Frontiers in genome editing最新文献_第10页

Base Editing in Peanut Using CRISPR/nCas9 利用CRISPR/nCas9对花生进行碱基编辑

Frontiers in genome editing Pub Date : 2022-05-12 DOI: 10.3389/fgeed.2022.901444

A. Neelakandan, B. Subedi, S. Traore, P. Binagwa, D. Wright, G. He

{"title":"Base Editing in Peanut Using CRISPR/nCas9","authors":"A. Neelakandan, B. Subedi, S. Traore, P. Binagwa, D. Wright, G. He","doi":"10.3389/fgeed.2022.901444","DOIUrl":"https://doi.org/10.3389/fgeed.2022.901444","url":null,"abstract":"Peanut (Arachis hypogaea L.), an allotetraploid legume of the Fabaceae family, is able to thrive in tropical and subtropical regions and is considered as a promising oil seed crop worldwide. Increasing the content of oleic acid has become one of the major goals in peanut breeding because of health benefits such as reduced blood cholesterol level, antioxidant properties and industrial benefits such as longer shelf life. Genomic sequencing of peanut has provided evidence of homeologous AhFAD2A and AhFAD2B genes encoding Fatty Acid Desaturase2 (FAD2), which are responsible for catalyzing the conversion of monounsaturated oleic acid into polyunsaturated linoleic acid. Research studies demonstrate that mutations resulting in a frameshift or stop codon in an FAD2 gene leads to higher oleic acid content in oil. In this study, two expression vectors, pDW3873 and pDW3876, were constructed using Cas9 fused to different deaminases, which were tested as tools to induce point mutations in the promoter and the coding sequences of peanut AhFAD2 genes. Both constructs harbor the single nuclease null variant, nCas9 D10A, to which the PmCDA1 cytosine deaminase was fused to the C-terminal (pDW3873) while rAPOBEC1 deaminase and an uracil glycosylase inhibitor (UGI) were fused to the N-terminal and the C-terminal respectively (pDW3876). Three gRNAs were cloned independently into both constructs and the functionality and efficiency were tested at three target sites in the AhFAD2 genes. Both constructs displayed base editing activity in which cytosine was replaced by thymine or other bases in the targeted editing window. pDW3873 showed higher efficiency compared to pDW3876 suggesting that the former is a better base editor in peanut. This is an important step forward considering introgression of existing mutations into elite varieties can take up to 15 years making this tool a benefit for peanut breeders, farmers, industry and ultimately for consumers.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43872135","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Potential of Genome Editing to Capture Diversity From Australian Wild Rice Relatives 基因组编辑获取澳大利亚野生稻近缘品种多样性的潜力

Frontiers in genome editing Pub Date : 2022-04-27 DOI: 10.3389/fgeed.2022.875243

Muhammad Abdullah, P. Okemo, A. Furtado, R. Henry

{"title":"Potential of Genome Editing to Capture Diversity From Australian Wild Rice Relatives","authors":"Muhammad Abdullah, P. Okemo, A. Furtado, R. Henry","doi":"10.3389/fgeed.2022.875243","DOIUrl":"https://doi.org/10.3389/fgeed.2022.875243","url":null,"abstract":"Rice, a staple food worldwide and a model crop, could benefit from the introduction of novel genetics from wild relatives. Wild rice in the AA genome group closely related to domesticated rice is found across the tropical world. Due to their locality outside the range of domesticated rice, Australian wild rice populations are a potential source of unique traits for rice breeding. These rice species provide a diverse gene pool for improvement that could be utilized for desirable traits such as stress resistance, disease tolerance, and nutritional qualities. However, they remain poorly characterized. The CRISPR/Cas system has revolutionized gene editing and has improved our understanding of gene functions. Coupled with the increasing availability of genomic information on the species, genes in Australian wild rice could be modified through genome editing technologies to produce new domesticates. Alternatively, beneficial alleles from these rice species could be incorporated into cultivated rice to improve critical traits. Here, we summarize the beneficial traits in Australian wild rice, the available genomic information and the potential of gene editing to discover and understand the functions of novel alleles. Moreover, we discuss the potential domestication of these wild rice species for health and economic benefits to rice production globally.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48479248","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Spatiotemporal Regulation of CRISPR/Cas9 Enables Efficient, Precise, and Heritable Edits in Plant Genomes CRISPR/Cas9的时空调控使植物基因组高效、精确和可遗传编辑成为可能

Frontiers in genome editing Pub Date : 2022-04-26 DOI: 10.3389/fgeed.2022.870108

Farhanur Rahman, Apurva Mishra, Archit Gupta, Rita Sharma

引用次数: 4

CRISPR-Mediated Activation of αV Integrin Subtypes Promotes Neuronal Differentiation of Neuroblastoma Neuro2a Cells crispr介导的αV整合素亚型激活促进神经母细胞瘤Neuro2a细胞的神经元分化

Frontiers in genome editing Pub Date : 2022-04-12 DOI: 10.3389/fgeed.2022.846669

Sara Riccardi, L. Cingolani, F. Jaudon

{"title":"CRISPR-Mediated Activation of αV Integrin Subtypes Promotes Neuronal Differentiation of Neuroblastoma Neuro2a Cells","authors":"Sara Riccardi, L. Cingolani, F. Jaudon","doi":"10.3389/fgeed.2022.846669","DOIUrl":"https://doi.org/10.3389/fgeed.2022.846669","url":null,"abstract":"Neuronal differentiation is a complex process whose dysfunction can lead to brain disorders. The development of new tools to target specific steps in the neuronal differentiation process is of paramount importance for a better understanding of the molecular mechanisms involved, and ultimately for developing effective therapeutic strategies for neurodevelopmental disorders. Through their interactions with extracellular matrix proteins, the cell adhesion molecules of the integrin family play essential roles in the formation of functional neuronal circuits by regulating cell migration, neurite outgrowth, dendritic spine formation and synaptic plasticity. However, how different integrin receptors contribute to the successive phases of neuronal differentiation remains to be elucidated. Here, we implemented a CRISPR activation system to enhance the endogenous expression of specific integrin subunits in an in vitro model of neuronal differentiation, the murine neuroblastoma Neuro2a cell line. By combining CRISPR activation with morphological and RT-qPCR analyses, we show that integrins of the αV family are powerful inducers of neuronal differentiation. Further, we identify a subtype-specific role for αV integrins in controlling neurite outgrowth. While αVβ3 integrin initiates neuronal differentiation of Neuro2a cells under proliferative conditions, αVβ5 integrin appears responsible for promoting a complex arborization in cells already committed to differentiation. Interestingly, primary neurons exhibit a complementary expression pattern for β3 and β5 integrin subunits during development. Our findings reveal the existence of a developmental switch between αV integrin subtypes during differentiation and suggest that a timely controlled modulation of the expression of αV integrins by CRISPRa provides a means to promote neuronal differentiation.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44371044","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4

Canadian Consumer Preferences Regarding Gene-Edited Food Products 加拿大消费者对基因编辑食品的偏好

Frontiers in genome editing Pub Date : 2022-04-11 DOI: 10.3389/fgeed.2022.854334

Oswaldo Vasquez, Hayley Hesseln, S. Smyth

{"title":"Canadian Consumer Preferences Regarding Gene-Edited Food Products","authors":"Oswaldo Vasquez, Hayley Hesseln, S. Smyth","doi":"10.3389/fgeed.2022.854334","DOIUrl":"https://doi.org/10.3389/fgeed.2022.854334","url":null,"abstract":"Innovations in food production and processing have largely remained “behind the scenes” for decades. The current nature of social media and calls for increased transparency regarding food results in a new landscape where consumer product demands are more important than ever, but are increasingly based on limited, or incorrect, information. One area where consumer awareness is rapidly emerging is the area of gene-edited food products. This article uses a consumer survey to gather perceptions regarding food safety, gene editing and willingness to consume for three gene-edited food products. Four factors were found to strongly influence consumer perceptions: trust in the Canadian food safety system; food technology neophobia scores; knowledge of genetics; and self-knowledge of gene editing. The survey of 497 Canadians found that 15% identified as neophobics and 12% as neophilics. The majority of participants identified as neutral. When presented with various food values, participants indicated that nutrition, price, and taste were the three most important values. A participants’ willingness to consume gene-edited food products strongly correlated with neophobic and neophilic preferences, with neophobics unwilling to consume and neophilics being uncertain. The only food value that strongly affects consumer willingness to consume is the environmental impact of a products’ production. Canadian consumers have a moderate to high level of trust in Canada’s food safety system, but this level of trust fails to carry over to food products produced through innovative technologies; however, consumers express a higher level of trust in gene-edited technology than genetically modified technology.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41852975","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Imaging Unique DNA Sequences in Individual Cells Using a CRISPR-Cas9-Based, Split Luciferase Biosensor 使用基于CRISPR-Cas9的分裂萤光素酶生物传感器成像单个细胞中的独特DNA序列

Frontiers in genome editing Pub Date : 2022-03-25 DOI: 10.3389/fgeed.2022.867390

Nicholas G. Heath, H. O’Geen, Nicole B. Halmai, J. Corn, D. Segal

{"title":"Imaging Unique DNA Sequences in Individual Cells Using a CRISPR-Cas9-Based, Split Luciferase Biosensor","authors":"Nicholas G. Heath, H. O’Geen, Nicole B. Halmai, J. Corn, D. Segal","doi":"10.3389/fgeed.2022.867390","DOIUrl":"https://doi.org/10.3389/fgeed.2022.867390","url":null,"abstract":"An extensive arsenal of biosensing tools has been developed based on the clustered regularly interspaced short palindromic repeat (CRISPR) platform, including those that detect specific DNA sequences both in vitro and in live cells. To date, DNA imaging approaches have traditionally used full fluorescent reporter-based fusion probes. Such “always-on” probes differentiate poorly between bound and unbound probe and are unable to sensitively detect unique copies of a target sequence in individual cells. Herein we describe a DNA biosensor that provides a sensitive readout for such low-copy DNA sequences through proximity-mediated reassembly of two independently optimized fragments of NanoLuc luciferase (NLuc), a small, bright luminescent reporter. Applying this “turn-on” probe in live cells, we demonstrate an application not easily achieved by fluorescent reporter-based probes, detection of individual endogenous genomic loci using standard epifluorescence microscopy. This approach could enable detection of gene edits during ex vivo editing procedures and should be a useful platform for many other live cell DNA biosensing applications.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48320541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System 使用双荧光稳定报告系统快速评估CRISPR转染效率和富集CRISPR诱导的突变

Frontiers in genome editing Pub Date : 2022-03-21 DOI: 10.3389/fgeed.2022.854866

Karim E Shalaby, Mustapha Aouida, Vijay Gupta, S. Ghanem, O. El‐Agnaf

{"title":"Rapid Assessment of CRISPR Transfection Efficiency and Enrichment of CRISPR Induced Mutations Using a Dual-Fluorescent Stable Reporter System","authors":"Karim E Shalaby, Mustapha Aouida, Vijay Gupta, S. Ghanem, O. El‐Agnaf","doi":"10.3389/fgeed.2022.854866","DOIUrl":"https://doi.org/10.3389/fgeed.2022.854866","url":null,"abstract":"The nuclease activity of the CRISPR-Cas9 system relies on the delivery of a CRISPR-associated protein 9 (Cas9) and a single guide RNA (sgRNA) against the target gene. CRISPR components are typically delivered to cells as either a Cas9/sgRNA ribonucleoprotein (RNP) complex or a plasmid encoding a Cas9 protein along with a sequence-specific sgRNA. Multiple transfection reagents are known to deliver CRISPR-Cas9 components, and delivery vectors are being developed for different purposes by several groups. Here, we repurposed a dual-fluorescence (RFP-GFP-GFP) reporter system to quantify the uptake level of the functional CRISPR-Cas9 components into cells and compare the efficiency of CRISPR delivery vectors. Using this system, we developed a novel and rapid cell-based microplate reader assay that makes possible real-time, rapid, and high throughput quantification of CRISPR nuclease activity. Cells stably expressing this dual-fluorescent reporter construct facilitated a direct quantification of the level of the internalized and functional CRISPR-Cas9 molecules into the cells without the need of co-transfecting fluorescently labeled reporter molecules. Additionally, targeting a reporter gene integrated into the genome recapitulates endogenous gene targeting. Thus, this reporter could be used to optimize various transfection conditions of CRISPR components, to evaluate and compare the efficiency of transfection agents, and to enrich cells containing desired CRISPR-induced mutations.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47822044","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Contributions of Genome Editing Technologies Towards Improved Nutrition, Environmental Sustainability and Poverty Reduction. 基因组编辑技术对改善营养、环境可持续性和减少贫困的贡献。

IF 4.9

Frontiers in genome editing Pub Date : 2022-03-17 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.863193

Stuart J Smyth

{"title":"Contributions of Genome Editing Technologies Towards Improved Nutrition, Environmental Sustainability and Poverty Reduction.","authors":"Stuart J Smyth","doi":"10.3389/fgeed.2022.863193","DOIUrl":"10.3389/fgeed.2022.863193","url":null,"abstract":"The Sustainable Development Goals (SDGs) were launched in 2015, with the top three goals being poverty eradication, improved food security and increased human health. All 17 SDGs have a target achievement date of 2030. These are ambitious and inspirational goals that require substantial innovation and technology adoption for successful achievement. Innovations in plant breeding have substantially contributed to transforming the efficiency of food production since the mid 20th century, with innovations emerging in the current millennium demonstrating enhanced potential to improve crop yields, the nutritional values of food crops and environmental impacts. These outcomes underpin several SDGs, but in particular the first three. As climate change is expected to become increasingly variable, with greater impacts on agriculture, the ability to ensure increased food production is going to be increasingly important, as higher yields directly contribute to reducing poverty. This article reviews recent reports of potential contributions from genome editing technologies in terms of increased yield, enhanced nutrition and greater sustainability, highlighting their importance for achieving the leading three SDGs.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"4 1","pages":"863193"},"PeriodicalIF":4.9,"publicationDate":"2022-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8968197/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"69928227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome. 基于CRISPR/ cas的DOCK8免疫缺陷综合征基因编辑策略

IF 4.9

Frontiers in genome editing Pub Date : 2022-03-17 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.793010

Sujan Ravendran, Sabina Sánchez Hernández, Saskia König, Rasmus O Bak

{"title":"CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome.","authors":"Sujan Ravendran, Sabina Sánchez Hernández, Saskia König, Rasmus O Bak","doi":"10.3389/fgeed.2022.793010","DOIUrl":"10.3389/fgeed.2022.793010","url":null,"abstract":"Defects in the DOCK8 gene causes combined immunodeficiency termed DOCK8 immunodeficiency syndrome (DIDS). DIDS previously belonged to the disease category of autosomal recessive hyper IgE syndrome (AR-HIES) but is now classified as a combined immunodeficiency (CID). This genetic disorder induces early onset of susceptibility to severe recurrent viral and bacterial infections, atopic diseases and malignancy resulting in high morbidity and mortality. This pathological state arises from impairment of actin polymerization and cytoskeletal rearrangement, which induces improper immune cell migration-, survival-, and effector functions. Owing to the severity of the disease, early allogenic hematopoietic stem cell transplantation is recommended even though it is associated with risk of unintended adverse effects, the need for compatible donors, and high expenses. So far, no alternative therapies have been developed, but the monogenic recessive nature of the disease suggests that gene therapy may be applied. The advent of the CRISPR/Cas gene editing system heralds a new era of possibilities in precision gene therapy, and positive results from clinical trials have already suggested that the tool may provide definitive cures for several genetic disorders. Here, we discuss the potential application of different CRISPR/Cas-mediated genetic therapies to correct the DOCK8 gene. Our findings encourage the pursuit of CRISPR/Cas-based gene editing approaches, which may constitute more precise, affordable, and low-risk definitive treatment options for DOCK8 deficiency.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"4 1","pages":"793010"},"PeriodicalIF":4.9,"publicationDate":"2022-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8969908/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41480667","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Base Editors for Citrus Gene Editing. 柑橘基因编辑的碱基编辑器

IF 4.9

Frontiers in genome editing Pub Date : 2022-02-28 eCollection Date: 2022-01-01 DOI: 10.3389/fgeed.2022.852867

Xiaoen Huang, Yuanchun Wang, Nian Wang

{"title":"Base Editors for Citrus Gene Editing.","authors":"Xiaoen Huang, Yuanchun Wang, Nian Wang","doi":"10.3389/fgeed.2022.852867","DOIUrl":"10.3389/fgeed.2022.852867","url":null,"abstract":"Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to the canker pathogen Xanthomonas citri subsp. citri (Xcc). In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene in citrus. ALS-edited plants were resistant to the herbicide chlorsulfuron. Two ALS-edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. The Cas9 gene was undetectable in the herbicide-resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"4 1","pages":"852867"},"PeriodicalIF":4.9,"publicationDate":"2022-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8919994/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41630584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0