scholarly journals Development of a gRNA-tRNA Array of CRISPR/Cas9 in Combination with Grafting Technique to Improve Gene Editing Efficiency of Sweet Orange

2021 ◽  
Author(s):  
Xiaomei Tang ◽  
Shulin Chen ◽  
Huiwen Yu ◽  
Xiongjie Zheng ◽  
Fei Zhang ◽  
...  
Keyword(s):  
Genetic Improvement ◽  
Gene Editing ◽  
Genome Engineering ◽  
Target Genes ◽  
Sweet Orange ◽  
Fruit Crops ◽  
Grafting Technique ◽  
Efficient Gene ◽  
Traditional Breeding ◽  
Selection Of

Abstract Sweet orange is one of the most popular fruit crops worldwide. Traditional breeding approaches in sweet orange is impracticable due to the apomixis and long juvenility, making it difficult to obtain hybrids and selection of ideal genotypes. The development of targeted genome engineering technologies made it possible for the precise modification of target genes. Recently, a more efficient gene editing tool has been emerged based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system (Bhaya et al. 2011). The development of CRISPR/Cas9 technology is promising to accelerate the process of genetic improvement in perennial crops.

scholarly journals Highly efficient generation of canker resistant sweet orange enabled by an improved CRISPR/Cas9 system

2021 ◽  
Author(s):  
Xiaoen Huang ◽  
Nian Wang
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Sweet Orange ◽  
Susceptibility Gene ◽  
Transformation Rate ◽  
Biallelic Mutation ◽  
Important Species ◽  
Citrus Production

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously (dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, CsU6 to drive sgRNA expression), we further improved our CRISPR/Cas9 system by choosing superior promoters (CmYLCV or CsUbi promoter) to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

Advances in therapeutic application of CRISPR-Cas9

2019 ◽  
Vol 19 (3) ◽  
pp. 164-174 ◽  
Author(s):  
Jinyu Sun ◽  
Jianchu Wang ◽  
Donghui Zheng ◽  
Xiaorong Hu
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Malignant Tumor ◽  
Gene Editing ◽  
Genome Engineering ◽  
Therapeutic Application ◽  
Adaptive Immune ◽  
Efficient Gene

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is one of the most versatile and efficient gene editing technologies, which is derived from adaptive immune strategies for bacteria and archaea. With the remarkable development of programmable nuclease-based genome engineering these years, CRISPR-Cas9 system has developed quickly in recent 5 years and has been widely applied in countless areas, including genome editing, gene function investigation and gene therapy both in vitro and in vivo. In this paper, we briefly introduce the mechanisms of CRISPR-Cas9 tool in genome editing. More importantly, we review the recent therapeutic application of CRISPR-Cas9 in various diseases, including hematologic diseases, infectious diseases and malignant tumor. Finally, we discuss the current challenges and consider thoughtfully what advances are required in order to further develop the therapeutic application of CRISPR-Cas9 in the future.

scholarly journals Scarless Enriched selection of Genome edited Human Pluripotent Stem Cells Using Induced Drug Resistance

2019 ◽  
Author(s):  
Keun-Tae Kim ◽  
Ju-Chan Park ◽  
Haeseung Lee ◽  
Hyeon-Ki Jang ◽  
Yan Jin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Gene Editing ◽  
Human Pluripotent Stem Cells ◽  
Cancer Drug ◽  
Efficient Gene ◽  
Anti Cancer ◽  
Low Efficiency ◽  
High Cytotoxicity ◽  
Selection Of

SummaryAn efficient gene editing technique for use in human pluripotent stem cells (hPSCs) would have great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs is a major technical hurdle that remains to be resolved. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to high expression of SLC35F2 in these cells. Consistent with this, knockout of SLC35F2 with CRISPR-Cas9 or depletion with siRNAs made hPSCs highly resistant to YM155. Simultaneous gene editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled genome-edited hPSCs to survive because YM155 resistance was temporarily induced, thereby achieving enriched selection of genome-edited clonal populations. This precise and efficient genome editing approach took as little as 3 weeks without cell sorting or introduction of additional genes.

scholarly journals Genetic editing for improvement of fruit and small fruit crops

2019 ◽  
pp. 10-15
Author(s):  
N. G. Tikhonova ◽  
E. K. Khlestkina
Keyword(s):  
Systematic Review ◽  
Gene Editing ◽  
Target Genes ◽  
Crop Improvement ◽  
Original Research ◽  
Fruit Crops ◽  
Internet Resources ◽  
Biotechnological Approach ◽  
Small Fruit ◽  
Present Systematic Review

Today the range of crops, which are studied and improved with the help of genetic editing technologies, in particular with CRISPR/Cas system, is actively expanding. High accuracy, ease of editing tools application and relative cheapness allow the use of this method in research in various fi elds of biology. The present systematic review summarizes the global experience of editing the genome of fruit and small fruit crops using the CRISPR/Cas9 tool and considers the prospect of using this newest biotechnological approach for crop improvement. The systematic review employed one of the most authoritative internet resources indexing the reviewed scientifi c publications, such as the Scopus database. By systematic search concerning 37 species of fruit and small fruit crops 115 publications were found, among which 26 described original research on CRISPR/Cas system application to fruit and small fruit crops. A total of 21 target genes have been edited in 8 crops. Some of the studies are consecrated to the approbation of the method or establishing/clarifying the functions of the target genes by their knockout. In the review we distinguished the publications describing application of gene editing tools for improving the properties of plants. By now 12 target genes have been successfully edited in fruit and small fruit plants. The main type of modifi cation is the knockout, which is aimed at negative regulators of the factors of resistance to pathogens or at changing the morphology of plants. In most cases the modifi ed plants have the desired characteristics and have been tested for resistance to pathogens by laboratory tests. It is discussed how much the target mutagenesis application to fruit and small fruit crops can be broadened in future.

scholarly journals Plant viral vectors: Expanding the Possibilities of Precise Gene Editing in Plant Genomes

2021 ◽  
Author(s):  
Stuti Kujur ◽  
Muthappa Senthil-Kumar ◽  
Rahul Kumar
Keyword(s):  
Genome Editing ◽  
Plant Transformation ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genome Engineering ◽  
Plant Cells ◽  
In Planta ◽  
Plant Genomes ◽  
Efficient Gene ◽  
Transformation Methods

Abstract The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

scholarly journals mSphere of Influence: Ushering in the CRISPR Revolution to Toxoplasma Biology

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Alfredo J. Guerra
Keyword(s):  
Toxoplasma Gondii ◽  
Structural Biology ◽  
Gene Disruption ◽  
Gene Editing ◽  
Genome Engineering ◽  
Content Type ◽  
Efficient Gene ◽  
Implementing Strategies ◽  
Molecular Parasitology

ABSTRACT Alfredo J. Guerra works in the field of molecular parasitology and structural biology. In this mSphere of Influence article, he reflects on how “Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9” by Bang Shen et al. (mBio 5:e01114-14, 2014, https://doi.org/10.1128/mBio.01114-14) and “Efficient Genome Engineering of Toxoplasma gondii using CRISPR/CAS9” by Saima M. Sidik et al. (PLoS One 9:e100450, 2014, https://doi.org/10.1371/journal.pone.0100450) made an impact on him by successfully implementing strategies to genetically manipulate T. gondii using CRISPR/CAS9 gene editing technology.

scholarly journals Highly Efficient Generation of Canker-Resistant Sweet Orange Enabled by an Improved CRISPR/Cas9 System

2022 ◽  
Vol 12 ◽  
Author(s):  
Xiaoen Huang ◽  
Yuanchun Wang ◽  
Nian Wang
Keyword(s):  
Genome Editing ◽  
Citrus Sinensis ◽  
Gene Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Sweet Orange ◽  
Susceptibility Gene ◽  
Transformation Rate ◽  
Biallelic Mutation ◽  
Important Species

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes, and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously [dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, citrus U6 (CsU6) to drive sgRNA expression], we further improved our CRISPR/Cas9 system by choosing superior promoters [Cestrum yellow leaf curling virus (CmYLCV) or Citrus sinensis ubiquitin (CsUbi) promoter] to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker-resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease-resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

scholarly journals New and TALENted Genome Engineering Toolbox

2013 ◽  
Vol 113 (5) ◽  
pp. 571-587 ◽  
Author(s):  
Jarryd M. Campbell ◽  
Katherine A. Hartjes ◽  
Timothy J. Nelson ◽  
Xiaolei Xu ◽  
Stephen C. Ekker
Keyword(s):  
Cardiovascular Disease ◽  
Gene Editing ◽  
Genome Engineering ◽  
Genetic Alterations ◽  
Model Systems ◽  
Patient Specific ◽  
Specific Gene ◽  
Genetic Defects ◽  
Throughput Model ◽  
Efficient Gene

Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.

Caracterização da Peneira Média em Clones de Coffea canephora

REVISTA FIMCA ◽  
2018 ◽  
Vol 5 (2) ◽  
pp. 28-31
Author(s):  
Darlan Darlan Sanches Barbosa Alves ◽  
Victor Mouzinho Spinelli ◽  
Marcos Santana Moraes ◽  
Carolina Augusto De Souza ◽  
Rodrigo da Silva Ribeiro ◽  
...  
Keyword(s):  
Genetic Improvement ◽  
Block Design ◽  
Coffea Canephora ◽  
Coffee Production ◽  
Randomized Block Design ◽  
Significant Difference ◽  
The Mean ◽  
Four Blocks ◽  
Selection Of

Introdução: O estado de Rondônia se destaca como tradicional produtor de café, sendo o segundo maior produtor brasileiro de C. canephora. No melhoramento genético de C. canephora, a seleção de plantas de elevada peneira média está associada à bebida de qualidade superior. Objetivos: O objetivo desse estudo foi avaliar a variabilidade genética de clones de C. canephora para o tamanho dos grãos, mensurado a partir da avaliação da peneira média (PM). Materiais e Métodos: Para isso, foi conduzido ao longo de dois anos agrícolas experimento no campo experimental da Embrapa no município de Ouro Preto do Oeste-RO, para a avaliação da peneira média de 130 genótipos (clones) com características das variedades botânicas Conilon, Robusta e híbridos intervarietais. O delineamento experimental utilizado foi de blocos ao acaso, com quatro repetições de quatro plantas por parcela. Resultados: Não houve resultados significativos para a interação clones X anos, indicando uma maior consistência no comportamento das plantas ao longo do tempo. Porém foram observadas diferenças significativas para o tamanho dos grãos entre os genótipos avaliados, possibilitando selecionar genótipos superiores. Conclusão: Os genótipos agruparam-se em cinco classes de acordo com o teste de média, subsidiando a caracterização de um gradiente de variabilidade da característica avaliada ABSTRACTIntroduction: Coffea canephora accounts for approximately 35% of the world's coffee production. The state of Rondônia stands out as a traditional coffee producer, being the second largest Brazilian producer of C. canephora. In the classical genetic improvement of C. anephora, the selection of plants of high average sieve is associated with a drink of superior quality. Objectives: The objective of this udy was to evaluate the genetic variability of Coffea canephora clones for the agronomic medium sieve (PM). Materials and Methods: The experiment was conducted in the experimental field of Embrapa, municipality of OuroPreto do Oeste-RO, located at coordinates 10º44'53 "S and 62º12'57". One hundred thirty genotypes (clones) of botanical characteristics Conilon, Robusta and intervarietal hybrids were evaluated in the agricultural years 2013-2014 and 2014-2015. The experimental design was a randomized block design with four blocks and four plants per plot, spacing 3.5 x 1.5 meters between plants. Results: Significant difference was found for the grain size. According to the F test, at 5% probability, the genotypes were grouped into five classes according to the mean test. Conclusion: The results obtained subsidized the characterization of a variability gradient of the evaluated trait.

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