RESUMO
CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genoma , DNA/genética , Pinças ÓpticasRESUMO
CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Edição de Genes/normas , Genoma/genética , Mutação , Especificidade por Substrato/genética , Animais , Proteínas Associadas a CRISPR/genética , Feminino , Humanos , Mutação INDEL , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/genética , Transgenes/genéticaRESUMO
Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.
Assuntos
Edição de Genes/métodos , Hepatócitos/transplante , Mutação , Doença da Deficiência de Ornitina Carbomoiltransferase/terapia , Ornitina Carbamoiltransferase/genética , Adulto , Idoso , Amônia/metabolismo , Animais , Células Cultivadas , Criança , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Hepatócitos/química , Hepatócitos/citologia , Humanos , Íntrons , Masculino , Camundongos , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Ácido Orótico/urina , Splicing de RNARESUMO
The mutation patterns at Cas9 targeted sites contain unique information regarding the nuclease activity and repair mechanisms in mammalian cells. However, analytical framework for extracting such information are lacking. Here, we present a novel computational platform called Rational InDel Meta-Analysis (RIMA) that enables an in-depth comprehensive analysis of Cas9-induced genetic alterations, especially InDels mutations. RIMA can be used to quantitate the contribution of classical microhomology-mediated end joining (c-MMEJ) pathway in the formation of mutations at Cas9 target sites. We used RIMA to compare mutational signatures at 15 independent Cas9 target sites in human A549 wildtype and A549-POLQ knockout cells to elucidate the role of DNA polymerase θ in c-MMEJ. Moreover, the single nucleotide insertions at the Cas9 target sites represent duplications of preceding nucleotides, suggesting that the flexibility of the Cas9 nuclease domains results in both blunt- and staggered-end cuts. Thymine at the fourth nucleotide before protospacer adjacent motif (PAM) results in a two-fold higher occurrence of single nucleotide InDels compared to guanine at the same position. This study provides a novel approach for the characterization of the Cas9 nucleases with improved accuracy in predicting genome editing outcomes and a potential strategy for homology-independent targeted genomic integration.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Reparo do DNA por Junção de Extremidades , Mutação INDEL , Software , Células A549 , Algoritmos , Sequência de Bases , Linhagem Celular , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/metabolismo , Conjuntos de Dados como Assunto , Francisella/enzimologia , Humanos , Motivos de Nucleotídeos , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/enzimologia , Especificidade por Substrato , DNA Polimerase tetaRESUMO
BACKGROUND: Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. RESULTS: To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. CONCLUSIONS: Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.
Assuntos
Colesterol/sangue , Hipercolesterolemia/genética , Fígado/metabolismo , Pró-Proteína Convertase 9/genética , Animais , Modelos Animais de Doenças , Edição de Genes , Genoma , Humanos , Hipercolesterolemia/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: Base Editing is a precise genome editing method that uses a deaminase-Cas9 fusion protein to mutate cytidine to thymidine in target DNA in situ without the generation of a double-strand break. However, the efficient enrichment of genetically modified cells using this technique is limited by the ability to detect such events. RESULTS: We have developed a Base Editing FLuorescent Activity REporter (BE-FLARE), which allows for the enrichment of cells that have undergone editing of target loci based on a fluorescence shift from BFP to GFP. We used BE-FLARE to evaluate the editing efficiency of APOBEC3A and APOBEC3B family members as alternatives deaminase domains to the rat APOBEC1 domain used in base editor 3 (BE3). We identified human APOBEC3A and APOBEC3B as highly efficient cytidine deaminases for base editing applications with unique properties. CONCLUSIONS: Using BE-FLARE to report on the efficiency and precision of editing events, we outline workflows for the accelerated generation of genetically engineered cell models and the discovery of alternative base editors.
Assuntos
Desaminase APOBEC-1/genética , Citidina Desaminase/genética , Edição de Genes/métodos , Engenharia Genética/métodos , Antígenos de Histocompatibilidade Menor/genética , Proteínas/genética , Animais , Humanos , RatosRESUMO
apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/agonistas , Apolipoproteínas E/agonistas , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacologia , Animais , Apolipoproteínas E/metabolismo , Aziridinas/química , Aziridinas/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Células Cultivadas , Humanos , Camundongos , Camundongos Knockout , Estrutura Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Antagonistas do Receptor Purinérgico P2X/química , Receptores Purinérgicos P2X7/deficiência , Bibliotecas de Moléculas Pequenas/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.
Assuntos
Sistemas CRISPR-Cas , Descoberta de Drogas/métodos , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Rim/fisiologia , Organoides/fisiologia , Podócitos/fisiologia , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Regulação da Expressão Gênica , Genótipo , Ensaios de Triagem em Larga Escala , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/ultraestrutura , Fenótipo , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/ultraestrutura , Fatores de Tempo , TranscriptomaRESUMO
Custom-designed nucleases (CDNs) greatly facilitate genetic engineering by generating a targeted DNA double-strand break (DSB) in the genome. Once a DSB is created, specific modifications can be introduced around the breakage site during its repair by two major DNA damage repair (DDR) mechanisms: the dominant but error-prone nonhomologous end joining (NHEJ) pathway, and the less-frequent but precise homologous recombination (HR) pathway. Here we describe ObLiGaRe, a new method for site-specific gene insertions that uses the efficient NHEJ pathway and acts independently of HR. This method is applicable with both zinc finger nucleases (ZFNs) and Tale nucleases (TALENs), and has enabled us to insert a 15-kb inducible gene expression cassette at a defined locus in human cell lines. In addition, our experiments have revealed the previously underestimated error-free nature of NHEJ and provided new tools to further characterize this pathway under physiological and pathological conditions.
Assuntos
Reparo do DNA por Junção de Extremidades , Endonucleases/genética , Engenharia Genética/métodos , Mutagênese Insercional , Recombinação Genética , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Endonucleases/metabolismo , Regulação da Expressão Gênica , Loci Gênicos , Genoma Humano , Células HCT116 , Humanos , Camundongos , Dedos de Zinco/genéticaRESUMO
Whereas bacterial artificial chromosomes (BACs) offer many advantages in studies of gene and protein function, generation of seamless, precisely mutated BACs has been difficult. Here we describe a counterselection-based recombineering method and its accompanying reagents. After identifying intramolecular recombination as the major problem in counterselection, we built a strategy to reduce these unwanted events by expressing Redß alone at the crucial step. We enhanced this method by using phosphothioated oligonucleotides, using a sequence-altered rpsL counterselection gene and developing online software for oligonucleotide design. We illustrated this method by generating transgenic mammalian cell lines carrying small interfering RNA-resistant and point-mutated BAC transgenes. Using this approach, we generated mutated TACC3 transgenes to identify phosphorylation-specific spindle defects after knockdown of endogenous TACC3 expression. Our results highlight the complementary use of precisely mutated BAC transgenes and RNA interference in the study of cell biology at physiological expression levels and regulation.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Mutagênese Sítio-Dirigida/métodos , Oligonucleotídeos/genética , Recombinação Genética/genética , Linhagem Celular Tumoral , Farmacorresistência Bacteriana , Proteínas de Escherichia coli/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Engenharia de Proteínas/métodos , Interferência de RNA , Proteína S9 Ribossômica , Proteínas Ribossômicas/genética , Software , TransgenesRESUMO
Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (â¼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery.
Assuntos
Marcação de Genes/métodos , Engenharia Genética/métodos , Variação Genética , Genoma Humano , Haplótipos , Recombinação Genética , Alelos , Linhagem Celular , Clonagem Molecular , Biblioteca Gênica , Genômica , HumanosRESUMO
Increased saturated fatty acid (SFA) levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased SFA content in cell membranes negatively impacts adipocyte insulin signaling. Preadipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking twofold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2-knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human preadipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high-saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.
Assuntos
Insulina , Fosfolipídeos , Camundongos , Humanos , Animais , Insulina/metabolismo , Adipócitos/metabolismo , Ácidos Graxos/metabolismo , Membrana Celular/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.
Assuntos
Pró-Proteína Convertase 9 , Translocação Genética , Animais , Camundongos , Pró-Proteína Convertase 9/genética , Sistemas CRISPR-Cas/genética , Mutação , Endonucleases/genética , Streptococcus pyogenes/genéticaRESUMO
Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (PolÏ´). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Proteínas Quinases/genética , Reparo do DNA/genética , DNA/genéticaRESUMO
The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.
RESUMO
Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. BAC (bacterial artificial chromosome) transgenes present a way to express a chosen protein at physiological levels with all regulatory elements in their native configurations, including cell cycle, alternative splicing and microRNA regulation. Recombineering has become the method of choice for modifying large constructs like BACs. Here, we present a method for protein tagging by recombineering BACs, transfecting cells and evaluating tagged protein expression.
Assuntos
Cromossomos Artificiais Bacterianos , Proteômica/métodos , Transgenes , Animais , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/análise , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , TransfecçãoRESUMO
Protein tagging offers many advantages for proteomic and regulomic research. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. Physiological expression of tagged proteins can be achieved by gene targeting to knock-in the protein tag or by BAC transgenesis. BAC transgenes usually retain the native gene architecture including all cis-regulatory elements as well as the exon-intron configurations. Consequently most BAC transgenes are authentically regulated (e.g. by transcription factors, cell cycle, miRNA) and can be alternatively spliced. Recombineering has become the method of choice for generating targeting constructs or modifying BACs. Here we present methods with detailed protocols for protein tagging by recombineering for BAC transgenesis and/or gene targeting, including the evaluation of tagged protein expression, the retrieval of associated protein complexes for mass spectrometry and the use of the tags in ChIP (chromatin immunoprecipitation).
Assuntos
Western Blotting/métodos , Imunoprecipitação da Cromatina/métodos , Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular/métodos , Imunoprecipitação/métodos , Proteínas Recombinantes de Fusão/biossíntese , Transgenes , Animais , Cromossomos Artificiais Bacterianos/metabolismo , Células-Tronco Embrionárias/metabolismo , Epitopos , Genótipo , Humanos , Espectrometria de Massas/métodos , Camundongos , Reação em Cadeia da Polimerase/métodos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genéticaRESUMO
Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing leads to increased levels of by-products, it can rescue pegRNAs that perform poorly with a nickase-based prime editor. We also present a small molecule approach that yields increased product purity of PEn editing. Next, we develop a homology-independent PEn editing strategy, which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Animais , Sistemas CRISPR-Cas , Reparo do DNA , Endonucleases/metabolismo , Edição de Genes , Mamíferos/genéticaRESUMO
The fatty acid composition of phosphatidylethanolamine (PE) determines cellular metabolism, oxidative stress, and inflammation. However, our understanding of how cells regulate PE composition is limited. Here, we identify a genetic locus on mouse chromosome 11, containing two poorly characterized genes Tlcd1 and Tlcd2, that strongly influences PE composition. We generated Tlcd1/2 double-knockout (DKO) mice and found that they have reduced levels of hepatic monounsaturated fatty acid (MUFA)-containing PE species. Mechanistically, TLCD1/2 proteins act cell intrinsically to promote the incorporation of MUFAs into PEs. Furthermore, TLCD1/2 interact with the mitochondria in an evolutionarily conserved manner and regulate mitochondrial PE composition. Lastly, we demonstrate the biological relevance of our findings in dietary models of metabolic disease, where Tlcd1/2 DKO mice display attenuated development of non-alcoholic steatohepatitis compared to controls. Overall, we identify TLCD1/2 proteins as key regulators of cellular PE composition, with our findings having broad implications in understanding and treating disease.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Fosfatidiletanolaminas , Animais , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfatidiletanolaminas/metabolismoRESUMO
How cells maintain vital membrane lipid homeostasis while obtaining most of their constituent fatty acids from a varied diet remains largely unknown. Here, we used transcriptomics, lipidomics, growth and respiration assays, and membrane property analyses in human HEK293 cells or human umbilical vein endothelial cells (HUVEC) to show that the function of AdipoR2 is to respond to membrane rigidification by regulating many lipid metabolism genes. We also show that AdipoR2-dependent membrane homeostasis is critical for growth and respiration in cells challenged with saturated fatty acids. Additionally, we found that AdipoR2 deficiency causes transcriptome and cell physiological defects similar to those observed in SREBP-deficient cells upon SFA challenge. Finally, we compared several genes considered important for lipid homeostasis, namely AdipoR2, SCD, FADS2, PEMT and ACSL4, and found that AdipoR2 and SCD are the most important among these to prevent membrane rigidification and excess saturation when human cells are challenged with exogenous SFAs. We conclude that AdipoR2-dependent membrane homeostasis is one of the primary mechanisms that protects against exogenous SFAs.