scholarly journals Efficient CRISPR/Cas9 Genome Editing in Alfalfa Using a Public Germplasm

2021 ◽  
Vol 3 ◽  
Author(s):  
Emilia Bottero ◽  
Gabriela Massa ◽  
Matías González ◽  
Margarita Stritzler ◽  
Hiromi Tajima ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Production Systems ◽  
Gene Knockout ◽  
Nitrogen Fertilizers ◽  
Public Programs ◽  
The Public ◽  
Cas9 Nuclease ◽  
Potential Applications ◽  
Different Doses

Because its ability to acquire large amounts of nitrogen by symbiosis, tetraploid alfalfa is the main source of vegetable proteins in meat and milk production systems in temperate regions. Alfalfa cultivation also adds fixed nitrogen to the soil, improving the production of non-legumes in crop rotation and reducing the use of nitrogen fertilizers derived from fossil fuel. Despite its economic and ecological relevance, alfalfa genetics remains poorly understood, limiting the development of public elite germplasm. In this brief article, we reported the high-efficiency of alfalfa mutagenesis by using the public clone C23 and the CRISPR/Cas9 system. Around half of the GUS overexpressing plants (35S-GUS under C23 genomic background) transformed with an editing plasmid containing two sgRNAs against the GUS gene and the Cas9 nuclease exhibited absence of GUS activity. Nucleotide analysis showed that the inactivation of GUS in CRISPR/Cas9-editing events were produced via different modifications in the GUS gene, including frameshift and non-sense mutations. Using the CRISPR/Cas9 system and two sgRNAs, we have also edited the alfalfa gene NOD26, generating plants with different doses of alleles at this locus, including complete gene knockout at high efficiency (11%). Finally, we discuss the potential applications of genome-editing technologies to polyploid research and to alfalfa improvement public programs.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Genome Editing in Agriculture: Technical and Practical Considerations

2019 ◽  
Vol 20 (12) ◽  
pp. 2888 ◽  
Author(s):  
Julia Jansing ◽  
Andreas Schiermeyer ◽  
Stefan Schillberg ◽  
Rainer Fischer ◽  
Luisa Bortesi
Keyword(s):  
Genome Editing ◽  
Mechanism Of Action ◽  
High Efficiency ◽  
Public Acceptance ◽  
Crop Species ◽  
Base Editing ◽  
The Public ◽  
Intact Plants ◽  
Efficient Delivery ◽  
Selection Of

The advent of precise genome-editing tools has revolutionized the way we create new plant varieties. Three groups of tools are now available, classified according to their mechanism of action: Programmable sequence-specific nucleases, base-editing enzymes, and oligonucleotides. The corresponding techniques not only lead to different outcomes, but also have implications for the public acceptance and regulatory approval of genome-edited plants. Despite the high efficiency and precision of the tools, there are still major bottlenecks in the generation of new and improved varieties, including the efficient delivery of the genome-editing reagents, the selection of desired events, and the regeneration of intact plants. In this review, we evaluate current delivery and regeneration methods, discuss their suitability for important crop species, and consider the practical aspects of applying the different genome-editing techniques in agriculture.

Productivity and quality of soybean seeds with different fertilization on podzolized chernozem

2020 ◽  
Vol 1 (97) ◽  
pp. 136-144
Author(s):  
H.M. Hospodarenko ◽  
◽  
I.V. Prokopchuk ◽  
K. P. Leonova ◽  
V.P. Boyko
Keyword(s):  
Crop Rotation ◽  
Crop Yields ◽  
Spring Barley ◽  
Mineral Fertilizer ◽  
Nitrogen Fertilizers ◽  
Mineral Fertilizers ◽  
Forest Steppe ◽  
Soybean Seeds ◽  
Different Doses

The productivity of agricultural crops is the most variable and integral indicator of their vital activity, which accumulates their genetic potential, soil fertility, weather conditions and components of agricultural technology. Soybean under optimal growing conditions (the reaction of the soil is close to neutral, sufficient phosphorus and potassium nutrition, the use of nitraginization) assimilates from the air about 70 % of the total nitrogen requirement. Therefore, it is believed that it is enough to apply only a starting dose of nitrogen fertilizers (20–40 kg/ha a. s.), to get a high yield with good indicators of grain quality. The results of studies of the influence of long-term (8 years) application of different doses and ratios of fertilizers in field crop rotation on podzolized chernozem in the conditions of the Right -Bank Forest-Steppe of Ukraine on the yield and quality of soybean seeds preceded by spring barley were presented. It was found that crop yields could be increased by 18–77 % owing to different doses, ratios and types of fertilizers. The highest indicators of seed yields for three years of the research (3,02 t/ha) were obtained under the application of mineral fertilizers at a dose of N110P60K80 per 1 ha of crop rotation area, including under soybean – N60P60K60. Exclusion of the nitrogen component from the complete fertilizer (N60P60K60) reduced its yield by 26 %, phosphorus – by 17, and potassium by 11 %. There was no significant decrease in soybean yield in the variant of the experiment with a decrease in the proportion of potassium in the composition of complete mineral fertilizer (N60P60K30) for three years of study. The largest mass of 1000 soybean seeds was formed at doses of N60К60 fertilizers, and their protein content — under the application of complete mineral fertilizer in doses of N60P60K60 and N60P60K30.

scholarly journals User knowledge factors that hinder the design of new home healthcare devices: investigating thirty-eight devices and their manufacturers

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fan Yang ◽  
Abdullah Al Mahmud ◽  
Tao Wang
Keyword(s):  
End Users ◽  
Home Healthcare ◽  
Success Factor ◽  
User Research ◽  
The Public ◽  
Root Cause ◽  
Potential Applications ◽  
User Knowledge ◽  
Key Success Factor ◽  
Innovation Processes

Abstract Background The demand for home healthcare devices arises; however, many home healthcare devices on the market are not designed to reflect the needs and features of the end-users. This study explored the user knowledge factors that hindered the design of new home healthcare devices and the interrelationships between the factors. Methods The abovementioned factors were identified from analysing the project documents of thirty-eight carefully selected home healthcare devices produced by five manufacturers; followed by interviewing the thirty stakeholders playing key roles in developing the devices. Results The design of the home healthcare devices was influenced by (1) the user insights utilised in formulating project strategies; (2) the sources of user information; (3) the execution of user research; and (4) the formulation of the manufacturers’ principal innovation processes. Conclusions The users’ characteristics and needs were not sufficiently reflected in developing new home healthcare devices. One root cause was that the end-users were not perceived by the manufacturers as a key success factor in most cases, given that most of the devices were initiated following the public sector’s requests. Actual or potential applications of this study include the facilitation of the appropriate application of human factors methods in developing new home healthcare devices and the improvement of the user performance of the end-devices.

scholarly journals Probiotics and Potential Applications for Alternative Poultry Production Systems

2021 ◽  
pp. 101156
Author(s):  
Rim El Jeni ◽  
Dana K. Dittoe ◽  
Elena G. Olson ◽  
Jeferson Lourenco ◽  
Nicolae Corcionivoschi ◽  
...  
Keyword(s):  
Production Systems ◽  
Poultry Production ◽  
Potential Applications

The French Nitrogen Industry during the Interwar Period: The Ambiguous Relationship between the State and Manufacturers

2021 ◽  
Vol 62 (1) ◽  
pp. 191-212
Author(s):  
Michael Llopart
Keyword(s):  
Market Competition ◽  
The State ◽  
Nitrogen Fertilizers ◽  
Private Industry ◽  
Public And Private ◽  
The Public ◽  
The First World War ◽  
French Case ◽  
International Cartel ◽  
First World

Abstract At the end of the First World War, the French government seized the opportunity to acquire the chemical processes of the German firm BASF, including the Haber-Bosch process. This patent made it possible to synthesize nitrogen from the air and thus produce nitrogen fertilizers in large quantities. French industrialists, however, refused to acquire these patents, and to make up for this lack of private sector involvement, the French Parliament decided in 1924 to create a national plant (ONIA), which became the first state-owned plant to be exposed to market competition. The intention was for the ONIA to supply the army with nitric acid in times of war, and, in peacetime, to sell fertilizers at the lowest possible prices in order to curb the monopoly of the private industry cartel. The purpose of this article is therefore to study the establishment and organisation of the French market for nitrogen fertilisers during the inter-war period by raising a number of questions about the ambiguous and complex relations between the state and private industry in this strategic sector. Why was the state policy initiated with the ONIA not successful at first? From 1927-1928, once the ONIA was operational, why and how did the public and private players jointly organise the marketing of fertilisers even though their interests were partially divergent? From the economic crisis of the 1930s onwards, how did the regulation of this mixed market evolve and how were public/private tensions overcome? In the French case, why did French producers leave the international cartel very early on in favour of state protectionism? And finally, to what extent can it be said that this “managed economy” framework succeeded in satisfying all the players in the French nitrogen industry?

scholarly journals Citizen views on genome editing: effects of species and purpose

2021 ◽  
Author(s):  
Gesa Busch ◽  
Erin Ryan ◽  
Marina A. G. von Keyserlingk ◽  
Daniel M. Weary
Keyword(s):  
Disease Resistance ◽  
Product Quality ◽  
Genome Editing ◽  
Total Sample ◽  
Moral Foundations ◽  
Perceived Risks ◽  
Public Response ◽  
The Public ◽  
Wide Range ◽  
The Us

AbstractPublic opinion can affect the adoption of genome editing technologies. In food production, genome editing can be applied to a wide range of applications, in different species and with different purposes. This study analyzed how the public responds to five different applications of genome editing, varying the species involved and the proposed purpose of the modification. Three of the applications described the introduction of disease resistance within different species (human, plant, animal), and two targeted product quality and quantity in cattle. Online surveys in Canada, the US, Austria, Germany and Italy were carried out with a total sample size of 3698 participants. Using a between-subject design, participants were confronted with one of the five applications and asked to decide whether they considered it right or wrong. Perceived risks, benefits, and the perception of the technology as tampering with nature were surveyed and were complemented with socio-demographics and a measure of the participants’ moral foundations. In all countries, participants evaluated the application of disease resistance in humans as most right to do, followed by disease resistance in plants, and then in animals, and considered changes in product quality and quantity in cattle as least right to do. However, US and Italian participants were generally more positive toward all scenarios, and German and Austrian participants more negative. Cluster analyses identified four groups of participants: ‘strong supporters’ who saw only benefits and little risks, ‘slight supporters’ who perceived risks and valued benefits, ‘neutrals’ who showed no pronounced opinion, and ‘opponents’ who perceived higher risks and lower benefits. This research contributes to understanding public response to applications of genome editing, revealing differences that can help guide decisions related to adoption of these technologies.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

scholarly journals Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

2019 ◽  
Vol 20 (15) ◽  
pp. 3623 ◽  
Author(s):  
Tobias Bruegmann ◽  
Khira Deecke ◽  
Matthias Fladung
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Constitutive Expression ◽  
Gc Content ◽  
Seed Region ◽  
Research Topics ◽  
Target Region ◽  
Cas9 Nuclease ◽  
Different Types ◽  
Poplar Hybrid

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

scholarly journals An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Namkha Nguyen ◽  
Morgan M. F. Quail ◽  
Aaron D. Hernday
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Gene Knockout ◽  
Strain Engineering ◽  
Molecular Genetic Analysis ◽  
Content Type ◽  
Highly Efficient ◽  
Discovery Research ◽  
Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

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