Abstract Functional genomics has been hampered by the paucity of efficient methods that connect genotype and metabolic phenotype at single-cell resolution. Using the industrial microalga Nannochloropsis oceanica as a model, we introduced a platform that comprises a genome-wide single-gene-edited mutant library and high-throughput Raman-activated Cell Sorting (RACS). The CRISPR/Cas-generated library consists of 3,567 microalgal mutants derived from 2,397 effective guide RNAs. Label-free sorting of the library for high carotenoid content by RACS unravels mutations in the violaxanthin de-epoxidase ( noVDE ) or in the proteasome assembly chaperone 4 ( noPAC4 ) genes. Knocking out all five known noVDE s reveal that the high carotenoid content is due to violaxanthin increase, whilst noPAC4 knockout boosted carotenoid content with elevations in violaxanthin, zeaxanthin, and β-carotene. Genetic and transcriptomic evidences suggest two previously unknown modes of carotenogenesis regulation mediated by noPAC4: epigenetic mechanisms via histone deacetylase (HDAC) and post-translational controls by the 26S proteasome. Therefore, by label-freely sorting single-cell metabolic phenotype and rapidly yet unambiguously tracing it to a genotype, this new forward-genetics approach can greatly accelerate the discovery of new genes and pathways.

Abstract Cancer therapy faces a critical need for treatments that selectively eliminate malignant cells without harming healthy tissue. The Proteus Project addresses this challenge by engineering a programmable gene circuit that couples CRISPR-based RNA sensing with an inducible cell death mechanism. Specifically, we repurpose a novel Type III-E CRISPR-Cas system (“Craspase”) – an RNA-guided protease complex – to detect cancer-specific RNA transcripts and, in response, cleave engineered gasdermin fusion proteins to trigger cell death (1)(2). We constructed the Proteus system in Saccharomyces cerevisiae as a surrogate model, integrating components that sense an oncogenic KRAS mutation and execute targeted pyroptosis (inflammatory apoptosis-like cell death). Preliminary results demonstrate successful assembly of the Craspase– gasdermin circuit, expression of key proteins, and proof-of-concept cell killing specifically in the presence of the oncogenic RNA trigger. This work, conducted as part of the iGEM 2025 SynBio Collective, showcases a modular synthetic biology approach for precision oncology therapeutics. The ongoing study underscores the potential of CRISPR-guided proteases in in situ cancer cell ablation and sets the stage for future validation in mammalian systems.

ABSTRACT CTCF-mediated chromatin loops are known to influence gene regulation, yet their role in pre-mRNA splicing remains incompletely understood. Here, we demonstrate that structural variants (SVs) at the anchors of intronic CTCF loops can modulate exon usage. By integrating high-resolution three-dimensional (3D) genome organization and gene expression datasets from C57BL/6J (B6) and 129S1/SvImJ (129S) mouse embryonic stem cells (ESCs), with structural variant (SV) maps from the 129S mouse, we identified thousands of intron-anchored CTCF loops. Our data indicate that SVs intersecting loop anchors are more frequently associated with differential exon inclusion events than with changes in overall gene expression. CRISPR/Cas9 deletion of two SV-harboring intronic CTCF sites in Numbl and Ireb2 validated the predicted splicing shifts observed between B6 and 129S ESC that correspond with diminished long-range chromatin looping. Our findings reveal a direct mechanistic link between 3D genome architecture and alternative splicing and highlight non-coding SVs as modulators of transcript diversity. Our study has thus identified a novel class of CTCF-bound regulatory elements regulating alternative splicing. Cataloging and validating these functional elements will elucidate molecular mechanisms underlying phenotypic variation within populations.

CHARGE syndrome is a developmental disorder that affects 1 in 10,000 births, and patients exhibit both physical and behavioral characteristics. De novo mutations in CHD7 (chromodomain helicase DNA binding protein 7) cause 67% of CHARGE syndrome cases. CHD7 is a DNA-binding chromatin remodeler with thousands of predicted binding sites in the genome, making it challenging to define molecular pathways linking loss of CHD7 to CHARGE phenotypes. To address this problem, here we used a previously characterized zebrafish CHARGE model to generate transcriptomic and proteomic datasets from larval zebrafish head tissue at two developmental time points. By integrating these datasets with differential expression, pathway, and upstream regulator analyses, we identified multiple consistently dysregulated pathways and defined a set of candidate genes that link loss of chd7 with disease-related phenotypes. Finally, to functionally validate the roles of these genes, CRISPR/Cas9-mediated knockdown of capgb , nefla , or rdh5 phenocopies behavioral defects seen in chd7 mutants. Our data provide a resource for further investigation of molecular mediators of CHD7 and a template to reveal functionally relevant therapeutic targets to alleviate specific aspects of CHARGE syndrome. Summary Statement We have identified Chd7 target genes capgb , nefla , and rdh5 that mediate CHARGE model phenotypes from transcriptomic and proteomic analysis of chd7 wild type, heterozygous, and homozygous mutant zebrafish brain tissue at two developmental time points.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of hematologic cancers. However, its efficacy in solid tumors, including pancreatic ductal adenocarcinoma (PDAC), has been limited. By integrating modular CRISPR screening with immunocompetent orthotopic models of PDAC, we identified unknown tumor-intrinsic modulators of CAR T-cell therapy response. Disruption of genes involved in oxidative and proteotoxic stress, particularly the Nrf2 target Slc33a1 , sensitizes PDAC tumors to CAR T-cell killing. Single cell gene expression analyses revealed that CAR-T resistant tumors exhibit reduced Nrf2 pathway activity. Mechanistically, we show that Nrf2 pathway hyperactivation by genetic ablation of Keap1 or expression of a tumor-derived Keap1 allele sensitized PDAC tumors to CAR T-cell therapy. Thus, cell-intrinsic molecular states accompanying malignant progression can sensitize tumor cells to cell-based immunotherapies. These molecular mechanisms could be exploited to augment both the efficacy of CAR-T cell therapy in solid malignancies, and may allow patient stratification by tumor genotype. Statement of significance CAR T-cell therapy remains an unsolved challenge for pancreatic cancer. The discovery of tumor-intrinsic mechanisms of resistance has been largely limited by current experimental models. Using large-scale genomic screening in an orthotopic, immunocompetent model of pancreatic cancer, we uncover a role for cell-intrinsic metabolic states in regulating CAR T-cell response.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by motor neuron deterioration. Genetic factors play a significant role in all cases, with 15 genome-wide significant study (GWAS) risk loci identified to date. Follow-up of these loci is a powerful strategy for research translation, as drug targets supported by genetic evidence are more likely to succeed in clinical development. Here, we focus on the RPSA-MOBP locus on chromosome 3 (lead SNP, rs631312, OR = 1.08 95% CI: 1.06–1.10, p = 3.3 × 10⁻¹²). We employ integrative in silico analyses to prioritise candidate genes, combining multiple ‘omics-based approaches, including Functional Mapping and Annotation (FUMA), Polygenic Priority Scoring (PoPS), Transcriptome-Wide Association across/within tissues (TWAS), gene-based test (mBAT-combo), chromatin interaction mapping (H-MAGMA), and Summary data Mendelian Randomisation (SMR), with GWAS data ( N cases = 29,612, N controls = 122,656). Both RPSA and MOBP were prioritised as candidate genes in multiple analyses. In-vivo expression analyses in ALS blood or iPSC-motor neurons were unremarkable for these genes but also other-relevant ALS genes. RPSA , highly conserved in zebrafish (92% homology), was selected for functional modelling, noting previously generated Mobp -ko mice show minimal phenotypic changes. CRISPR/Cas9-induced rpsa loss-of-function (LOF) in zebrafish triggers progressive and severe phenotypes mimicking pathology observed in SMN- and TDP43-deficient zebrafish, two key proteins/genes associated with diseases of the motor neurons. RPSA -deficient animals exhibit marked motor neuron axon pathology, progressive loss of motor function and rapid decline culminating with premature death at around 7 days- post-fertilisation. These phenotypes were notably similar to those observed in SMN and TDP-43 zebrafish models, together with prominent cardiovascular abnormalities. This study identifies RPSA as a critical gene for motor neuron health, with implications for ALS pathogenesis. The RPSA/MOBP locus is also associated with other neurodegenerative diseases including frontotemporal dementia/FTD, corticobasal degeneration/CBD and progressive supranuclear palsy/PSP, highlighting its potential as a therapeutic target for multiple conditions.

TRIB members (TRIB1, TRIB2 and TRIB3) represent atypical members of the serine/threonine kinase superfamily and are involved in multiple biological processes such as cell proliferation and differentiation. TRIB roles in GC are not fully investigated. We hypothesized that TRIB members play crucial roles in regulating GC activity and may activate separate signaling pathways. TRIB1 and TRIB3 are induced in GC of ovulatory follicles (OF) following hCG injection as compared to dominant follicles (DF) whereas TRIB2 is suppressed by hCG in OF. Protein analyses of cultured primary GC showed that luteinizing hormone (LH) treatment inhibited TRIB2 and induced TRIB3, while follicle-stimulating hormone (FSH) treatment increased TRIB2 and TRIB3 expression but at different times. These results demonstrate a different regulation of TRIB members during follicular development and in response to gonadotropins. TRIB3 inhibition via CRISPR/Cas9 showed a positive effect on AKT signaling pathway while showing negative effects on P38 MAPK signaling. Overall, TRIBs may play crucial roles in regulating GC function and activity and may activate separate signaling pathways, which impact follicular development, ovulation and luteinization.

Foodborne pathogens represent a class of pathogenic microorganisms capable of causing food poisoning or serving as foodborne vectors, constituting a major source of food safety concerns. With increasing demands for rapid diagnostics, conventional culture-based methods and PCR assays face limitations due to prolonged turnaround times and specialized facility requirements. While CRISPR-based detection has emerged as a promising rapid diagnostic platform, its inherent inability to detect low-abundance targets necessitates coupling with isothermal amplification, thereby increasing operational complexity. In this study, we developed a novel amplification-free Cascade-CRISPR detection system utilizing hairpin DNA amplifier. This method achieves detection sensitivity as low as 10 fM for DNA targets within 30 minutes without requiring pre-amplification, with background signal suppression achieved through optimized NaCl concentration. Validation using artificially contaminated food samples demonstrated the platform's robust performance for both Toxoplasma gondii(T. gondii) and Listeria monocytogenes(L. monocytogenes) detection, confirming broad applicability. In summary, this study establishes an amplification-free Cascade-CRISPR detection platform that achieves high sensitivity and rapid turnaround, demonstrating strong potential for on-site screening of foodborne pathogens.

Interactions between drugs and their targets impact efficacy and, when altered by mutation, can result in resistance 1-3 . Assessing and understanding the impacts of all possible mutations at a drug binding site remain challenging, however 4-6 . Here, we used Multiplex Oligo Targeting (MOT) for mutational profiling, and computational modelling, to decode efficacy and resistance space at the otherwise native binding site for a low nanomolar potency, anti-trypanosomal, proteasome inhibitor 7 . We saturation-edited twenty codons in the Trypanosoma brucei proteasome β5 subunit and subjected the resulting MOT libraries to stepwise drug selection. Amplicon sequencing, and codon variant scoring, yielding dose-response profiles for >100 resistance-conferring mutants, among 1,280 possible codon variants. Codon variant scores were predictive of relative resistance observed using a bespoke set of mutants, while fitness profiling revealed otherwise extensive constraints on mutational fitness and resistance space. The resistance profile that emerged allowed us to readily predict routes to spontaneous drug resistance observed within ‘accessible’, single nucleotide mutational space. In silico analysis of β5 subunit mutations predicted impacts on ligand affinity via steric effects, hydrogen-bonding and lipophilicity, which when combined with predictions of proteasome function perturbing mutations, were closely aligned with observed impacts on drug resistance. We conclude that MOT-library profiling facilitates assessment of all possible mutations at a drug binding site. Further decoding of drug target structure-activity relationships and drug resistance space will facilitate the design of more effective and durable drugs.

ABSTRACT Since their discovery, CRISPR–Cas systems have been widely applied in areas ranging from genome editing to biosensing, owing to their specific, RNA-guided target recognition. Their performance in complex biological environments has been extensively studied, particularly to optimize guide RNA design and minimize off-target cleavage. Here, we focus on the kinetic inhibition of the interaction between Cas12a - a Class 2, Type V effector - and its target, caused by interference from non-cognate background nucleic acids. This effect is particularly relevant for sensing applications in complex mixtures or cellular contexts, where genome- and transcriptome-derived sequences may impede CRISPR–Cas activity. Using in vitro assays under defined conditions, we systematically examine the influence of background single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) on reaction kinetics. We find that both the purine-to-pyrimidine ratio and the GC content of the guide RNA seed region significantly affect kinetic inhibition by background polynucleotides. Guide RNAs with low GC content and a high purine fraction in the seed region were least affected by background sequences. Experiments with dCas12a-based gene activation in living cells indicate that our in vitro findings may also be relevant for in vivo applications.

Understanding toxin resistance in insects is key to appreciate niche adaptations and for pest management, but remains challenging due to complex genetics. To investigate how Drosophila sechellia evolved resistance to toxic octanoic acid (OA) of noni fruit, its exclusive habitat, we experimentally-evolved the closely-related Drosophila simulans with increased OA resistance, identifying multiple loci under selection. Cross-referencing these with a genome-wide, OA-resistance CRISPR screen in Drosophila melanogaster cells highlighted two proteins: Kraken, a putative detoxification enzyme in digestive and renal tissues, and Alkbh7, a mitochondrial protein linked to fatty acid metabolism. Both genes show elevated expression in D. sechellia and OA-resistant D. simulans . In D. melanogaster , kraken mutants are more OA-sensitive, while Alkbh7 overexpression increased OA resistance. Importantly, mutations of these genes in D. sechellia reduced OA tolerance. Our identification of genes underlying OA resistance in lab and natural contexts demonstrates how complementary, cross-species selection approaches can elucidate toxin adaptation mechanisms.

Phage therapy has been proposed as an alternative to antibiotics to treat resistant infections. However, we have a limited understanding of how antibiotic resistance genes (ARGs) associate with bacterial phage defense systems (PDSs). Here, we explore the relationship between ARGs and PDSs in a sample of 2,559 plasmids originating from 1,044 E. coli isolates, representing a snapshot of clinical and non-clinical diversity in Oxfordshire, UK (2008-2020). In total, we identify 3,193 ARGs and 14,013 PDSs (180 unique types). We demonstrate that E. coli plasmids are enriched for ARGs and PDSs (both p<0.001), with a bias towards toxin-antitoxin/abortive-infection, TIR-domain and CBASS systems (all q<0.025). We proceed to show that ARGs and PDSs are physically linked by plasmids ( p <0.001). Together, our results suggest that phage therapy may inadvertently select for antibiotic resistant bacteria, and that antibiotic use may similarly drive resistance to phage.

Ubiquitin E3 ligases play crucial roles in the DNA damage response (DDR) by modulating the turnover, localization, activation, and interactions of DDR and DNA replication proteins. To gain further insight into how the ubiquitin system regulates the DDR, we performed a CRISPR-Cas9 knockout screen focused on E3 ligases and related proteins with the DNA topoisomerase I inhibitor, camptothecin. This uncovered the CTLH ubiquitin E3 ligase complex — and particularly one of its core subunits, MAEA — as a critical regulator of the cellular response to single-ended DNA double-strand breaks (seDSBs) and replication stress. In tandem, we identified patients with variants in MAEA who present with neurodevelopmental deficits including global developmental delay, dysmorphic facial features, brain abnormalities, intellectual disability, and abnormal movement. Analysis of patient-derived cell lines and mutation modeling reveal an underlying defect in HR-dependent DNA repair and replication fork restart as a likely cause of disease. We propose that MAEA dysfunction hinders DNA repair by reducing the efficiency of RAD51 loading at sites of DNA damage, which compromises genome integrity and cell division during development.

Honey bees visit food sources up to several kilometers away from their hives, which is underpinned by their sophisticated learning and memory, and cognitive abilities. However, the molecular and neural bases for these advanced brain functions remain obscure. Here, we focused on mKast , a gene preferentially expressed in the optic lobes, a visual center, and a specific Kenyon cell subtype in the mushroom bodies, a higher-order center, in the honey bee brain. We successfully produced homozygous mutant honey bee workers by crossing individuals mutated with CRISPR/Cas9 targeting mKast . Through behavioral analyses of mKast mutants using a new conditioning paradigm and a visual response assay, we found that mKast functions in bimodal learning and memory based on olfactory and visual information, and direction-specific motion sensing. We also found that mKast homozygous mutants have defects in survival outside the colony. These findings suggest that mKast modulate brain functions underlying homing ability that is essential for nidificating hymenopteran species.

A new frontier in functional genomics is to construct “Perturbation Atlas” that systematically profiles the effects of perturbation of all the mammalian genes across various cell types in health and disease. To this end, we have previously developed an in situ CRISPR screening platform in mice named iMAP, where Cas9 guides are “stored” in germline-transmitted transgenes but inducibly “released” (expressed) via Cre-mediated recombination. A major limitation of iMAP is a strong bias in guide representation following the recombination. We now report that this bias was effectively mitigated by eliminating a recombination hotspot at the transgene. We next profiled, across 46 tissues, the abundance of the guides targeting 71 (52%) genes encoding writers, readers and erasers of RNA modifications, revealing their context- specific functions. Furthermore, in a tumor model, we identified regulators of NK cell activation, which was validated in human NK cells, thus offering potential targets for improving cancer immunotherapy. Finally, a public database was established for accessing and analyzing iMAP data. Our study marks an important step toward the creation of the Perturbation Atlas.

Despite advances in disease treatment, our understanding of how damaged organs recover and the mechanisms governing this process remain poorly defined. Here, we mapped the transcriptional and regulatory landscape of human cardiac recovery using single cell multiomics. Macrophages emerged as the most reprogrammed cell type. Deep learning identified the transcription factor RUNX1 as a key regulator of this process. Macrophage-specific Runx1 deletion recapitulated the human cardiac recovery phenotype in a chronic heart failure model. Runx1 deletion reprogrammed macrophages to a reparative phenotype, reduced fibrosis, and promoted cardiomyocyte adaptation. RUNX1 chromatin profiling revealed a conserved regulon that diminished during recovery. Mechanistically, the epigenetic reader BRD4 controlled Runx1 expression in macrophages. Chromatin activity mapping, combined with CRISPR perturbations, identified the precise regulatory element governing Runx1 expression. Therapeutically, small molecule Runx1 inhibition was sufficient to promote cardiac recovery. Our findings uncover a druggable RUNX1 epigenetic mechanism that orchestrates recovery of heart function.

The Y1 mouse adrenocortical carcinoma cell line presents amplification of the KRas oncogene and high-basal levels of KRAS-GTP mediated by the GEF SOS. In this research, we developed a dynamic model based on ordinary differential equations of the KRAS-GTP activation mediated by SOS in Y1 cells, which showed that SOS only is not sufficient to reach the high-basal levels of KRAS-GTP experimentally observed for this cell line. Interestingly, a modification in this system, which added another GEF in the model, made the model reach the expected levels of KRAS activation, leading to the hypothesis that there was a missing element in this system. To find this missing element, a PCR panel of RasGEFs was performed and the GEF Rasgrp4 was found highly expressed in parental Y1 cell lines, indicating that this was the missing element in the system. Finally, tumor growth assays in Balb/c-NUDE mice with the Y1 cell versus RASGRP4 CRISPR depleted Y1 cells, showed reduced tumor growth and frequency for the RASGRP4 depleted cells.

The regenerative response of retinal cells to injury and aging depends on the epigenomic plasticity that enables the dedifferentiation and neuronal differentiation capacities of Müller glial cells (MG). In mammals, this regenerative ability is extremely limited, and disruptions in epigenetic mechanisms, particularly those involving DNA methylation and demethylation, may underlie this restricted potential. To explore this possibility, we aimed to develop DNA methylation-targeting molecular tools to enhance the dedifferentiation and neurogenic capacity of primary MG cultures derived from mouse retina. Using CRISPR/dCas9-based gene regulation technology, we selectively and transiently inhibited Dnmt3a , a de novo DNA methyltransferase previously implicated in maintaining transcriptional repression. Our results show that Dnmt3a knockdown leads to sustained upregulation of pluripotency-associated genes, including Ascl1 , Lin28 , and Nestin , as measured by RT-qPCR and immunofluorescence. This epigenetic modulation also promoted increased cell proliferation and migration, both hallmarks of a regenerative response. Furthermore, Dnmt3a knockdown, either alone or in combination with neurogenic stimuli, induced MG to acquire neuronal-like morphologies and express the early neuronal marker βIII-tubulin. These findings suggest that Dnmt3a acts as a repressive regulator of MG plasticity, likely serving as an epigenetic barrier that counteracts injury-induced demethylation events. Overall, our study identifies Dnmt3a as a critical modulator of MG fate and highlights the potential of its targeted downregulation to facilitate reprogramming. By prolonging the transient progenitor-like state of MG, DNMT3a inhibition may serve as a complementary approach to unlock the neurogenic and regenerative potential of the mammalian retina, offering promising avenues for future therapeutic strategies. Author Summary Retinal damage caused by injury, disease, or aging can lead to vision loss and ultimately blindness. Unlike mammals, the small freshwater zebrafish possesses a remarkable ability to regenerate its retina and restore vision after injury. Extensive research has focused on uncovering the molecular mechanisms behind this regenerative process in zebrafish, with the goal of understanding what is absent or suppressed in the mammalian retina. It is now well established that this regenerative capacity depends on a specific type of retinal cell: Müller glia. These cells can undergo dedifferentiation, a process in which they lose their specialized function, morphology, and gene expression profile. This is followed by neuronal differentiation, allowing them to replace lost neurons with newly generated ones. In recent years, numerous molecules and molecular pathways have been identified that may limit regenerative potential in mammals. In this study, we developed a molecular tool to specifically block one of these inhibitory factors, DNMT3a, a DNA methyltransferase involved in epigenetic repression. We demonstrate that in the absence of DNMT3a, mouse Müller glia can more efficiently dedifferentiate and subsequently adopt neuronal-like characteristics. These findings suggest that DNMT3a acts as a barrier to retinal regeneration and may represent a promising target for future therapeutic strategies aimed at promoting neural repair in the mammalian retina.

We know little about the fitness landscapes of bacterial operators, regulatory DNA elements that are crucial to regulate metabolic genes like those of the lac operon for lactose utilization. For example, we do not know whether adaptive evolution could easily create strong operators from weak ones or from non-regulatory DNA. To find out, we used CRISPR-Cas-assisted genome editing, bulk competition, and high-throughput sequencing to map the fitness landscape of more than 140,000 lac operator variants in two chemical environments that harbor lactose or glycerol as sole carbon sources. Both landscapes are highly rugged and contain thousands of fitness peaks, which allow only 2 percent of evolving populations to reach a high fitness peak. The landscapes share only 15 percent of fitness peaks. Our work illustrates that landscape ruggedness caused by epistasis can represent an important obstacle to adaptive evolution of regulatory sequences. It also shows that a simple environmental change can substantially affect fitness landscape topography.

Recent advances in prime editing technologies using CRISPR modules fused with reverse transcriptase (RT) have enabled efficient and precise reprogramming of target genomic sequences. Twin prime editing using two coordinated prime editor complexes is a promising strategy for inducing extensive genomic modifications via reverse transcribed complementary templates. However, current twin prime editing systems still require improvements in editing efficiency, accuracy, and intended edit predictability. Here, efficiency and precision of twin prime editing were enhanced via engineering and optimizing conventional SpCas9(H840A)-RT-based prime editor components. A La domain–fused prime editor (La-SpCas9(H840A)-RT) and optimized pegRNAs were developed, achieving a 1.75 ± 0.21-fold increase in gene editing efficiency at multiple genomic loci in human-derived cell lines without increasing off-target activity. La-SpCas9(H840A)-RT facilitated efficient ∼2.8 kb GFP transgene knock-in at target loci and eliminated the expanded polyQ tract in ATXN3 in patient-derived mutant cell lines modeling spinocerebellar ataxia type 3. The advanced twin prime editing platform expands genome engineering capabilities beyond existing CRISPR-based systems and holds great promise for diverse biotechnological and therapeutic applications.

Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling modulation of protein-protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargos to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent anti-leukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications. One-Sentence Summary The establishment of modular platform for synthesizing bio-orthogonal macromolecular conjugates (BMC) enabled fast and targeted delivery of membrane-impermeant macromolecular drugs.

Plant cysteine oxidases (PCOs) are O 2 -sensing enzymes that play an important role in plant responses to low oxygen (hypoxia). PCO-catalysed dioxygenation of the N-terminal Cys of substrates, including Group VII Ethylene Response Factors (ERVIIs), targets them for degradation via the Cys/Arg N-degron pathway, however these substrates are stabilized in hypoxia due to reduced PCO activity. When plants are flooded, submergence-induced hypoxia results in ERFVII-mediated upregulation of hypoxia responsive genes that reconfigure plant metabolism and allow short-term resilience to the conditions. However, the increasing frequency and duration of flood events requires strategies to improve plant flood resilience, particularly amongst agronomic crops. One possibility is to prolong the stability of ERFVIIs by engineering the PCOs to catalyse their oxidation less efficiently. We report a structure-guided kinetic and biophysical investigation of Arabidopsis thaliana PCO4 that reveals residues important for substrate-binding and catalysis. We subsequently selected At PCO4 variants Y183F and C173A, with severe and mild impacts on At PCO4 activity, respectively, to complement Arabidopsis pco1pco2pco4pco5 plants and investigate their impact on submergence resilience. Both variants appeared to be beneficial for survival and recovery after 2.5 and 3.5 days of dark submergence when compared to control plants, indicating that engineering PCOs can be used as a strategy to improve flood tolerance in plants.

Motivation Approximate string matching (ASM) is the problem of finding all occurrences of a pattern P in a text T while allowing up to k errors. ASM was researched extensively around the 1990s, but with the rise of large-scale datasets, focus shifted towards inexact approaches based on seed-chain-extend . These methods often provide large speedups in practice, but do not guarantee finding all matches with ≤ k errors. However, many applications, such as CRISPR off-target detection, require exhaustive results with no false negatives. Methods We introduce Sassy, a library and tool for ASM of short (up to ≈1000 bp) patterns in large texts. Sassy builds on earlier tools that use Myers’ bitpacking, such as Edlib. Algorithmically, the two main novelties are to split each sequence into 4 parts that are searched in parallel, and to use bitvectors in the text direction ( horizontally ) rather than the pattern direction ( vertically ). This allows significant speedups for short queries, especially when k is small, as has complexity O ( k⌈n/W⌉ ) when searching random text, where W = 256 is the SIMD width. Practically speaking, Sassy is the only recent index-free tool that is designed specifically for ASM, rather than the more common semi-global alignment. In addition, Sassy supports the IUPAC alphabet, which is essential for primer design and for matching ambiguous bases in assemblies. Separately, we also introduce the concept of overhang cost a variant of ‘overlap’ alignment where e.g. a suffix of the pattern is matched against a prefix of the text, where each character of the pattern that extends beyond the text incurs a cost of e.g. α = 0.5. This is important when matches are near contig or read ends. Results Compared to Edlib, Sassy is 4 × to 15 × faster for sequences up to length 1000, and has throughputs exceeding 2 GB/s, whereas Edlib remains below 130 MB/s. Likewise, Sassy is up to 10 × faster than parasail when searching short strings. Sassy is also readily applicable to biological problems such as CRISPR off-target detection. When searching 61 guide sequences in a human genome, Sassy is 100 × faster than SWOffinder and only slightly slower (for k ≤ 3) than CHOPOFF, for which building its index takes 20 minutes. Sassy also scales well to larger k ∈ {4, 5}, unlike CHOPOFF whose index took over 10 hours to build. Availibility Sassy is available as Rust library and binary at https://github.com/RagnarGrootKoerkamp/sassy .

Abstract Tomato high pigment-2 (hp-2dg, hp-2, and hp-2j) mutant lines are characterized by mutations in the DE-ETIOLATED1(SlDET1; Solyc01g056340) gene. SlDET1 is responsible for encoding a nuclear protein that acts as a negative regulator involved in light signaling, repressing photomorphogenesis. These tomato mutant lines are known for increased levels of antioxidant pigments in fruits, such as flavonoids and carotenoids, compared to the wild-type fruits. In this study, CRISPR/Cas9 followed by the non-homologous end joining mechanism of repair (NHEJ), was used to target the SlDET1gene and investigate whether the effects of these mutations could mimic the effects of hp-2 mutant lines, improving the nutritional features of tomato fruits. Our results indicated that mutations generated by CRISPR/Cas9 NHEJ in the hp-2and hp-2j regions (exon 11) resulted in significant changes in the SlDET1 coding and protein sequences, causing a low survival rate of edited sprouts and regenerated plants with very compromised capacity of allelic heritability of these mutations for the following generations. However, regenerated plants containing these site-specific mutations in the SlDET1 gene showed higher levels of phytochemicals in ripe fruits. Furthermore, these edited plants also showed an upregulation of structural genes involved in the synthesis of these biocompounds. Although the SlDET1 gene could be considered an interesting target gene for the nutritional improvement of tomato fruit, our results showed that mutations within its exon 11 are quite critical and can induce severe perturbations in plant metabolism, physiology, and survival, with a compromised possibility to develop new stable edited lines.

Abstract Corneal neovascularization is a sight-threatening condition for which current treatments such as anti-VEGF agents are limited by invasiveness and side effects. We present the first non-viral, CRISPR/Cas9-based gene therapy delivered via topical eye drops that penetrates the cornea and inhibits pathological neovascularization. Cas9 ribonucleoproteins (RNPs) targeting the Vegfa gene were complexed with a liposomal carrier (lipofectamine) and administered to mice after alkali burn injury to the cornea. This approach achieved approximately 2% gene editing at the Vegfa locus in vivo, which significantly reduced local VEGF-A expression. Consequently, treated corneas showed markedly decreased macrophage infiltration and robust suppression of both hemangiogenesis and lymphangiogenesis compared to untreated controls. These findings demonstrate that even modest in vivo gene editing can yield a strong therapeutic effect, highlighting a clinically relevant strategy for controlling corneal angiogenesis. Our study introduces a feasible and safe topical CRISPR therapy for corneal diseases, offering a potential alternative to invasive or virus-based gene delivery methods.