RESUMO
Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.
Assuntos
Metilenotetra-Hidrofolato Desidrogenase (NADP) , Neoplasias , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Ácido Fólico/metabolismo , Formiatos , Purinas , Tetra-HidrofolatosRESUMO
Determining the functional role of thousands of genetic sequence variants (mutations) associated with genetic diseases is a major challenge. Here we present clustered regularly interspaced short palindromic repeat (CRISPR)-SelectTIME, CRISPR-SelectSPACE and CRISPR-SelectSTATE, a set of flexible knock-in assays that introduce a genetic variant in a cell population and track its absolute frequencies relative to an internal, neutral control mutation as a function of time, space or a cell state measurable by flow cytometry. Phenotypically, CRISPR-Select can thereby determine, for example, pathogenicity, drug responsiveness/resistance or in vivo tumor promotion by a specific variant. Mechanistically, CRISPR-Select can dissect how the variant elicits the phenotype by causally linking the variant to motility/invasiveness or any cell state or biochemical process with a flow cytometry marker. The method is applicable to organoids, nontransformed or cancer cell lines. It is accurate, quantitative, fast and simple and works in single-well or 96-well higher throughput format. CRISPR-Select provides a versatile functional variant assay for research, diagnostics and drug development for genetic disorders.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genéticaRESUMO
BACKGROUND: The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient. RESULTS: Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12-14% for the two genes, which translated into 7-8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis. CONCLUSION: Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Isoantígenos/genética , Linfócitos T/metabolismo , Antígeno CD52/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Eletroporação , Edição de Genes/métodos , Genômica , Células HEK293 , Humanos , Mutação INDEL , PlasmídeosRESUMO
Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.
Assuntos
Sistemas CRISPR-Cas , Reparo do DNA , DNA/genética , Edição de Genes/métodos , Genoma , Mutação INDEL , Animais , Clonagem de Organismos/métodos , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Técnicas de Inativação de Genes , Humanos , Camundongos , Ovinos/genética , Solanum tuberosum/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismoRESUMO
The ARHGAP35 gene encoding p190A RhoGAP (p190A) is significantly altered by both mutation and allelic deletion in human cancer, but the functional implications of such alterations are not known. Here, we demonstrate for the first time that p190A is a tumor suppressor using a xenograft mouse model with carcinoma cells harboring defined ARHGAP35 alterations. In vitro, restoration of p190A expression in carcinoma cells promotes contact inhibition of proliferation (CIP) through activation of LATS kinases and phosphorylation of the proto-oncogenic transcriptional co-activator YAP. In contrast, p190A forms harboring recurrent cancer mutations exhibit loss of function in modulating the Hippo pathway, inducing CIP, as well as attenuated suppression of tumor growth in mice. We determine that p190A promotes mesenchymal to epithelial transition (MET) and elicits expression of a cassette of epithelial adherens junction-associated genes in a cell density-dependent manner. This cassette includes CDH1 encoding E-cadherin, which amplifies p190A-mediated LATS activation and is necessary for CIP. Oppositely, we establish that p190A is obligatory for E-cadherin to activate LATS kinases and induce CIP. Collectively, this work defines a novel mechanism by which p190A and E-cadherin cooperate in modulating Hippo signaling to suppress tumor cell growth.
Assuntos
Antígenos CD/biossíntese , Caderinas/biossíntese , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/patologia , Proteínas Repressoras/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CRISPR/Cas9 technology allows facile modification of the genome in virtually any desired way through the use of easily designed plasmid constructs that express a gRNA targeting a genomic site-of-interest and Cas9. Hydrodynamic tail vein injection, on the other hand, is a simple method to deliver "naked" plasmid DNA to 5-40% of the hepatocytes of the liver of adult mice. Here, we describe how these two techniques can be combined to create a workflow for fast, easy, and cost-efficient in vivo genome editing of the adult mouse liver. Using this method, large cohorts of mice with genetically modified livers can be established within 3 weeks to generate models for gene function in normal physiology and diseases of the liver.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Animais , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos , Plasmídeos/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
ARHGAP35 encoding p190A RhoGAP is a cancer-associated gene with a mutation spectrum suggestive of a tumor-suppressor function. In this study, we demonstrate that loss of heterozygosity for ARHGAP35 occurs in human tumors. We sought to identify tumor-suppressor capacities for p190A RhoGAP (p190A) and its paralog p190B in epithelial cells. We reveal an essential role for p190A and p190B to promote contact inhibition of cell proliferation (CIP), a function that relies on RhoGAP activity. Unbiased mRNA sequencing analyses establish that p190A and p190B modulate expression of genes associated with the Hippo pathway. Accordingly, we determine that p190A and p190B induce CIP by repressing YAP-TEAD-regulated gene transcription through activation of LATS kinases and inhibition of the Rho-ROCK pathway. Finally, we demonstrate that loss of a single p190 paralog is sufficient to elicit nuclear translocation of YAP and perturb CIP in epithelial cells cultured in Matrigel. Collectively, our data reveal a novel mechanism consistent with a tumor-suppressor function for ARHGAP35.
Assuntos
Proliferação de Células/fisiologia , Inibição de Contato/fisiologia , Células Epiteliais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neoplasias/patologia , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Cães , Proteínas Ativadoras de GTPase/genética , Regulação Neoplásica da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Via de Sinalização Hippo , Humanos , Células Madin Darby de Rim Canino , Neoplasias/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAP , Quinases Associadas a rho/metabolismoRESUMO
Aberrant expression of O-glycans is a hallmark of epithelial cancers. Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Here, we investigated the expression patterns of all of the GalNAc-Ts in colon cancer by analyzing transcriptomic data. We found that GalNAc-T6 was highly up-regulated in colon adenocarcinomas but absent in normal-appearing adjacent colon tissue. These results were verified by immunohistochemistry, suggesting that GalNAc-T6 plays a role in colon carcinogenesis. To investigate the function of GalNAc-T6 in colon cancer, we used precise gene targeting to produce isogenic colon cancer cell lines with a knockout/rescue system for GALNT6 GalNAc-T6 expression was associated with a cancer-like, dysplastic growth pattern, whereas GALNT6 knockout cells showed a more normal differentiation pattern, reduced proliferation, normalized cell-cell adhesion, and formation of crypts in tissue cultures. O-Glycoproteomic analysis of the engineered cell lines identified a small set of GalNAc-T6-specific targets, suggesting that this isoform has unique cellular functions. In support of this notion, the genetically and functionally closely related GalNAc-T3 homolog did not show compensatory functionality for effects observed for GalNAc-T6. Taken together, these data strongly suggest that aberrant GalNAc-T6 expression and site-specific glycosylation is involved in oncogenic transformation.
Assuntos
Adenocarcinoma/enzimologia , Diferenciação Celular , Colo/enzimologia , Neoplasias do Colo/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/enzimologia , N-Acetilgalactosaminiltransferases/biossíntese , Proteínas de Neoplasias/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Glicosilação , Humanos , Mucosa Intestinal/patologia , N-Acetilgalactosaminiltransferases/genética , Proteínas de Neoplasias/genéticaRESUMO
BACKGROUND & AIMS: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development. METHODS: We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. RESULTS: Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1-Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC. CONCLUSIONS: Using CRISPR/Cas9 technology, we found generation of the Dnajb1-Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1-PRKACA might be developed as therapeutics for this form of liver cancer.
Assuntos
Biomarcadores Tumorais/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/genética , Transformação Celular Neoplásica/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Edição de Genes/métodos , Fusão Gênica , Proteínas de Choque Térmico HSP40/genética , Neoplasias Hepáticas/genética , Animais , Biomarcadores Tumorais/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Proteínas de Choque Térmico HSP40/metabolismo , Neoplasias Hepáticas/metabolismo , Camundongos , Fenótipo , Fatores de TempoRESUMO
This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.
Assuntos
Análise Mutacional de DNA/métodos , Desoxirribonucleases/metabolismo , Citometria de Fluxo/métodos , Edição de Genes/métodos , Genômica/métodos , Mutação INDEL , Animais , Células CHO , Cricetinae , Cricetulus , Técnicas de Introdução de Genes , Técnicas de Inativação de GenesRESUMO
Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment.
Assuntos
Endolina/genética , Animais , Sequência de Bases , Linhagem Celular , Códon sem Sentido/genética , Surdez/genética , Dinamarca , Família , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Repetições de Microssatélites/genética , Órgão Espiral/metabolismo , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNARESUMO
Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferase genes controlling N-glycosylation in CHO cells and constructed a design matrix that facilitates the generation of desired glycosylation, such as human-like α2,6-linked sialic acid capping. This engineering approach will aid the production of glycoproteins with improved properties and therapeutic potential.
Assuntos
Glicoproteínas , Engenharia de Proteínas/métodos , Proteínas Recombinantes , Animais , Células CHO , Cricetinae , Cricetulus , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.
Assuntos
Análise Mutacional de DNA/métodos , Mutação INDEL , Animais , Células CHO , Sistemas CRISPR-Cas , Linhagem Celular , Cricetulus , Eletroforese Capilar , Marcação de Genes , Cobaias , Humanos , Camundongos , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30-70% at high transfection efficiencies and â¼ 2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with â¼ 1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9.
Assuntos
Proteínas Associadas a CRISPR/genética , Desoxirribonucleases/genética , Técnicas de Introdução de Genes , Proteínas Luminescentes/genética , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular Tumoral , Separação Celular , Desoxirribonucleases/metabolismo , Citometria de Fluxo , Corantes Fluorescentes , Genoma , Humanos , Células K562 , Proteínas Luminescentes/metabolismo , Peptídeos/química , Plasmídeos/genética , Dedos de ZincoRESUMO
BACKGROUND: During epithelial morphogenesis, a complex comprising the ßPIX (PAK-interacting exchange factor ß) and class I PAKs (p21-activated kinases) is recruited to adherens junctions. Scrib, the mammalian ortholog of the Drosophila polarity determinant and tumor suppressor Scribble, binds ßPIX directly. Scrib is also targeted to adherens junctions by E-cadherin, where Scrib strengthens cadherin-mediated cell-cell adhesion. Although a role for the Scrib-ßPIX-PAK signaling complex in promoting membrane protrusion at wound edges has been elucidated, a function for this complex at adherens junctions remains unknown. RESULTS: Here, we establish that Scrib targets ßPIX and PAK2 to adherens junctions where a ßPIX-PAK2 complex counterbalances apoptotic stimuli transduced by Scrib and elicited by cadherin-mediated cell-cell adhesion. Moreover, we show that this signaling pathway regulates cell survival in response to osmotic stress. Finally, we determine that in suspension cultures, the Scrib-ßPIX-PAK2 complex functions to regulate anoikis elicited by cadherin engagement, with Scrib promoting and the ßPIX-PAK2 complex suppressing anoikis, respectively. CONCLUSIONS: Our findings demonstrate that the Scrib-ßPIX-PAK2 signaling complex functions as an essential modulator of cell survival when localized to adherens junctions of polarized epithelia. The activity of this complex at adherens junctions is thereby essential for normal epithelial morphogenesis and tolerance of physiological stress. Furthermore, when localized to adherens junctions, the Scrib-ßPIX-PAK2 signaling complex serves as a key determinant of anoikis sensitivity, a pivotal mechanism in tumor suppression. Thus, this work also reveals the need to expand the definition of anoikis to include a central role for adherens junctions.
Assuntos
Apoptose/fisiologia , Caderinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Quinases Ativadas por p21/metabolismo , Junções Aderentes/metabolismo , Animais , Anoikis , Sobrevivência Celular , Cães , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/metabolismo , Pressão Osmótica , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Quinases Ativadas por p21/genéticaRESUMO
Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.
Assuntos
Acrilamidas/química , Alcenos/química , Cisteína/química , Nitrilas/química , Desdobramento de Proteína , Proteólise , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Compostos de Sulfidrila/químicaRESUMO
Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions.
Assuntos
DNA de Cadeia Simples/genética , Marcação de Genes/métodos , Engenharia Genética/métodos , Linhagem Celular , Endonucleases/genética , Deleção de Genes , Humanos , Mutagênese Insercional/métodos , Oligodesoxirribonucleotídeos/genética , Mutação Puntual/genética , Dedos de Zinco/genéticaRESUMO
The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.
Assuntos
Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sítios de Ligação , Linhagem Celular , Humanos , Modelos Moleculares , Fosforilação , Ligação ProteicaRESUMO
The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during development, tissue regeneration, and carcinoma progression, often via promoting the epithelial-mesenchymal transition (EMT). Many mechanisms by which ERK exerts this control remain elusive. We demonstrate that the ERK-activated kinase RSK is necessary to induce mesenchymal motility and invasive capacities in nontransformed epithelial and carcinoma cells. RSK is sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK is to induce transcription of a potent promotile/invasive gene program by FRA1-dependent and -independent mechanisms. The program enables RSK to coordinately modulate the extracellular environment, the intracellular motility apparatus, and receptors mediating communication between these compartments to stimulate motility and invasion. These findings uncover a mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanate multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties.