Intestinal stem cells (ISCs) face the challenge of integrating metabolic demands with unique regenerative functions. Studies have shown an intricate interplay between metabolism and stem cell capacity, however it is still not understood how this process is regulated. Combining ribosome profiling and CRISPR screening in intestinal organoids, we show that RNA translation is at the root of this interplay. We identify the nascent polypeptide-associated complex (NAC) as a key mediator of this process, and show that it regulates ISC metabolism by relocalizing ribosomes to the mitochondria. Upon NAC inhibition, intestinal cells show decreased import of mitochondrial proteins, which are needed for oxidative phosphorylation, and, consequently, enable the cell to maintain a stem cell identity. Furthermore, we show that overexpression of NACα is sufficient to drive mitochondrial respiration and promote ISC identity. Ultimately, our results reveal the pivotal role of ribosome localization in regulating mitochondrial metabolism and ISC function.

Barth syndrome (BTHS) is a rare mitochondrial disease caused by pathogenic variants in the gene TAFAZZIN, which leads to abnormal cardiolipin (CL) metabolism on the inner mitochondrial membrane. Although TAFAZZIN is ubiquitously expressed, BTHS involves a complex combination of tissue specific phenotypes including cardiomyopathy, neutropenia, skeletal myopathy, and growth delays, with a relatively minimal neurological burden. To understand both the developmental and functional effects of TAZ-deficiency in different tissues, we generated isogenic TAZ knockout (TAZ-KO) and WT cardiomyocytes (CMs) and neural progenitor cells (NPCs) from CRISPR-edited induced pluripotent stem cells (iPSCs). In TAZ-KO CMs we discovered evidence of dysregulated mitophagy including dysmorphic mitochondria and mitochondrial cristae, differential expression of key autophagy-associated genes, and an inability of TAZ-deficient CMs to properly initiate stress-induced mitophagy. In TAZ-deficient NPCs we identified novel phenotypes including a reduction in CIV abundance and CIV activity in the CIII2&CIV2 intermediate complex. Interestingly, while CL acyl chain manipulation was unable to alter mitophagy defects in TAZ-KO CMs, we found that linoleic acid or oleic acid supplementation was able to partially restore CIV abundance in TAZ-deficient NPCs. Taken together, our results have implications for understanding the tissue-specific pathology of BTHS and potential for tissue-specific therapeutic targeting. Moreover, our results highlight an emerging role for mitophagy in the cardiac pathophysiology of BTHS and reveal a potential neuron-specific bioenergetic phenotype.

The grain size in cereals is one of the main component traits contributing to yield. Previous studies showed that loss-of-function (LOF) mutations in GS3, encoding Gγ subunit of the multimeric G protein complex, increase grain size and weight in rice. While association between allelic variation in GS3 homologs of wheat and grain weight/size was detected previusly, the effects of LOF alleles on these traits remain unknown. We used genome editing to create the TaGS3 mutant lines with the LOF homeo-allele dosage variation. Contrary to results obtained for rice, editing of all three TaGS3 copies result in significant decrease in grain length, width, grain area and weight, without affecting number of grains per spike. Compared to wild type, the highest increase in grain weight and area was observed in mutants with the intermediate dosage of the LOF alleles, indicating that suppressive effects of TaGS3 on grain size and weight in wheat are dosage-dependent and non-additive. Our results suggest that TaGS3 likely represents a functionally diverged homolog of GS3 evolved in the wheat lineage. The newly developed LOF alleles of TaGS3 expand the set of CRISPR-Cas9-induced variants of yield component genes that could be used for increasing grain weight in wheat.

Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ~664 kb from the ONECUT1 promoter, as coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hPSCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. This enhancer contains a confidently fine-mapped type 2 diabetes (T2D) associated variant (rs528350911) which disrupts a GATA motif. Introduction of the risk variant into hPSCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a slight reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.

The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two single-point mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal only modest functional differences in on-target activity, and similar specificity. These data highlight how guide RNA interactions can be tuned in the absence of major functional losses, but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic S. pyogenes Cas9. A K267E/R332A double mutant did modestly enhance GeoCas9 specificity, highlighting the robust evolutionary tolerance of Cas9 and species-dependent complexity. Ultimately, this work provides an avenue by which to modulate the structure, motion, and nucleic acid interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism.

CRISPR/Cas9 technology in conjunction with somatic cell nuclear transplantation (SCNT) provides the primary approach to producing gene-edited pigs, and targeting nuclear donors with CRISPR/Cas9 is crucial. Gene-edited nuclear donors are inefficient due to poor editing efficiency and low delivery efficiency, which are highly associated with CRISPR/Cas9 form selection. Nevertheless, there is not a straightforward method to evaluate CRISPR/Cas9 editing efficiency on the porcine genome. In this study, a fluorescence report signal and micropattern arrays-based platform was developed to visually assess the efficiency of CRISPR/Cas9 editing. Based on the quantity and state of cells grown on micropattern arrays, 200 μm in diameter and 150 μm in spacing were optimal specifications for culturing porcine cells. The editing efficiency of three different CRISPR/Cas9 system forms: DNA, mRNA, and Ribonucleoprotein (RNP) were rapidly evaluated using this platform, with mRNA proving the most effective. Subsequently, four homozygotes with β4GalNT2 gene knockout were quickly obtained by mRNA-based form, which lays the groundwork for the subsequent generation of gene-edited pigs. This platform makes gene knockout efficiency evaluation rapid, intuitive, and efficient. It also holds great promise for customizing evaluation platforms for different cell types, evaluating delivery techniques, and swiftly testing innovative gene editing tools.

Background: The gene for apolipoprotein E4 (ApoE4 E4) confers an increased risk for development and lowers the age of onset of Alzheimers disease (AD). It is a highly suitable target for CRISPR-based editing because ApoE4 differs from ApoE3 by a single nucleotide polymorphism in the codon for residue 112 that codes for arginine (CGC) in E4 and cysteine (TGC) in E3. Editing of E4 to E3 could lower the risk of AD or ameliorate E4-related AD phenotypes. For AD, in order to deliver CRISPR components across the blood-brain barrier to the brain, we have developed a delivery platform termed Synthetic Exosomes (SEs) microfluidically-synthesized deformable nanovesicles approximately the size of natural exosomes that have the ability to cross the BBB and deliver cargo to the brain. Here, we describe our use of SEs carrying CRISPR to successfully edit E4 to E3 in brain tissue of an E4-expressing mouse model. Methods. Several CRISPR guide RNAs (gRNA) and Cytosine Base Editor (CBE) mRNAs were synthesized by chemical and in vitro transcription syntheses, respectively. Four combinations of gRNA and CBE mRNA were tested in vitro for their relative activity to edit the E4 (cytosine) to E3 (thymine) in E4-expressing neuroblastoma (E4-N2A) and human Kelly-neuroblastoma cells, to assess which combination produced the highest E4 to E3 base editing efficiency. The CRISPR RNA combination with the highest efficiency was encapsulated in SEs and injected intravenously (IV) via the tail vein into an AD model E4-expressing (E4-5XFAD) transgenic mouse; as a negative control, an E4-5XFAD mouse was injected with empty SEs. Five days after injection, mice were euthanized and brain, liver, and buffy coat (white blood cells (WBC)) collected to determine the editing of E4 to E3 measured by Next Generation Sequencing. In addition, E3 mRNA was measured in the brain and liver and compared to the %E3 gene editing. Results. The highest gRNA+CBE mRNA editing efficiency was ~50% in E4-N2A cells and the same gRNA+CBE combination delivered in SEs to Kelly neuroblastoma cells showed 6.5% editing efficiency. In the E4-5XFAD mouse in vivo, five days after IV delivery of a single dose of the highest-activity SE-CRISPR gRNA+CBE mRNA, the percent of E4 edited to E3 was 0.14% in brain, 0.8% in liver, and 0.36 % in WBCs. As evidence of functional editing, SE-CRISPR-treated mice had 0.03% E3 mRNA in brain and 0.09% E3 mRNA in liver. Conclusions. While this level of ApoE4 to E3 editing achieved five days after a single IV injection of SE-CRISPR is small, it provides initial in vivo proof-of-concept that the ApoE4 gene can be successfully edited and editing results in functional expression of ApoE3 mRNA. The findings presented herein supports further optimization of the SE-CRISPR approach to increase the level of editing in brain as part of clinical development of SE-CRISPR as a powerful novel therapeutic approach for AD.

Mutating replication-dependent (RD) histone genes is an important tool for understanding chromatin-based epigenetic regulation. Deploying this tool in metazoan models is particularly challenging because RD histones in these organisms are typically encoded by many genes, often located at multiple loci. Such RD histone gene arrangements make the ability to generate homogenous histone mutant genotypes by site-specific gene editing quite difficult. Drosophila melanogaster provides a solution to this problem because the RD histone genes are organized into a single large tandem array that can be deleted and replaced with transgenes containing mutant histone genes. In the last ~15 years several different RD histone gene replacement platforms have been developed using this simple strategy. However, each platform contains weaknesses that preclude full use of the powerful developmental genetic capabilities available to Drosophila researchers. Here we describe the development of a newly engineered platform that rectifies many of these weaknesses. We used CRISPR to precisely delete the RD histone gene array (HisC), replacing it with a multifunctional cassette that permits site-specific insertion of either one or two synthetic gene arrays using selectable markers. We designed this cassette with the ability to selectively delete each of the integrated gene arrays in specific tissues using site-specific recombinases. We also present a method for rapidly synthesizing histone gene arrays of any genotype using Golden Gate cloning technologies. These improvements facilitate generation of histone mutant cells in various tissues at different stages of Drosophila development and provide an opportunity to apply forward genetic strategies to interrogate chromatin structure and gene regulation.

ABSTRACT Background Dopamine is a powerful neuromodulator of diverse brain functions, including movement, motivation, reward, and cognition. D1-type dopamine receptors (D1DRs) are the most prevalently expressed dopamine receptors in the brain. Neurons expressing D1DRs are heterogeneous and involve several subpopulations. Studying these neurons has been limited by current animal models, especially when considering their integration with conditional or intersectional genetic tools. New method To address this limitation, we developed a novel Drd1-P2A-Flpo (Drd1-Flpo) mouse line in which the Flpo gene was knocked in immediately after the Drd1 gene using CRISPR-Cas9. We validated the Drd1-Flpo line by confirming Flp expression and functionality specific to D1DR+ neurons. Comparison with existing methods: The Drd1-Flpo line is useful resource for studying subpopulation of D1DR+ neurons with intersectional genetic tools. Conclusions We demonstrated brain-wide GFP expression driven by Drd1-Flpo, suggesting that this mouse line may be useful for comprehensive anatomical and functional studies in many brain regions. The Drd1-Flpo model will advance the study of dopaminergic signaling by providing a new tool for investigating the diverse roles of D1DR+ neurons and their subpopulations in brain disease. Significance Statement The roles of dopamine in the brain are mediated by dopamine receptors. D1-type dopamine receptors (D1DRs) and D1DR-expressing (D1DR+) neurons play important roles in various brain functions. We generated a Drd1-Flpo mouse line that expresses Flp recombinase in D1DR+ neurons. This novel Drd1-Flpo mouse facilitates investigation of specific roles of D1DR+ neurons in various brain areas including the striatum, frontal cortex, and cerebellum, and it provides an alternative to existing Drd1-Cre mice. In addition, the Drd1-Flpo mouse line provides a tool for intersectional genetic studies, when used with existing transgenic Cre lines. The Drd1-Flpo mouse line can help unravel the specific contributions of D1DR+ neuron subpopulations to brain function and dysfunction.

Mutations in the GBA gene, which encodes the lysosomal enzyme β-glucocerebrosidase (GCase), are the most prevalent genetic susceptibility factor for Parkinson's disease (PD). However, only approximately 20% of carriers develop the disease, suggesting the presence of genetic modifiers influencing the risk of developing PD in the presence of GBA mutations. Here we screened 1,634 human transcription factors (TFs) for their effect on GCase activity in cell lysates of the human glioblastoma line LN-229, into which we introduced the pathogenic GBA L444P variant via adenine base editing. Using a novel arrayed CRISPR activation library, we uncovered 11 TFs as regulators of GCase activity. Among these, activation of MITF and TFEC increased lysosomal GCase activity in live cells, while activation of ONECUT2 and USF2 decreased it. Conversely, ablating USF2 increased GBA mRNA and led to enhanced levels of GCase protein and activity. While MITF, TFEC, and USF2 affected GBA transcription, ONECUT2 was found to control GCase trafficking by modulating the guanine exchange factors PLEKHG4 and PLEKHG4B. Hence, our study provides a systematic approach to identifying modulators of GCase activity, expands the transcriptional landscape of GBA regulation, and deepens our understanding of the mechanisms involved in influencing GCase activity.

From an ENU-mutated mangrove killifish line R228, we have identified and isolated a novel mutant line, no-fin-ray/ nfr in which fin ray development is largely reduced. Besides the reduction of the fin, the nfr mutant also exhibited other phenotype associated with ectodermal cell lineages including loss of scales, deformation in the gill structure such as decreasing the number of gill filaments, the reduction in the number of jaw teeth, pharyngeal teeth and gill rakers. Illumina RNAseq with 12 embryos each from mutants, siblings and the parental WT strain Hon9 identified a mutation in the edaradd in a highly conserved C-terminal death domain. Edaradd is known as a cytoplasmic accessory protein for the Ectodysplasin A ( EDA ) signalling pathway. To confirm the crucial role of edaradd during fin development, CRISPR RNAs were designed to knock out the gene in another killifish species, Arabian killifish. Indeed, Arabian killifish edaradd crispants showed a potent reduction of the fin development with 100% frequency. Furthermore, EDA crispants also showed identical phenotypes to that of edaradd crispants, confirming the fin defect in the mutants/crispants is caused by the signalling pathway of the EDA in the killifish species. These data demonstrate a powerful genetic approach using isogenic self-fertilising mangrove killifish as a tool for identifying mutants and their mutation, and revealed the crucial role of edaradd in the fish fin development and other ectoderm derived epithelial tissues.

Dengue virus (DENV) can hijack non-neutralizing IgG antibodies to facilitate its uptake into target cells expressing Fc gamma receptors (FcgR) - a process known as antibody-dependent enhancement (ADE) of infection. Beyond a requirement for FcgR, host dependency factors for this non-canonical infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout screens in an in vitro system permissive to infection only in the presence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates binding and internalization of IgG-bound DENV but is not required for canonical infection. Additionally, we identified host factors with no previously described role in DENV infection, including TBC1D24 and SV2B, both of which have known functions in regulated secretion. Using genetic knockout and trans-complemented cells, we validated a functional requirement for these host factors in ADE assays performed with monoclonal antibodies and polyclonal sera in multiple cell lines and using all four DENV serotypes. We show that knockout of TBC1D24 or SV2B impaired binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that are required for ADE of DENV infection. Our findings represent a first step towards advancing fundamental knowledge behind the biology of ADE that can ultimately be exploited to inform vaccination and therapeutic approaches.

Resistance to chemotherapy has been a major hurdle that limits therapeutic benefits for many types of cancer. Here we systematically identify genetic drivers underlying chemoresistance by performing 30 genome-scale CRISPR knockout screens for seven chemotherapeutic agents in multiple cancer cells. Chemoresistance genes vary between conditions primarily due to distinct genetic background and mechanism of action of drugs, manifesting heterogeneous and multiplexed routes towards chemoresistance. By focusing on oxaliplatin and irinotecan resistance in colorectal cancer, we unravel that evolutionarily distinct chemoresistance can share consensus vulnerabilities identified by 26 second-round CRISPR screens with druggable gene library. We further pinpoint PLK4 as a therapeutic target to overcome oxaliplatin resistance in various models via genetic ablation or pharmacological inhibition, highlighting a single-agent strategy to antagonize evolutionarily distinct chemoresistance. Our study not only provides resources and insights into the molecular basis of chemoresistance, but also proposes potential biomarkers and therapeutic strategies against such resistance.

Current technologies for upregulation of endogenous genes use targeted artificial transcriptional activators but stable gene activation requires persistent expression of these synthetic factors. Although general “hit-and-run” strategies exist for inducing long-term silencing of endogenous genes using targeted artificial transcriptional repressors, to our knowledge no equivalent approach for gene activation has been described to date. Here we show stable gene activation can be achieved by harnessing endogenous transcription factors ( EndoTF s) that are normally expressed in human cells. Specifically, EndoTFs can be recruited to activate endogenous human genes of interest by using CRISPR-based gene editing to introduce EndoTF DNA binding motifs into a target gene promoter. This Precision Editing of Regulatory Sequences to Induce Stable Transcription-On ( PERSIST-On ) approach results in stable long-term gene activation, which we show is durable for at least five months. Using a high-throughput CRISPR prime editing pooled screening method, we also show that the magnitude of gene activation can be finely tuned either by using binding sites for different EndoTF or by introducing specific mutations within such sites. Our results delineate a generalizable framework for using PERSIST-On to induce heritable and fine-tunable gene activation in a hit-and-run fashion, thereby enabling a wide range of research and therapeutic applications that require long-term upregulation of a target gene.

The availability of large databases of biological sequences presents an opportunity for in-depth exploration of gene diversity and function. Bacterial defense systems are a rich source of diverse, but difficult to annotate genes with biotechnological applications. In this work, we present Domainator, a flexible and modular software suite for domain-based gene neighborhood and protein search, extraction, and clustering. We demonstrate the utility of Domainator through three examples related to bacterial defense systems. First, we cluster CRISPR-associated Rossman fold (CARF) containing proteins with difficult to annotate effector domains, classifying most of them as likely transcriptional regulators and a subset as likely RNAses. Second, we extract and cluster P4-like phage satellite defense hotspots and identify an abundant system related to Lamassu phage defense systems. Third, we integrate a protein language model into Domainator and use it to identify restriction enzymes with low homology to known reference sequences, validating the activity of one example in-vitro. Domainator is made available as an open-source package with detailed documentation and usage examples.

The introduction of genome engineering technology has transformed biomedical research, making it possible to make precise changes to genetic information. However, creating an efficient gene-editing system requires a deep understanding of CRISPR technology, and the complex experimental systems under investigation. While Large Language Models (LLMs) have shown promise in various tasks, they often lack specific knowledge and struggle to accurately solve biological design problems. In this work, we introduce CRISPR-GPT, an LLM agent augmented with domain knowledge and external tools to automate and enhance the design process of CRISPR-based gene-editing experiments. CRISPR-GPT leverages the reasoning ability of LLMs to facilitate the process of selecting CRISPR systems, designing guide RNAs, recommending cellular delivery methods, drafting protocols, and designing validation experiments to confirm editing outcomes. We showcase the potential of CRISPR-GPT for assisting non-expert researchers with gene-editing experiments from scratch and validate the agent’s effectiveness in a real-world use case. Furthermore, we explore the ethical and regulatory considerations associated with automated gene-editing design, highlighting the need for responsible and transparent use of these tools. Our work aims to bridge the gap between biological researchers across various fields with CRISPR genome engineering technology and demonstrate the potential of LLM agents in facilitating complex biological discovery tasks.

CRISPR/Cas9-based targeted gene editing is a fundamental technique for studying gene functions in various organisms. In plants, the introduction of a T-DNA construct harboring Cas9 nuclease and single guide RNA (sgRNA) sequences induces sequence-specific DNA double-strand breaks, inducing the loss of gene function. Arabidopsis thaliana is a model for CRISPR/Cas9 system development and gene function studies; the introduction of Cas9 under the egg or zygote promoter and multiple sgRNA modules generates heritable or non-mosaic mutants for multiple targets in the T1 generation of A. thaliana . Recent reports reflect use of several CRISPR/Cas9 vectors in generating single– and higher-order mutants; however, the development of a reliable, cost-effective, and high-throughput CRISPR/Cas9 platform is necessary for targeting highly duplicated gene families. In this study, we have developed a simple and user-friendly construction system for the CRISPR/Cas9 vector series with improved gene editing efficiency by simply inserting a single intron into Cas9 , and effectively demonstrated the simultaneous knockout of multiple genes involved in A. thaliana sexual reproduction. An unbiased PCR-mediated mutant identification in the T1 generation revealed that our CRISPR/Cas9 system can support a > 70 kb deletion of > 30 tandemly duplicated synergid-specific genes and simultaneous knockout of five redundant genes essential for double fertilization. We performed a one-shot knockout of seven homologous pollen tube receptor-like kinase genes and identified their specific and overlapping roles in pollen tube growth and guidance. Our system can potentially facilitate further research in experimental plant biology to search for genetically unidentified components using reverse genetic candidate approaches.

Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world’s population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.

CRISPR prime editing offers unprecedented versatility and precision for the installation of genetic edits in situ . Here we describe the development and characterization of the Multiplexing Of Site-specific Alterations for In situ Characterization ( MOSAIC ) method, which leverages a non-viral PCR-based prime editing method to enable rapid installation of thousands of defined edits in pooled fashion. We show that MOSAIC can be applied to perform in situ saturation mutagenesis screens of: (1) the BCR-ABL1 fusion gene, successfully identifying known and potentially new imatinib drug-resistance variants; and (2) the IRF1 untranslated region (UTR), re-confirming non-coding regulatory elements involved in transcriptional initiation. Furthermore, we deployed MOSAIC to enable high-throughput, pooled screening of hundreds of systematically designed prime editing guide RNA ( pegRNA ) constructs for a large series of different genomic loci. This rapid screening of >18,000 pegRNA designs identified optimized pegRNAs for 89 different genomic target modifications and revealed the lack of simple predictive rules for pegRNA design, reinforcing the need for experimental optimization now greatly simplified and enabled by MOSAIC. We envision that MOSAIC will accelerate and facilitate the application of CRISPR prime editing for a wide range of high-throughput screens in human and other cell systems.

Abstract Genome editing in plants using CRISPR/ Cas9 typically involves integrating transgenic constructs into plant genome. However, a challenge arises after the target gene is successfully edited, transgene elements such as Cas9 , gRNA cassette, and selective marker genes remain integrated. This integration of transgenes causes regulatory and environmental concerns, particularly for commercialization. In addressing this issue, we present the establishment of a transgene-free genome editing system in sorghum, achieved through transient gene expression without selection. We selected the phytoene desaturase ( PDS ) gene as the target due to its capacity to induce a visible phenotypic change, namely albinism, upon mutation. Following microprojectile co-transformation with maize optimised Cas9 vector and a gRNA cassette with kanamycin resistance gene, immature embryo (IE) derived tissues were divided into two groups (selection and non-selection) and deployed as parallel experiments. Remarkably, 4 out of 18 homozygous/biallelic editing lines in the non-selection group were identified as transgene-free lines in the T 0 generation, with no traceable transgenes. Conversely, no transgene-free editing line was achieved in the selection group. This strategy not only enables to regenerate transgene-free genome-edited lines more efficiently but also saves one generation of time by eliminating the need for self-crossing or out-crossing. Our results displayed the feasibility of achieving transgene-free genome-edited plants within a single generation in sorghum. Furthermore, this approach opens avenues for vegetatively propagated crops like pineapple, sugarcane, and banana to obtain transgene-free genome-edited lines, facilitating their commercialization.

MARK2 , a member of the evolutionarily conserved PAR1/MARK serine/threonine kinase family, has been identified as a novel risk gene for autism spectrum disorder (ASD) based on the enrichment of de novo loss-of-function (Lof) variants in large-scale sequencing studies of ASD individuals. However, the features shared by affected individuals and the molecular mechanism of MARK2 variants during early neural development remained unclear. Here, we report 31 individuals carrying heterozygous MARK2 variants and presenting with ASD, other neurodevelopmental disorders, and typical facial dysmorphisms. Lof variants predominate (81%) in affected individuals, while computational analysis and in vitro transfection assay also point to MARK2 loss resulting from missense variants. Using patient-derived and CRISPR-engineered isogenic induced pluripotent stem cells (iPSCs), and Mark 2 +/- (HET) mice, we show that MARK2 loss leads to systemic neurodevelopmental deficits, including anomalous polarity in neural rosettes, imbalanced proliferation and differentiation in neural progenitor cells (NPCs), abnormal cortical development and ASD-like behaviors in mice. Further using RNA-Seq and lithium treatment, we link MARK2 loss to the downregulated WNT/β-catenin signaling pathway and identify lithium as a potential drug for treating MARK2 -related ASD.

SUMMARY MYC is a potent oncogene that is frequently overexpressed in human tumors arising in different tissues. To date there are no approved therapies to directly antagonize oncogenic MYC and its role in driving tumorigenesis. As an alternative approach we employed genetic screens using CRISPR and shRNA to identify the genes that are required for the survival and growth of cells harboring high levels of MYC expression. We find that cells with elevated MYC require the expression of many pro-growth and metabolic pathways including genes involved in mitochondrial citrate production and transport. This citrate producing pathway is critical for cells with elevated MYC to generate the necessary acetyl-CoA to drive the lipid synthesis required for increased proliferation. Inhibition of this pathway results in reduced proliferation and in vivo tumor growth providing a potential therapeutic strategy to target MYC-driven cancers. HIGHLIGHTS – CRISPR and shRNA screens identify synthetic lethal interactions with overexpressed MYC – MYC overexpressing cells are more sensitive to disruption of citrate production and transport – Inhibition of SLC25A1 reduces growth of MYC driven tumors

ABSTRACT The human pathogens Plasmodium and Schistosoma are each responsible for over 200 million infections annually, being particularly problematic in low- and middle-income countries. There is a pressing need for new drug targets for these diseases, driven by emergence of drug-resistance in Plasmodium and the overall dearth of new drug targets for Schistosoma . Here, we explored the opportunity for pathogen-hopping by evaluating a series of quinoxaline-based anti-schistosomal compounds for activity against P. falciparum . We identified compounds with low nanomolar potency against 3D7 and multidrug-resistant strains. Evolution of resistance using a mutator P. falciparum line revealed a low propensity for resistance. Only one of the series, compound 22, yielded resistance mutations, including point mutations in a non-essential putative hydrolase pfqrp1, as well as copy-number amplification of a phospholipid-translocating ATPase, pfatp2 , a potential target. Notably, independently generated CRISPR-edited mutants in pfqrp1 also showed resistance to compound 22 and a related analogue. Moreover, previous lines with pfatp2 copy-number variations were similarly less susceptible to challenge with the new compounds. Finally, we examined whether the predicted hydrolase activity of PfQRP1 underlies its mechanism of resistance, showing that both mutation of the putative catalytic triad and a more severe loss of function mutation elicited resistance. Collectively, we describe a compound series with potent activity against two important pathogens and their potential target in P. falciparum .

Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multi-scale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution, and the visualization of macromolecular complexes in situ , especially if these molecules are expressed with appropriately-engineered and easily-detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our technology encompasses a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion (HITI). As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express 6 fluorescently-tagged proteins and simultaneously visualize 5 different subcellular compartments. Our protocol leads from the in-silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.

Abstract Most Epstein–Barr virus-associated gastric carcinoma (EBVaGC) harbor non-silent mutations that activate phosphoinositide 3 kinase (PI3K) to drive downstream metabolic signaling. To gain insights into PI3K/mTOR pathway dysregulation in this context, we performed a human genome-wide CRISPR/Cas9 screen for hits that synergistically blocked EBVaGC proliferation together with the PI3K antagonist alpelisib. Multiple subunits of carboxy terminal to LisH (CTLH) E3 ligase, including the catalytic MAEA subunit, were among top screen hits. CTLH negatively regulates gluconeogenesis in yeast, but not in higher organisms. Instead, we identified that the CTLH substrates MKLN1 and ZMYND19, which highly accumulated upon MAEA knockout, associated with one another and with lysosomes to inhibit mTORC1. ZMYND19/MKLN1 bound Raptor and RagA/C, but rather than perturbing mTORC1 lysosomal recruitment, instead blocked a late stage of its activation, independently of the tuberous sclerosis complex. Thus, CTLH enables cells to rapidly tune mTORC1 activity at the lysosomal membrane via the ubiquitin/proteasome pathway.