RESUMEN
Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery.
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Sistemas CRISPR-Cas , Técnicas de Sustitución del Gen , Animales , Sistemas CRISPR-Cas/genética , Ratones , Masculino , Técnicas de Sustitución del Gen/métodos , Línea Celular , Testículo/citología , Testículo/metabolismo , Edición Génica/métodos , Técnicas de Cultivo de Célula/métodos , Células Madre Germinales Adultas/metabolismo , Células Germinativas/metabolismo , Células Germinativas/citologíaRESUMEN
We generated a human induced pluripotent stem cell (hiPSC) line (CMCi014-A-78) expressing a GFP reporter in the 3'-UTR region of the KLOTHO locus using CRISPR/Cas9-mediated homologous recombination to screen for candidates regulating KLOTHO. The established cell line exhibits a normal karyotype, typical stem cell morphology, expression of pluripotency markers, and the ability to differentiate into the three germ layers. Consequently, this hiPSC line could serve as a valuable resource for screening KLOTHO regulators in hiPSC-derived target cells or organoids.
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Regiones no Traducidas 3' , Glucuronidasa , Proteínas Fluorescentes Verdes , Células Madre Pluripotentes Inducidas , Proteínas Klotho , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Glucuronidasa/metabolismo , Glucuronidasa/genética , Línea Celular , Sistemas CRISPR-Cas , Genes Reporteros , Diferenciación Celular , Técnicas de Sustitución del Gen/métodos , Sitios GenéticosRESUMEN
The organic anion transporting polypeptide (OATP) 2B1 is considered an emerging drug transporter that is found expressed in pharmacokinetically relevant organs such as the liver, small intestine, and kidney. Despite its interaction with various substrate drugs, the understanding of its in vivo relevance is still limited. In this study, we first validated the interaction of atorvastatin with rat OATP2B1 using transiently transfected HeLa cells. Moreover, we characterized our rSlco2b1-knockout and SLCO2B1-knockin rats for mRNA, protein expression, and localization of OATP2B1 in the liver, small intestine, and kidney. The transporter showed the highest expression in the liver followed by the small intestine. In humanized rats, human OATP2B1 is localized on the sinusoidal membrane of hepatocytes. In enterocytes of wild-type and humanized rats, the transporter was detected in the luminal membrane with the vast majority being localized subapical. Subsequently, we assessed atorvastatin pharmacokinetics in male wild-type, rSlco2b1-knockout, and SLCO2B1-knockin rats after a single-dose administration (orally and intravenously). Investigating the contribution of rat OATP2B1 or human OATP2B1 to oral atorvastatin pharmacokinetics revealed no differences in concentration-time profiles or pharmacokinetic parameters. However, when comparing the pharmacokinetics of atorvastatin after intravenous administration in SLCO2B1-humanized rats and knockout animals, notable differences were observed. In particular, the systemic exposure (area under the curve) decreased by approximately 40% in humanized animals, whereas the clearance was 57% higher in animals expressing human OATP2B1. These findings indicate that human OATP2B1 influences pharmacokinetics of atorvastatin after intravenous administration, most likely by contributing to the hepatic uptake. SIGNIFICANCE STATEMENT: Wild-type, rSlco2b1-knockout, and SLCO2B1-humanized Wistar rats were characterized for the expression of rat and human SLCO2B1/OATP2B1. Pharmacokinetic studies of atorvastatin over 24 hours were conducted in male wild-type, rSlco2b1-knockout, and SLCO2B1-humanized rats. After a single-dose intravenous administration, a lower systemic exposure and an increase in clearance were observed in SLCO2B1-humanized rats compared with knockout animals indicating a contribution of OATP2B1 to the hepatic clearance.
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Atorvastatina , Hígado , Transportadores de Anión Orgánico , Animales , Atorvastatina/farmacocinética , Atorvastatina/administración & dosificación , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Humanos , Masculino , Ratas , Hígado/metabolismo , Células HeLa , Ratas Transgénicas , Intestino Delgado/metabolismo , Técnicas de Inactivación de Genes/métodos , Riñón/metabolismo , Técnicas de Sustitución del Gen/métodos , Administración Oral , Administración Intravenosa , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Hepatocitos/metabolismo , Distribución TisularRESUMEN
The integration of CRISPR/Cas9 genome editing techniques with organoid technology has revolutionized the field of tumor modeling, enabling the creation of diverse tumor models with distinct mutational profiles. This protocol details the application of CRISPR knock-ins to engineer tumor organoids with reporter cassettes, which are regulated by endogenous promoters of specific genes of interest. This approach facilitates the precise fluorescent labeling, isolation, and subsequent manipulation of targeted tumor cell subpopulations. The utilization of these knock-in reporter cassettes not only allows the visualization and purification of specific tumor cell subsets but also enables conditional cell ablation and lineage tracing studies. In this chapter, we provide a comprehensive guide for the design, construction, delivery, and validation of CRISPR/Cas9 tools tailored for knock-in reporter cassette integration into specific marker genes of interest. By following this protocol, researchers can harness the potential of engineered tumor organoids to decipher intricate tumor heterogeneity, track metastatic trajectories, and unveil novel therapeutic vulnerabilities linked to specific tumor cell subpopulations.
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Sistemas CRISPR-Cas , Edición Génica , Técnicas de Sustitución del Gen , Organoides , Organoides/metabolismo , Organoides/patología , Humanos , Técnicas de Sustitución del Gen/métodos , Edición Génica/métodos , Animales , Neoplasias/genética , Neoplasias/patología , Genes ReporterosRESUMEN
Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.
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Aptámeros de Nucleótidos , Sistemas CRISPR-Cas , Edición Génica , Técnicas de Inactivación de Genes , Linfocitos T , Humanos , Edición Génica/métodos , Aptámeros de Nucleótidos/genética , Linfocitos T/metabolismo , Linfocitos T/inmunología , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Técnicas de Sustitución del Gen/métodos , TransgenesRESUMEN
CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQR and enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce 'recoding' mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM's design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.
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Sistemas CRISPR-Cas , Técnicas de Sustitución del Gen , ARN Guía de Sistemas CRISPR-Cas , Programas Informáticos , Técnicas de Sustitución del Gen/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Humanos , Algoritmos , Internet , Edición Génica/métodos , Reparación del ADN por Recombinación , Mutación , Mutagénesis InsercionalRESUMEN
Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating tardigrades have long been desired. Despite our prior success in somatic cell gene editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades.
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Sistemas CRISPR-Cas , Edición Génica , Homocigoto , Partenogénesis , Tardigrada , Animales , Tardigrada/genética , Edición Génica/métodos , Partenogénesis/genética , Femenino , Técnicas de Sustitución del Gen/métodos , Técnicas de Inactivación de Genes , AlelosRESUMEN
Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3'-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.
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Sistemas CRISPR-Cas , Edición Génica , Humanos , Edición Génica/métodos , Células HEK293 , Técnicas de Sustitución del Gen/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismoRESUMEN
Forkhead box protein J1 (FOXJ1), a member of the forkhead family, is an important transcription factor regulating multiciliated cell differentiation and motile ciliogenic program. Here, we established a FOXJ1- EGFP knock-in human embryonic stem cell (hESC) line by inserting a P2A-EGFP gene cassette of FOXJ1 using CRISPR/Cas9 system. The reporter cell line retained a normal karyotype, expressed comparable pluripotent marker genes, and maintained differentiation potential. This reporter cell line enables live identification of multiciliated cells during the general lung differentiation and will be a valuable tool for studying the multiciliated cell differentiation, ciliogenesis and mechanism of related pulmonary diseases.
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Sistemas CRISPR-Cas , Factores de Transcripción Forkhead , Células Madre Embrionarias Humanas , Humanos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Línea Celular , Diferenciación Celular , Técnicas de Sustitución del Gen/métodos , Marcación de Gen/métodos , Genes ReporterosRESUMEN
HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have generated Hand2Dre, a knock-in allele expressing Dre recombinase. To avoid disrupting Hand2 function, the Dre cDNA is inserted at the 3' end of the Hand2 coding sequence following a viral 2A peptide. Hand2Dre homozygotes can therefore be used in complex crosses to increase the proportion of useful genotypes among offspring. Dre expression in mid-gestation Hand2Dre embryos is indistinguishable from wild-type Hand2 expression, and HandDre efficiently recombines rox target sites in vivo. In combination with existing Cre and Flp mouse lines, Hand2Dre will therefore extend the ability to perform genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by developmental expression of Hand2.
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Alelos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Técnicas de Sustitución del Gen , Animales , Femenino , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Técnicas de Sustitución del Gen/métodos , Integrasas/genética , Integrasas/metabolismo , MasculinoRESUMEN
Gene knock-in refers to the insertion of exogenous functional genes into a target genome to achieve continuous expression. Currently, most knock-in tools are based on site-directed nucleases, which can induce double-strand breaks (DSBs) at the target, following which the designed donors carrying functional genes can be inserted via the endogenous gene repair pathway. The size of donor genes is limited by the characteristics of gene repair, and the DSBs induce risks like genotoxicity. New generation tools, such as prime editing, transposase, and integrase, can insert larger gene fragments while minimizing or eliminating the risk of DSBs, opening new avenues in the development of animal models and gene therapy. However, the elimination of off-target events and the production of delivery carriers with precise requirements remain challenging, restricting the application of the current knock-in treatments to mainly in vitro settings. Here, a comprehensive review of the knock-in tools that do not/minimally rely on DSBs and use other mechanisms is provided. Moreover, the challenges and recent advances of in vivo knock-in treatments in terms of the therapeutic process is discussed. Collectively, the new generation of DSBs-minimizing and large-fragment knock-in tools has revolutionized the field of gene editing, from basic research to clinical treatment.
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Roturas del ADN de Doble Cadena , Técnicas de Sustitución del Gen , Terapia Genética , Técnicas de Sustitución del Gen/métodos , Animales , Humanos , Terapia Genética/métodos , Edición Génica/métodosRESUMEN
Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.
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Linaje de la Célula , Técnicas de Sustitución del Gen , Células Madre Pluripotentes , Humanos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Linaje de la Célula/genética , Técnicas de Sustitución del Gen/métodos , Genes Reporteros , Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas/genética , Línea Celular , Edición Génica/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Precise insertion of fluorescent tags by CRISPR-Cas9-mediated homologous recombination (HR) in mammalian genes is a powerful tool allowing to study gene function and protein gene products. Here, we present a protocol for efficient HR-mediated targeted insertion of fluorescent markers in the genome of hard-to-transfect erythroid cell lines MEL (mouse erythroleukemic) and MEDEP (mouse ES cell-derived erythroid progenitor line). We describe steps for plasmid construction, electroporation, amplification, and verification of genome editing. We then detail procedures for isolating positive clones and validating knockin clones. For complete details on the use and execution of this protocol, please refer to Deleuze et al.1.
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Sistemas CRISPR-Cas , Células Eritroides , Edición Génica , Técnicas de Sustitución del Gen , Sistemas CRISPR-Cas/genética , Animales , Ratones , Células Eritroides/metabolismo , Células Eritroides/citología , Técnicas de Sustitución del Gen/métodos , Edición Génica/métodos , Transfección/métodos , Línea Celular , Recombinación Homóloga/genética , Electroporación/métodosRESUMEN
Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is caused by a loss-of-function mutation in CDKL5 gene, encoding a serine-threonine kinase highly expressed in the brain. CDD manifests with early-onset epilepsy, autism, motor impairment and severe intellectual disability. While there are no known treatments for CDD, the use of cannabidiol has recently been introduced into clinical practice for neurodevelopmental disorders. Given the increased clinical utilization of cannabidiol, we examined its efficacy in the CDKL5R59X knock-in (R59X) mice, a CDD model based on a human mutation that exhibits both lifelong seizure susceptibility and behavioural deficits. We found that cannabidiol pre-treatment rescued the increased seizure susceptibility in response to the chemoconvulsant pentylenetetrazol (PTZ), attenuated working memory and long-term memory impairments, and rescued social deficits in adult R59X mice. To elucidate a potential mechanism, we compared the developmental hippocampal and cortical expression of common endocannabinoid (eCB) targets in R59X mice and their wild-type littermates, including cannabinoid type 1 receptor (CB1R), transient receptor potential vanilloid type 1 (TRPV1) and 2 (TRPV2), G-coupled protein receptor 55 (GPR55) and adenosine receptor 1 (A1R). Many of these eCB targets were developmentally regulated in both R59X and wild-type mice. In addition, adult R59X mice demonstrated significantly decreased expression of CB1R and TRPV1 in the hippocampus, and TRPV2 in the cortex, while TRPV1 was increased in the cortex. These findings support the potential for dysregulation of eCB signalling as a plausible mechanism and therapeutic target in CDD, given the efficacy of cannabidiol to attenuate hyperexcitability and behavioural deficits in this disorder.
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Cannabidiol , Proteínas Serina-Treonina Quinasas , Convulsiones , Animales , Cannabidiol/farmacología , Convulsiones/tratamiento farmacológico , Convulsiones/genética , Convulsiones/metabolismo , Ratones , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Síndromes Epilépticos/genética , Síndromes Epilépticos/tratamiento farmacológico , Pentilenotetrazol , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen/métodos , Masculino , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Endocannabinoides/metabolismo , Conducta Animal/efectos de los fármacos , Ratones Endogámicos C57BL , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Espasmos Infantiles , Receptores de CannabinoidesRESUMEN
Transgenic mice expressing human major histocompatibility complex class II (MHCII) risk alleles are widely used in autoimmune disease research, but limitations arise due to non-physiologic expression. To address this, physiologically relevant mouse models are established via knock-in technology to explore the role of MHCII in diseases like rheumatoid arthritis. The gene sequences encoding the ectodomains are replaced with the human DRB1*04:01 and 04:02 alleles, DRA, and CD74 (invariant chain) in C57BL/6N mice. The collagen type II (Col2a1) gene is modified to mimic human COL2. Importantly, DRB1*04:01 knock-in mice display physiologic expression of human MHCII also on thymic epithelial cells, in contrast to DRB1*04:01 transgenic mice. Humanization of the invariant chain enhances MHCII expression on thymic epithelial cells, increases mature B cell numbers in spleen, and improves antigen presentation. To validate its functionality, the collagen-induced arthritis (CIA) model is used, where DRB1*04:01 expression led to a higher susceptibility to arthritis, as compared with mice expressing DRB1*04:02. In addition, the humanized T cell epitope on COL2 allows autoreactive T cell-mediated arthritis development. In conclusion, the humanized knock-in mouse faithfully expresses MHCII, confirming the DRB1*04:01 alleles role in rheumatoid arthritis and being also useful for studying MHCII-associated diseases.
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Alelos , Antígenos de Diferenciación de Linfocitos B , Artritis Reumatoide , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Antígenos de Histocompatibilidad Clase II , Ratones Endogámicos C57BL , Ratones Transgénicos , Animales , Ratones , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/inmunología , Humanos , Técnicas de Sustitución del Gen/métodos , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Artritis Experimental/genética , Artritis Experimental/inmunología , Cadenas HLA-DRB1/genética , Cadenas HLA-DRB1/inmunología , Colágeno Tipo II/genética , Colágeno Tipo II/inmunologíaRESUMEN
BACKGROUND: The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route. RESULTS: We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods. CONCLUSIONS: Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9-4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.
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Sistemas CRISPR-Cas , Dependovirus , Ratones , Animales , Dependovirus/genética , Reproducibilidad de los Resultados , Cigoto , Marcación de Gen , Técnicas de Sustitución del Gen/métodosRESUMEN
Various DNA breaks created via programmable CRISPR/Cas9 nuclease activity results in different intracellular DNA break repair pathways. Based on the cellular repair pathways, CRISPR-based gene knock-in methods can be categorized into two major strategies: 1) Homology-independent strategies which are targeted insertion events based on non-homologous end joining, and 2) Homology-dependent strategies which are targeted insertion events based on the homology-directed repair. This review elaborates on various gene knock-in methods in mammalian cells using the CRISPR/Cas9 system and in sync with DNA-break repair pathways. Gene knock-in methods are applied in functional genomics and gene therapy. To compensate or correct genetic defects, different CRISPR-based gene knock-in strategies can be used. Thus, researchers need to make a conscious decision about the most suitable knock-in method. For a successful gene-targeted insertion, some determinant factors should be considered like cell cycle, dominant DNA repair pathway, size of insertions, and donor properties. In this review, different aspects of each gene knock-in strategy are discussed to provide a framework for choosing the most appropriate gene knock-in method in different applications.
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Reparación del ADN/genética , Reparación del ADN/fisiología , Técnicas de Sustitución del Gen/métodos , Animales , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , ADN/química , ADN/metabolismo , Roturas del ADN/efectos de los fármacos , Reparación del ADN por Unión de Extremidades/genética , Edición Génica/métodos , Humanos , Reparación del ADN por Recombinación/genéticaRESUMEN
OBJECTIVE: The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding. METHODS: Using a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding. RESULTS: We found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior. CONCLUSIONS: Our results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation.
Asunto(s)
Núcleo Hipotalámico Paraventricular/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , Animales , Regulación de la Temperatura Corporal/fisiología , Encéfalo/metabolismo , Núcleo Hipotalámico Dorsomedial/metabolismo , Técnicas de Sustitución del Gen/métodos , Hipotálamo/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Neuronas/metabolismo , Receptor de Melanocortina Tipo 4/fisiología , Médula Espinal/metabolismoAsunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Técnicas de Sustitución del Gen/métodos , Transgenes/genética , Línea Celular , Reparación del ADN por Unión de Extremidades/genética , Terapia Genética , Células HEK293 , Humanos , Selección de Paciente , Reparación del ADN por Recombinación/genéticaRESUMEN
Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR era in the field of avian genome engineering.