption, a sequence of each system was selected for further in vivo experiments. Although both TALENs and CRISPR-Cas9 systems led to a drastic reduction of ATXN3 aggregates in the striatum of a lentiviral-based mouse model of MJD/SCA3, only CRISPR-Cas9 system allowed the improvement of key neuropathological markers of the disease. Importantly, the administration of the engineered system in YAC-MJD84.2/84.2 mice mediated a delay in disease progression, when compared with non-treated littermates. These data provide the first in vivo evidence of the efficacy of a CRISPR-Cas9-based approach to permanently inactivate the ATXN3 gene in the brain of two mouse models of the disease, supporting its potential as a new therapeutic avenue in the context of MJD/SCA3. ### Competing Interest Statement N.E.S. is an adviser to Qiagen and a cofounder and adviser of TruEdit Bio and OverT Bio. P.D.H. acknowledges outside interest in Terrain Biosciences, Stylus Medicine, Spotlight Therapeutics, Arbor Biosciences, Varda Space, Vial Health, and Veda Bio, where he holds various roles including as co-founder, director, scientific advisory board member, or consultant.

Ageing erodes human immunity, in part by reshaping the T cell repertoire, leading to increased vulnerability to infection, malignancy and vaccine failure 1–3 . Attempts to rejuvenate immune function have yielded only modest results and are limited by toxicity or lack of clinical feasibility 1,3–5 . Here we show that the liver can be transiently repurposed to restore age-diminished immune cues and improve T cell function in aged mice. These immune cues were found by performing multi-omic mapping across central and peripheral niches in young and aged animals, leading to the identification of Notch and Fms-like tyrosine kinase 3 ligand (FLT3L) pathways, together with interleukin-7 (IL-7) signalling, as declining with age. Delivery of mRNAs encoding Delta-like ligand 1 (DLL1), FLT3L and IL-7 to hepatocytes expanded common lymphoid progenitors, boosted de novo thymopoiesis without affecting haematopoietic stem cell (HSC) composition, and replenished T cells while enhancing dendritic cell abundance and function. Treatment with these mRNAs improved peptide vaccine responses and restored antitumour immunity in aged mice by increasing tumour-specific CD8 + infiltration and clonal diversity and synergizing with immune checkpoint blockade. These effects were reversible after dosing ceased and did not breach self-tolerance, in contrast to the inflammatory and autoimmune liabilities of recombinant cytokine treatments 6,7 . These findings underscore the promise of mRNA-based strategies for systemic immune modulation and highlight the potential of interventions aimed at preserving immune resilience in ageing populations.

Despite ongoing efforts to study CRISPR systems, the evolutionary origins giving rise to reprogrammable RNA-guided mechanisms remain poorly understood. Here, we describe an integrated sequence/structure evolutionary tracing approach to identify the ancestors of the RNA-targeting CRISPR-Cas13 system. We find that Cas13 likely evolved from AbiF, which is encoded by an abortive infection-linked gene that is stably associated with a conserved non-coding RNA (ncRNA). We further characterize a miniature Cas13, classified here as Cas13e, which serves as an evolutionary intermediate between AbiF and other known Cas13s. Despite this relationship, we show that their functions substantially differ. Whereas Cas13e is an RNA-guided RNA-targeting system, AbiF is a toxin-antitoxin (TA) system with an RNA antitoxin. We solve the structure of AbiF using cryoelectron microscopy (cryo-EM), revealing basic structural alterations that set Cas13s apart from AbiF. Finally, we map the key structural changes that enabled a non-guided TA system to evolve into an RNA-guided CRISPR system.

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz’s mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.