ABSTRACT Non-viral gene editing offers a practical alternative to viral delivery for durable biologics production. Clinical trials have shown that adeno-associated virus encoding broadly neutralizing antibodies can protect against HIV, but result in limited, short-lived responses. The development of non-viral gene editing approaches in hematopoietic stem and progenitor cells holds promise for long-term antibody production. In this study, we evaluated CRISPR/Cas9 and CRISPR/Cas12a for gene knock-in at the immunoglobulin heavy chain locus in non-human primate hematopoietic stem and progenitor cells. Delivering the nuclease as a protein alongside a custom DNA template, we optimized editing with Cas12a and demonstrated higher knock-in efficiency and fewer non-specific edits than Cas9. Transplantation of edited non-human primate hematopoietic stem and progenitor cells into MISTRG mice led to engraftment, B cell differentiation, and transgene expression of a reporter transgene or anti-HIV antibody after HIV immunization with detectable anti-HIV antibody titers in peripheral blood circulation. These findings demonstrate the feasibility of using non-viral gene editing in HSPC as a potential strategy for sustained biologics production in the treatment of chronic diseases such as HIV. Future work will assess the efficacy of this model in a non-human primate model of HIV infection.

CRISPR–Cas9 gene editing holds transformative promise for genetic therapies, but is hindered by off-target effects that undermine its precision and safety. To address this, we developed CRISMER, a hybrid deep-learning architecture that uses multi-branch convolutional neural networks to extract k-mer features and transformer blocks to capture long-range dependencies. This hybrid approach enhances the prediction and optimization of single-guide RNA (sgRNA) designs. CRISMER was trained on Change-seq and Site-seq datasets, using a 20 × 16 sparse one-hot encoding scheme, and evaluated on independent datasets including Circle-seq, Guide-seq, Surro-seq, and TTISS. CRISMER outperformed existing tools, achieving an F1 score of 0.7092 and a PR-AUC of 0.8006 on the CRISPR-DIPOff dataset. It also excelled in measuring sgRNA specificity and optimizing designs for genes, such as PCSK9 and BCL11A, yielding sgRNAs with reduced off-target activity. For example, a G-to-C mutation at position 12 in the sgRNA for PCSK9 and at position 11 for BCL11A led to significant improvements in off-target profiles. Interpretability analysis via integrated gradients confirmed the model’s focus on critical PAM-proximal regions and mismatch patterns. These results demonstrate that CRISMER significantly improves the accuracy and safety of CRISPR-Cas9, advancing its reliability for therapeutic applications.

Abstract ​Spatially resolved in vivo CRISPR screening integrates gene editing with spatial transcriptomics to examine how genetic perturbations alter gene expression within native tissue environments. However, current methods are limited to small perturbation panels and the detection of a narrow subset of protein-coding RNAs. We present Perturb-DBiT, a distinct and versatile approach for the simultaneous co-sequencing of spatial total RNA whole-transcriptome and single-guide RNAs (sgRNAs), base-by-base, on the same tissue section. This method enables unbiased discovery of how genetic perturbations influence RNA regulation, cellular dynamics, and tissue architecture in situ. Applying Perturb-DBiT to a human cancer metastatic colonization model, we mapped large panels of sgRNAs across tumor colonies in consecutive tissue sections alongside their corresponding total RNA transcriptomes. This revealed novel insights into how perturbations affect long non-coding RNA (lncRNA) co-variation, microRNA–mRNA interactions, and global and distinct tRNA alterations in amino acid metabolism linked to tumor migration and growth. By integrating transcriptional pseudotime trajectories, we further uncovered the impact of perturbations on clonal dynamics and cooperation. In an immune-competent syngeneic mouse model, Perturb-DBiT enabled investigation of genetic perturbations within the tumor immune microenvironment, revealing distinct and synergistic effects on immune infiltration and suppression. Perturb-DBiT provides a spatially resolved comprehensive view of how genetic knockouts influence diverse molecular and cellular responses including small and large RNA regulation, tumor proliferation, migration, metastasis, and immune interactions, offering a panoramic perspective on perturbation responses in complex tissues.

The evolution of genome engineering technologies has transformed biomedical research, enabling precise and efficient modification of genetic material Doudna and Charpentier, 2014. Among these, CRISPR-Cas9 stands out as a revolutionary gene-editing tool, though it often requires extensive expertise and technical knowledge Cong et al., 2013; J. G. Doench et al., 2016. We propose GeneFix-AI, an Artificial Intelligence (AI)-driven platform for real-time prediction and correction of genetic mutations in non-human species. Developed using cutting-edge models inspired by recent advances at Harvard and Peking University Chen et al., 2021; Wu et al., 2020, GeneFix-AI integrates machine learning to predict mutations, design optimal guide RNAs, and evaluate editing outcomes. This system aims to automate the CRISPR-Cas9 workflow, making high-precision gene editing more accessible to researchers without extensive molecular biology backgrounds Liu et al., 2019. We present the system architecture, training methodology, and potential impact of GeneFix-AI in democratizing genome editing and accelerating discoveries in genetics.

Abstract Background: CRISPR-Cas9 technology is a powerful tool for precise genome editing and is increasingly applied to correct genetic mutations associated with various diseases, including cancer. This system utilizes a single-guide RNA (sgRNA), typically 20 base pairs long and complementary to the target DNA sequence, to direct the Cas9 nuclease for targeted gene activation (knock-in) or repression (knockout). In recent advancements in cancer immunotherapy, CRISPR-Cas9 has been extensively used to enhance the efficacy of Chimeric Antigen Receptor (CAR) T-cell therapy. The development of universal CAR T cells involves the knockout of key genes such as TRAC (T-cell receptor alpha chain), B2M (Beta-2 microglobulin), and PDCD1 (Programmed cell death protein 1), which improves T-cell persistence, immune evasion, and anti-tumor function. Method: In this study, sgRNAs targeting PDCD1, B2M, and TRAC were designed using nine widely recognized AI-driven bioinformatics tools: CHOPCHOP, CRISPOR, GenScript, Benchling, Cas-Designer, E-CRISP, CRISPR-ERA, CRISPRscan, and ATUM gRNA Tool. These platforms use various algorithms and genomic datasets to predict sgRNA candidates with high on-target activity and minimal off-target effects. The selected sgRNAs were assessed based on criteria including GC content, self-complementarity, and exon targeting. Results: The sgRNA design tools consistently identified high-confidence target sites within exon 1 of the PDCD1, TRAC, and B2M genes. For PDCD1 (PD-1), the sgRNA sequence (5′-CACGAAGCTCTCCGATGTGT-3′) was selected as the most optimal candidate, showing strong consensus across all platforms. Similarly, for TRAC, the sgRNA (5′-TCTCTCAGCTGGTACACGGC-3′) targeting exon 1 was chosen based on its high predicted efficiency and specificity. In the case of B2M, the sgRNA (5′-GAGTAGCGCGAGCACAGCTA-3′) was identified as an ideal target site within exon 1, a region critical for MHC class I expression and immune evasion. These sgRNAs demonstrated favorable characteristics including appropriate GC content, minimal self-complementarity, and low predicted off-target activity. To ensure their functional reliability, all selected sgRNAs were validated through an extensive review of scientific literature and previously published patent data, confirming their utility in gene knockout studies related to CAR T-cell enhancement. Conclusion: Among the tools evaluated, CHOPCHOP, Benchling, and CRISPOR emerged as the most comprehensive, offering robust information on GC content, self-complementarity, exon identification, and detailed off-target predictions. Additionally, this study compiled a list of relevant clinical trials involving gene knockouts of PDCD1, TRAC, and B2M to further support the therapeutic relevance of these targets in CAR T-cell development.

Streptococcus mutans is a major cause of dental caries worldwide. Targeted therapeutic strategies to eradicate S. mutans include oral phage rinses. In this study, we investigated how phage resistance develops in S. mutans . As a model phage, we used ɸAPCM01, which is known to infect a serotype e strain. We isolated and sequenced the genomes of 15 spontaneous resistant mutants and found that 10 had acquired novel CRISPR spacers targeting the phage, with a total of 18 new spacers identified. Additionally, eight strains contained mutations in rhamnose-glucose polysaccharide (RGP) biosynthetic genes, three of which also acquired spacers. Only the rgp mutants exhibited defects in phage absorption, supporting the role of these cell surface glycans as the phage receptor. Mutations in rgpF and the newly identified gene rgpX led to severe cell division defects and impaired biofilm formation, the latter of which shared by the rgpD mutant. Thus, rgp mutations confer phage resistance but impose severe fitness costs, limiting pathogenic potential. Surprisingly, we found that ɸAPCM01 was capable of binding to and injecting its genome into UA159, a model serotype c strain. However, UA159 was resistant to infection due to an unknown post-entry defense mechanism. Consequently, ɸAPCM01 has the potential to infect both major serotypes associated with dental caries. Repositories The genome sequence of Streptococcus mutans DPC6143 was deposited at NCBI with the accession number NZ_CP172847.1.

ABSTRACT Neuromodulators such as the monoamines are known to differ from classical neurotransmitters like glutamate in the time scale of signaling due to activation of slower G protein-coupled receptors. Recent work has suggested that the mode of release also differs between classical and modulatory transmitters. Although many components of neurotransmitter release machinery have been identified, we still understand little about the mechanisms responsible for differences in release. In this study, we address the differences between release of dopamine and glutamate by comparing the composition of synaptic vesicles (SVs) that contain the vesicular monoamine transporter 2 (VMAT2) versus vesicular glutamate transporter 2 (VGLUT2). Previous work has shown that these SV populations differ in frequency dependence, recycling kinetics and biogenesis. Taking advantage of a CRISPR-generated knock-in mouse with a cytoplasmic hemagglutinin (HA) tag at the N-terminus of VMAT2 to immunoisolate monoamine SVs, we find differences in the abundance and isoform expression of many SV protein families. Validation in primary neurons and in brain tissue confirms these differences in SV protein abundance between dopamine and glutamate release sites. Functional analysis reveals that the loss of differentially expressed SCAMP5 selectively impairs the recycling of VGLUT2 SVs, sparing VMAT2 vesicles in the same neuronal population. These findings provide new insights into the molecular diversity of SVs and the mechanisms that regulate the release of dopamine and glutamate, with implications for the physiological role of these transmitters and behavior.

ABSTRACT Receptor-interacting protein kinase 2 (RIPK2) has emerged as a promising drug target in various cancers, including prostate cancer (PC). However, the absence of reliable biomarkers to assess RIPK2 activity limits both patient selection for anti-RIPK2 therapies and treatment monitoring. To address this gap, we performed RNA-Seq analysis on PC cell lines (22Rv1, DU145, and PC3) with CRISPR/Cas9-mediated RIPK2 knockout ( RIPK2 -KO) using two independent guide RNAs. This analysis identified 13 candidate RIPK2-regulated genes, of which eight were validated by reverse transcription quantitative PCR (RT-qPCR). Furthermore, treatment with two distinct RIPK2 inhibitors significantly reduced RIPK2 signature scores in five independent PC cell lines in a dose- and/or time-dependent manner. Clinical association analyses revealed that high RIPK2 signature scores correlate with metastasis and worse biochemical recurrence-free, progression-free, disease-free, and overall survival, outperforming RIPK2 mRNA levels as a prognostic biomarker. This study establishes, for the first time, a RIPK2-regulated gene signature as a potential biomarker for RIPK2 activity and PC prognosis, warranting further validation in clinical specimens to provide a much-needed tool for patient stratification and response monitoring in RIPK2-targeted therapies.

Abstract CRISPR/Cas9-mediated genome editing is a powerful tool for producing animal models of human diseases. However, it often encounters challenges related to low efficiency of donor DNA templates insertion through homology-directed repair (HDR) pathway or unwanted insertions and/or multiplications. Here, we present findings from multiple targeting experiments aimed at generating a Nup93 conditional knockout (cKO) mouse model. Injection of CRISPR/Cas9 components into over two thousand zygotes, resulted in 270 founder animals. Our study revealed various obstacles associated with the use of single-stranded (ssDNA) and double-stranded DNA (dsDNA) templates during cKO generation, highlighting the critical role of denaturation of long 5’-monophosphorylated dsDNA templates in enhancing precise genome editing and reducing template multiplications. Application of RAD52 protein increased HDR efficiency of ssDNA integration almost 4-fold, albeit with an associated increase in template multiplication. Targeting the antisense strand of DNA using two crRNAs demonstrated better efficacy in HDR-mediated precise genome editing when compared to targeting the sense or sense-antisense strands. In addition, the application of 5’-end biotin-modified donor DNA resulted in up to a 8-fold increase in HDR-mediated single-copy template integration compered to unmodified dsDNA donor. Furthermore, application of 5’-end C3 spacer modified template resulted in up to a 20-fold increase in correctly HDR modified mice independent from ssDNA or dsDNA template employment. This study underscores potential pitfalls in CRISPR/Cas9-mediated genome editing and offers simple practical solutions to refine this potent tool. These findings highlight various strategies to enhance CRISPR/Cas9 HDR efficiency, providing a framework for improving precision in the generation of conditional knockout models.

CRISPR and their associated Cas proteins provide adaptive immunity in prokaryotes, protecting against invading genetic elements. These systems are categorized into types and are highly diverse. Genomes often harbor multiple CRISPR arrays varying in length and distance from Cas loci. However, the ecological roles of multiple CRISPR arrays and their interactions with multiple Cas loci remain poorly understood. We present a comprehensive analysis of CRISPR systems that uncovers variation between diverse Cas types regarding the occurrence of multiple arrays, the distribution of their lengths and positions relative to Cas loci, and the diversity of their repeat sequences. Some types tend to occurr as the sole Cas present, but typically comprise two or more arrays, especially for types I-E and I-F. Multiple Cas types are also common, with some systems showing a preference for specific co-occurrence. Distinct array distributions and orientations around Cas loci indicate substantial differences in functionality and transcriptional behavior among Cas types. Our analysis suggests that arrays with identical repeats in the same genome acquire new spacers at comparable rates, irrespective of their proximity to the Cas locus. Furthermore, repeat similarities in our data set indicate that arrays of systems that often co-occur with other systems tend to have more diverse repeats than those mostly appearing alongside solitary systems within the genome. Our analysis suggests that co-occurring Cas type pairs might not only collaborate in spacer acquisition but also maintain independent and complementary functions and that CRISPR systems distribute their defensive spacer repertoire equally across multiple CRISPR arrays.

Proteins of the cytohesin family are known for their guanine-nucleotide exchange factor function for ARF-GTPases, mainly for ARF1 and ARF6. While Arf1 and Arf6 deficiency results in embryonic lethality, in vivo functions of cytohesins are rarely described and mostly inconspicuous. We analyzed the role of cytohesin-2 in vivo and in vitro and found that cytohesin-2 full knockout mice die within one day after birth. Mass spectrometry-based organellar proteomics in wildtype and CRISPR-Cas9-generated cytohesin-2 -/- C2 myoblasts revealed a markedly altered Golgi compartment. Golgi volumes were reduced in different cytohesin-2 -/- cell lines compared to wildtype cells as revealed by immunofluorescence. Reduced Golgi volumes were rescued by introducing cytohesin-2. Finally, we observed that typical functions of the Golgi apparatus were disrupted in cytohesin-2-deficient cells. Cytohesin2 -/- C2 myoblasts exhibited significant changes in the galactose / N-acetyl-galactosamine glycosylation on the cell surface compared to wildtype cells when stained with peanut agglutinin. Further, protein secretion was overall reduced in neonatal cytohesin-2 -/- mice compared to wildtype as determined by mass spectrometry-based proteomics. This study describes the essential role of cytohesin-2 in neonatal development and a novel function of the protein in Golgi regulation.

RNA-binding proteins (RBPs) are important regulators of post-transcriptional gene expression. Understanding which and how RBPs promote cancer progression is crucial for cancers that lack effective targeted therapies such as triple negative breast cancer (TNBC). Here, we employ both in vitro and in vivo pooled CRISPR/Cas9 screening to identify 50 RBP candidates that are essential for TNBC cell survival. Integrated eCLIP and RNA-sequencing analysis identify that poly(U)-binding splicing factor 60 (PUF60) drives exon inclusion within proliferation-associated transcripts that, when mis-spliced, induce cell cycle arrest and DNA damage. Furthermore, disrupting PUF60 interactions with 3’ splice sites via a substitution in its RNA-binding domain causes widespread exon skipping, leading to downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. We demonstrate that loss of PUF60-RNA interactions inhibits TNBC cell proliferation and shrinks tumor xenografts, revealing the molecular mechanism by which PUF60 supports cancer progression. Significance Our work demonstrates functional in vivo screening of RBPs as an effective strategy for identifying unexpected cancer regulators. Here, we reveal a crucial role for PUF60-mediated splicing activity in supporting oncogenic proliferation rates and highlight its potential as a therapeutic target in triple negative breast cancer.

Genome wide association studies have identified multiple loci that mediate the risk of developing late-onset Alzheimer’s Disease (LOAD). The gene WW-domain containing oxidoreductase ( WWOX ) has been identified in recent LOAD risk meta-analyses, yet its function in the brain is poorly understood. Using Drosophila, we discovered that knockdown of the highly conserved Wwox gene impacts longevity and sleep, having roles in both neuronal and glial subtypes. In an amyloid beta 42 (Aβ 42 ) transgenic model of AD, RNAi-mediated knockdown of Wwox significantly decreased both lifespan and locomotion whilst elevating soluble Aβ 42 . Transcriptomic and metabolomic analyses revealed that these effects were accompanied by elevated lactate dehydrogenase ( Ldh ) mRNA and lactate levels, downstream of an increase in the key unfolded protein response protein Atf4. Strikingly, we found that upregulation of Wwox in the Aβ 42 model through CRISPR activation significantly reduced amyloid load, improved longevity and locomotion. Multi-omics analysis revealed Wwox upregulation partially reversed several key Aβ 42 -induced transcriptional pathways in the brain and reduced levels of L-methionine and associated enzymes. These findings support a role for reduced WWOX levels in the genetic risk of developing LOAD via pyruvate metabolism and point towards WWOX activation as a protective therapeutic strategy.

ABSTRACT Human induced pluripotent stem cells (iPSCs) offer immense potential as a source for cell therapy in spinal cord injury (SCI) and other diseases. The development of hypoimmunogenic, universal cells that could be transplanted to any recipient without requiring a matching donor, could significantly enhance their therapeutic potential and accelerate clinical translation. To create off-the-shelf hypoimmunogenic cells, we used CRISPR-Cas9 to delete B2M (HLA class I) and CIITA (master regulator of HLA class II). Double-knockout (DKO) iPSC-derived neural progenitor cells (NPCs) evaded T cell-mediated immune rejection in vitro and after grafting into the injured spinal cord of athymic rats and humanized mice. However, loss of HLA class I heightened susceptibility to host natural killer (NK) cell attack, limiting graft survival. To counter this negative effect, we engineered DKO NPCs to overexpress macrophage migration inhibitory factor (MIF), an NK cell checkpoint ligand. MIF expression markedly reduced NK cell-mediated cytotoxicity and improved long-term engraftment and integration of NPCs in the animal models for spinal cord injury. These findings demonstrate that MIF overexpression, combined with concurrent B2M and CIITA deletion, generates hiPSC neural derivatives that escape both T- and NK-cell surveillance. This strategy provides a scalable route to universal donor cells for regenerative therapies in SCI and potentially other disorders.

Bacillus methanolicus represents a thermophilic methylotroph whose methanol utilization depends on plasmid-encoded genes. It serves as a unique model for deciphering plasmid-dependent methylotrophy and an ideal chassis for low-carbon biomanufacturing using CO2-derived C1 substrates. Despite its evolutionary uniqueness and industrial potential, the lack of synthetic biology tools has hindered both mechanistic understanding and strain engineering. Here, we present a comprehensive synthetic biology platform comprising a high-efficiency electroporation protocol, a CRISPR method enabling robust and multiplex genome editing, diverse neutral loci for gene integration and overexpression, and a cloud-based genome-scale metabolic model iBM822 for user-friendly biodesign. Leveraging this toolkit, we systematically dissected plasmid-dependent methylotrophy, host restriction-modification systems, and functional significance of the chromosomal methylotrophic genes through targeted deletion. To address plasmid loss-induced strain degeneration, we integrated the large endogenous plasmid pBM19 into the chromosome for stable and intact methylotrophic growth. Finally, by integrating metabolic modeling with CRISPR editing, we engineered L-arginine feedback regulation to achieve the first L-arginine biosynthesis from methanol. This study establishes a synthetic biology framework for B. methanolicus, promoting mechanistic exploration of methylotrophy and low-carbon biomanufacturing.

Patients who are recipients of allogeneic transplants or have underlying autoimmune disease require immune suppression, often with calcineurin inhibitors (CNI). There is an expanding repertoire of immune effector cell (IEC) therapies, including CD19 CAR-T cells and viral-specific T cells (VSTs), deployed in these patients; however, ongoing CNI therapy may be detrimental to IEC function. We thus developed a CRISPR/Cas9-based approach to engineer dual CNI [cyclosporine (CsA) and voclosporin (VCS)] resistant IEC therapies by targeting PPIA (encoding cyclophilin A - CypA), a critical binding partner for both drugs. Because CypA has several homeostatic functions in T cells, a complete CypA knock-out could impair cell viability. To avoid this, we edited the last exon of the PPIA gene, corresponding to the C-terminus of CypA, selectively disrupting amino acids that mediate CsA/VCS-based inhibition, while leaving the majority of CypA intact. Unlike an edit in an upstream exon, which was detrimental to cell survival and rapidly selected out, C-terminal editing was stable throughout expansion and preserved CypA protein expression. This edit was then introduced into two types of IECs. Edited CD19 CAR-T cells retained in vitro effector function in the presence of CsA/VCS, including preserved proliferation, target cell killing, and cytokine production. Edited CMV-specific T cells demonstrated antigen-specific proliferation and cytokine production in the presence of CsA/VCS. This report of site-specific CypA modification offers a promising avenue for developing next-generation IECs that should function effectively in patients receiving CsA/VCS and thus expand applications for adoptive cell therapies in multiple clinical settings. Key Points CRISPR editing of the last exon of PPIA retains CypA expression but with an altered C-terminus that disrupts CsA and VCS interactions PPIA Δ C immune effector cells demonstrate retained proliferation and function in the presence of CsA and VCS

Trichoderma atroviride is a well-known mycoparasitic fungus widely used for the biological control of fungal plant pathogens. Expanding its genetic toolbox is essential to facilitate efficient genetic manipulation, including successive transformations and multiple or reusable selection markers for consecutive gene deletions. We applied CRISPR/Cas9 via ribonucleoprotein (RNP) complexes for gene editing in T. atroviride and successfully deleted three target genes, i.e., pks4 (involved in spore pigment production), pyr4 (pyrimidine biosynthesis), and pex5 (receptor for peroxisomal matrix protein import). Although double-strand breaks induced by Cas9 can be repaired via homology-directed repair (HDR), using donor templates, the most effective gene deletions in our case were achieved via non-homologous end joining (NHEJ), by co-transforming a transiently stable telomere vector carrying the hygromycin-resistance gene ( hph ), which was rapidly lost under non-selective conditions. This strategy promoted NHEJ repair and resulted in the efficient deletion of open reading frames between two Cas9 target sites. Our results demonstrate that combining CRISPR/Cas9 RNP delivery with transient telomere vectors provides a fast and reliable method for marker-free gene deletion and vector recycling in T. atroviride , advancing the reverse genetic toolkit available for this important biocontrol fungus.

Abstract Background: Thermus thermophilus HB27 is a promising thermophilic chassis for recombinant thermostable protein production, owing to its high optimal growth temperature, which can simplify downstream processing and reduce contamination risks. However, maximizing its potential requires optimized genetic tools and host strains. Key limitations include a shortage of well-characterized strong constitutive promoters and potential degradation of recombinant proteins by proteases. To address these, we established a β-galactosidase reporter system (endogenous TTP0042) to screen for strong constitutive promoters and investigated the impact of deleting specific protease genes on protein expression. Results: Screening of 13 endogenous promoter regions identified P0984 as exhibiting significantly 13-fold higher activity than the control promoter driving the reporter gene. Constructing a plasmid-free strain (HB27ΔpTT27) successfully minimized 270 kb of the genome; it exhibited auxotrophy for cobalamin (requiring 0.1 μg/ml AdoCbl for growth) and a slightly reduced growth rate compared to the wild-type, while its transformation efficiency remained comparable. Notably, a CRISPR-deficient precursor strain (HB27ΔIII-ABΔI-CΔ CRF3 ) showed a significant (~100-fold) increase in transformation efficiency compared to the wild-type, facilitating subsequent genetic manipulations. Systematic knockout of 16 predicted non-essential protease loci was performed. Characterization revealed that deletion of TTC0264 (putative ClpY/HslU) and TTC1905 (putative HhoB) significantly reduced extracellular proteolytic activity. Iterative deletion based on phenotypic analysis led to strain DSP9 (10 protease loci deletions), which maintained robust growth and exhibited enhanced accumulation of the β-galactosidase reporter protein compared to the parental strains. Conclusions: This study provides foundational advancements for T. thermophilus HB27 chassis development, and genetic tools represent valuable resources for optimizing T. thermophilus as a platform for heterologous thermostable protein production and ideas for antibiotic-free systems.

Cdc42 is a Rho-family GTPase conserved across eukaryotes, where it plays essential roles in cell polarization. In single-celled yeast systems, Cdc42 is a key driver of symmetry breaking and polarized growth, forming zones of activity that locally recruit eRectors to organize the cytoskeleton and polarize secretion. Here we show that Cdc42 also functions in cell-cell fusion during Schizosaccharomyces pombe sexual reproduction but concentrates at the fusion site through mechanisms distinct from those proposed in Saccharomyces cerevisiae . Notably, the cdc42-mCherry SW allele (but not the cdc42-sfGFP SW allele), which is functional for cell polarization and has been used across organisms for dynamic studies, exhibits a strong fusion defect. These cells block fusion before cell wall digestion but after actin fusion focus formation, indicating that Cdc42 is required to translate the vesicle cluster into polarized cargo delivery. We trace the defect to instability of Cdc42-mCherry SW and demonstrate that cell fusion requires higher Cdc42 protein levels than mitotic polarized growth. Remarkably, by constructing an allelic series driving Cdc42 expression over a 5-fold range, we discover that polarized growth responds linearly to Cdc42 protein levels, whereas sexual reproduction exhibits a sharp switch-like response. Thus, the topology of the Cdc42 regulatory network is distinct for its polarization and mating functions.

The subcellular positioning of organelles is critical to their function and is dynamically adapted to changes in cell morphology. Yet, how cells sense shifts in their dimensions and redistribute organelles accordingly remains unclear. Here we reveal that cell-size-scaling of mitochondria distribution and function is directed by polarised trafficking of mRNAs. We identify a 29bp 3’UTR motif in mRNA encoding TRAK2, a key determinant of mitochondria retrograde transport, that promotes cell-size-dependent targeting of TRAK2 mRNA to distal sites of cell protrusions. Cell-size-scaled mRNA polarisation in turn scales mitochondria distribution by defining the precise site of TRAK2-MIRO1 retrograde transport complex assembly. Consequently, 3’UTR motif excision perturbs size-regulated transport and eradicates scaling of mitochondria positioning, triggering distal accumulation of mitochondria and progressive hypermotility as cells increase size. Together, our results reveal an RNA-driven mechanistic basis for the cell-size-scaling of organelle distribution and function that is critical to homeostatic control of motile cell behaviour.

Photosynthesis and respiration are fundamental metabolic processes in plants, tightly connected through shared substrates, energy dynamics, and redox balance. Arabidopsis is the key genetic model for plants but monitoring these sorts of physiological processes presents significant challenges using traditional gas-exchange or fluorescence-based techniques due to the small size of intact Arabidopsis thaliana (arabidopsis) seedlings. Here, we validate and characterize the use of Clark-type oxygen electrodes, specifically the Hansatech Oxytherm+P system, to quantify both photosynthetic and respiratory activity in intact arabidopsis seedlings. By monitoring oxygen evolution in dark and light phases, we demonstrate that oxygen consumption and production correspond to mitochondrial respiration and photosynthesis, respectively. These processes were modulated by tissue biomass, light intensity, developmental stage, and stress conditions. Specific inhibitors such as potassium cyanide and paraquat confirmed that the recorded changes in oxygen concentrations reflected mitochondrial cytochrome oxidase activity and photosystem electron transport-dependent oxygen production, respectively. Moreover, oxygen evolution increased significantly with bicarbonate supplementation, validating the system's sensitivity to carbon fixation. We further showed that photosynthetic activity measured with this method correlates with a quantitative green index and responds dynamically to de-etiolation, abiotic stress (salt, osmotic, oxidative), and temperature shifts. Our study lays the groundwork for measuring photosynthesis based on oxygen evolution and respiration in arabidopsis knockout mutants, CRISPR lines, overexpression lines and ecotypes using Clark-type oxygen electrodes and highlights key considerations and limitations to consider when applying this approach. This platform could also be adapted for many other small tissue plant samples.

In Drosophila melanogaster, bag of marbles ( bam ) encodes a protein essential for germline stem cell daughter (GSC) differentiation in early gametogenesis. Despite its essential role in D. melanogaster , direct functional evaluation of bam in other closely related Drosophila species reveal this essential function is not necessarily conserved. In D. teissieri , for example, bam is not essential for GSC daughter differentiation. Here, we generated bam null alleles using CRISPR-Cas9 in a species more distantly related to D. melanogaster, D. americana , to interrogate whether bam ’s essential GSC differentiation function is novel to the melanogaster species group or a function more basal to the Drosophila genus. To further characterize the extent of the functional flexibility of other GSC regulating genes, we generated a gene ortholog dataset for 366 GSC regulating genes essential in D. melanogaster across 15 additional Drosophila and two outgroup species. We find that bam ’s essential GSC function is conserved between D. melanogaster and D. americana and therefore originated prior to the formation of the melanogaster species group. Additionally, we find that ∼8% of the 366 GSC genes essential in D. melanogaster are absent in at least one of the 17 species in our ortholog dataset. These results indicate that developmental systems drift (DSD), in which the specific genes regulating a function may change, but the final phenotype is retained, occurs in stem cell regulation and the production of gametes across Drosophila species. Article summary Results: from CRISPR induced bam null mutants in D. americana and comparative ortholog analysis of essential GSC regulating genes indicate that the evolutionary origin of bam ’s essential GSC differentiation function is likely basal to the Drosophila genus, and there is functional flexibility in at least ∼8% of the 366 GSC regulating genes across the 17 included species.

Macrophages in the tumor microenvironment exert potent anti-tumorigenic activity through phagocytosis. Yet therapeutics that enhance macrophage phagocytosis have not improved outcomes in clinical trials for patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). To systematically identify regulators of phagocytosis, we performed genome-scale CRISPR knockout screens in human leukemia cells co-cultured with human monocyte-derived macrophages. Surprisingly, we found that whereas the classic “don’t eat me” signal CD47 inhibited mouse macrophages, it did not inhibit phagocytosis by human macrophages. In contrast, the O-linked glycosylation and sialylation pathways were strong negative regulators of phagocytosis. In AML, the cell surface O-linked glycoprotein CD43 was the major effector of the O-linked glycosylation and sialylation pathways. Genetic deletion or antibody blockade of CD43 enhanced macrophage phagocytosis. This work highlights the importance of using human platforms to identify immune checkpoints, and nominates CD43 as a glyco-immune regulator of human macrophage phagocytosis.

Most genetic variants associated with human traits and diseases lie in noncoding regions of the genome 1 , and a key challenge is determining which genes they affect 2,3 . A common approach has been to leverage associations between natural genetic variation and gene expression to identify expression quantitative trait loci (eQTLs) in the population 4,5 . At the same time, a newer method uses pooled CRISPR interference (CRISPRi) perturbations of noncoding loci with single-cell transcriptome sequencing 6,7 . Here, we systematically compared the results from these approaches across hundreds of genomic regions associated with blood cell traits. We find that while the two approaches often identify the same target genes, they also capture distinct features of gene regulation. CRISPRi tends to detect genes that are physically closer to regulatory variants and more constrained, whereas eQTL studies are sensitive to detecting multiple, often distal, genes. By comparing these discoveries to a gold-standard set of genes linked to blood traits, we demonstrate that the two approaches provide highly complementary insights with distinct strengths and caveats. Our results offer guidance for improved design of CRISPRi and eQTL studies and highlight their potential as a powerful toolkit for interpreting disease-associated loci.

Human congenital anomalies account for twice the mortality of childhood cancer. Despite advancements in genome sequencing and transgenic mouse models that have aided in understanding their pathogenesis, significant gaps remain. Through a forward genetics approach, we previously discovered the hypo-morphic anteater allele of Cse1l which displayed variable craniofacial phenotypes. To circumvent the variability seen in this model, we generated a conditional allele of Cse1l and genetically ablated it in the dorsal midline giving rise to portions of the nervous system and the cranial neural crest cells using the Wnt1-Cre 2 driver. Our analysis revealed that Wnt1-Cre2; Cse1l CRISPR/flox embryos exhibited severe malformations in the forebrain, midbrain, and hindbrain, accompanied by a dramatic hypoplasia of the frontonasal, maxillary, and mandibular processes, and the second pharyngeal arch. Wnt1-Cre2; Cse1l CRISPR/flox embryos were embryonic lethal by E11.5 likely due to defects in the ventricular myocardium. Wnt1-Cre2; Cse1l CRISPR/flox embryos exhibited consistently increased apoptosis at E9.5 in the affected tissues along with an increase in p53 expression. These data together show a previously unknown critical function of CSE1L in neural crest cell survival during development. Summary Statement Cse1l is critical for neural crest cell survival and genetic ablation of Cse1l in neural crest cells resulted in dramatic apoptosis with increase in p53 expression.