RESUMEN
Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.
Asunto(s)
Neoplasias Endometriales , Sarcoma Estromático Endometrial , Sarcoma , Cromatina , Proteínas de Unión al ADN/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Femenino , Histonas/metabolismo , Humanos , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Sarcoma/genética , Sarcoma Estromático Endometrial/genética , Sarcoma Estromático Endometrial/metabolismo , Sarcoma Estromático Endometrial/patología , Translocación Genética/genéticaRESUMEN
In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins.
Asunto(s)
Bacteriófagos/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/metabolismo , Streptococcus thermophilus/enzimología , Proteínas Virales/metabolismo , Regulación Alostérica , Bacteriófagos/genética , Sitios de Unión , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/ultraestructura , ADN/genética , ADN/ultraestructura , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Células K562 , Cinética , Mutación , Unión Proteica , Conformación Proteica , Streptococcus thermophilus/genética , Relación Estructura-Actividad , Proteínas Virales/genética , Proteínas Virales/ultraestructuraRESUMEN
The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Acetilación , Sitios de Unión , Unión Competitiva , Sistemas CRISPR-Cas , Proteínas Cromosómicas no Histona/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Histona Acetiltransferasas/genética , Histonas/genética , Humanos , Células K562 , Lisina Acetiltransferasa 5 , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Transfección , Proteína 1 de Unión al Supresor Tumoral P53/genética , UbiquitinaciónRESUMEN
Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Streptococcus thermophilus/enzimología , Streptococcus thermophilus/genética , Animales , Proteína 9 Asociada a CRISPR/química , Línea Celular , Células Cultivadas , División del ADN , Humanos , Mamíferos , Ratones , Ratones Noqueados , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
BACKGROUND AIMS: The culture and ex vivo engineering of red blood cells (RBCs) can help characterize genetic variants, model diseases, and may eventually spur the development of applications in transfusion medicine. In the last decade, improvements to the in vitro production of RBCs have enabled efficient erythroid progenitor proliferation and high enucleation levels from several sources of hematopoietic stem and progenitor cells (HSPCs). Despite these advances, there remains a need for refining the terminal step of in vitro human erythropoiesis, i.e., the terminal maturation of reticulocytes into erythrocytes, so that it can occur without feeder or accessory cells and animal-derived components. METHODS: Here, we describe the near-complete erythroid differentiation of cultured RBCs (cRBCs) from adult HSPCs in accessory-cell-free and xeno-free conditions. RESULTS: The approach improves post-enucleation cell integrity and cell survival, and it enables subsequent storage of cRBCs for up to 42 days in classical additive solution conditions without any specialized equipment. CONCLUSIONS: We foresee that these improvements will facilitate the characterization of RBCs derived from gene-edited HSPCs.
Asunto(s)
Eritrocitos , Células Madre Hematopoyéticas , Animales , Adulto , Humanos , Diferenciación Celular/genética , EritropoyesisRESUMEN
Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.
Asunto(s)
Sistemas CRISPR-Cas/genética , Células Cultivadas/fisiología , Reparación del ADN/genética , Edición Génica/métodos , Mutagénesis Sitio-Dirigida , Marcadores Genéticos/genética , HumanosRESUMEN
Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.
Asunto(s)
Albúminas/genética , Terapia de Reemplazo Enzimático , Terapia Genética , Genoma , Hígado/metabolismo , Transgenes/fisiología , Albúminas/metabolismo , Animales , Dependovirus/genética , Endonucleasas , Enfermedad de Fabry/genética , Enfermedad de Fabry/terapia , Factor IX/genética , Factor VIII/genética , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/terapia , Vectores Genéticos/administración & dosificación , Hemofilia A/genética , Hemofilia A/terapia , Hemofilia B/genética , Hemofilia B/terapia , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lisosomas/enzimología , Ratones , Ratones Endogámicos C57BL , Mucopolisacaridosis I/genética , Mucopolisacaridosis I/terapia , Mucopolisacaridosis II/genética , Mucopolisacaridosis II/terapia , Regiones Promotoras Genéticas/genética , Edición de ARN , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Dedos de ZincRESUMEN
Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.
Asunto(s)
Reparación del ADN/genética , Modelos Animales de Enfermedad , Marcación de Gen/métodos , Terapia Genética/métodos , Genoma/genética , Hemofilia B/genética , Hemostasis , Animales , Secuencia de Bases , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Endonucleasas/química , Endonucleasas/genética , Endonucleasas/metabolismo , Exones/genética , Factor IX/análisis , Factor IX/genética , Vectores Genéticos/genética , Células HEK293 , Hemofilia B/fisiopatología , Humanos , Intrones/genética , Hígado/metabolismo , Regeneración Hepática , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Fenotipo , Homología de Secuencia , Dedos de ZincRESUMEN
The HBO1 HAT protein is the major source of histone H4 acetylation in vivo and has been shown to play critical roles in gene regulation and DNA replication. A distinctive characteristic of HBO1 HAT complexes is the presence of three PHD finger domains in two different subunits: tumor suppressor proteins ING4/5 and JADE1/2/3. Biochemical and functional analyses indicate that these domains interact with histone H3 N-terminal tail region, but with a different specificity toward its methylation status. Their combinatorial action is essential in regulating chromatin binding and substrate specificity of HBO1 complexes, as well as cell growth. Importantly, localization analyses on the human genome indicate that HBO1 complexes are enriched throughout the coding regions of genes, supporting a role in transcription elongation. These results underline the importance and versatility of PHD finger domains in regulating chromatin association and histone modification crosstalk within a single protein complex.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Acetilación , Sitios de Unión , Células Cultivadas , Proteínas de Unión al ADN/genética , Células HeLa , Histona Acetiltransferasas/genética , Histonas/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Metilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
In this chapter, we intend to review gene therapy concepts applied to the potential treatment of tyrosinemia for parents and pediatricians. Therefore, our main objective is to give general informations in a comprehensible manner. Considering the nature of tyrosinemia and the current state of technology, a particular focus will be put on strategies using viral delivery of DNA to the liver. In light of the recent development of the CRISPR technology and the revival of promises for previously unavailable therapeutical tools, the present chapter aims at presenting up to date facts and potential pitfalls towards an application for metabolic diseases, in particular tyrosinemia.
Asunto(s)
Tirosinemias/genética , Tirosinemias/terapia , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Hígado/metabolismoRESUMEN
Spinal cord injury (SCI) causes the release of danger signals by stressed and dying cells, a process that leads to neuroinflammation. Evidence suggests that inflammation plays a role in both the damage and repair of injured neural tissue. We show that microglia at sites of SCI rapidly express the alarmin interleukin (IL)-1α, and that infiltrating neutrophils and macrophages subsequently produce IL-1ß. Infiltration of these cells is dramatically reduced in both IL-1α(-/-) and IL-1ß(-/-) mice, but only IL-1α(-/-) mice showed rapid (at day 1) and persistent improvements in locomotion associated with reduced lesion volume. Similarly, intrathecal administration of the IL-1 receptor antagonist anakinra restored locomotor function post-SCI. Transcriptome analysis of SCI tissue at day 1 identified the survival factor Tox3 as being differentially regulated exclusively in IL-1α(-/-) mice compared with IL-1ß(-/-) and wild-type mice. Accordingly, IL-1α(-/-) mice have markedly increased Tox3 levels in their oligodendrocytes, beginning at postnatal day 10 (P10) and persisting through adulthood. At P10, the spinal cord of IL-1α(-/-) mice showed a transient increase in mature oligodendrocyte numbers, coinciding with increased IL-1α expression in wild-type animals. In adult mice, IL-1α deletion is accompanied by increased oligodendrocyte survival after SCI. TOX3 overexpression in human oligodendrocytes reduced cellular death under conditions mimicking SCI. These results suggest that IL-1α-mediated Tox3 suppression during the early phase of CNS insult plays a crucial role in secondary degeneration. SIGNIFICANCE STATEMENT: The mechanisms underlying bystander degeneration of neurons and oligodendrocytes after CNS injury are ill defined. We show that microglia at sites of spinal cord injury (SCI) rapidly produce the danger signal interleukin (IL)-1α, which triggers neuroinflammation and locomotor defects. We uncovered that IL-1α(-/-) mice have markedly increased levels of the survival factor Tox3 in their oligodendrocytes, which correlates with the protection of this cell population, and reduced lesion volume, resulting in unprecedented speed, level, and persistence of functional recovery after SCI. Our data suggest that central inhibition of IL-1α or Tox3 overexpression during the acute phase of a CNS insult may be an effective means for preventing the loss of neurological function in SCI, or other acute injuries such as ischemia and traumatic brain injuries.
Asunto(s)
Interleucina-1alfa/biosíntesis , Degeneración Nerviosa/fisiopatología , Oligodendroglía/metabolismo , Receptores de Progesterona/biosíntesis , Traumatismos de la Médula Espinal/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Eliminación de Gen , Proteínas del Grupo de Alta Movilidad , Humanos , Immunoblotting , Inmunohistoquímica , Interleucina-1alfa/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Recuperación de la Función/fisiología , Transactivadores , Regulación hacia ArribaRESUMEN
Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.
Asunto(s)
Endonucleasas/metabolismo , Neoplasias/enzimología , Neoplasias/genética , Translocación Genética , Dedos de Zinc , Línea Celular , Puntos de Rotura del Cromosoma , Humanos , Nucleofosmina , Proteínas Tirosina Quinasas/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismoRESUMEN
Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here, we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system, we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to â¼1%-8%. Interestingly, ZFNickases targeting the AAVS1 "safe harbor" locus revealed similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ, but stimulated far lower levels of gene addition-suggesting that other, yet to be identified mediators of nick-induced gene targeting exist. Introduction of site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e., HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Marcación de Gen/métodos , Genoma Humano , Proteínas Recombinantes de Fusión/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Dominio Catalítico , Línea Celular Transformada , Línea Celular Tumoral , Clonación Molecular , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Reparación del ADN por Unión de Extremidades , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos , Histonas/metabolismo , Humanos , Mutación INDEL , Datos de Secuencia Molecular , Ingeniería de Proteínas/métodos , Receptores CCR5/genética , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transformación GenéticaRESUMEN
Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.
Asunto(s)
Endonucleasas/genética , Factor IX/biosíntesis , Terapia Genética/métodos , Vectores Genéticos , Genoma , Hemofilia B/terapia , Dedos de Zinc/genética , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Endonucleasas/metabolismo , Factor IX/genética , Factor IX/metabolismo , Hemofilia B/genética , Hemofilia B/patología , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Multimerización de ProteínaRESUMEN
Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.
Asunto(s)
Biotecnología/métodos , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Marcación de Gen/métodos , Genoma de Planta/genética , Zea mays/genética , Dedos de Zinc , Desoxirribonucleasas/genética , Alimentos Modificados Genéticamente , Genes de Plantas/genética , Resistencia a los Herbicidas/genética , Herbicidas/farmacología , Herencia , Fosfatos de Inositol/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Plantas Modificadas Genéticamente , Recombinación Genética/genética , Reproducibilidad de los ResultadosRESUMEN
Zinc-finger nucleases (ZFNs) drive efficient genome editing by introducing a double-strand break into the targeted gene. Cleavage is induced when two custom-designed ZFNs heterodimerize upon binding DNA to form a catalytically active nuclease complex. The importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization. Here we report the development and application of a yeast-based selection system designed to functionally interrogate the ZFN dimer interface. We identified critical residues involved in dimerization through the isolation of cold-sensitive nuclease domains. We used these residues to engineer ZFNs that have superior cleavage activity while suppressing homodimerization. The improvements were portable to orthogonal domains, allowing the concomitant and independent cleavage of two loci using two different ZFN pairs. These ZFN architectures provide a general means for obtaining highly efficient and specific genome modification.
Asunto(s)
Endonucleasas/metabolismo , Dedos de Zinc/fisiología , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Endonucleasas/genética , Genoma , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Dedos de Zinc/genéticaRESUMEN
The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus.
Asunto(s)
Alelos , Proteínas Portadoras/genética , Desoxirribonucleasas/genética , Marcación de Gen/métodos , Proteínas de Xenopus/genética , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/metabolismo , Desoxirribonucleasas/metabolismo , Xenopus , Proteínas de Xenopus/metabolismo , Dedos de ZincRESUMEN
Zinc-finger nucleases (ZFNs) are powerful tools for editing the genomes of cell lines and model organisms. Given the breadth of their potential application, simple methods that increase ZFN activity, thus ensuring genome modification, are highly attractive. Here we show that transient hypothermia generally and robustly increased the level of stable, ZFN-induced gene disruption, thereby providing a simple technique to enhance the experimental efficacy of ZFNs.
Asunto(s)
Desoxirribonucleasas/metabolismo , Marcación de Gen/métodos , Dedos de Zinc , Frío , Células HeLa , Humanos , Células K562RESUMEN
Bacterial community collapse due to phage infection is a major risk in cheese making processes. As virulent phages are ubiquitous and diverse in milk fermentation factories, the use of phage-resistant lactic acid bacteria (LAB) is essential to obtain high-quality fermented dairy products. The LAB species Streptococcus thermophilus contains two type II-A CRISPR-Cas systems (CRISPR1 and CRISPR3) that can effectively protect against phage infection. However, virulent streptococcal phages carrying anti-CRISPR proteins (ACR) that block the activity of CRISPR-Cas systems have emerged in yogurt and cheese environments. For example, phages carrying AcrIIA5 can impede both CRISPR1 and CRISPR3 systems, while AcrIIA6 stops only CRISPR1. Here, we explore the activity and diversity of a third streptococcal phage anti-CRISPR protein, namely AcrIIA3. We were able to demonstrate that AcrIIA3 is efficiently active against the CRISPR3-Cas system of S. thermophilus. We used AlphaFold2 to infer the structure of AcrIIA3 and we predicted that this new family of functional ACR in virulent streptococcal phages has a new α-helical fold, with no previously identified structural homologs. Because ACR proteins are being explored as modulators in genome editing applications, we also tested AcrIIA3 against SpCas9. We found that AcrIIA3 could block SpCas9 in bacteria but not in human cells. Understanding the diversity and functioning of anti-defence mechanisms will be of importance in the design of long-term stable starter cultures.
Asunto(s)
Bacteriófagos , Fagos de Streptococcus , Humanos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Fagos de Streptococcus/genética , Sistemas CRISPR-Cas/genética , Edición GénicaRESUMEN
Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we design a robust co-selection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). We readily engineer highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process reveals that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific co-selection strategies.