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1.
Annu Rev Genet ; 2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34530641

RESUMO

CRISPR-based genome editing holds promise for genome engineering and other applications in diverse organisms. Defining and improving the genome-wide and transcriptome-wide specificities of these editing tools are essential for realizing their full potential in basic research and biomedical therapeutics. This review provides an overview of CRISPR-based DNA- and RNA-editing technologies, methods to quantify their specificities, and key solutions to reduce off-target effects for research and improve therapeutic applications. Expected final online publication date for the Annual Review of Genetics, Volume 55 is November 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

2.
Nat Commun ; 12(1): 4902, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385461

RESUMO

Efficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.


Assuntos
Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , Edição de Genes/métodos , Aprendizado de Máquina , Uracila-DNA Glicosidase/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Códon/genética , Citidina Desaminase/genética , Escherichia coli/genética , Feminino , Biblioteca Gênica , Células HEK293 , Humanos , Camundongos , Reprodutibilidade dos Testes , Uracila-DNA Glicosidase/genética
3.
J Mol Cell Biol ; 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34097054

RESUMO

Tumor development is a process involving loss of the differentiation phenotype and acquisition of stem-like characteristics, which is driven by intracellular rewiring of signaling network. The measurement of network reprogramming and disorder would be challenging due to the complexity and heterogeneity of tumors. Here, we proposed signaling entropy to assess the degree of tumor network disorder. We calculated signaling entropy for 33 tumor types in The Cancer Genome Atlas database based on transcriptomic and proteomic data. The signaling entropy of tumors was significantly higher than that of normal samples and was highly correlated with cell stemness, cancer type, tumor grade, and metastasis. We further demonstrated the sensitivity and accuracy of using local signaling entropy in prognosis prediction and drug response evaluation. Overall, signaling entropy could reveal cancer network disorders related to tumor malignant potency, clinical prognosis, and drug response.

5.
Nat Cell Biol ; 23(1): 99-108, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398178

RESUMO

Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (<0.001 relative to Gapdh; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions.


Assuntos
Células-Tronco Embrionárias Murinas/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Regiões Promotoras Genéticas , RNA Guia/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Animais , Sistemas CRISPR-Cas , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Neuroblastoma/patologia , RNA Guia/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética
6.
Aging (Albany NY) ; 12(23): 23849-23871, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33221766

RESUMO

Hepatocellular carcinoma (HCC) is a heterogeneous disease with various genetic and epigenetic abnormalities. Previous studies of HCC driver genes were primarily based on frequency of mutations and copy number alterations. Here, we performed an integrative analysis of genomic and epigenomic data from 377 HCC patients to identify driver genes that regulate gene expression in HCC. This integrative approach has significant advantages over single-platform analyses for identifying cancer drivers. Using this approach, HCC tissues were divided into four subgroups, based on expression of the transcription factor E2F and the mutation status of TP53. HCC tissues with E2F overexpression and TP53 mutation had the highest cell cycle activity, indicating a synergistic effect of E2F and TP53. We found that overexpression of the identified driver genes, stratifin (SFN) and SPP1, correlates with tumor grade and poor survival in HCC and promotes HCC cell proliferation. These findings indicate SFN and SPP1 function as oncogenes in HCC and highlight the important role of enhancers in the regulation of gene expression in HCC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Biologia Computacional , Genômica , Neoplasias Hepáticas/genética , Integração de Sistemas , Proteínas 14-3-3/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Proliferação de Células , Variações do Número de Cópias de DNA , Metilação de DNA , Bases de Dados Genéticas , Fatores de Transcrição E2F/genética , Epigênese Genética , Exorribonucleases/genética , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Predisposição Genética para Doença , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Mutação , Gradação de Tumores , Osteopontina/genética , Fenótipo , Proteína Supressora de Tumor p53/genética
7.
EMBO J ; 39(22): e104741, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33058229

RESUMO

Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE-S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine-specific C-to-U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high-specificity versions of CURE to create fewer missense mutations than RESCUE-S at the off-targets transcriptome-wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE-S being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear-localized version of CURE, but not that of RESCUE-S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility.


Assuntos
Citidina Desaminase/genética , Proteínas/genética , Edição de RNA , Núcleo Celular/metabolismo , Células HEK293 , Humanos , RNA/química , RNA/metabolismo , RNA Guia , Transcriptoma
8.
Cancer Cell ; 38(5): 734-747.e9, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32888432

RESUMO

We integrate the genomics, proteomics, and phosphoproteomics of 480 clinical tissues from 146 patients in a Chinese colorectal cancer (CRC) cohort, among which 70 had metastatic CRC (mCRC). Proteomic profiling differentiates three CRC subtypes characterized by distinct clinical prognosis and molecular signatures. Proteomic and phosphoproteomic profiling of primary tumors alone successfully distinguishes cases with metastasis. Metastatic tissues exhibit high similarities with primary tumors at the genetic but not the proteomic level, and kinase network analysis reveals significant heterogeneity between primary colorectal tumors and their liver metastases. In vivo xenograft-based drug tests using 31 primary and metastatic tumors show personalized responses, which could also be predicted by kinase-substrate network analysis no matter whether tumors carry mutations in the drug-targeted genes. Our study provides a valuable resource for better understanding of mCRC and has potential for clinical application.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Genômica/métodos , Metástase Neoplásica/tratamento farmacológico , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteômica/métodos , Animais , Antineoplásicos/farmacologia , China , Estudos de Coortes , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Terapia de Alvo Molecular , Metástase Neoplásica/genética , Fosforilação , Medicina de Precisão , Prognóstico , Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Sci Adv ; 6(29): eaba1773, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32832622

RESUMO

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5'-CC-3' motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.

10.
Nat Protoc ; 15(9): 3009-3029, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32796939

RESUMO

Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq (https://github.com/sydaileen/GOTI-seq) for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.


Assuntos
Embrião de Mamíferos/metabolismo , Edição de Genes/métodos , Animais , Feminino , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação
11.
Nat Methods ; 17(6): 600-604, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424272

RESUMO

Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant-YE1-BE3-FNLS-that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits.


Assuntos
Proteína 9 Associada à CRISPR/genética , Citosina/metabolismo , DNA/genética , Edição de Genes/métodos , RNA/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Células HEK293 , Humanos , Mutação , Mutação Puntual
12.
Cell ; 181(3): 590-603.e16, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32272060

RESUMO

Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.


Assuntos
Neurogênese/fisiologia , Neuroglia/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Sistemas CRISPR-Cas/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Modelos Animais de Doenças , Dopamina/metabolismo , Regulação da Expressão Gênica/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças do Sistema Nervoso/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Células Ganglionares da Retina/fisiologia
13.
FASEB J ; 34(3): 4764-4782, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32027432

RESUMO

Damage or degeneration of inner ear spiral ganglion neurons (SGNs) causes hearing impairment. Previous in vitro studies indicate that cochlear glial cells can be reprogrammed into SGNs, however, it remains unknown whether this can occur in vivo. Here, we show that neonatal glial cells can be converted, in vivo, into SGNs (defined as new SGNs) by simultaneous induction of Neurog1 (Ngn1) and Neurod1. New SGNs express SGN markers, Tuj1, Map2, Prox1, Mafb and Gata3, and reduce glial cell marker Sox10 and Scn7a. The heterogeneity within new SGNs is illustrated by immunostaining and transcriptomic assays. Transcriptomes analysis indicates that well reprogrammed SGNs are similar to type I SGNs. In addition, reprogramming efficiency is positively correlated with the dosage of Ngn1 and Neurod1, but declined with aging. Taken together, our in vivo data demonstrates the plasticity of cochlear neonatal glial cells and the capacity of Ngn1 and Neurod1 to reprogram glial cells into SGNs. Looking ahead, we expect that combination of Neurog1 and Neurod1 along with other factors will further boost the percentage of fully converted (Mafb+/Gata3+) new SGNs.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/metabolismo , Animais , Sequência de Bases , Imunofluorescência , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição MafB/genética , Fator de Transcrição MafB/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
14.
Genomics Proteomics Bioinformatics ; 18(5): 525-538, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33450402

RESUMO

The estrogen receptor (ER)-negative breast cancer subtype is aggressive with few treatment options available. To identify specific prognostic factors for ER-negative breast cancer, this study included 705,729 and 1034 breast invasive cancer patients from the Surveillance, Epidemiology, and End Results (SEER) and The Cancer Genome Atlas (TCGA) databases, respectively. To identify key differential kinase-substrate node and edge biomarkers between ER-negative and ER-positive breast cancer patients, we adopted a network-based method using correlation coefficients between molecular pairs in the kinase regulatory network. Integrated analysis of the clinical and molecular data revealed the significant prognostic power of kinase-substrate node and edge features for both subtypes of breast cancer. Two promising kinase-substrate edge features, CSNK1A1-NFATC3 and SRC-OCLN, were identified for more accurate prognostic prediction in ER-negative breast cancer patients.

15.
Elife ; 82019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31829937

RESUMO

Conserved proteins drive clathrin-mediated endocytosis (CME), which from yeast to humans involves a burst of actin assembly. To gain mechanistic insights into this process, we performed a side-by-side quantitative comparison of CME in two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae is more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast. In both yeasts, accumulation of ~70 WASp molecules activates the Arp2/3 complex to drive membrane invagination. In contrast to budding yeast, WASp-mediated actin nucleation plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, although the assembly of two zones of actin filaments is specific for fission yeast and not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations with broad implications that are expected to extend from yeast to humans.


Assuntos
Clatrina/metabolismo , Endocitose , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas Fúngicas/metabolismo , Microscopia Intravital
16.
Nature ; 571(7764): 275-278, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181567

RESUMO

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9-13. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA12, and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.


Assuntos
DNA/genética , Edição de Genes/métodos , Mutagênese , Mutação , Nucleosídeo Desaminases/genética , Engenharia de Proteínas , RNA/genética , Adenina/metabolismo , Aminoidrolases/genética , Aminoidrolases/metabolismo , Citosina/metabolismo , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Células HEK293 , Humanos , Nucleosídeo Desaminases/metabolismo , Especificidade por Substrato , Transfecção
17.
Genome Biol ; 20(1): 101, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118069

RESUMO

Base editing installs a precise nucleotide change in specific gene loci without causing a double-strand break. Its efficiency in human embryos is generally low, limiting its utility in functional genetic studies. Here, we report that injecting base editors into human cleaving two-cell and four-cell embryos results in much higher (up to 13-fold) homozygotic nucleotide substitution efficiency as opposed to MII oocytes or zygotes. Furthermore, as a proof-of-principle study, a point mutation can be efficiently corrected by our method. Our study indicates that human cleaving embryos provide an efficient base editing window for robust gene disruption and correction.


Assuntos
Pesquisas com Embriões , Embrião de Mamíferos , Edição de Genes/métodos , Humanos
18.
Science ; 364(6437): 289-292, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30819928

RESUMO

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.


Assuntos
Blastômeros , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Citosina , Edição de Genes/métodos , Polimorfismo de Nucleotídeo Único , Animais , Análise Mutacional de DNA , Embrião de Mamíferos , Feminino , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação
19.
Cancer Med ; 7(11): 5488-5496, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30306725

RESUMO

Postoperative chemotherapy has been widely used in the treatment of early-staged ovarian cancer patients underwent unilateral resection, but the clinical decision mainly depends on the doctor's experience without a well-defined guideline. This study used propensity score matching to analyze the effect of postoperative chemotherapy for early-staged ovarian cancer patients underwent unilateral resection on prognosis. Patients of age 50 or younger than 50 with early-staged ovarian cancer were explored from the Surveillance, Epidemiology, and End Results program database during 2000-2018. Propensity score matching was used to randomize the dataset and reduce the selection biases. Univariate and multivariate cox proportional hazards models were utilized to estimate the necessity of chemotherapy. In univariate analysis of matched population, both the overall survival and cancer-specific survival analysis showed that chemotherapy had no effect on the prognosis of early-staged young ovarian cancer patients (Overall survival, P = 0.477; Cancer-specific survival, P = 0.950). In propensity-adjusted multivariate analysis, chemotherapy still had no effect on both the overall and cancer-specific survival probability after excluding the effect of all the confounding factors (HR = 0.863, CI = 0.587-1.269, P = 0.455; HR = 1.009, CI = 0.633-1.607, P = 0.970). Our study suggested that postoperative chemotherapy is not necessary for early-staged young ovarian cancer patients with unilateral resection, as indicated by both the overall survival and cancer-specific survival.


Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/cirurgia , Adulto , Quimioterapia Adjuvante , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Prognóstico , Programa de SEER , Análise de Sobrevida
20.
Development ; 145(20)2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30275281

RESUMO

In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Zigoto/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Sequência de Bases , Cóclea/metabolismo , Surdez/genética , Surdez/fisiopatologia , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Motores Moleculares/metabolismo , Mutação/genética
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