RESUMO
Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.
Assuntos
Citidina Desaminase , Edição de Genes , Humanos , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/metabolismo , Dedos de Zinco , Citidina/genética , Sistemas CRISPR-CasRESUMO
Neuronal tau reduction confers resilience against ß-amyloid and tau-related neurotoxicity in vitro and in vivo. Here, we introduce a novel translational approach to lower expression of the tau gene MAPT at the transcriptional level using gene-silencing zinc finger protein transcription factors (ZFP-TFs). Following a single administration of adeno-associated virus (AAV), either locally into the hippocampus or intravenously to enable whole-brain transduction, we selectively reduced tau messenger RNA and protein by 50 to 80% out to 11 months, the longest time point studied. Sustained tau lowering was achieved without detectable off-target effects, overt histopathological changes, or molecular alterations. Tau reduction with AAV ZFP-TFs was able to rescue neuronal damage around amyloid plaques in a mouse model of Alzheimer's disease (APP/PS1 line). The highly specific, durable, and controlled knockdown of endogenous tau makes AAV-delivered ZFP-TFs a promising approach for the treatment of tau-related human brain diseases.
Assuntos
Doença de Alzheimer , Fatores de Transcrição , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Camundongos , Placa Amiloide/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Some camouflaged animals hide colour signals and display them only transiently. These hidden colour signals are often conspicuous and are used as a secondary defence to warn or startle predators (deimatic displays) and/or to confuse them (flash displays). The hidden signals used in these displays frequently resemble typical aposematic signals, so it is possible that prey with hidden signals have evolved to employ colour patterns of a form that predators have previously learned to associate with unprofitability. Here, we tested this hypothesis by conducting two experiments that examined the effect of predator avoidance learning on the efficacy of deimatic and flash displays. We found that the survival benefits of both deimatic and flash displays were substantially higher against predators that had previously learned to associate the hidden colours with unprofitability than against naive predators. These findings help explain the phenological patterns we found in 1568 macro-lepidopteran species on three continents: species with hidden signals tend to occur later in the season than species without hidden signals.
Assuntos
Aprendizagem da Esquiva , Pigmentação , Comportamento Predatório , Animais , Cor , Sinais (Psicologia) , Estações do AnoRESUMO
Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.
Assuntos
Clivagem do DNA , Edição de Genes/métodos , Linfócitos T , Sequência de Bases , DNA/genética , DNA/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas , Humanos , Células K562 , Domínios Proteicos , RNA MensageiroRESUMO
Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.
Assuntos
Alelos , Proteína Huntingtina/genética , Doença de Huntington/terapia , Mutação , Transcrição Gênica , Dedos de Zinco , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Doença de Huntington/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Neuroproteção , Repetições de TrinucleotídeosRESUMO
It has previously been shown that engineered zinc finger nucleases (ZFNs) can be packaged into adeno-associated viruses (AAVs) and delivered intravenously into mice, non-human primates, and most recently, humans to induce highly efficient therapeutic genome editing in the liver. Lipid nanoparticles (LNPs) are synthetic delivery vehicles that enable repeat administration and are not limited by the presence of preexisting neutralizing antibodies in patients. Here, we show that mRNA encoding ZFNs formulated into LNP can enable >90% knockout of gene expression in mice by targeting the TTR or PCSK9 gene, at mRNA doses 10-fold lower than has ever been reported. Additionally, co-delivering mRNA-LNP containing ZFNs targeted to intron 1 of the ALB locus with AAV packaged with a promoterless human IDS or FIX therapeutic transgene can result in high levels of targeted integration and subsequent therapeutically relevant levels of protein expression in mice. Finally, we show repeat administration of ZFN mRNA-LNP after a single AAV donor dose results in significantly increased levels of genome editing and transgene expression compared to a single dose. These results demonstrate LNP-mediated ZFN mRNA delivery can drive highly efficient levels of in vivo genome editing and can potentially offer a new treatment modality for a variety of diseases.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Edição de Genes/métodos , Nanopartículas/administração & dosagem , RNA Mensageiro/administração & dosagem , Nucleases de Dedos de Zinco/administração & dosagem , Animais , Células Cultivadas , Dependovirus/genética , Feminino , Técnicas de Inativação de Genes , Vetores Genéticos , Hepatócitos/metabolismo , Íntrons/genética , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pré-Albumina/genética , Pró-Proteína Convertase 9/genética , RNA Mensageiro/genética , Transgenes/genética , Nucleases de Dedos de Zinco/farmacologiaRESUMO
Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Edição de Genes/métodos , Genoma Humano , Engenharia de Proteínas/métodos , Nucleases de Dedos de Zinco/genética , Pareamento de Bases , Sequência de Bases , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Loci Gênicos , Biblioteca Genômica , Humanos , Mutação INDEL , Células K562 , Biblioteca de Peptídeos , Plasmídeos/química , Plasmídeos/metabolismo , Transformação Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Nucleases de Dedos de Zinco/metabolismoRESUMO
Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein-protein interactions, modifying protein-DNA interactions is more difficult. This may be related to the structural features of protein-DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein-DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Pareamento de Bases , DNA/química , Clivagem do DNA , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desoxirribonucleases/química , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Edição de Genes , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Recombinases/química , Recombinases/genética , Recombinases/metabolismo , Efetores Semelhantes a Ativadores de Transcrição/química , Efetores Semelhantes a Ativadores de Transcrição/genética , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Dedos de ZincoRESUMO
Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.
Assuntos
Desoxirribonucleases/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Receptores CCR5/metabolismo , Dedos de Zinco , Animais , Proteínas de Ligação a DNA/genética , Desoxirribonucleases/genética , Genes Reporter , Genoma , Humanos , Biblioteca de Peptídeos , Receptores CCR2/metabolismoRESUMO
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.
Assuntos
Albuminas/genética , Terapia de Reposição de Enzimas , Terapia Genética , Genoma , Fígado/metabolismo , Transgenes/fisiologia , Albuminas/metabolismo , Animais , Dependovirus/genética , Endonucleases , Doença de Fabry/genética , Doença de Fabry/terapia , Fator IX/genética , Fator VIII/genética , Doença de Gaucher/genética , Doença de Gaucher/terapia , Vetores Genéticos/administração & dosagem , Hemofilia A/genética , Hemofilia A/terapia , Hemofilia B/genética , Hemofilia B/terapia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lisossomos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Mucopolissacaridose I/genética , Mucopolissacaridose I/terapia , Mucopolissacaridose II/genética , Mucopolissacaridose II/terapia , Regiões Promotoras Genéticas/genética , Edição de RNA , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dedos de ZincoRESUMO
Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.
Assuntos
Regulação da Expressão Gênica/fisiologia , Genoma , Edição de RNA/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , DNA/genética , Ensaio de Imunoadsorção Enzimática , Marcadores Genéticos , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Endonucleases/genética , Fator IX/biossíntese , Terapia Genética/métodos , Vetores Genéticos , Genoma , Hemofilia B/terapia , Dedos de Zinco/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Endonucleases/metabolismo , Fator IX/genética , Fator IX/metabolismo , Hemofilia B/genética , Hemofilia B/patologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Multimerização ProteicaRESUMO
Long-term engraftment of allogeneic cells necessitates eluding immune-mediated rejection, which is currently achieved by matching for human leukocyte antigen (HLA) expression, immunosuppression, and/or delivery of donor-derived cells to sanctuary sites. Genetic engineering provides an alternative approach to avoid clearance of cells that are recognized as "non-self" by the recipient. To this end, we developed designer zinc finger nucleases and employed a "hit-and-run" approach to genetic editing for selective elimination of HLA expression. Electro-transfer of mRNA species coding for these engineered nucleases completely disrupted expression of HLA-A on human T cells, including CD19-specific T cells. The HLA-A(neg) T-cell pools can be enriched and evade lysis by HLA-restricted cytotoxic T-cell clones. Recognition by natural killer cells of cells that had lost HLA expression was circumvented by enforced expression of nonclassical HLA molecules. Furthermore, we demonstrate that zinc finger nucleases can eliminate HLA-A expression from embryonic stem cells, which broadens the applicability of this strategy beyond infusing HLA-disparate immune cells. These findings establish that clinically appealing cell types derived from donors with disparate HLA expression can be genetically edited to evade an immune response and provide a foundation whereby cells from a single donor can be administered to multiple recipients.
Assuntos
Desoxirribonucleases/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Transplante de Células-Tronco/métodos , Transplante Homólogo , Antígenos CD19/metabolismo , Sequência de Bases , Diferenciação Celular , Citotoxicidade Imunológica/imunologia , Eletroporação , Células-Tronco Embrionárias/citologia , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Leucócitos Mononucleares/citologia , Dados de Sequência Molecular , Engenharia de Proteínas , Linfócitos T/imunologia , Dedos de ZincoRESUMO
Medical schools conduct research, provide clinical care, and educate future physicians and scientists. Each school has its own unique mix of revenue sources and expense sharing among the medical school, faculty practice plan(s), parent university, and affiliated hospital(s). Despite these differences, revenues from clinical care subsidize the money-losing research and education missions at every medical school.In this perspective, the authors discuss the flow of funds among a medical school, its faculty practice plan(s), parent university, and affiliated hospital(s). They summarize where medical school revenues come from, how revenues and expenses flow within a medical school and between a medical school and its partners, and why understanding this process is crucial to leading and managing such an enterprise. They conclude with recommendations for medical schools to consider in developing funds flow models that meet their individual needs and circumstances: (1) understand economic drivers, (2) reward desired behaviors, (3) enable every unit to generate a positive margin, (4) communicate budget priorities, financial performance, and the use of institutional resources, and (5) establish principles for sharing resources and allocating expenses among entities within the institution.Medical schools should develop funds flow models that are transparent, aligned with their strategic priorities, and reward the behaviors necessary to produce effective collaboration within and across mission areas.
Assuntos
Administração Financeira/organização & administração , Faculdades de Medicina/economia , Centros Médicos Acadêmicos/economia , Centros Médicos Acadêmicos/organização & administração , Educação de Graduação em Medicina/economia , Docentes de Medicina , Humanos , Renda , Objetivos Organizacionais , Salários e Benefícios , Faculdades de Medicina/organização & administração , Estados UnidosRESUMO
Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR(+) T cells to eliminate expression of the endogenous αß T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or ß TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR(+)TCR(neg) T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.
Assuntos
Antígenos CD19/imunologia , Epitopos/imunologia , Engenharia Genética , Imunoterapia/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Adulto , Células Apresentadoras de Antígenos/imunologia , Antígenos de Neoplasias/imunologia , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Células Cultivadas , Endonucleases/metabolismo , Técnicas de Inativação de Genes , Humanos , Células K562 , Ativação Linfocitária/imunologia , Dedos de ZincoRESUMO
Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola ß-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Brassica napus/enzimologia , Brassica napus/genética , Engenharia Genética/métodos , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Dedos de Zinco/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Sequência de Bases , Cruzamentos Genéticos , DNA Complementar/genética , Ativação Enzimática , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Dados de Sequência Molecular , Folhas de Planta/enzimologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/metabolismo , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Marcação de Genes/métodos , Genoma Humano , Proteínas Recombinantes de Fusão/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Domínio Catalítico , Linhagem Celular Transformada , Linhagem Celular Tumoral , Clonagem Molecular , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Reparo do DNA por Junção de Extremidades , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos , Histonas/metabolismo , Humanos , Mutação INDEL , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Receptores CCR5/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transformação GenéticaRESUMO
Zinc-finger nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.
Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Integrase de HIV/genética , Dedos de Zinco/genética , Sítios de Ligação , Linhagem Celular Tumoral , Análise por Conglomerados , Endodesoxirribonucleases/metabolismo , Marcação de Genes , Loci Gênicos , Vetores Genéticos/genética , Integrase de HIV/metabolismo , Humanos , Lentivirus/genética , Ligação Proteica , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).