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Epidermal growth factor receptor (EGFR) mutation status differs according to ethnicity, gender, smoking history, and histology types. The present study aimed to evaluate EGFR mutation status in patients with non-small cell lung cancer (NSCLC) and further explore its association with clinical characteristics and prognosis in advanced NSCLC patients (Stage IIIB-IV). 238 NSCLC patients were enrolled in this study from October 2016 through December 2019. Patient characteristics and clinical data including age, gender, smoking history, histology types, tumor stage, survival status, and time were collected via electronic medical record system or telephone. 21 somatic mutations which spanned exons 18-21 of EGFR were detected using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method, followed by analysis of links to clinical characteristics, progression-free survival (PFS) and overall survival (OS). 103 patients were detected harboring EGFR mutations among the 238 cases tested (43.3%), and exons 19 and 21 were the highest mutation frequencies, with 20.6% and 19.3% respectively. The EGFR mutation rate was much higher in female versus male (57.4% vs 31.5%, p <0.001), in non-smokers compared to smokers (56.8% vs 25.9%, p <0.001), and in those with adenocarcinoma than other histology types (48.3% vs 3.7%, p <0.001). For patients in advanced stage, median PFS was 11 months in patients harboring EGFR mutations, versus 4 months in patients with wild type EGFR (p <0.001); median OS was 24 versus 12 months (p <0.001). Never smoking (p = 0.042) and adenocarcinoma (p = 0.007) were independent favorable factors for EGFR mutations. Our data strengthen the findings of high prevalence of EGFR mutations in Asian patients with NSCLC. Mutations are prevalent in those patients who are female, adenocarcinoma, and have never smoked. Moreover, advanced EGFR mutation-positive patients have better PFS and OS than those with wild type EGFR.
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BACKGROUND: No standard of care for mucosal melanoma (MM) in the adjuvant setting has been established. Meanwhile, relapse-free survival (RFS) is only â¼5 months after surgery alone. This phase II trial aimed to compare toripalimab versus high-dose interferon-α2b (HDI) as an adjuvant therapy for resected MM. PATIENTS AND METHODS: From July 2017 to May 2019, 145 patients with resected MM were randomized (1 : 1) to receive HDI (n = 72) or toripalimab (n = 73) for 1 year until disease relapse/distant metastasis, unacceptable toxicity, or withdrawal of consent. The primary endpoint was RFS. The secondary endpoints included distant metastasis-free survival (DMFS), overall survival (OS), and safety. RESULTS: After a median follow-up of 26.3 months, the number of RFS, OS, and DMFS events was 51 versus 46, 33 versus 29, and 49 versus 44 in the toripalimab arm and the HDI arm, respectively. The median RFS was 13.6 [95% confidence interval (CI) 8.31-19.02] months and 13.9 (95% CI 8.28-19.61) months in the toripalimab arm and the HDI arm, respectively. The DMFS was not significantly different between the two arms [hazard ratio (HR) 1.00; 95% CI 0.65-1.54]. The median OS was 35.1 months (95% CI 27.93 months-not reached) in the toripalimab arm, with no significant difference in all-cause death (HR 1.11, 95% CI 0.66-1.84) for the two arms. The median sums of the patients' actual infusion doses were 3672 mg and 1054.5 MIU in the toripalimab arm and the HDI arm, respectively. The incidence of treatment-emergent adverse events with a grade ≥3 was much higher in the HDI arm than in the toripalimab arm (87.5% versus 27.4%). CONCLUSIONS: Toripalimab showed a similar RFS and a more favorable safety profile than HDI, both better than historical data, suggesting that toripalimab might be the better treatment option. However, additional translational studies and better treatment regimens are still warranted to improve the clinical outcome of MM.
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Melanoma , Recurrencia Local de Neoplasia , Anticuerpos Monoclonales Humanizados , Humanos , Interferón alfa-2/uso terapéutico , Interferón-alfa/efectos adversos , Melanoma/patología , Recurrencia Local de Neoplasia/inducido químicamente , Recurrencia Local de Neoplasia/tratamiento farmacológicoRESUMEN
The ability to control translation of endogenous or exogenous RNAs in eukaryotic cells would facilitate a variety of biotechnological applications. Current strategies are limited by low fold changes in transgene output and the size of trigger RNAs (trRNAs). Here we introduce eukaryotic toehold switches (eToeholds) as modular riboregulators. eToeholds contain internal ribosome entry site sequences and form inhibitory loops in the absence of a specific trRNA. When the trRNA is present, eToeholds anneal to it, disrupting the inhibitory loops and allowing translation. Through optimization of RNA annealing, we achieved up to 16-fold induction of transgene expression in mammalian cells. We demonstrate that eToeholds can discriminate among viral infection status, presence or absence of gene expression and cell types based on the presence of exogenous or endogenous RNA transcripts.
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Biosíntesis de Proteínas , ARN , Animales , Mamíferos/genética , Biosíntesis de Proteínas/genética , ARN Viral/genéticaRESUMEN
OBJECTIVE: To investigate the effect of fatty acid synthase (FASN) gene silencing on lipid metabolism and biological behaviors of human hepatoblastoma HepG2 cells. OBJECTIVE: Small interfering RNA (siRNA) targeting FASN gene or a negative control siRNA sequence (NC-siRNA) was transfected into HepG2 cells, and the gene silencing efficiency was evaluated with qRT-PCR and Western blotting. Triglyceride level in the cells was detected using enzyme-linked immunosorbent assay, and Oil red O staining was performed to examine intracellular lipid droplets. The proliferation ability of the transfected cells was tested by CCK-8 assay, and cell apoptosis was evaluated using annexin V-FITC/PI apoptosis detection kit. Wound healing assay and Transwell assay were performed to assess the migration ability of the transfected cells. OBJECTIVE: Transfection of the cells with FASN-siRNA, but not NC-siRNA, significantly lowered FASN expression at both the mRNA and protein level (P < 0.001) and decreased the number of lipid droplets (P < 0.001) and triglyceride level (P < 0.01) in the cells. FASN gene silencing significantly inhibited proliferation, increased apoptosis rate and suppressed migration of HepG2 cells (P < 0.001). OBJECTIVE: FASN gene silencing inhibits proliferation and migration and promotes apoptosis of HepG2 cells possibly by suppressing lipid synthesis in the cells.
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Hepatoblastoma , Neoplasias Hepáticas , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Ácido Graso Sintasas/genética , Silenciador del Gen , Células Hep G2 , Hepatoblastoma/genética , Humanos , Metabolismo de los Lípidos , Neoplasias Hepáticas/genética , ARN Interferente Pequeño/genéticaRESUMEN
OBJECTIVE: Patients with cancer are usually immunosuppressive and susceptible to COVID-19 infection. Asymptomatic COVID-19 cases are infective and cannot be identified by symptom-based screening. There is an urgent need to control virus spread by asymptomatic carriers at cancer centres. We aim to describe the characteristics, screening methods, and outcomes of cancer patients with asymptomatic COVID-19 infection and to further explore anti-tumour treatment for this population. PATIENTS AND METHODS: We reviewed patients with cancer who were admitted to Hubei Cancer Hospital in Wuhan from February 1, 2020, to April 4, 2020. We collected demographic data, laboratory findings, treatment information, nucleic acid and serum test results, chest computed tomography (CT) information and survival status of cancer patients diagnosed with asymptomatic COVID-19 infection. RESULTS: A total of 16 cancer patients with asymptomatic COVID-19 infection were confirmed. The most common cancer type was breast cancer. The blood cell counts of most patients were in the normal range. Lymphocytes of 100% of asymptomatic carriers were in the normal range. Thirteen (81.3%) patients were positive for virus-specific IgM antibodies, and three (18.8%) were positive by PCR; only one (6.3%) patient showed novel coronavirus pneumonia features on CT. Three (18.3%) patients died, and the cause of death was considered malignancy caused by delaying anti-tumour treatment. CONCLUSIONS: Our study shows that the lymphocytes of 100% of asymptomatic carriers were in the normal range. This result indicates that the host immunity of asymptomatic carriers is not significantly disrupted by COVID-19. Single PCR detection is not sufficient to screen among asymptomatic individuals, and a combination of PCR tests, serological tests and CT is of great importance. Unless the tumour is life-threatening or rapidly progressing, we advise restarting active anti-tumour therapy after PCR tests become negative.
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Enfermedades Asintomáticas/epidemiología , Instituciones Oncológicas/estadística & datos numéricos , Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Anciano , Betacoronavirus , COVID-19 , China/epidemiología , Infecciones por Coronavirus/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Pandemias , Neumonía Viral/complicaciones , SARS-CoV-2 , Tasa de SupervivenciaRESUMEN
The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering.
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Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Campylobacter jejuni/enzimología , Endonucleasas/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Proteínas Asociadas a CRISPR/química , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Endonucleasas/química , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Ultralow-frequency (ULF) Raman spectroscopy becomes increasingly important in the area of two-dimensional (2D) layered materials; however, such measurement usually requires expensive and nonstandard equipment. Here, the measurement of ULF Raman signal down to 10 cm(-1) has been realized with high throughput by combining a kind of longpass edge filters with a single monochromator, which are verified by the Raman spectrum of L-cystine using three laser excitations. Fine adjustment of the angle of incident laser beam from normal of the longpass edge filters and selection of polarization geometry are demonstrated how to probe ULF Raman signal with high signal-to-noise. Davydov splitting of the shear mode in twisted (2+2) layer graphenes (t(2+2)LG) has been observed by such system in both exfoliated and transferred samples. We provide a direct evidence of twist-angle dependent softening of the shear coupling in t(2+2)LG, while the layer-breathing coupling at twisted interfaces is found to be almost identical to that in bulk graphite. This suggests that the exfoliation and transferring techniques are enough good to make a good 2D heterostructures to demonstrate potential device application. This Raman system will be potentially applied to the research field of ULF Raman spectroscopy.
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Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD.
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Sistemas CRISPR-Cas , Distrofina/genética , Exones/genética , Terapia Genética/métodos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Dependovirus , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Eliminación de SecuenciaRESUMEN
Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.
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Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Células Satélite del Músculo Esquelético/metabolismo , Transducción Genética/métodos , Animales , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Dependovirus , Modelos Animales de Enfermedad , Exones , Mutación del Sistema de Lectura , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Miocardio/metabolismo , ARN Mensajero/genética , Eliminación de SecuenciaRESUMEN
The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer adjacent motif (PAM) and complementarity to the guide RNA. Recently, we harnessed Staphylococcus aureus Cas9 (SaCas9), which is significantly smaller than Streptococcus pyogenes Cas9 (SpCas9), to facilitate efficient in vivo genome editing. Here, we report the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets, containing the 5'-TTGAAT-3' PAM and the 5'-TTGGGT-3' PAM, at 2.6 and 2.7 Å resolutions, respectively. The structures revealed the mechanism of the relaxed recognition of the 5'-NNGRRT-3' PAM by SaCas9. A structural comparison of SaCas9 with SpCas9 highlighted both structural conservation and divergence, explaining their distinct PAM specificities and orthologous sgRNA recognition. Finally, we applied the structural information about this minimal Cas9 to rationally design compact transcriptional activators and inducible nucleases, to further expand the CRISPR-Cas9 genome editing toolbox.
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Proteínas Bacterianas/química , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Sistemas CRISPR-Cas , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Ingeniería Genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/metabolismo , Alineación de Secuencia , Streptococcus pyogenes/enzimologíaRESUMEN
The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.
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Proteínas Asociadas a CRISPR/metabolismo , Ingeniería Genética/métodos , Genoma/genética , Staphylococcus aureus/enzimología , Animales , Secuencia de Bases , Proteínas Asociadas a CRISPR/genética , Colesterol/sangre , Colesterol/metabolismo , Marcación de Gen , Hígado/metabolismo , Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Proproteína Convertasa 9 , Proproteína Convertasas/biosíntesis , Proproteína Convertasas/sangre , Proproteína Convertasas/deficiencia , Proproteína Convertasas/genética , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/sangre , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Staphylococcus aureus/genética , Especificidad por SustratoRESUMEN
Little is known about how human genetic variation affects the responses to environmental stimuli in the context of complex diseases. Experimental and computational approaches were applied to determine the effects of genetic variation on the induction of pathogen-responsive genes in human dendritic cells. We identified 121 common genetic variants associated in cis with variation in expression responses to Escherichia coli lipopolysaccharide, influenza, or interferon-ß (IFN-ß). We localized and validated causal variants to binding sites of pathogen-activated STAT (signal transducer and activator of transcription) and IRF (IFN-regulatory factor) transcription factors. We also identified a common variant in IRF7 that is associated in trans with type I IFN induction in response to influenza infection. Our results reveal common alleles that explain interindividual variation in pathogen sensing and provide functional annotation for genetic variants that alter susceptibility to inflammatory diseases.
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Células Dendríticas/inmunología , Interacción Gen-Ambiente , Interacciones Huésped-Patógeno/genética , Factor 7 Regulador del Interferón/genética , Factores de Transcripción STAT/genética , Adulto , Enfermedades Autoinmunes/genética , Enfermedades Transmisibles/genética , Células Dendríticas/efectos de los fármacos , Escherichia coli , Femenino , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Virus de la Influenza A , Interferón beta/farmacología , Lipopolisacáridos/inmunología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Transcriptoma , Adulto JovenRESUMEN
The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.
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Proteínas Asociadas a CRISPR/química , Cristalografía por Rayos X , Endonucleasas/química , ARN Bacteriano/química , Streptococcus pyogenes/química , Secuencia de Aminoácidos , Bacterias/enzimología , Proteínas Asociadas a CRISPR/metabolismo , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Endonucleasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Bacteriano/metabolismo , Alineación de Secuencia , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/metabolismo , ARN Pequeño no TraducidoRESUMEN
The microbial CRISPR-Cas adaptive immune system can be harnessed to facilitate genome editing in eukaryotic cells (Cong L et al., Science 339, 819-823, 2013; Mali P et al., Science 339, 823-826, 2013). Here we describe a protocol for the use of the RNA-guided Cas9 nuclease from the Streptococcus pyogenes type II CRISPR system to achieve specific, scalable, and cost-efficient genome editing in mammalian cells.
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Genoma , Edición de ARN , Animales , Línea Celular , Clonación Molecular , Reparación del ADN por Unión de Extremidades , Orden Génico , Marcación de Gen/métodos , Vectores Genéticos , Humanos , Transfección , ARN Pequeño no TraducidoRESUMEN
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ingeniería Genética/métodos , Genoma , Secuencia de Bases , Técnicas de Cultivo de Célula , Línea Celular , Reparación del ADN por Unión de Extremidades , Análisis Mutacional de ADN , Reparación del ADN , Desoxirribonucleasas/química , Desoxirribonucleasas/genética , Técnicas de Genotipaje , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutagénesis , TransfecciónRESUMEN
Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.
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Roturas del ADN de Doble Cadena , Marcación de Gen/métodos , Genoma , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Cigoto/metabolismo , ARN Pequeño no TraducidoRESUMEN
In this study, heparin-like poly(ethersulfone) (HLPES) was synthesized by a combination of polycondensation and post-carboxylation methods, and was characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance hydrogen spectrum and gel permeation chromatography. Owing to the similar backbone structure, the synthesized HLPES could be directly blended with pristine PES at any ratios to prepare PES/HLPES membranes. After the introduction of HLPES, the microscopic structure of the modified PES membranes was changed, while the hydrophilicity was significantly enhanced. Bovine serum albumin and bovine serum fibrinogen adsorption, activated partial thromboplastin time, thromb time and platelet adhesion for the modified PES membranes were investigated. The results indicated that the blood compatibility of the PES/HLPES membranes was significantly improved compared with that of pristine PES membrane. For the PES/HLPES membranes, obvious decreases in platelet activation on PF-4 level, in complement activation on C3a and C5a levels, and in leukocytes activation on CD11b levels were observed compared with those for the pristine PES membrane. The improved blood compatibility of the PES/HLPES membrane might due to the existence of the hydrophilic groups (-SO3Na, -COONa). Furthermore, the modified PES membranes showed good cytocompatibility. Hepatocytes cultured on the PES/HLPES membranes presented improved growth in terms of SEM observation, MTT assay and confocal laser scanning microscope observation compared with those on the pristine PES membrane. These results indicate that the PES/HLPES membranes present great potential in blood-contact fields such as hemodialysis and bio-artificial liver supports.
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Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/farmacología , Heparina/síntesis química , Ensayo de Materiales , Polímeros/síntesis química , Sulfonas/síntesis química , Adsorción , Adulto , Animales , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/ultraestructura , Antígeno CD11b/metabolismo , Bovinos , Forma de la Célula/efectos de los fármacos , Cromatografía en Gel , Activación de Complemento/efectos de los fármacos , Complemento C3a/metabolismo , Fibrinógeno/metabolismo , Heparina/química , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/ultraestructura , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Membranas Artificiales , Microscopía Fluorescente , Tiempo de Tromboplastina Parcial , Adhesividad Plaquetaria/efectos de los fármacos , Polímeros/química , Albúmina Sérica Bovina/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Sulfonas/química , Tiempo de Coagulación de la Sangre TotalRESUMEN
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
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ADN/genética , Desoxirribonucleasas/genética , Ingeniería Genética/métodos , Factores de Transcripción/genética , Proteínas Bacterianas/genética , Disparidad de Par Base , Secuencia de Bases , Datos de Secuencia Molecular , Streptococcus pyogenes/genética , ARN Pequeño no TraducidoRESUMEN
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.