CRISPR-Cas12a effects RNA-guided cleavage of dsDNA in cis , after which it remains catalytically active and non-specifically cleaves ssDNA in trans . Native host-defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well characterised Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage, and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in E. coli , and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-NUC interactions. We generated and tested REC2 and NUC mutants, including a hitherto unstudied ‘NUC loop’, integrity of which is critical for the function of Cas12 orthologues. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications. Graphical abstract

Dictyostelids are a species-rich clade of cellular slime molds that are widely found in soils and have been studied for over a century. Most research focusses on Dictyostelium discoideum, which - due to its ease of culturing and genetic tractability - has been adopted as a model species in the fields of developmental biology, cell biology and microbiology. Over decades, genome editing methods in D. discoideum have steadily improved but remain relatively time-consuming and limited in scope, effective in a few species only. Here, we introduce a CRISPR-Cas9 editing protocol that is cloning-free, selection-free, highly-efficient, and effective across Dictyostelid species. After optimizing our protocol in D. discoideum, we obtained knock-out efficiencies of ~80% and knock-in efficiencies of ~30% without antibiotic selection. Efficiencies depend on template concentrations, insertion sizes, homology arms and target sites. Since our protocol is selection-free, we can isolate mutants as soon as one day post-transfection, vastly expediting the generation of knock-outs, fusion proteins and expression reporters. Our protocol also makes it possible to generate several knock-in mutations simultaneously in the same cells. Boosted by cell-sorting and fluorescent microscopy, we could readily apply our CRISPR-Cas9 editing protocol to phylogenetically distant Dictyostelid species, which diverged hundreds of millions of years ago and have never been genome edited before. Our protocol therefore opens the door to performing broad-scale genetic interrogations across Dicyostelids.

We introduced two targeted DNA double-strand breaks on the same Arabidopsis chromosome using CRISPR-Cas9 and replaced the FLOWERING LOCUS T ( FT ) promoter with that of a histone variant gene through chromosomal inversion. The resulting lines misexpressed FT and flowered early, like FT -overexpressing transgenic plants. This system can be used to create gain-of-function mutations that modify target gene expression as desired without incorporating foreign DNA sequences.

ABSTRACT Insufficient infiltration of cytotoxic lymphocytes to solid tumors limits the efficacy of immunotherapies and cell therapies. Here, we report a programmable mechanism to mobilize Natural Killer (NK) and T cells to breast cancer tumors by engineering these cells to express orphan and metabolite-sensing G protein-coupled receptors (GPCRs). First, in vivo and in vitro CRISPR activation screens in NK-92 cells identified GPR183 , GPR84 , GPR34 , GPR18 , FPR3 , and LPAR2 as top enhancers of both tumor infiltration and chemotaxis to breast cancer. These genes equip NK and T cells with the ability to sense and migrate to chemoattracting metabolites such as 7α,25-dihydroxycholesterol and other factors released from breast cancer. Based on Perturb-seq and functional investigations, GPR183 also enhances effector functions, such that engineering NK and CAR NK cells to express GPR183 enhances their ability to migrate to, infiltrate, and control breast cancer tumors. Our study uncovered metabolite-based tumor immune recruitment mechanisms, opening avenues for spatially targeted cell therapies.

Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction (SVF) of mice using ribosome profiling (Ribo-Seq). Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting (FACS) of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. Among these, we validated a novel microprotein as a key regulator of adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation. Significance Obesity and its associated metabolic disorders pose significant public health challenges, yet the molecular mechanisms regulating adipocyte function remain incompletely understood. Small open reading frames (smORFs) and their encoded microproteins represent an emerging class of regulatory elements with potential roles in metabolism. Here, we leveraged CRISPR/Cas9 screening to functionally characterize smORFs in adipocytes, identifying novel regulators of cell proliferation and lipid metabolism. Our findings demonstrate that conservation is not a prerequisite for smORF function, as we validated a mouse-specific microprotein that modulates adipocyte differentiation. This work establishes a robust pipeline for unbiased smORF discovery and highlights the potential for species-specific microproteins to regulate adipose biology. Future studies in human adipocytes may uncover additional microproteins with therapeutic relevance for obesity and metabolic disease.

Summary The ability to maintain invariant developmental phenotypes across disparate environments is termed canalization, but few examples of canalization mechanisms are described. In plants, robust flower production across environmental gradients contributes to reproductive success and agricultural yields. Flowers are produced by the shoot apical meristem (SAM) in an auxin-dependent manner following the switch from vegetative growth to the reproductive phase. While the timing of this phase change, called the floral transition, is sensitized to numerous environmental and endogenous signals, flower formation itself is remarkably invariant across environmental conditions. Previously we found that CLAVATA peptide signaling promotes auxin-dependent flower primordia formation in cool environments, but that high temperatures can restore primordia formation through unknown mechanisms. Here, we show that heat promotes floral primordia patterning and formation in SAMs not by increased auxin production, but through the production of the mobile flowering signal, florigen, in leaves. Florigen, which includes FLOWERING LOCUS T ( FT ) and its paralog TWIN SISTER OF FT ( TSF ) in Arabidopsis thaliana , is necessary and sufficient to buffer flower production against the loss of CLAVATA signaling and promotes heat-mediated primordia formation through specific SAM expressed transcriptional regulators. We find that sustained florigen production is necessary for continuous flower primordia production at warmer temperatures, contrasting florigen’s switch-like control of floral transition. Lastly, we show that CLAVATA signaling and florigen synergize to canalize flower production across broad temperature ranges. This work sheds light on the mechanisms governing the canalization of plant development and provides potential targets for engineering crop plants with improved thermal tolerances.

Abstract Glioblastoma remains an incurable and highly aggressive brain tumor due to its immune-suppressive microenvironment. In this paper, we propose a novel theoretical approach using CRISPR-engineered macrophages to infiltrate, recognize, and eradicate glioblastoma.

Abstract Inherited genetic disorders impact at least 300 million individuals worldwide, presenting a significant therapeutic challenge. Although CRISPR-based genome editing offers a promising avenue for targeted interventions, the pathogenic and likely pathogenic variants amenable to such treatments have yet to be fully delineated. Here, we present the mEdit platform (https://igimedit.org) alongside a library of 179,819 pathogenic and likely pathogenic germline variants across 4,659 genes that include their potential correctability using CRISPR-based approaches. mEdit assesses the therapeutic editability of these variants by reporting mutation-specific guide RNAs (gRNA), efficiency scoring, off-target evaluation, and variant annotations from different databases. Our analysis reveals that >95% of these variants are targetable by at least one CRISPR tool, including >14% suitable for base editing strategies. Furthermore, we introduce the concept of histoetiology to assign the root-cause tissues of these variants, providing crucial insights into their clinical editing tractability. This study establishes a strategic roadmap for prioritizing CRISPR-based therapeutic development, highlighting the opportunities and gaps in the current landscape of gene-editable human mutations. Our findings underscore the potential of CRISPR technologies to address a vast array of genetic disorders, paving the way for future advances in genetic medicine. In particular, given the established clinical tractability of gene editing in the eye, the hematopoietic system, and the liver, our analysis nominates, for the first time, ~25% of the human mutome as directly amenable to in-the-clinic CRISPR gene editing therapeutic development.

Spinal cord injury (SCI) remains a major clinical challenge, with limited therapeutic options for restoring lost neurological function. While efforts to mitigate secondary damage have improved early-phase management, achieving sustained neurorepair and functional recovery remains elusive. Advances in stem cell engineering and regenerative medicine have opened new avenues for targeted interventions, particularly through the transplantation of neural stem/progenitor cells (NSPCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs). However, patient-specific factors such as cellular senescence, genetic and epigenetic variability, injury microenvironment, and comorbidities influence the efficacy of stem cell therapies by affecting graft survival and differentiation. Overcoming these challenges necessitates cutting-edge technologies, including single-cell transcriptomics, CRISPR-mediated hypoimmunogenic engineering, and biomaterial-based delivery platforms, which enable personalized and precision-driven SCI repair. Leveraging these advancements may help stem cell therapies overcome translational barriers and establish clinically viable regenerative solutions. This review explores the intersection of patient-specific variability, bioengineering innovations, and transcriptomic-guided precision medicine to define the next frontier in SCI therapy.

Liposomal drug delivery has transformed contemporary medicine with the supply of targeted, controlled, and effective drug release systems. Liposomal nanocarriers maximize drug bioavailability and reduce systemic toxicity, making them especially beneficial for cancer therapy and precision medicine. The paper points out development and progress in liposomal drug delivery with emphasis on mechanisms for targeted release of drugs and growing application of nanocarriers in contemporary therapeutics. Despite their advantages, the liposomal formulations are subjected to some strong disadvantages including rapid immune system clearance, tumor heterogeneity, and high-scale production. Despite these drawbacks, certain new developments by way of PEGylation, ligand-grafted liposomes, and hybrid lipid-polymer nanocarriers have proved promising and effective in bringing improvements to liposome stability and target specificity. In addition, artificial intelligence predictive modeling is becoming an effective approach for optimizing liposomal formulations to tailor treatment regimens to the patient.Also, liposomal nanoparticle use in the treatment of cancer has provided avenues for the development of new chemotherapy and gene therapy approaches for facilitating precision medicine. The review also illustrates current trends, including theranostic liposomes to facilitate real-time drug delivery and imaging and CRISPR-based liposomal gene therapy. The future is conjugating nanotechnology, bioengineering, and artificial intelligence in formulating the next generation of intelligent nanocarriers. Despite current limitations, liposomal preparations have unmatched promise to revolutionize drug delivery systems and precision medicine.

Retinitis pigmentosa (RP) is a genetically diverse blinding disorder lacking broadly effective therapies. We performed a genome-wide in vivo CRISPR knockout screen in mice carrying the P23H rhodopsin mutation (the most common cause of autosomal dominant RP in the United States) to systematically identify neuroprotective genes. We discovered multiple knockouts that accelerated rod photoreceptor loss, validated top candidates, and showed that overexpressing two genes-UFD1 and UXT-preserved rods and cones, maintained retinal function, and improved visual behaviors. To accelerate translation, we developed a human P23H RP model in adult retinal explants, recreating key disease features. UFD1 and UXT augmentation prevented photoreceptor loss in human P23H retinas. Our findings establish a pipeline for systematic identification and translational testing of neuroprotective genes in mouse and human RP models, provide a novel set of validated candidate genes, and underscore the therapeutic promise of UFD1 and UXT as mutation-agnostic strategies to preserve vision.

Spinocerebellar ataxias (SCAs) are autosomal dominantly inherited neurodegenerative disorders with no effective treatment. Aberrant signalling through the metabotropic glutamate receptor (mGluR1) has been implicated in several SCAs. However, whether disease is caused through decreased or increased mGluR1 signalling remains controversial. Here, we generate the first mouse model of enhanced mGluR1 function by introducing a gain-of-function mutation (p.Y792C) that causes SCA44 in the metabotropic glutamate receptor 1 (Grm1 ) gene. Grm1 mutant mice recapitulate key pathophysiological aspects of SCA, including progressive motor deficits, altered climbing fibre innervation and perturbed Purkinje cell spontaneous activity. We report that changes in synaptic innervation and intrinsic Purkinje cell activity upon overactive mGluR1 signalling manifest in a lobule- and disease-stage-specific manner. Our findings demonstrate that enhanced mGluR1 function is a direct and specific driver of Purkinje cell dysfunction and pathology and provide a mechanism for understanding the selective vulnerability of different Purkinje cell populations in SCA.

Enhancers are the epicentres of tissue-specific gene regulation. In this study, we have used the central nervous system (CNS) specific expression of the Drosophila grainyhead ( grh ) gene to make a case for deleting the enhancers in a sensitised background of other enhancer deletion, to functionally validate their role in tissue-specific gene regulation. We identified novel enhancers for grh and subsequently deleted two of them, to establish their collective importance in regulating grh expression in CNS. This showed that grh relies on multiple enhancers for its robust expression in neural stem cells (NSCs), with different combinations of enhancers playing a critical role in regulating its expression in various subset of these cells. We also found that these enhancers and the grh gene show epigenetic synchrony across the three cell types (NSCs, intermediate progenitors and neurons) of the developing CNS; and grh is not transcribed in intermediate progenitor cells, which inherits the Grh protein from the NSCs. We propose that this could be a general mechanism for regulating the expression of cell fate determinant protein in intermediate progenitor cells. Lastly, our results underline that enhancer redundancy results in phenotypic robustness in grh gene expression, which seems to be a consequence of the cumulative activity of multiple enhancers.

Chikungunya virus (CHIKV) infection imposes a significant socio-economic burden due to the absence of effective antiviral treatments and vaccines. Host factors are essential for the CHIKV life cycle, making them promising targets for antiviral therapy. Previous studies have identified Mitogen-activated protein kinase activated protein kinase 2 (MK2) and Mitogen-activated protein kinase activated protein kinase 3 (MK3) as key host factors in CHIKV infection; however, their role in an animal model remain unclear. This study highlights the critical roles of MK2 and MK3 host factors in an animal model following CHIKV infection. To investigate their functions, mk2b (mk2b-/-) , mk3 (mk3-/-) , and mk2b-mk3 (mk2b-/-mk3-/-) double knockout zebrafish were generated using the CRISPR-Cas9 technique. A significant high viral titer of CHIKV was observed in case of all knockout groups compared to the wild-type (WT) control using plaque assay, RT-qPCR and immunofluorescence assay. Among the knockout groups, mk3-/- displayed the highest susceptibility to CHIKV, followed by mk2b-/-. In contrast, the mk2b-/-mk3-/- double knockout exhibited the lowest susceptibility to CHIKV infection. Additionally, severe symptoms such as bent body, impaired response to physical stimuli, and increased mortality were most pronounced in mk3-/- larvae compared to other knockouts and the WT. The expression levels of infɸ1 and rsad2 were also elevated in all knockout groups during the early days of infection indicating higher interferon response in the absence of mk2b and mk3 during CHIKV infection. In conclusion, this study confirms that the mk2b and mk3 host proteins are essential in controlling the CHIKV infection in organism level and subsequently may contribute in designing antiviral therapeutics in future. Furthermore, the knockout model of mk2b and mk3 in zebrafish could serve as a valuable tool for studying their roles in other viral infections. Author Summary CHIKV, transmitted by Aedes aegypti and Aedes albopictus mosquitoes, causes febrile illness and has spread across Africa, Asia, Europe, and America. Despite extensive research, effective antiviral drugs and vaccines are yet to be commercially available. This study examines the role of mk2b and mk3 host factors following CHIKV infection. For this purpose, mk2b and mk3 single as well as double knockout have been generated in zebrafish using the CRISPR-Cas-9 technique. Findings suggest that zebrafish exhibit high CHIKV susceptibility in the absence of mk2b and mk3 , confirming its importance during infection. Moreover, these three knockout models could serve as a valuable platform for examine the role of mk2b and mk3 in the presence of other viral infection.

Evaluating the impact of bacteriophages on bacterial communities is required to assess the future utility of phage therapy. Methods able to study bacterial polycultures in the presence of phages are useful to mimic evolutionary pressures found in natural environments and recapitulate complex ecological contexts. Bacteriophages can drive rapid genetic and phenotypic changes in host cells. However, the presence of other bacteria can also impact bacterial densities and community structure, and classical methods remain lengthy and resource intensive. Here, we introduce a microdroplet-based encapsulation method in which bacterial co-cultures are imaged using Z-stack brightfield microscopy. The method relies on automated droplet imaging using a novel AI-based autofocus function, coupled with morphology-based deep learning models for accurate identification of two morphologically distinct bacterial species. We show that we can monitor the relative growth dynamics of P. aeruginosa and S. aureus growing in 11 picolitre droplets for up to 24 hours. We demonstrate quantification of growth rates, bacterial densities and lysis dynamics of the two species without the need for plating. We show that a potent lytic phage of P. aeruginosa can either fully lyse the initial P. aeruginosa population or keep its density low long-term when in the presence of S. aureus .

Summary After invasion and replication, intracellular pathogens must egress from infected host cells . Toxoplasma gondii facilitates this process by permeabilizing host cells by releasing perforin-like protein 1 (PLP1) through induced microneme secretion. However, the precise mechanism of host cell permeabilization remains enigmatic. Here, we identified the secretory microneme protein MIC11 as a key factor for membrane disruption. A CRISPR-based in vivo screen revealed several genes including MIC11 as an essential gene for virulence. Deletion of MIC11 resulted in severe defects in both membrane rupture and egress. Scanning mutagenesis identified functional motifs in MIC11, and mechanistic analyses demonstrated that MIC11 directly associates with PLP1, regulating its activity in membrane disruption. The MIC11 paralogue MIC22 compensated for MIC11 deletion, suggesting a conserved mechanism of egress in the feline-restricted stages of T. gondii . The discovery of MIC11 advances the understanding of how parasites disrupt host cells to facilitate rapid egress and successful dissemination.

ABSTRACT Regulation of LDLR gene expression plays an important role in the development of atherosclerotic diseases including heart attack and stroke. Although LDLR regulation by sterol response elements has been well characterized, the functional significance of other noncoding regions at the LDLR locus remains poorly defined. We developed and applied a high throughput CRISPR screen to test the functional significance of candidate LDLR cis -regulatory elements in their native genomic context. Analysis of our screen results revealed a discrete region in the first intron of LDLR with a significant impact on cellular LDL uptake. We validated the presence of enhancer activity in this region by confirming that its disruption reduced endogenous LDLR expression while its insertion upstream of a minimal promoter augmented reporter gene expression. We then applied a massively parallel reporter assay to fine map enhancer activity in this region to a 129 bp interval that is highly conserved among vertebrates, exhibits biochemical hallmarks of enhancer activity, is enriched for transcription factor binding motifs, and contains a common genetic variant (rs57217136) that has been associated with human LDL cholesterol levels by genome-wide association studies. Overall, these findings demonstrate the power of CRISPR screening to interrogate candidate CREs and support the functional significance of an enhancer in the first intron of LDLR .

We identified N -ethylmaleimide-sensitive factor attachment protein beta ( NAPB ) as a potential risk gene for autism and epilepsy. Notably, Qatari monozygotic triplets with loss of function mutations in NAPB exhibit early onset epileptic encephalopathy and varying degrees of autism. In this study, we generated NAPB zebrafish model using CRISPR-Cas9-sgRNAs technology for gene editing of the two orthologs napba and napbb . We observed that napb crispants (CR) show shorter motor neuron axons length together with altered locomotion behavior, including significant increases in larvae total distance traveled, swimming velocity, and rotation frequency, indicating that these behavioral changes effectively mimic the human epileptic phenotype. We applied microelectrode array (MEA) technology to monitor neural activity and hyperexcitability in the zebrafish model. The napb CR shows hyperexcitability in the brain region. By combining behavioral tests with electrophysiological MEA assays, the established NAPB zebrafish model can be employed to study the pathophysiological mechanisms of ASD and epilepsy to screen potential therapeutic drugs.

ABSTRACT The rapid evolution of novel functions requires targeted mutagenesis to avoid harmful mutations. Diversity-generating retroelements (DGRs) are natural systems that accelerate the evolution of diverse bacterial functions through targeted hypermutation. Here, we establish a method utilizing DGRs coupled to recombineering (DGRec), enabling the diversification of any sequence of interest in E. coli . DGRec can programmably diversify specific residues by leveraging the high error rate of the DGR reverse-transcriptase at adenines. We perform a detailed characterization of the reverse-transcriptase biases, highlighting how it maximizes the exploration of the sequence space while avoiding nonsense mutations. Applied to the phage λ GpJ receptor binding domain, and to its lamB receptor, DGRec created diverse variants enabling E. coli to evade infection, and λ to reinfect lamB mutants.

We describe a scalable and cost-effective sgRNA synthesis workflow that reduces costs by over 70% through the use of large pools of microarray-derived oligos encoding unique sgRNA spacers. These sub-pool oligos are assembled into full-length dsDNA templates via Golden Gate Assembly before in vitro transcription with T7 RNA polymerase. RNA-seq analysis reveals severe biases in spacer representation, with some spacers being highly overrepresented while others are completely absent. Consistent with previous studies, we identify guanine-rich sequences within the first four nucleotides of the spacer, immediately downstream of the T7 promoter, as the primary driver of this bias. To address this issue, we introduced a guanine tetramer upstream of all spacers, which reduced bias by an average of 19% in sgRNA libraries containing 389 spacers. However, this modification also increased the presence of high-molecular-weight RNA species after transcription. We also tested two alternative bias-reduction strategies: compartmentalizing spacers within emulsions and optimizing DNA input and reaction volumes. Both methods independently reduced bias in 2,626-plex sgRNA libraries, though to a lesser extent than the guanine tetramer approach. These advancements enhance both the affordability and uniformity of sgRNA libraries, with broad implications for improving CRISPR-Cas9 screens and optimizing guide RNA design for other CRISPR and nuclease systems.

Abstract Phosphorus (P) is a crucial macronutrient and its deficiency severely limits plant growth and yield. Although multiple inorganic phosphate (Pi) signaling regulators have been identified, the function of them in plant development and flowering time regulatory remains inadequately characterized in C4 model species like Setaria italica . Here, CRISPR/Cas9-generated SiPHO2 knockout lines exhibited disrupted Pi homeostasis, and the lines showed shoot Pi accumulation, leaf tip necrosis, modified root architecture and reduced yield compared with wildtype (Ci846) under Pi deficient conditions. Transcriptome analysis suggested these phenotypic abnormalities might due to expression patterns alteration of Pi starvation-responsive genes. Notably, SiPHO2 knockout lines displayed earlier heading date under Pi deficiency but delayed heading date under normal conditions compared to Ci846 plants. Expression profiling and transgenic functional verification revealed that the heading date reversal correlated with the expression pattern of FLOWERING LOCUS T c ( SiFTc ), rather than SiFTa , which is the closest homolog of Heading date 3a ( OsHd3a ). This study identifies a novel flowering regulator as a potential target for coordinating phosphorus-mediated heading date regulation and yield production. Our findings elucidate genetic mechanisms underlying phosphorus-dependent developmental regulation and propose a strategic approach for improving crop yield under Pi starvation.

ABSTRACT Long non-coding RNAs (lncRNAs) can regulate gene expression. Some are essential for organismal development and physiology and can contribute to diseases including cancer. Whilst most lncRNAs exhibit little sequence similarity, conservation of lncRNA transcription relative to neighbouring protein-coding genes suggests potential functional significance. Most positionally equivalent lncRNAs are uncharacterized and it remains unclear whether they exert similar roles in distant species. Here, we identified syntenic melanoma-associated lncRNAs predicted to be components of the MITF gene regulatory network in human melanoma, with positionally equivalent transcripts in zebrafish. We prioritized Differentiation Antagonizing Non-Protein Coding RNA ( DANCR ), a cancer-associated lncRNA critical for maintaining somatic progenitor cells in human models, for functional investigation. Dancr is a multi-exonic, cytoplasmically-enriched lncRNA transcribed from syntenic regions in the human and zebrafish genomes. MITF and c-MYC, key melanoma transcription factors, regulate human DANCR expression and melanoma patients with high DANCR display significantly decreased survival. DANCR is a melanoma oncogene that controls cancer-associated gene expression networks and promotes human melanoma cell proliferation and migration. Zebrafish dancr is dynamically expressed across multiple different cell types in the developing embryo, regulates genes involved in cell death, and is essential for embryonic development. Our work suggests that cancer-critical lncRNAs such as DANCR , expressed from similar regions in vertebrate genomes, may regulate related genes and processes involved in both embryonic development and tumorigenesis across species.

Pooled processing, in which cells from multiple sources are cultured or captured together, is an increasingly popular strategy for droplet-based single cell sequencing studies. This design allows efficient scaling of experiments, isolation of cell-intrinsic differences, and mitigation of batch effects. We present CellBouncer, a computational toolkit for demultiplexing and analyzing single-cell sequencing data from pooled experiments. We demonstrate that CellBouncer can separate and quantify multi-species and multi-individual cell mixtures, identify unknown mitochondrial haplotypes in cells, assign treatments from lipid-conjugated barcodes or CRISPR sgRNAs, and infer pool composition, outperforming existing methods. We also introduce methods to quantify ambient RNA contamination per cell, infer individual donors’ contributions to the ambient RNA pool, and determine a consensus doublet rate harmonized across data types. Applying these tools to tetraploid composite cells, we identify a competitive advantage of human over chimpanzee mitochondria across 10 cell fusion lines and provide evidence for inter-mitochondrial incompatibility and mito-nuclear incompatibility between species.

Cystic fibrosis (CF) is caused by homozygous mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, resulting in multi-organ dysfunction and decreased lifespan and quality of life. A durable cure for CF will likely require a gene therapy approach to correct CFTR. Rapid advancements in genome editing technologies such as CRISPR/Cas9 have already resulted in successful FDA approval for cell-based gene editing therapies, providing new therapeutic avenues for many rare diseases. However, immune responses to gene therapy delivery vectors and editing tools remain a challenge, especially for strategies targeting complex in vivo tissues such as the lung. Previous findings in non-CF healthy individuals reported pre-existing antibody and T cell dependent immune responses to recombinant Cas9 proteins, suggesting potential additional obstacles for incorporation of CRISPR/Cas9 technologies in gene therapies. To determine if pre-existing immunity to Cas9 from S. aureus or S. pyogenes was present or augmented in people with CF (PwCF), anti-Cas9 IgG levels and Cas9-specific T cell responses were determined from peripheral blood samples of PwCF and non-CF healthy controls. Overall, non-CF controls and PwCF displayed evidence of pre-existing antibody and T cell responses to both S. aureus and S. pyogenes Cas9, although there were no significant differences between the two populations. However, we observed global changes in activation of Th1 and CD8 T cell responses as measured by IFNγ and TNF that warrant further investigation and mechanistic understanding as this finding has implications not only for CRISPR/Cas9 gene therapy for PwCF, but also for protection against infectious disease.

Although two-thirds of cancers arise from loss-of-function mutations in tumor suppressor genes, there are few approved targeted therapies linked to these alterations. Synthetic lethality offers a promising strategy to treat such cancers by targeting vulnerabilities unique to cancer cells with these mutations. To identify clinically relevant synthetic lethal interactions, we analyzed genome-wide CRISPR/Cas9 knock-out (KO) viability screens from the Cancer Dependency Map and evaluated their clinical relevance in patient tumors through mutual exclusivity, a pattern indicative of synthetic lethality. Indeed, we found significant enrichment of mutual exclusivity for interactions involving cancer driver genes compared to non-driver mutations. To identify therapeutic opportunities, we integrated drug sensitivity data to identify inhibitors that mimic the effects of CRISPR-mediated KO. This approach revealed potential drug repurposing opportunities, including BRD2 inhibitors for bladder cancers with ARID1A mutations and SIN3A -mutated cell lines showing sensitivity to nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. However, we discovered that pharmacological inhibitors often fail to phenocopy KO of matched drug targets, with only a small fraction of drugs inducing similar effects. This discrepancy reveals fundamental differences between pharmacological and genetic perturbations, emphasizing the need for approaches that directly assess the interplay of loss-of-function mutations and drug activity in cancer models. Author Summary Synthetic lethality is an emerging approach for targeting a biological dependency in cancer cells that does not harm normal cells. This strategy is particularly valuable for targeting loss-of-function mutations in tumor suppressor genes, which are more challenging to directly target. In an effort to accelerate treatments for cancer patients, we aimed to map out these dependencies and overlap them with responses to available drugs. We discovered different outcomes when a protein is targeted by a drug versus when that same target is disrupted genetically. Thus, if a drug is to be effectively repurposed as synthetic lethal agent, feasibility studies must capture drug biology, ideally by test the drug empirically in relevant cancer models. A second notable discovery is that in vitro synthetic lethal interactions involving cancer driver genes are significantly more likely to exhibit consistent patterns, such as mutual exclusivity in human tumor samples. This is important since selection of relevant cell lines is often critical in drug development to maximize potential for translation to clinical responses.