ABSTRACT
HIV-1 remains a global health crisis1, highlighting the need to identify new targets for therapies. Here, given the disproportionate HIV-1 burden and marked human genome diversity in Africa2, we assessed the genetic determinants of control of set-point viral load in 3,879 people of African ancestries living with HIV-1 participating in the international collaboration for the genomics of HIV3. We identify a previously undescribed association signal on chromosome 1 where the peak variant associates with an approximately 0.3 log10-transformed copies per ml lower set-point viral load per minor allele copy and is specific to populations of African descent. The top associated variant is intergenic and lies between a long intergenic non-coding RNA (LINC00624) and the coding gene CHD1L, which encodes a helicase that is involved in DNA repair4. Infection assays in iPS cell-derived macrophages and other immortalized cell lines showed increased HIV-1 replication in CHD1L-knockdown and CHD1L-knockout cells. We provide evidence from population genetic studies that Africa-specific genetic variation near CHD1L associates with HIV replication in vivo. Although experimental studies suggest that CHD1L is able to limit HIV infection in some cell types in vitro, further investigation is required to understand the mechanisms underlying our observations, including any potential indirect effects of CHD1L on HIV spread in vivo that our cell-based assays cannot recapitulate.
Subject(s)
DNA Helicases , DNA-Binding Proteins , Genetic Variation , HIV Infections , HIV-1 , Viral Load , Humans , Cell Line , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HIV Infections/genetics , HIV-1/growth & development , HIV-1/physiology , Viral Load/genetics , Africa , Chromosomes, Human, Pair 1/genetics , Alleles , RNA, Long Noncoding/genetics , Virus ReplicationABSTRACT
Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP:
Subject(s)
Genetic Techniques , Mice, Knockout , Phenotype , Animals , Disease/genetics , Disease Models, Animal , Female , Genes, Essential , Genome-Wide Association Study , Male , MiceABSTRACT
With the wide adoption of genomic sequencing in children having seizures, an increasing number of SCN2A genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent SCN2A genetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENT A mounting number of SCN2A genetic variants have been identified from patients with epilepsy, but how SCN2A variants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurring SCN2A variant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform for in vitro drug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenic SCN2A variant.
Subject(s)
Epilepsy/genetics , Epilepsy/physiopathology , Gene Editing/methods , NAV1.2 Voltage-Gated Sodium Channel/genetics , Neurons/pathology , Cerebral Cortex/physiopathology , Humans , Induced Pluripotent Stem Cells/pathology , Induced Pluripotent Stem Cells/physiology , MutationABSTRACT
Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis.
Subject(s)
Brain Neoplasms/genetics , CRISPR-Cas Systems , Gene Targeting/methods , Glioma/genetics , Neural Stem Cells/cytology , Alleles , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Brain Neoplasms/metabolism , Epitope Mapping , Epitopes , Glioma/metabolism , Green Fluorescent Proteins/metabolism , Homologous Recombination , Humans , Mice , Mice, Knockout , Mutation , Nerve Tissue Proteins/genetics , Oligodendrocyte Transcription Factor 2 , Oligonucleotides/genetics , Point Mutation , Recombination, Genetic , Regenerative Medicine , TransgenesABSTRACT
Loss-of-function studies are key for investigating gene function, and CRISPR technology has made genome editing widely accessible in model organisms and cells. However, conditional gene inactivation in diploid cells is still difficult to achieve. Here, we present CRISPR-FLIP, a strategy that provides an efficient, rapid and scalable method for biallelic conditional gene knockouts in diploid or aneuploid cells, such as pluripotent stem cells, 3D organoids and cell lines, by co-delivery of CRISPR-Cas9 and a universal conditional intronic cassette.
Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Embryonic Stem Cells/cytology , Gene Editing/methods , Gene Knockout Techniques/methods , beta Catenin/genetics , Animals , Cell Line , Genome/genetics , HEK293 Cells , Humans , MiceABSTRACT
The generation of induced pluripotent stem cell models of human disease requires efficient modification of one or both alleles depending on dominant or recessive inheritance of the disease. To faithfully recapitulate many disease variants, the introduction of a single base change is required. The introduction of additional silent mutations designed to prevent re-cutting of the modified allele by Cas9 is not an optimal strategy, particularly for non-coding variants. Here, we developed an improved protocol for efficient engineering of single nucleotide variants in human iPS cells. Using a fluorescent BFP->GFP assay to monitor the incorporation of a single base pair change, we optimized the protocol to achieve HDR in 70% of unselected human iPS cells. The additive effects of cold shock, a small molecule enhancer of HDR and chemically modified ssODN dramatically shift the bias of repair in favor of HDR, resulting in a seven-fold higher ratio of HDR to NHEJ from 0.5 to 3.7.
Subject(s)
Gene Editing/methods , Induced Pluripotent Stem Cells , Recombinational DNA Repair , CRISPR-Cas Systems/genetics , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cell Line , Culture Media/chemistry , DNA Breaks, Double-Stranded , DNA End-Joining Repair , HEK293 Cells , Humans , Oligonucleotides/genetics , Polymorphism, Single Nucleotide , RNA, Guide, Kinetoplastida/genetics , Transfection/methodsABSTRACT
Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.
Subject(s)
Alleles , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Gene Knockout Techniques/methods , Animals , Cell Line , Chromosomal Proteins, Non-Histone/genetics , Gene Expression , Genetic Vectors , Histone-Lysine N-Methyltransferase/genetics , Mice , Polycomb Repressive Complex 2/geneticsABSTRACT
Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.
Subject(s)
Biomedical Research/methods , Cardiovascular Agents/pharmacology , Cell Lineage , Drug Discovery/methods , Heart Diseases/drug therapy , High-Throughput Screening Assays , Induced Pluripotent Stem Cells/physiology , Myocytes, Cardiac/physiology , Toxicity Tests/methods , Cardiovascular Agents/toxicity , Cell Differentiation , Cell Proliferation , Cells, Cultured , Genotype , Heart Diseases/chemically induced , Heart Diseases/metabolism , Heart Diseases/pathology , Heart Diseases/physiopathology , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Phenotype , Risk AssessmentABSTRACT
Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.
Subject(s)
CRISPR-Cas Systems , Deoxyribonuclease I/metabolism , Endonucleases/metabolism , Genome, Bacterial , Animals , Base Sequence , Deoxyribonuclease I/genetics , Embryo, Mammalian , Gene Expression Regulation, Enzymologic , HeLa Cells , Humans , Mice , Mutation , RNA, BacterialABSTRACT
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
Subject(s)
Gene Deletion , Gene Knockout Techniques/methods , Genes/genetics , Genetic Association Studies/methods , Genome/genetics , Mice, Knockout/genetics , Alleles , Animals , Computational Biology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Genes, Lethal/genetics , Genetic Vectors/genetics , Genomics , Genotype , Humans , Mice , Mice, Inbred C57BL , Mutagenesis, Insertional/methods , Phenotype , Polymerase Chain Reaction , RatsABSTRACT
UNLABELLED: The rapid development of CRISPR-Cas9 mediated genome editing techniques has given rise to a number of online and stand-alone tools to find and score CRISPR sites for whole genomes. Here we describe the Wellcome Trust Sanger Institute Genome Editing database (WGE), which uses novel methods to compute, visualize and select optimal CRISPR sites in a genome browser environment. The WGE database currently stores single and paired CRISPR sites and pre-calculated off-target information for CRISPRs located in the mouse and human exomes. Scoring and display of off-target sites is simple, and intuitive, and filters can be applied to identify high-quality CRISPR sites rapidly. WGE also provides a tool for the design and display of gene targeting vectors in the same genome browser, along with gene models, protein translation and variation tracks. WGE is open, extensible and can be set up to compute and present CRISPR sites for any genome. AVAILABILITY AND IMPLEMENTATION: The WGE database is freely available at www.sanger.ac.uk/htgt/wge CONTACT: : vvi@sanger.ac.uk or skarnes@sanger.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Databases, Factual , Gene Expression Regulation , Genetic Vectors , Genome , RNA Editing/genetics , Animals , Humans , Mice , SoftwareABSTRACT
The International Mouse Phenotyping Consortium (IMPC) web portal (http://www.mousephenotype.org) provides the biomedical community with a unified point of access to mutant mice and rich collection of related emerging and existing mouse phenotype data. IMPC mouse clinics worldwide follow rigorous highly structured and standardized protocols for the experimentation, collection and dissemination of data. Dedicated 'data wranglers' work with each phenotyping center to collate data and perform quality control of data. An automated statistical analysis pipeline has been developed to identify knockout strains with a significant change in the phenotype parameters. Annotation with biomedical ontologies allows biologists and clinicians to easily find mouse strains with phenotypic traits relevant to their research. Data integration with other resources will provide insights into mammalian gene function and human disease. As phenotype data become available for every gene in the mouse, the IMPC web portal will become an invaluable tool for researchers studying the genetic contributions of genes to human diseases.
Subject(s)
Databases, Genetic , Mice, Knockout , Phenotype , Animals , Biological Ontologies , Internet , MiceABSTRACT
Gracia-Diaz and colleagues analysed high-density DNA microarray and whole genome sequencing (WGS) data from the KOLF2.1J 'reference' human induced pluripotent stem cell (hiPSC) line1, and report the presence of five high-confidence heterozygous copy number variants (CNVs) at least 100kbp in length2. Since three of these CNVs span coding genes, some of which have been associated with neurodevelopmental disease, the authors raise the concern that these CNVs may compromise the utility of KOLF2.1J for neurological disease modelling. We appreciate their thorough analysis and thoughtful interpretation, and agree that potential users of this line should be made aware of all cases where KOLF2.1J differs from the reference genome. However, we believe that the benefits from the widespread use of KOLF2.1J outweigh the potential risks that might arise from the identified CNVs.
ABSTRACT
Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.
Subject(s)
Amyotrophic Lateral Sclerosis , Induced Pluripotent Stem Cells , Neurodegenerative Diseases , Humans , Amyotrophic Lateral Sclerosis/metabolism , Microglia/metabolism , Induced Pluripotent Stem Cells/metabolism , Profilins/metabolism , MutationABSTRACT
The Sanger Mouse Genetics Project generates knockout mice strains using the EUCOMM/KOMP-CSD embryonic stem (ES) cell collection and characterizes the consequences of the mutations using a high-throughput primary phenotyping screen. Upon achieving germline transmission, new strains are subject to a panel of quality control (QC) PCR- and qPCR-based assays to confirm the correct targeting, cassette structure, and the presence of the 3' LoxP site (required for the potential conditionality of the allele). We report that over 86 % of the 731 strains studied showed the correct targeting and cassette structure, of which 97 % retained the 3' LoxP site. We discuss the characteristics of the lines that failed QC and postulate that the majority of these may be due to mixed ES cell populations which were not detectable with the original screening techniques employed when creating the ES cell resource.
Subject(s)
Embryonic Stem Cells/cytology , Germ Cells/cytology , Mice, Mutant Strains/genetics , Animals , Breeding , Mice , Quality ControlSubject(s)
CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Animals , Breeding , CRISPR-Associated Proteins/genetics , Deoxyribonuclease I/genetics , Deoxyribonuclease I/metabolism , Female , Gene Expression Regulation, Enzymologic , Genome , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence AlignmentABSTRACT
We have developed dual recombinase-mediated cassette exchange (dRMCE) to efficiently re-engineer the thousands of available conditional alleles in mouse embryonic stem cells. dRMCE takes advantage of the wild-type loxP and FRT sites present in these conditional alleles and in many gene-trap lines. dRMCE is a scalable, flexible tool to introduce tags, reporters and mutant coding regions into an endogenous locus of interest in an easy and highly efficient manner.
Subject(s)
Embryonic Stem Cells/metabolism , Genetic Engineering/methods , Recombinases/physiology , Alleles , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Carrier Proteins/physiology , Intracellular Signaling Peptides and Proteins , Mice , Nerve Tissue Proteins/physiology , Nuclear Proteins/physiology , Promoter Regions, Genetic , Recombination, Genetic , Smad4 Protein/geneticsABSTRACT
We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations were reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable high-throughput modification of many genes in mouse ESCs.