Application Of Genome Editing Technologies Research Articles

In planta Genome Editing in Commercial Wheat Varieties.

Limitations for the application of genome editing technologies on elite wheat (Triticum aestivum L.) varieties are mainly due to the dependency on in vitro culture and regeneration capabilities. Recently, we developed an in planta particle bombardment (iPB) method which has increased process efficiency since no culture steps are required to create stably genome-edited wheat plants. Here, we report the application of the iPB method to commercially relevant Japanese elite wheat varieties. The biolistic delivery of gold particles coated with plasmids expressing CRISPR/Cas9 components designed to target TaQsd1 were bombarded into the embryos of imbibed seeds with their shoot apical meristem (SAM) exposed. Mutations in the target gene were subsequently analyzed within flag leaf tissue by using cleaved amplified polymorphic sequence (CAPS) analysis. A total of 9/358 (2.51%) of the bombarded plants (cv. “Haruyokoi,” spring type) carried mutant alleles in the tissue. Due to the chimeric nature of the T0 plants, only six of them were inherited to the next (T1) generation. Genotypic analysis of the T2 plants revealed a single triple-recessive homozygous mutant of the TaQsd1 gene. Compared to wild type, the homozygous mutant exhibited a 7 days delay in the time required for 50% seed germination. The iPB method was also applied to two elite winter cultivars, “Yumechikara” and “Kitanokaori,” which resulted in successful genome editing at slightly lower efficiencies as compared to “Haruyokoi.” Taken together, this report demonstrates that the in planta genome editing method through SAM bombardment can be applicable to elite wheat varieties that are otherwise reluctant to callus culture.

Policies on Application of Genome Editing Technology in Human Embryo in Japanese Government

Policies on Application of Genome Editing Technology in Human Embryo in Japanese Government

Improving Nutritional and Functional Quality by Genome Editing of Crops: Status and Perspectives.

Genome-editing tools including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats (CRISPR) system have been applied to improve the quality of staple, oilseed, and horticultural crops with great accuracy and efficiency compared to conventional breeding. In particular, the CRISPR method has proven to be a feasible, cost-effective and versatile tool allowing precise and efficient editing of plant genomes in recent years, showing great potential in crop improvement. Until now, various genome-edited crops with enhanced commercial value have been developed by not only global companies but also small laboratories in universities, suggesting low entry barriers with respect to manpower and capital. In this study, we review the current applications of genome editing technologies to improve the nutritional and functional quality and preferred traits of various crops. Combining this rapidly advancing genome-editing technology and conventional breeding will greatly extend the potential of genome-edited crops and their commercialization.

Human genome editing: how to prevent rogue actors

BackgroundHuman genome editing technologies offer much potential benefit. However, central to any conversation relating to the application of such technologies are certain ethical, legal, and social difficulties around their application. The recent misuse, or inappropriate use, by certain Chinese actors of the application of genome editing technologies has been, of late, well noted and described. Consequently, caution is expressed by various policy experts, scientists, bioethicists, and members of the public with regard to the appropriate use of human germline genome editing and its possible future effect on future generations.Main textAs concerns about the applications of heritable genome editing have grown, so too have the questions around what is to be done to curtail ‘rogue actors’. This paper explores various ways in which to regulate genomic editing that are socially beneficial, while being cognisant of legal and ethical principles and rights values. This is done by evolving regulatory frameworks across jurisdictions in an attempt to raise issues, address common principles, and set responsible standards for stewardship of the novel technology.ConclusionsIt is suggested that robust and concrete regulatory measures be introduced that are culturally and contextually sensitive, inclusive, appropriate, and trustworthy – and are based on public empowerment and human rights objectives. Doing so will ensure that we are perfectly positioned to harness and promote the benefits that novel technologies have to offer, while safeguarding public health and curtailing the ambitions of rogue actors. This it is acknowledged is no easy task, so, as a point of departure, this paper sets out a path forward by means of certain, practical recommendations – by constructing genome editing regulation in a manner that both fulfils the desire to better progress human health and that can withstand legal and ethical scrutiny.The following observations and recommendations are made: Firstly, that a solution of effective, legitimate governance should consist of a combination of national and supranational legislative regulation or ‘hard’ law, in combination with ‘soft’ ethics, firmly anchored in and underpinned by human rights values. Second, that efforts to support legal and ethical solutions should be rigorous, practical, and robust, contribute to a reaffirmation of human rights in a contextually sensitive manner, and be transnational in reach. Lastly, that greater harmonisation across jurisdictions and increased public engagement be sought. This it is proposed will address the question of how to implement a normative framework which in turn can prevent future rogue actors.

Introducing Chemistry Students to Emerging Technologies in Gene Editing, Their Applications, and Ethical Considerations

Genome editing has widespread influence in many fields, including medicine, biotechnology, and agriculture. Despite their origins in chemistry laboratories, these techniques are currently under utilized in chemistry curricula. Introducing future generations of chemists to these emerging technologies is essential in chemistry classrooms. The purpose of this tutorial review is to introduce chemistry audiences—particularly undergraduate chemistry students and educators—to the emerging field of genome editing. Indeed, as the applications of genome editing technologies continue to grow, it will be important for chemistry students to gain a working knowledge of their underlying principles as well as some of their limitations. Here, we aim to provide a general overview of genome editing technologies and highlight some practical ways in which these technologies can be integrated into the undergraduate chemistry curriculum. In the overview, we describe the key players in—and the modes of action of—the three major genome editing technologies, namely, (1) zinc finger nucleases (ZFNs), (2) transcription activator-like effector nucleases (TALENs) and (3) the clustered regularly interspaced short palindromic repeats/CRISPR-associated system (CRISPR/Cas) system. We then highlight several applications of genome editing technologies, particularly the CRISPR/Cas system, and their potential impact on various fields, including basic research, medicine, biotechnology, and the food industry. In this context, we present potential ethical issues associated with the application of genome editing technologies. Finally, by presenting a case study, we highlight the ways that genome editing technologies can be integrated into the undergraduate chemistry curriculum. This review can be added to biochemistry or organic chemistry courses as reading material.

Affected Genome Editing Crops: The Consequences of Genome-Edited Babies in China.

Though genome editing (GE) is a powerful technology, germline GE engineering is strongly objectionable for a huge ethical challenge. The after-effects of the genome-edited babies incident have been emerging in China, whether technology or ethics. It is very noticeable that the case has been adverse effects on the application of GE technology in other fields, especially in GE crops. After the incident, research and development of GE crops was affected obviously. It is clear that GE crops and other basic research and application related to GE technology should be encouraged and should not be treated differently. An ethically acceptable development route of GE technology needs to be established in China.

Applications of Genome Editing Technology in Research on Chromosome Aneuploidy Disorders.

Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated that aneuploidies can be rescued to a normal diploid state using genetic engineering in cultured cells. Here, we summarize a series of studies mainly applying genome editing to eliminate an extra copy of human chromosome 21, the cause of the most common constitutional aneuploidy disorder Down syndrome. We also present findings on induced pluripotent stem cell reprogramming, which has been shown to be one of the most promising technologies for converting aneuploidies into normal diploidy without the risk of genetic alterations such as genome editing-mediated off-target effects.

The development and application of genome editing technology in ruminants: a review

The development and application of genome editing technology in ruminants: a review

Application of genome editing technology in human gene therapy

Application of genome editing technology in human gene therapy

Genome Editing: Current State of Research and Application to Animal Husbandry

The creation and development of genome editing (GE) technologies bring new opportunities for the genetic engineering of farm mammals and poultry. The present review characterizes GE systems based on ZFN, TALEN, and CRISPR/Cas9 and directions for their improvement in relation to farm animals. The fields for the application of GE technologies in animal husbandry and poultry farming and the objectives and prospects for their further development are discussed.

The application of genome editing technology

Genome editing technology is currently the most effective tool for accurately manipulating genomes at specific locations. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats associated Cas9 (CRISPR/Cas9) system were used genome editing technologies. Both editing done with ZFNs and TALENs consist of DNA-binding domains which are fused to endonucleases. This is done to enable a broad range of genetic modifications by inducing DNA double-strand breaks (DSB) that stimulate the error-prone Non-Homologous End-Joining (NHEJ) or a homology-directed repair (HDR) at specific genomic locations. Different to ZFNs and TALENs, the CRISPR/Cas system is an RNA-mediated specific recognition process which DSB been introduced after the sgRNA binds to the targeted DNA sequence. The ZFNs, TALENs and CRISPR/Cas9 systems modify these genomic characteristics precisely through these repair mechanisms, and have been successfully used to manipulate the genome in human cells. These genome editing tools can be used to investigate gene function, to explore the genetic mechanisms of a disease, to discover new therapeutic targets, and to develop new disease models. Moreover, these genome editing technologies have a great potential in gene therapy. Here, we review the latest advances in the application of genome editing technology for the study and treatment of genetic diseases [Duchenne muscular dystrophy (DMD), hemophilia], cancers [chimeric antigen receptor T-cell immunotherapy (CAR-T) technology], viral infections [human immunodeficiency virus (HIV), hepatitis B virus (HBV)], bio-agricultures, and microorganisms.

ГЕНОМНОЕ РЕДАКТИРОВАНИЕ РИСА ПРИ ИСПОЛЬЗОВАНИИ СИСТЕМЫ CRISPR/Cas

Genome editing using the CRISPR/Cas system is a breakthrough tech­nology in plant genetics and breeding. The most large-scale application of this new technology on crop species is observed for rice. This fact is explained not only by the significance of this crop, but also by the relatively high transformation amenability. Although the end result of genome editing is a non-transgenic plant with desired mutation (muta­tions), an unavoidable step in the process of creating such a new mutant is the use of genetic engineering methods. To date, the CRISPR/Cas sys­tem has been tested on dozens of rice target genes, of which mutations in more than 30 genes have led to the desired improvement of economically important traits. The remaining experiments are related mainly to the verification of the genes’ functions, and belong to the field of reverse genetics. Improvement or acquisition of new properties is associated with mutations in the genes that affect productivity, grain fragrance and chemical composition, flowering time, the resistance to biotic and abiotic stress factors, and herbicides, as well as pollination control needed in hybrid breeding. These achievements are reviewed in the current article. It is important to note that about fifty different genotypes are already involved in improving rice varieties with the help of genome editing. This creates the prerequisites for a wide practical application of genome editing technologies in rice breeding programs

Genome editing for plant disease resistance: applications and perspectives.

Diseases severely affect crop yield and quality, thereby threatening global food security. Genetic improvement of plant disease resistance is essential for sustainable agriculture. Genome editing has been revolutionizing plant biology and biotechnology by enabling precise, targeted genome modifications. Editing provides new methods for genetic improvement of plant disease resistance and accelerates resistance breeding. Here, we first summarize the challenges for breeding resistant crops. Next, we focus on applications of genome editing technology in generating plants with resistance to bacterial, fungal and viral diseases. Finally, we discuss the potential of genome editing for breeding crops that present novel disease resistance in the future. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

Genome editing in human embryos: ethical concerns and practical applications

As genome editing techniques are developed, they have been applied to somatic cells in clinical use. Regarding the adaptation of these techniques in human zygotes, advances in single-cell analysis technology are expected to enhance knowledge at the molecular level, even in human preimplantation embryo development, and the application of genome editing technology for molecular functional analysis is expected. However, the use of genetic modification techniques on germlines, including zygotes, spreads to all cells of an individual and is subsequently passed down through generations. Here we explore the possibilities and challenges of applying genome editing techniques to better understand the scientific aspects of the human germline.

Engagement and Education of the Sickle Cell Disease Community: What Sickle Cell Disease Patients and Parents Want to Know about CRISPR Genome Editing

Introduction:One of the first therapeutic targets of CRISPR genome editing will likely be sickle cell disease (SCD), the most commonly inherited blood disorder in the U.S.SCD affects 100,000 individuals in the U.S. and more than 250 million individuals globally. Two targets of genome editing in SCD currently under investigation are HBB and BCL11A. The first approach (HBB) focuses on correcting the Glu6Val mutation in patient-derived stem and progenitor cells to promote differentiation into normal erythrocytes. The second approach involves disrupting BCL11A, a repressor of fetal hemoglobin. While preliminary results from research using both mechanisms show promise, risks related to insertional mutagenesis and host immune responses remain. As the research advances, limited knowledge exists regarding SCD stakeholders' (patients, parents and physicians) understanding of genome editing and their educational needs.Methods:We conducted a mixed methods study, which included a CRISPR genome editing educational video, pre- and post-video surveys, and fifteen, moderated focus groups: 6 patient groups, 6 parent groups, and 3 physician groups. The surveys included an assessment of participants' knowledge of CRISPR gene editing and the Genetic Literacy and Comprehension (GLAC) instrument. Eligible participants were patients with a diagnosis of SCD, parents of a child with SCD, and physicians who have delivered care to at least five adult or pediatric patients living with SCD for a minimum of 12 months. Differences between the pre- and post-video score of CRISPR technology knowledge were assessed using paired t-tests. NVivo 11 qualitative software tool was used to assist in data analysis.Results:For each of the eight complex genetic terms in the GLAC, patients and parents were asked to rank their familiarity on a scale from 1-7, and to complete fill-in-the-blank, multiple choice questions aimed at assessing their understanding of the term. Baseline genetic literacy levels were high for both stakeholder groups. Patients and parents had an average familiarity score of 6.34, and 6.88, respectively. 89% of both groups scored >70% on the fill-in-the-blank questions. As shown in Figure 1, the educational video tool was effective at increasing CRISPR genome editing knowledge among patients and parents - the average CRISPR genome editing knowledge scores of both groups increased by over two points (p<0.0001 and p=0.05, respectively). Prior to viewing the video, patients and parents had lower CRISPR genome-editing knowledge scores than physicians (p<0.0002 and p<0.0002, respectively). However, after viewing the video, there were no significant differences in scores between patients, parents, and physicians. Several qualitative themes emerged related to patients' and parents' interest in understanding CRISPR genome editing. These themes included the need for easily accessible scientific information regarding: 1) CRISPR's mechanism of action; 2) potential adverse reactions to CRISPR; 3) effects of genome editing on future generations; and 4) burden of participating in a genome editing clinical trial.Discussion:After watching the video tool, patients' and parents' understanding of CRISPR genome editing improved significantly. This suggests that video education tools may be an effective method to educate individuals living with SCD and parents about genome editing. Furthermore, this study reveals patients and parents were not only familiar with complex genetic terms, but also correctly defined them, suggesting a good baseline genetic literacy. The themes indicate the importance of keeping patients and parents informed and supporting their desires to be educated about the applications of genome editing technology which will further the advancement of clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Genome editing in Bombyx mori: New opportunities for silkworm functional genomics and the sericulture industry.

In recent years, research in life sciences has been remarkably revolutionized owing to the establishment, development and application of genome editing technologies. Genome editing has not only accelerated fundamental research but has also shown promising applications in agricultural breeding and therapy. In particular, the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology has become an indispensable tool in molecular biology owing to its high efficacy and simplicity. Genome editing tools have also been established in silkworm (Bombyx mori), a model organism of Lepidoptera insects with high economic importance. This has remarkably improved the level and scope of silkworm research and could reveal new mechanisms or targets in basic entomology and pest management studies. In this review, we summarize the progress and potential of genome editing in silkworm and its applications in functional genomic studies for generating novel genetic materials.

Genome Editing in Stem Cells for Disease Therapeutics.

Programmable nucleases including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein have tremendous potential biological and therapeutic applications as novel genome editing tools. These nucleases enable precise modification of the gene of interest by disruption, insertion, or correction. The application of genome editing technology to pluripotent stem cells or hematopoietic stem cells has the potential to remarkably advance the contribution of this technology to life sciences. Specifically, disease models can be generated and effective therapeutics can be developed with great efficiency and speed. Here we review the characteristics and mechanisms of each programmable nuclease. In addition, we review the applications of these nucleases to stem cells for disease therapies and summarize key studies of interest.

Transient Expression of CRISPR/Cas9 Machinery Targeting TcNPR3 Enhances Defense Response in Theobroma cacao.

Theobroma cacao, the source of cocoa, suffers significant losses to a variety of pathogens resulting in reduced incomes for millions of farmers in developing countries. Development of disease resistant cacao varieties is an essential strategy to combat this threat, but is limited by sources of genetic resistance and the slow generation time of this tropical tree crop. In this study, we present the first application of genome editing technology in cacao, using Agrobacterium-mediated transient transformation to introduce CRISPR/Cas9 components into cacao leaves and cotyledon cells. As a first proof of concept, we targeted the cacao Non-Expressor of Pathogenesis-Related 3 (TcNPR3) gene, a suppressor of the defense response. After demonstrating activity of designed single-guide RNAs (sgRNA) in vitro, we used Agrobacterium to introduce a CRISPR/Cas9 system into leaf tissue, and identified the presence of deletions in 27% of TcNPR3 copies in the treated tissues. The edited tissue exhibited an increased resistance to infection with the cacao pathogen Phytophthora tropicalis and elevated expression of downstream defense genes. Analysis of off-target mutagenesis in sequences similar to sgRNA target sites using high-throughput sequencing did not reveal mutations above background sequencing error rates. These results confirm the function of NPR3 as a repressor of the cacao immune system and demonstrate the application of CRISPR/Cas9 as a powerful functional genomics tool for cacao. Several stably transformed and genome edited somatic embryos were obtained via Agrobacterium-mediated transformation, and ongoing work will test the effectiveness of this approach at a whole plant level.

Genome Editing Advances the Structural Study of Silk.

We first applied the genome edited silkworm silk (GE-silk) to interpret X-ray fiber diagram, and implied a great potential for the application of genome editing technology to the structural study of silk. The origin of a weak meridional layer-line streak with a spacing of ∼21 Å, observed in the X-ray fiber diagram of Bombyx mori silkworm silk, has been widely believed but not experimentally proven to be a period of the pseudostructure associated with the occurrence of serine residues at regular intervals in a hexapeptide repeating unit -G-A-G-A-G-S-. The above hypothesis was experimentally demonstrated from X-ray measurements of GE-silk.

Application of genome editing technologies in rats for human disease models.

Laboratory rats and mice are representative experimental animals for models of human disease. The emergence of genome editing technologies has enabled us to produce a variety of genetically modified animals, including rats, as a means of elucidating the in vivo functions of the gene of interest and characterizing the molecular mechanisms of human disease. Several advanced techniques for knock-in methodologies in rats are currently in development, which permit researchers to introduce precise nucleotide modifications at target sites in the rat's genome. Furthermore, recent studies with knock-out rats have revealed that observed disease phenotypes are often more similar than mouse models to those of humans. In this article, we introduce the methodologies for efficient gene manipulation in rats using genome editing technologies, and describe the advances made using rats for human disease models. We also discuss the importance of gene manipulation in animal models for the better understanding of fundamental processes among different species.