RESUMO
The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Linfócitos T/metabolismo , Proteína 9 Associada à CRISPR/fisiologia , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/fisiologia , DNA/genética , Endonucleases/genética , Edição de Genes/métodos , Técnicas Genéticas , Genoma/genética , Genômica/métodos , Humanos , Leucócitos Mononucleares/metabolismo , Conformação Molecular , RNA Guia de Cinetoplastídeos/genética , Relação Estrutura-Atividade , Linfócitos T/fisiologiaRESUMO
The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Clivagem do DNA , DNA/química , Endonucleases/química , Engenharia Genética/métodos , RNA Guia de Cinetoplastídeos/química , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células HEK293 , Humanos , Conformação de Ácido Nucleico , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/ultraestruturaRESUMO
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Assuntos
Sistemas CRISPR-Cas/genética , Mapeamento Cromossômico/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNARESUMO
The transcription factor GATA3 is the master regulator that drives mammary luminal epithelial cell differentiation and maintains mammary gland homeostasis. Loss of GATA3 is associated with aggressive breast cancer development. We have identified ZNF503/ZEPPO2 zinc-finger elbow-related proline domain protein 2 (ZPO2) as a transcriptional repressor of GATA3 expression and transcriptional activity that induces mammary epithelial cell proliferation and breast cancer development. We show that ZPO2 is recruited to GATA3 promoter in association with ZBTB32 (Repressor of GATA, ROG) and that ZBTB32 is essential for down-regulation of GATA3 via ZPO2. Through this modulation of GATA3 activity, ZPO2 promotes aggressive breast cancer development. Our data provide insight into a mechanism of GATA3 regulation, and identify ZPO2 as a possible candidate gene for future diagnostic and therapeutic strategies.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fator de Transcrição GATA3/genética , Regulação Neoplásica da Expressão Gênica , Animais , Sítios de Ligação , Biópsia , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Análise por Conglomerados , Modelos Animais de Doenças , Progressão da Doença , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Metástase Neoplásica , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Amplification of 8p11-12 in human breast cancers is associated with increased proliferation and tumor grade and reduced metastasis-free patient survival. We identified Zeppo1 (zinc finger elbow-related proline domain protein 1) (FLJ14299/ZNF703) within this amplicon as a regulator of cell adhesion, migration, and proliferation in mammary epithelial cells. Overexpression of Zeppo1 reduces cell-cell adhesion and stimulates migration and proliferation. Knockdown of Zeppo1 induces adhesion and lumen formation. Zeppo1 regulates transcription, complexing with Groucho and repressing E-cadherin expression and Wnt and TGFß reporter expression. Zeppo1 promotes expression of metastasis-associated p120-catenin isoform 1 and alters p120-catenin localization upon cell contact with the extracellular matrix. Significantly, Zeppo1 overexpression in a mouse breast cancer model increases lung metastases, while reducing Zeppo1 expression reduces both tumor size and the number of lung metastases. These results indicate that Zeppo1 is a key regulator of breast cancer progression.
Assuntos
Caderinas/metabolismo , Cateninas/metabolismo , Regulação Neoplásica da Expressão Gênica , Metástase Neoplásica/genética , Transporte Proteico/genética , Proteínas Repressoras/metabolismo , Células 3T3 , Animais , Adesão Celular/genética , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/genética , delta CateninaRESUMO
The NET (nocA, Nlz, elB, TLP-1) subfamily of zinc finger proteins is an important mediator during developmental processes. The evolutionary conserved zinc finger protein ZNF503/Zeppo2 (zinc finger elbow-related proline domain protein 2, Zpo2) plays critical roles during embryogenesis. We found that Zpo2 is expressed in adult tissue and examined its function. We found that ZPO2 is a nuclearly targeted transcriptional repressor that is expressed in mammary epithelial cells. Elevated Zpo2 levels increase mammary epithelial cell proliferation. Zpo2 promotes cellular invasion through down-regulation of E-cadherin and regulates the invasive phenotype in a RAC1-dependent manner. We detect elevated Zpo2 expression during breast cancer progression in a MMTV-PyMT transgenic mouse model. Tumor transplant experiments indicated that overexpression of Zpo2 in MMTV-PyMT mammary tumor cell lines enhances lung metastasis. Our findings suggest that Zpo2 plays a significant role in mammary gland homeostasis and that deregulation of Zpo2 may promote breast cancer development.
Assuntos
Proteínas de Transporte/metabolismo , Proliferação de Células , Células Epiteliais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Movimento Celular , Células Epiteliais/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Mamárias Experimentais/patologia , Camundongos , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
How breast cancers are able to disseminate and metastasize is poorly understood. Using a hyperplasia transplant system, we show that tumor dissemination and metastasis occur in discrete steps during tumor progression. Bioinformatic analysis revealed that loss of the transcription factor GATA-3 marked progression from adenoma to early carcinoma and onset of tumor dissemination. Restoration of GATA-3 in late carcinomas induced tumor differentiation and suppressed tumor dissemination. Targeted deletion of GATA-3 in early tumors led to apoptosis of differentiated cells, indicating that its loss is not sufficient for malignant conversion. Rather, malignant progression occurred with an expanding GATA-3-negative tumor cell population. These data indicate that GATA-3 regulates tumor differentiation and suppresses tumor dissemination in breast cancer.
Assuntos
Neoplasias da Mama/patologia , Diferenciação Celular , Fator de Transcrição GATA3/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Células Epiteliais/patologia , Feminino , Fator de Transcrição GATA3/deficiência , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hiperplasia , Imuno-Histoquímica , Glândulas Mamárias Animais , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologiaRESUMO
BACKGROUND: Human pancreatic islet structure poses challenges to investigations that require specific modulation of gene expression. Yet dissociation of islets into individual cells destroys cellular interactions important to islet physiology. Approaches that improve transient targeting of gene expression in intact human islets are needed in order to effectively perturb intracellular pathways to achieve biological effects in the most relevant tissue contexts. RESULTS: Electroporation of intact human cadaveric islets resulted in robust and specific suppression of gene expression. Two genes were simultaneously suppressed by 80% from baseline levels. When multiple (up to 5) genes were simultaneously targeted, effective suppression of 3 of 5 genes occurred. Enzymatic pretreatment of islets was not required. Simultaneous targeting of RB and p53 pathway members resulted in cell cycle reentry as measured by EDU incorporation in 10% of islet nuclei. CONCLUSIONS: At least three genes can be effectively suppressed simultaneously in cultured intact human pancreatic islets without disruption of islet architecture or overt alterations in function. This enabled the effective modulation of two central growth control pathways resulting in the phenotypic outcome of cell cycle reentry in postmitotic islet cells. Transient exposure to multiple siRNAs is an effective approach to modify islets for study with the potential to aid clinical applications.
Assuntos
Ciclo Celular , Inativação Gênica , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Proteína do Retinoblastoma/genética , Proteína Supressora de Tumor p53/genética , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Insulina/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Escherichia coli , Genoma/genética , Células HEK293 , Humanos , Modelos GenéticosRESUMO
Netrin and its receptor Neogenin are thought to be regulators of axonal guidance in the nervous system. A recent report suggests they also play a role in epithelial morphogenesis.
Assuntos
Adesão Celular/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteínas de Membrana/fisiologia , Fatores de Crescimento Neural/fisiologia , Animais , Epitélio , Glândulas Mamárias Animais/fisiologia , Camundongos , Morfogênese , Netrina-1 , Proteínas Supressoras de TumorRESUMO
The GATA family of transcription factors plays fundamental roles in cell-fate specification. However, it is unclear if these genes are necessary for the maintenance of cellular differentiation after development. We identified GATA-3 as the most highly enriched transcription factor in the mammary epithelium of pubertal mice. GATA-3 was found in the luminal cells of mammary ducts and the body cells of terminal end buds (TEBs). Upon conditional deletion of GATA-3, mice exhibited severe defects in mammary development due to failure in TEB formation during puberty. After acute GATA-3 loss, adult mice exhibited undifferentiated luminal cell expansion with basement-membrane detachment, which led to caspase-mediated cell death in the long term. Further, FOXA1 was identified as a downstream target of GATA-3 in the mammary gland. This suggests that GATA-3 actively maintains luminal epithelial differentiation in the adult mammary gland, which raises important implications for the pathogenesis of breast cancer.