RESUMO
Prime editors have high potential for disease modelling and regenerative medicine efforts including those directed at the muscle-wasting disorder Duchenne muscular dystrophy (DMD). However, the large size and multicomponent nature of prime editing systems pose substantial production and delivery issues. Here, we report that packaging optimized full-length prime editing constructs in adenovector particles (AdVPs) permits installing precise DMD edits in human myogenic cells, namely, myoblasts and mesenchymal stem cells (up to 80% and 64%, respectively). AdVP transductions identified optimized prime-editing reagents capable of correcting DMD reading frames of â¼14% of patient genotypes and restoring dystrophin synthesis and dystrophin-ß-dystroglycan linkages in unselected DMD muscle cell populations. AdVPs were equally suitable for correcting DMD iPSC-derived cardiomyocytes and delivering dual prime editors tailored for DMD repair through targeted exon 51 deletion. Moreover, by exploiting the cell cycle-independent AdVP transduction process, we report that 2- and 3-component prime-editing modalities are both most active in cycling than in post-mitotic cells. Finally, we establish that combining AdVP transduction with seamless prime editing allows for stacking chromosomal edits through successive delivery rounds. In conclusion, AdVPs permit versatile investigation of advanced prime editing systems independently of their size and component numbers, which should facilitate their screening and application.
Assuntos
Distrofina , Terapia Genética , Distrofia Muscular de Duchenne , Humanos , Sistemas CRISPR-Cas/genética , Distrofina/genética , Distrofina/metabolismo , Edição de Genes , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Mioblastos/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and 'double-cut' designs, respectively. Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs), in contrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Humanos , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Genômica , Células-Tronco Pluripotentes Induzidas , MamíferosRESUMO
Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs.
Assuntos
Distrofina , Edição de Genes , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Sistemas CRISPR-Cas/genética , Distrofina/genética , Distrofina/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Humanos , Células Musculares/metabolismo , Distrofia Muscular de Duchenne/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Prime editing is a recent precision genome editing modality whose versatility offers the prospect for a wide range of applications, including the development of targeted genetic therapies. Yet, an outstanding bottleneck for its optimization and use concerns the difficulty in delivering large prime editing complexes into cells. Here, we demonstrate that packaging prime editing constructs in adenoviral capsids overcomes this constrain resulting in robust genome editing in both transformed and non-transformed human cells with up to 90% efficiencies. Using this cell cycle-independent delivery platform, we found a direct correlation between prime editing activity and cellular replication and disclose that the proportions between accurate prime editing events and unwanted byproducts can be influenced by the target-cell context. Hence, adenovector particles permit the efficacious delivery and testing of prime editing reagents in human cells independently of their transformation and replication statuses. The herein integrated gene delivery and gene editing technologies are expected to aid investigating the potential and limitations of prime editing in numerous experimental settings and, eventually, in ex vivo or in vivo therapeutic contexts.
Assuntos
Adenoviridae/genética , Edição de Genes/métodos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Deleção de Genes , Células HEK293 , Células HeLa , HumanosRESUMO
RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by 'tiptoeing' over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.
Assuntos
Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Desoxirribonuclease I/metabolismo , Edição de Genes/métodos , Proteínas de Bactérias/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Células Clonais , Desoxirribonuclease I/genética , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Reporter , Técnicas de Genotipagem , Células HEK293 , Células HeLa , Heterocromatina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas , Polimorfismo Genético , RNA Guia de Cinetoplastídeos/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/enzimologia , Especificidade por Substrato , TransfecçãoRESUMO
Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.
Assuntos
Sistemas CRISPR-Cas/genética , DNA/genética , Desoxirribonuclease I/química , Edição de Genes/métodos , Sequência de Bases , DNA/química , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Desoxirribonuclease I/genética , Endonucleases/química , Marcação de Genes/métodos , Genoma/genética , Humanos , Mutação/genética , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Enhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Terapia Genética , Vetores Genéticos/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles.
Assuntos
Distrofina/metabolismo , Endonucleases/metabolismo , Mioblastos/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Adenoviridae/genética , Alelos , Sequência de Bases , Western Blotting , Sistemas CRISPR-Cas , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Distrofina/genética , Endonucleases/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Células HEK293 , Células HeLa , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Mutação , RNA Guia de Cinetoplastídeos/genética , Transdução GenéticaRESUMO
Transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases derived from clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems have become ubiquitous genome editing tools. Despite this, the impact that distinct high-order chromatin conformations have on these sequence-specific designer nucleases is, presently, ill-defined. The same applies to the relative performance of TALENs and CRISPR/Cas9 nucleases at isogenic target sequences subjected to different epigenetic modifications. Here, to address these gaps in our knowledge, we have implemented quantitative cellular systems based on genetic reporters in which the euchromatic and heterochromatic statuses of designer nuclease target sites are stringently controlled by small-molecule drug availability. By using these systems, we demonstrate that TALENs and CRISPR/Cas9 nucleases are both significantly affected by the high-order epigenetic context of their target sequences. In addition, this outcome could also be ascertained for S. pyogenes CRISPR/Cas9 complexes harbouring Cas9 variants whose DNA cleaving specificities are superior to that of the wild-type Cas9 protein. Thus, the herein investigated cellular models will serve as valuable functional readouts for screening and assessing the role of chromatin on designer nucleases based on different platforms or with different architectures or compositions.
Assuntos
Sistemas CRISPR-Cas/genética , Cromatina/genética , Edição de Genes , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Epigênese Genética/genética , Engenharia Genética , Genótipo , Humanos , Conformação Molecular , Streptococcus pyogenes/genéticaRESUMO
Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.
Assuntos
Adenoviridae/genética , DNA Viral/genética , DNA/química , Desoxirribonucleases/química , Engenharia Genética/métodos , Linhagem Celular , Separação Celular , Marcação de Genes/métodos , Genoma , Células HEK293 , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Reprodutibilidade dos TestesRESUMO
The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.
Assuntos
Adenoviridae/genética , Proteínas de Bactérias/genética , Desoxirribonucleases/genética , HIV-1/genética , Cromossomos Humanos , Quebras de DNA de Cadeia Dupla , Loci Gênicos , Vetores Genéticos , Células HEK293 , Células HeLa , Humanos , Transdução GenéticaRESUMO
BACKGROUND: The AdEasy system has acquired preeminence amongst the various methods for producing first-generation, early region 1 (E1)-deleted human adenovirus (HAdV) vectors (AdVs) as a result of the fast and reproducible recovery of full-length AdV genomes via homologous recombination in Escherichia coli. METHODS: From the classical AdEasy system, a new production platform was derived to assemble first- and second-generation [i.e. E1- plus early region 2A (E2A)-deleted] AdVs displaying on their surface HAdV serotype 5 (HAdV5) fibers (F5) or chimeric fibers (F5/50) comprising the tail of F5 and the fiber shaft and knob of HAdV serotype 50 (HAdV50). The CD46-interacting chimeric fibers allow for the high-level transduction of various human primary cell types of clinical interest with low or no surface expression of the Coxsackievirus and adenovirus receptor. RESULTS: A new set of pAdEasy plasmid 'backbones' with or without E2A and encoding F5 or F5/50 was constructed and recombined in E. coli strain BJ5183 with a 'shuttle' plasmid coding for ß-galactosidase. The resulting clones yielded AdV preparations with similar high titers following their rescue and propagation in producer cells. The AdVs with F5/50 were superior to those carrying F5 with respect to transducing human skeletal myocytes and mesenchymal stem cells. CONCLUSIONS: In the present study, an AdEasy system tailored for the production of not only first-, but also second-generation AdVs equipped with the receptor-interacting fiber domains of the prototypic species C HAdV5 or of the species B member HAdV50 is presented. This system expands the range of applications for this robust and versatile AdV production platform.
Assuntos
Adenovírus Humanos/genética , Enterovirus/genética , Vetores Genéticos/genética , Proteína Cofatora de Membrana/genética , Tropismo Viral , Western Blotting , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Perfilação da Expressão Gênica , Células HeLa , Humanos , Células-Tronco Mesenquimais/metabolismo , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Transdução Genética , beta-Galactosidase/genéticaRESUMO
Genome editing based on dual CRISPR-Cas9 complexes (multiplexes) permits removing specific genomic sequences in living cells leveraging research on functional genomics and genetic therapies. Delivering the required large and multicomponent reagents in a synchronous and stoichiometric manner remains, however, challenging. Moreover, uncoordinated activity of independently acting CRISPR-Cas9 multiplexes increases the complexity of genome editing outcomes. Here, we investigate the potential of fostering precise multiplexing genome editing using high-capacity adenovector particles (AdVPs) for the delivery of Cas9 ortholog fusion constructs alone (forced Cas9 heterodimers) or together with their cognate guide RNAs (forced CRISPR-Cas9 heterodimers). We demonstrate that the efficiency and accuracy of targeted chromosomal DNA deletions achieved by single AdVPs encoding forced CRISPR-Cas9 heterodimers is superior to that obtained when the various components are delivered separately. Finally, all-in-one AdVP delivery of forced CRISPR-Cas9 heterodimers triggers robust DMD exon 51 splice site excision resulting in reading frame restoration and selection-free detection of dystrophin in muscle cells derived from Duchenne muscular dystrophy patients. In conclusion, AdVPs promote precise multiplexing genome editing through the integrated delivery of forced CRISPR-Cas9 heterodimer components, which, in comparison with split conventional CRISPR-Cas9 multiplexes, engage target sequences in a more coordinated fashion.
RESUMO
There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a predictable and efficient manner remains challenging. Here, we investigated the principle of combining domains from different lineage-specific TFs to improve directed cellular differentiation. As proof-of-concept, we engineered the whole-human TF MyoDCD, which has the NH(2)-terminal transcription activation domain (TAD) and adjacent DNA-binding motif of MyoD COOH-terminally fused to the TAD of myocardin (MyoCD). We found via reporter gene and marker protein assays as well as by a cell fusion readout system that, targeting the TAD of MyoCD to genes normally responsive to the skeletal muscle-specific TF MyoD enforces more robust myogenic reprogramming of nonmuscle cells than that achieved by the parental, prototypic master TF, MyoD. Human mesenchymal stem cells (hMSCs) transduced with a codon-optimized microdystrophin gene linked to a synthetic striated muscle-specific promoter and/or with MyoD or MyoDCD were evaluated for complementing the genetic defect in Duchenne muscular dystrophy (DMD) myocytes through heterotypic cell fusion. Cotransduction of hMSCs with MyoDCD and microdystrophin led to chimeric myotubes containing the highest dystrophin levels.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Fatores de Transcrição/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Fatores de Transcrição/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo DMD-correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective DMD alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in E1 and E2A. Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% ± 13% of defective DMD alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both DMD gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of ß-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of DMD gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of DMD-correcting agents.
Assuntos
Adenoviridae/genética , Edição de Genes/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , Distrofia Muscular de Duchenne/genética , Humanos , Distrofia Muscular de Duchenne/patologiaRESUMO
JNK and ERK MAP kinases regulate cellular responses to genotoxic stress in a cell type and cell context-dependent manner. However, the factors that determine and execute JNK- and ERK-controlled stress responses are only partly known. In this study, we investigate the roles of the AP-1 components ATF3 and Fra1 in JNK- and ERK-dependent cell cycle arrest and apoptosis. We show that the anti-cancer drug cisplatin or UV light activates both JNK and ERK in human glioblastoma cells lacking functional p53. Inhibition experiments of JNK or ERK activities revealed that the ERK pathway strongly promotes cisplatin- and UV-induced apoptosis in these glioblastoma cells. Furthermore, JNK but not ERK is required for ATF3 induction, and both ERK and JNK are necessary for post-transcriptional induction of Fra1 in response to cisplatin or UV. Knock-down of ATF3 and Fra1 results in increased and decreased cisplatin-induced apoptosis, respectively, indicating that ATF3 is an anti-apoptotic JNK effector and Fra1 is a pro-apoptotic ERK/JNK effector. Knock-down experiments also revealed that ATF3 and Fra1, respectively, enhance and reduce S-phase arrest through differential modulation of the Chk1-Cdk2 pathway. Thus, we identify novel reciprocal functions of ATF3 and Fra1 in JNK- and ERK-dependent DNA damage responses.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Dano ao DNA , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator 3 Ativador da Transcrição/genética , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Northern Blotting , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Quinase 1 do Ponto de Checagem , Cisplatino/farmacologia , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Lentivirus/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Fase S/efeitos dos fármacos , Fase S/efeitos da radiação , Fator de Transcrição AP-1 , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Gene editing based on homology-directed repair (HDR) depends on donor DNA templates and programmable nucleases, e.g., RNA-guided CRISPR-Cas9 nucleases. However, next to inducing HDR involving the mending of chromosomal double-stranded breaks (DSBs) with donor DNA substrates, programmable nucleases also yield gene disruptions, triggered by competing non-homologous end joining (NHEJ) pathways. It is, therefore, imperative to identify parameters underlying the relationship between these two outcomes in the context of HDR-based gene editing. Here we implemented quantitative cellular systems, based on epigenetically regulated isogenic target sequences and donor DNA of viral, non-viral, and synthetic origins, to investigate gene-editing outcomes resulting from the interaction between different chromatin conformations and donor DNA structures. We report that, despite a significantly higher prevalence of NHEJ-derived events at euchromatin over Krüppel-associated box (KRAB)-impinged heterochromatin, HDR frequencies are instead generally less impacted by these alternative chromatin conformations. Hence, HDR increases in relation to NHEJ when open euchromatic target sequences acquire a closed heterochromatic state, with donor DNA structures determining, to some extent, the degree of this relative increase in HDR events at heterochromatin. Finally, restricting nuclease activity to HDR-permissive G2 and S phases of the cell cycle through a Cas9-Geminin construct yields lower, hence more favorable, NHEJ to HDR ratios, independently of the chromatin structure.
RESUMO
Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.
Assuntos
DNA/genética , Edição de Genes , Sistemas CRISPR-Cas , Linhagem Celular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Reparo do DNA por Junção de Extremidades , Genoma Humano , Humanos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
The nucleotide excision repair (NER) system consists of two sub-pathways, global genome repair (GGR) and transcription-coupled repair (TCR), which exhibit distinct functions in the cellular response to genotoxic stress. Defects in TCR result in prolonged UV light-induced stalling of RNA polymerase II and hypersensitivity to apoptosis induced by UV and certain chemotherapeutic drugs. Here, we show that low doses of UV trigger delayed activation of the stress-induced MAPkinase JNK and its proapoptotic targets c-Jun and ATF-3 in TCR-deficient primary human fibroblasts from Xeroderma Pigmentosum (XP) and Cockayne syndrome (CS) patients. This delayed activation of the JNK pathway is not observed in GGR-deficient TCR-proficient XP cells, is independent of functional p53, and is established through repression of the JNK-phosphatase MKP-1 rather than by activation of the JNK kinases MKK4 and 7. Enzymatic reversal of UV-induced cyclobutane pyrimidine dimers (CPDs) by CPD photolyase abrogated JNK activation, MKP-1 repression, and apoptosis in TCR-deficient XPA cells. Ectopic expression of MKP-1 inhibited DNA-damage-induced JNK activity and apoptosis. These results identify both MKP-1 and JNK as sensors and downstream effectors of persistent DNA damage in transcribed genes and suggest a link between the JNK pathway and UV-induced stalling of RNApol II.
Assuntos
Apoptose/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas Imediatamente Precoces/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transcrição Gênica , Linhagem Celular Transformada , Transformação Celular Viral , Células Cultivadas , Síndrome de Cockayne/genética , Reparo do DNA , Fosfatase 1 de Especificidade Dupla , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Humanos , Proteína Fosfatase 1 , Fator de Transcrição AP-1/metabolismo , Raios Ultravioleta , Xeroderma Pigmentoso/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Mutations disrupting the reading frame of the ~2.4 Mb dystrophin-encoding DMD gene cause a fatal X-linked muscle-wasting disorder called Duchenne muscular dystrophy (DMD). Genome editing based on paired RNA-guided nucleases (RGNs) from CRISPR/Cas9 systems has been proposed for permanently repairing faulty DMD loci. However, such multiplexing strategies require the development and testing of delivery systems capable of introducing the various gene editing tools into target cells. Here, we investigated the suitability of adenoviral vectors (AdVs) for multiplexed DMD editing by packaging in single vector particles expression units encoding the Streptococcus pyogenes Cas9 nuclease and sequence-specific gRNA pairs. These RGN components were customized to trigger short- and long-range intragenic DMD excisions encompassing reading frame-disrupting exons in patient-derived muscle progenitor cells. By allowing synchronous and stoichiometric expression of the various RGN components, we demonstrate that dual RGN-encoding AdVs can correct over 10% of target DMD alleles, readily leading to the detection of Becker-like dystrophin proteins in unselected muscle cell populations. Moreover, we report that AdV-based gene editing can be tailored for removing mutations located within the over 500-kb major DMD mutational hotspot. Hence, this single DMD editing strategy can in principle tackle a broad spectrum of mutations present in more than 60% of patients with DMD.