scholarly journals Highly efficient generation of bacteria leaf blight resistance and transgene-free rice using genome editing and multiplexed selection system

2020 ◽  
Author(s):  
Kun Yu ◽  
Zhiqiang Liu ◽  
Huaping Gui ◽  
Lizhao Geng ◽  
Juan Wei ◽  
...  
Keyword(s):  
Protein Binding ◽  
Genome Editing ◽  
Leaf Blight ◽  
Genomic Research ◽  
Blight Resistance ◽  
Selection System ◽  
Transcription Activator ◽  
Efficient Generation ◽  
A Genome ◽  
Selection Of

Abstract Background Rice leaf blight is a worldwide devastating disease caused by bacteria Xanthomonas oryzae pv. Oryzae (Xoo). The UPT (up-regulated by transcription activator-like 1 effector) box in promoter region of the rice Xa13 gene played a key role in Xoo pathogenicity. Mutation of key bacterial protein binding site in UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistant to bacterial.Highly efficient generation and selection transgene-free, edited plants helpful to shorten and simple the gene editing breeding process. Selective elimination of transgenic pollen of E0 plants can enrich proportion of E1 transgene-free offspring and expression of the color mark gene in seeds makes the selection of E2 plants is very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacteria leaf blight resistance and transgene-free rice plants.Results We introduced site specific mutations into the UPT box using CRISPR/Cas12a technology to hamper TAL (Transcription-Activator Like effectors) protein binding and gene activation, and generated genome edited rice with improved bacteria blight resistance. Transgenic pollens of E0 plants were eliminated by pollen specific expression of α-amylase gene Zmaa1, the proportion of transgene-free plants were enriched from 25% to 50% in single T-DNA insertion events in E1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced 50% cost and achieved up to 98.64% of accuracy for selection of transgene-free edited plants. Conclusion We demonstrated core nucleotide deletion in the UPT box of Xa13 promoter conferred resistance to rice blight and selection of transgene-free plants were boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

scholarly journals Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Kun Yu ◽  
Zhiqiang Liu ◽  
Huaping Gui ◽  
Lizhao Geng ◽  
Juan Wei ◽  
...  
Keyword(s):  
Protein Binding ◽  
Genome Editing ◽  
Leaf Blight ◽  
Bacterial Leaf Blight ◽  
Selection System ◽  
Transcription Activator ◽  
Efficient Generation ◽  
A Genome ◽  
Transgenic Pollen ◽  
Selection Of

Abstract Background Rice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria. Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants. Results We introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants. Conclusion We demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

scholarly journals Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing

Plants ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 687 ◽  
Author(s):  
Chang Ho Ahn ◽  
Mummadireddy Ramya ◽  
Hye Ryun An ◽  
Pil Man Park ◽  
Yae-Jin Kim ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genetically Modified Organisms ◽  
Ornamental Plants ◽  
Safety Evaluation ◽  
Evaluation Process ◽  
Vase Life ◽  
Transcription Activator ◽  
A Genome ◽  
Dna Specificity

Biotechnological approaches have been used to modify the floral color, size, and fragrance of ornamental plants, as well as to increase disease resistance and vase life. Together with the advancement of whole genome sequencing technologies, new plant breeding techniques have rapidly emerged in recent years. Compared to the early versions of gene editing tools, such as meganucleases (MNs), zinc fingers (ZFNs), and transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) is capable of altering a genome more efficiently and with higher accuracy. Most recently, new CRISPR systems, including base editors and prime editors, confer reduced off-target activity with improved DNA specificity and an expanded targeting scope. However, there are still controversial issues worldwide for the recognition of genome-edited plants, including whether genome-edited plants are genetically modified organisms and require a safety evaluation process. In the current review, we briefly summarize the current progress in gene editing systems and also introduce successful/representative cases of the CRISPR system application for the improvement of ornamental plants with desirable traits. Furthermore, potential challenges and future prospects in the use of genome-editing tools for ornamental plants are also discussed.

TALEN-mediated genome editing: prospects and perspectives

2014 ◽  
Vol 462 (1) ◽  
pp. 15-24 ◽  
Author(s):  
David A. Wright ◽  
Ting Li ◽  
Bing Yang ◽  
Martin H. Spalding
Keyword(s):  
Genome Editing ◽  
Double Strand Breaks ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Strand Breaks ◽  
Natural Processes ◽  
Current State ◽  
A Genome ◽  
Dna Dsbs ◽  
Repair Mechanisms

Genome editing is the practice of making predetermined and precise changes to a genome by controlling the location of DNA DSBs (double-strand breaks) and manipulating the cell's repair mechanisms. This technology results from harnessing natural processes that have taken decades and multiple lines of inquiry to understand. Through many false starts and iterative technology advances, the goal of genome editing is just now falling under the control of human hands as a routine and broadly applicable method. The present review attempts to define the technique and capture the discovery process while following its evolution from meganucleases and zinc finger nucleases to the current state of the art: TALEN (transcription-activator-like effector nuclease) technology. We also discuss factors that influence success, technical challenges and future prospects of this quickly evolving area of study and application.

scholarly journals Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Wei Shen ◽  
Jun Zhang ◽  
Binan Geng ◽  
Mengyue Qiu ◽  
Mimi Hu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Zymomonas Mobilis ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Strain Improvement ◽  
Genomic Research ◽  
Francisella Novicida ◽  
A Genome

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

scholarly journals Genome Editing Technology and Its Application Potentials in the Industrial Filamentous Fungus Aspergillus oryzae

2021 ◽  
Vol 7 (8) ◽  
pp. 638
Author(s):  
Jun-ichi Maruyama
Keyword(s):  
Genome Editing ◽  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
Genetic Manipulation ◽  
Wild Strain ◽  
Soy Sauce ◽  
Transcription Activator ◽  
Gene Modification ◽  
A Genome ◽  
Industrial Strains

Aspergillus oryzae is a filamentous fungus that has been used in traditional Japanese brewing industries, such as the sake, soy sauce, and miso production. In addition, A. oryzae has been used in heterologous protein production, and the fungus has been recently used in biosynthetic research due to its ability to produce a large amount of heterologous natural products by introducing foreign biosynthetic genes. Genetic manipulation, which is important in the functional development of A. oryzae, has mostly been limited to the wild strain RIB40, a genome reference suitable for laboratory analysis. However, there are numerous industrial brewing strains of A. oryzae with various specialized characteristics, and they are used selectively according to the properties required for various purposes such as sake, soy sauce, and miso production. Since the early 2000s, genome editing technologies have been developed; among these technologies, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) have been applied to gene modification in A. oryzae. Notably, the CRISPR/Cas9 system has dramatically improved the efficiency of gene modification in industrial strains of A. oryzae. In this review, the development of genome editing technology and its application potentials in A. oryzae are summarized.

scholarly journals Inducible Plasmid Self-Destruction (IPSD) assisted genome engineering in lactobacilli and bifidobacteria

2019 ◽  
Author(s):  
Fanglei Zuo ◽  
Zhu Zeng ◽  
Lennart Hammarström ◽  
Harold Marcotte
Keyword(s):  
Synthetic Biology ◽  
Homologous Recombination ◽  
Genetic Engineering ◽  
Genome Editing ◽  
Genome Engineering ◽  
Bacterial Species ◽  
Knock Out ◽  
Recombination System ◽  
A Genome ◽  
Selection Of

ABSTRACTGenome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria but still, have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called Inducible Plasmid Self-Destruction (IPSD) was successfully used to perform gene knock-out and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.

scholarly journals In Vivo Genome Editing as a Therapeutic Approach

2018 ◽  
Vol 19 (9) ◽  
pp. 2721 ◽  
Author(s):  
Beatrice Ho ◽  
Sharon Loh ◽  
Woon Chan ◽  
Boon Soh
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Genetic Diseases ◽  
Gene Mutations ◽  
Genetic Mutation ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Therapeutic Benefits ◽  
A Genome

Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo genome engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed.

scholarly journals Multiplex Genome-Editing Technologies for Revolutionizing Plant Biology and Crop Improvement

2021 ◽  
Vol 12 ◽  
Author(s):  
Mohamed Abdelrahman ◽  
Zheng Wei ◽  
Jai S. Rohila ◽  
Kaijun Zhao
Keyword(s):  
Genome Editing ◽  
Crop Improvement ◽  
Plant Molecular Biology ◽  
Plant Biology ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Simultaneous Generation ◽  
Multiple Loci ◽  
A Genome ◽  
Improvement Programs

Multiplex genome-editing (MGE) technologies are recently developed versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. These genome-editing tools have greatly increased the feasibility of introducing desired changes at multiple nucleotide levels into a target genome. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) [CRISPR/Cas] system-based MGE tools allow the simultaneous generation of direct mutations precisely at multiple loci in a gene or multiple genes. MGE is enhancing the field of plant molecular biology and providing capabilities for revolutionizing modern crop-breeding methods as it was virtually impossible to edit genomes so precisely at the single base-pair level with prior genome-editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Recently, researchers have not only started using MGE tools to advance genome-editing applications in certain plant science fields but also have attempted to decipher and answer basic questions related to plant biology. In this review, we discuss the current progress that has been made toward the development and utilization of MGE tools with an emphasis on the improvements in plant biology after the discovery of CRISPR/Cas9. Furthermore, the most recent advancements involving CRISPR/Cas applications for editing multiple loci or genes are described. Finally, insights into the strengths and importance of MGE technology in advancing crop-improvement programs are presented.

scholarly journals Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies

2021 ◽  
Vol 22 (19) ◽  
pp. 10355
Author(s):  
Nicolás González González Castro ◽  
Jan Bjelic ◽  
Gunya Malhotra ◽  
Cong Huang ◽  
Salman Hasan Alsaffar
Keyword(s):  
Genome Editing ◽  
Zinc Finger Nucleases ◽  
Clinical Settings ◽  
Transcription Activator ◽  
Entire Genome ◽  
Rational Decision Making ◽  
Programmable Nucleases ◽  
High Degree ◽  
Detection Strategies ◽  
Selection Of

Recent advances in programmable nucleases including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) have propelled genome editing from explorative research to clinical and industrial settings. Each technology, however, features distinct modes of action that unevenly impact their applicability across the entire genome and are often tested under significantly different conditions. While CRISPR-Cas is currently leading the field due to its versatility, quick adoption, and high degree of support, it is not without limitations. Currently, no technology can be regarded as ideal or even applicable to every case as the context dictates the best approach for genetic modification within a target organism. In this review, we implement a four-pillar framework (context, feasibility, efficiency, and safety) to assess the main genome editing platforms, as a basis for rational decision-making by an expanding base of users, regulators, and consumers. Beyond carefully considering their specific use case with the assessment framework proposed here, we urge stakeholders interested in genome editing to independently validate the parameters of their chosen platform prior to commitment. Furthermore, safety across all applications, particularly in clinical settings, is a paramount consideration and comprehensive off-target detection strategies should be incorporated within workflows to address this. Often neglected aspects such as immunogenicity and the inadvertent selection of mutants deficient for DNA repair pathways must also be considered.

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