Era Of Genome Editing Research Articles

Breeding of ornamental orchids with focus on Phalaenopsis: current approaches, tools, and challenges for this century.

Ornamental orchid breeding programs have been conducted to develop commercially valuable cultivars with improved characteristics of commercial interest, such as size, flower color, pattern, shape, and resistance to pathogens. Conventional breeding, including sexual hybridization followed by selection of desirable characteristics in plants, has so far been the main method for ornamental breeding, but other techniques, including mutation induction by polyploidization and gamma irradiation, and biotechnological techniques, such as genetic transformation, have also been studied and used in ornamental breeding programs. Orchids are one of the most commercially important families in floriculture industry, having very particular reproductive biology characteristics and being a well-studied group of ornamentals in terms of genetic improvement. The present review focuses on the conventional and biotechnological techniques and approaches specially employed in breeding Phalaenopsis orchids, the genus with highest worldwide importance as an ornamental orchid, highlighting the main limitations and strengths of the approaches. Furthermore, new opportunities and future prospects for ornamental breeding in the CRISPR/Cas9 genome editing era are also discussed. We conclude that conventional hybridization remains the most used method to obtain new cultivars in orchids. However, the emergence of the first biotechnology-derived cultivars, as well as the new biotechnological tools available, such as CRISPR-Cas9, rekindled the full potential of biotechnology approaches and their importance for improve ornamental orchid breeding programs.

Tuberculosis Treatment in The Human Genome Editing Era: Unbundling the Right of Access to Healthcare Services in South Africa

Tuberculosis (TB) is both preventable and curable, yet it is the top killer disease in South Africa. In 2018, about 300,000 people in South Africa fell ill with TB and about 63,000 people died of TB. This demonstrates that the incidence of TB in South Africa is far greater than in other continents. This calls for a paradigm shift in the response to fighting the TB epidemic. Specifically, this necessitates promoting access to progressive treatment methods to fight the age-old disease that is still affecting a greater fraction of the South African population. Treating TB with monotherapy has, in most instances, proven to be ineffective. The burden of the TB epidemic as witnessed in South Africa is a compelling reason for alternative treatment options such as human genome editing. The main purpose of this paper is to examine to what extent the legal and policy framework in South Africa enables the government to provide TB treatment to patients using human genome editing. The paper concludes that the right approach to accessing this technology is important because it provides a solid ground to demand access.

CRISPR/Cas9 editing of transformer2 gene of the Oriental fruit Fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) leads to intersex phenotype

The Oriental fruit fly, Bactrocera dorsalis is a serious, highly invasive pest of fruits affecting global trade. Areawide management of B. dorsalis by release of sterile males obtained through irradiation suffers from the disadvantage of somatic mutations. In this regard, insect pest management in the post genome editing era poised for producing precise mutations based on CRISPR/Cas for example precision guided sterile insect technique and gene drive. Development of these technologies depend on the genes in sex determination pathway and spermatogenesis in target species. In the sex determination pathway of B. dorsalis, transformer (tra & tra2) are involved in female specific splicing of double sex (dsx). CRISPR/Cas9 mediated loss-of-function of tra has been shown to influence sex determination while it is lacking in tra2. In the present study we have edited transformer2 gene through embryonic microinjection of CRISPR/Cas9 ribonucleprotein (Cas9+sgRNA) targeting the sixth exon. Among the emergent G0 individuals, two showed an intersex phenotype, with a male-specific dorsal bristles in the third tergite on the left side of the abdomen and a degenerate ovipositor. The third individual manifested an unique phenotype, with a complete absence of the male specific dorsal bristles but a complete degenerate ovipositor. Sequencing results revealed both addition and deletions in all the three intersexes. Additionally, molecular karyotyping of these individuals, through Maleness on Y (MoY) PCR revealed a XX karyotype for all of them. Hence, we have shown that editing of the transformer2 locus in B. dorsalis interferes with the normal sex determination.

Creating novel ornamentals via new strategies in the era of genome editing.

Ornamental breeding has traditionally focused on improving novelty, yield, quality, and resistance to biotic or abiotic stress. However, achieving these goals has often required laborious crossbreeding, while precise breeding techniques have been underutilized. Fortunately, recent advancements in plant genome sequencing and editing technology have opened up exciting new frontiers for revolutionizing ornamental breeding. In this review, we provide an overview of the current state of ornamental transgenic breeding and propose four promising breeding strategies that have already proven successful in crop breeding and could be adapted for ornamental breeding with the help of genome editing. These strategies include recombination manipulation, haploid inducer creation, clonal seed production, and reverse breeding. We also discuss in detail the research progress, application status, and feasibility of each of these tactics.

Accelerated Breeding for Helianthus annuus (Sunflower) through Doubled Haploidy: An Insight on Past and Future Prospects in the Era of Genome Editing.

The aim of any breeding process is to fully express the targeted, superior/desirable parent characteristic in the progeny. Hybrids are often used in this dynamic, and complex process for which homozygous parents-which may require up to eight generations of back crossing and selection-are required. Doubled haploid (DH) technologies can facilitate the production of true breeding lines faster and in a more efficient manner than the traditional back crossing and selection strategies. Sunflower is the third most important oilseed crop in the world and has no available double haploid induction procedure/technique that can be efficiently used in breeding programs. A reproducible and efficient doubled haploid induction method would be a valuable tool in accelerating the breeding of new elite sunflower varieties. Although several attempts have been made, the establishment of a sunflower doubled haploid induction protocol has remained a challenge owing recalcitrance to in vitro culture regeneration. Approaches for haploid development in other crops are often cultivar specific, difficult to reproduce, and rely on available tissue culture protocols-which on their own are also cultivar and/or species specific. As an out-crossing crop, the lack of a double haploid system limits sunflower breeding and associated improvement processes, thereby delaying new hybrid and trait developments. Significant molecular advances targeting genes, such as the centromeric histone 3 (CenH3) and Matrilineal (MTL) gene with CRISPR/Cas9, and the successful use of viral vectors for the delivery of CRISPR/Cas9 components into plant cells eliminating the in vitro culture bottleneck, have the potential to improve double haploid technology in sunflower. In this review, the different strategies, their challenges, and opportunities for achieving doubled haploids in sunflower are explored.

Nanocarriers: A novel strategy for the delivery of CRISPR/Cas systems.

In recent decades, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) has become one of the most promising genome-editing tools for therapeutic purposes in biomedical and medical applications. Although the CRISPR/Cas system has truly revolutionized the era of genome editing, the safe and effective delivery of CRISPR/Cas systems represents a substantial challenge that must be tackled to enable the next generation of genetic therapies. In addition, there are some challenges in the in vivo delivery to the targeted cells/tissues. Nanotechnology-based drug delivery systems can be employed to overcome this issue. This review discusses different types and forms of CRISPR/Cas systems and the current CRISPR/Cas delivery systems, including non-viral carriers such as liposomes, polymeric, and gold particles. The focus then turns to the viral nanocarriers which have been recently used as a nanocarrier for CRISPR/Cas delivery.

Book review: “The New Microbiology: From Microbiomes to CRISPR”

At the time when owing to COVID-19 pandemic, the public is more interested in infectious diseases and pathogens than in any other period, a book titled “The New Microbiology: From Microbiomes to CRISPR” by the renowned microbiologist Pascale Cossart was translated and published by our colleague. In this book, the author describes important discoveries and new conceptual advances in the field of microbiology over the past century. The author emphasizes that bacteria form a microbiome and lead a social life and explains the impact and importance of the microbiome on human health and ecosystem. Further, the discovery of ‘CRISPR,’ which led to the era of genome editing and gene therapy, has been described in details. This book will dramatically change our perspective regarding all living things, including bacteria, plants, animals, and even insects, as well as our eating habits and daily life, based on the new understanding of microorganisms. I recommend reading this book as I am sure that it will broaden the perspectives of both clinical microbiologists and health care workers.

Breaking the Rules

GEN EdgeVol. 4, No. 1 CRISPRBreaking the RulesA gripping new film -- Make People Better – dissects the scandal surrounding the birth of the gene-edited CRISPR babies in 2018, offering some surprising insights into the highly publicized story and some teasing hints at where we go next.Kevin DaviesKevin DaviesKevin Davies PhD is the Executive Editor of The CRISPR Journal and GEN Biotechnology, and the author of Editing Humanity: The CRISPR Revolution and the New Era of Genome Editing (Pegasus Books, 2020). This essay was originally published in The CRISPR Journal.Search for more papers by this authorPublished Online:23 Dec 2022https://doi.org/10.1089/genedge.4.1.166AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 4Issue 1Jan 2022 Information© 2022 by GEN PublishingTo cite this article:Kevin Davies.Breaking the Rules.GEN Edge.Jan 2022.982-986.http://doi.org/10.1089/genedge.4.1.166Published in Volume: 4 Issue 1: December 23, 2022PDF download

Tropical crops enter the era of genome editing

Tropical crops enter the era of genome editing

The Development of Mitochondrial Gene Editing Tools and Their Possible Roles in Crop Improvement for Future Agriculture.

We are living in the era of genome editing. Nowadays, targeted editing of the plant nuclear DNA is prevalent in basic biological research and crop improvement since its first establishment a decade ago. However, achieving the same accomplishment for the plant mitochondrial genome has long been deemed impossible. Recently, the pioneer studies on editing plant mitogenome have been done using the mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) in rice, rapeseed, and Arabidopsis. It is well documented that mitochondria play essential roles in plant development and stress tolerance, particularly, in cytoplasmic male sterility widely used in production of hybrids. The success of mitochondrial genome editing enables studying the fundamentals of mitochondrial genome. Furthermore, mitochondrial RNA editing (mostly by nuclear-encoded pentatricopeptide repeat (PPR) proteins) in a sequence-specific manner can simultaneously change the production of translatable mitochondrial mRNA. Moreover, direct editing of the nuclear-encoding mitochondria-targeted factors required for plant mitochondrial genome dynamics and recombination may facilitate genetic manipulation of plant mitochondria. Here, the present state of knowledge on editing the plant mitochondrial genome is reviewed.

A Reliable Regeneration Method in Genome-Editable Bell Pepper ‘Dempsey’

A reliable regeneration technique is critical for the improvement of pepper traits in the genome editing era. Recently, we reported that peppers were successfully and specifically edited using CRISPR tools, CRISPR/Cas9 and CRISPR/Cas12a (LbCpf1). Although genome-editing tools can be applied to modify peppers at the cellular level, feasible pepper regeneration techniques have not been developed. Therefore, we studied a pepper regeneration protocol for Capsicum annuum L. ‘Dempsey’, a bell pepper species that has been proven to be genome-editable. Three explant types were used in this study, including the first leaves, cotyledons and hypocotyls of pepper seedlings. The shoot buds of the tested explants were produced using 8 mg/L 6-benzylaminopurine (BAP)- and 6 mg/L indole-3-acetic acid (IAA)-containing shoot induction medium (SIM). The first leaves of the ‘Dempsey’ seedlings showed an average shooting rate of 69.8%, whereas the hypocotyls and cotyledons had approximately 25.5% and 19.5% shooting rates, respectively. The regenerated ‘Dempsey’ plants exhibited no alterations in fruit and fertile seed phenotypes. Furthermore, the parent ‘Dempsey’ and progenies of the regenerants were cytogenetically stable with the same chromosome numbers (2n = 24). Therefore, this regeneration protocol enables the precise molecular breeding of ‘Dempsey’ peppers when coupled with CRISPR tools.

Genome Editing Revolution in Life Sciences

Programmable nucleases—ZFNs, TALENs and CRISPR-Cas9—have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with great implications across life sciences: biology, agriculture, ecology and medicine. Nucleases-based genome editing (aka gene editing) depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences within a mammalian genome, could be addressed by protein engineering, ushering in the era of genome editing. ZFNs that are fusions of zinc finger proteins (ZFPs) and FokI cleavage domain, resulted from the basic research on Type IIs FokI restriction enzyme, which showed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain. Studies on 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target recognition sequence from 9 to 18 bp that is large enough to specify a unique genomic locus in plant and mammalian cells, including human cells. Subsequently, a ZFN-induced DSB was shown to stimulate homologous recombination in frog eggs. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to FokI cleavage domain expanded the capability. ZFNs and TALENs have been successfully used to modify a multitude of recalcitrant organisms and cell types that were unapproachable previously attesting to the success of protein engineering, long before the arrival of CRISPR. The recent technique to deliver a targeted DSB to cellular genomes are RNA-guided nucleases as exemplified by the Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system, which include ease of RNA design for new targets and dependence on a single constant Cas9 protein, have led to its wide adoption by research labs around the world. The 2020 Nobel Prize for Chemistry was awarded to Jennifer Doudna and Emmanualle Charpentier for harnessing CRISPR-Cas9 system to provide a simplified technique for genome editing. The programmable nucleases have also been shown to cut at off-target sites with mutagenic consequences, which is a serious concern for human therapeutic applications. Therefore, applications of genome editing technologies to human therapeutics will ultimately depend on risk versus benefit analysis and informed consent

Fruit crops in the era of genome editing: closing the regulatory gap.

The conventional breeding of fruits and fruit trees has led to the improvement of consumer-driven traits such as fruit size, yield, nutritional properties, aroma and taste, as well as the introduction of agronomic properties such as disease resistance. However, even with the assistance of modern molecular approaches such as marker-assisted selection, the improvement of fruit varieties by conventional breeding takes considerable time and effort. The advent of genetic engineering led to the rapid development of new varieties by allowing the direct introduction of genes into elite lines. In this review article, we discuss three such case studies: the Arctic® apple, the Pinkglow pineapple and the SunUp/Rainbow papaya. We consider these events in the light of global regulations for the commercialization of genetically modified organisms (GMOs), focusing on the differences between product-related systems (the USA/Canada comparative safety assessment) and process-related systems (the EU "precautionary principle" model). More recently, genome editing has provided an efficient way to introduce precise mutations in plants, including fruits and fruit trees, replicating conventional breeding outcomes without the extensive backcrossing and selection typically necessary to introgress new traits. Some jurisdictions have reacted by amending the regulations governing GMOs to provide exemptions for crops that would be indistinguishable from conventional varieties based on product comparison. This has revealed the deficiencies of current process-related regulatory frameworks, particularly in the EU, which now stands against the rest of the world as a unique example of inflexible and dogmatic governance based on political expediency and activism rather than rigorous scientific evidence.

Substrains matter in phenotyping of C57BL/6 mice.

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.

The progress of CAR-T therapy in cancer and beyond

Chimeric antigen receptor T (CAR-T) technology is the pinnacle of modern immunology, system biology, synthetic biology and cellular engineering. It is the powerful new player of cancer therapy since the concept of adoptive cell therapy. Clinical success of CAR-T cells targeting antigen unique to B cell leukemia has made it the focus of new development in cancer therapy. The latest success is reported in a clinical case of using CAR-T cells to treat pediatric acute lymphoblastic leukemia. However, the major challenges are to make CAR-T cells a reliable, controllable, safe and effective platform that could apply to diverse cancer types including solid tumors. In this review we summarize the recent research progress to tackle the challenges and discuss the broader application of CAR-T cells beyond cancer in the context of genome editing era.

Proteolistics: A Protein Delivery Method.

Intracellular protein delivery in plant tissues is becoming an important tool for addressing both basic and applied research questions by plant biologists, especially in the era of genome editing. The ability to deliver proteins or protein/RNA complexes into cells allows for producing gene-edited plants that are free of transgene integration in the genome. Here we describe a protocol for the delivery of a protein/gold particle mixture in plant cells through biolistics. The key for the delivery is the drying of the protein/gold suspension directly onto the gene-gun cartridge or macrocarrier. The intracellular protein delivery into plant cells is achieved through the bombardment using the Bio-Rad PDS-1000/He particle delivery device. We termed this methodology "proteolistics."

Animal transgenesis now and beyond in the era of genome editing: Snapshots from the 15th Transgenic Technology Meeting (TT2019) in Kobe, Japan.

The 15th Transgenic Technology (TT) Meeting of the International Society for Transgenic Technologies (ISTT) was held for the first time in Japan in April of 2019 (TT2019). Delegates from around the world, including researchers, technicians and trainees, spent an exciting 4days, engaging in scientific and technical discussions, information sharing and networking. In the era of genome editing, CRISPR technologies prevailed as popular subjects of discussion throughout the meeting on topics ranging from their applications in the mouse and growing number of other research and industrial animal species, technical challenges in the production of genome-edited animals, to ethical considerations in biomedical research. In particular, impressive progress was reported on the use of CRISPR technologies in nonconventional animal models and large mammalian species in biomedical and industrial settings, indicating areas of expanding frontiers in animal transgenesis. Amid growing excitement with genome editing technologies, reports on conventional genetic engineering approaches and reproductive biology techniques demonstrated that these techniques remain essential to meet the demanding needs of generating complex genome modifications, complementing genome editing approaches. TT2019 was a great success, concluding with a widely shared appreciation of the current field and hints for future directions of animal transgenesis.

CAR T Cell Immunotherapy in Human and Veterinary Oncology: Changing the Odds Against Hematological Malignancies.

The advent of the genome editing era brings forth the promise of adoptive cell transfer using engineered chimeric antigen receptor (CAR) T cells for targeted cancer therapy. CAR T cell immunotherapy is probably one of the most encouraging developments for the treatment of hematological malignancies. In 2017, two CAR T cell therapies were approved by the US Food and Drug Administration: one for the treatment of pediatric acute lymphoblastic leukemia (ALL) and the other for adult patients with advanced lymphomas. However, despite significant progress in the area, CAR T cell therapy is still in its early days and faces significant challenges, including the complexity and costs associated with the technology. B cell lymphoma is the most common hematopoietic cancer in dogs, with an incidence approaching 0.1% and a total of 20-100 cases per 100,000 individuals. It is a widely accepted naturally occurring model for human non-Hodgkin's lymphoma. Current treatment is with combination chemotherapy protocols, which prolong life for less than a year in canines and are associated with severe dose-limiting side effects, such as gastrointestinal and bone marrow toxicity. To date, one canine study generated CAR T cells by transfection of mRNA for CAR domain expression. While this was shown to provide a transient anti-tumor activity, results were modest, indicating that stable, genomic integration of CAR modules is required in order to achieve lasting therapeutic benefit. This commentary summarizes the current state of knowledge on CAR T cell immunotherapy in human medicine and its potential applications in animal health, while discussing the potential of the canine model as a translational system for immuno-oncology research.

Loss-of-function approaches in comparative physiology: is there a future for knockdown experiments in the era of genome editing?

Loss-of-function technologies, such as morpholino- and RNAi-mediated gene knockdown, and TALEN- and CRISPR/Cas9-mediated gene knockout, are widely used to investigate gene function and its physiological significance. Here, we provide a general overview of the various knockdown and knockout technologies commonly used in comparative physiology and discuss the merits and drawbacks of these technologies with a particular focus on research conducted in zebrafish. Despite their widespread use, there is an ongoing debate surrounding the use of knockdown versus knockout approaches and their potential off-target effects. This debate is primarily fueled by the observations that, in some studies, knockout mutants exhibit phenotypes different from those observed in response to knockdown using morpholinos or RNAi. We discuss the current debate and focus on the discrepancies between knockdown and knockout phenotypes, providing literature and primary data to show that the different phenotypes are not necessarily a direct result of the off-target effects of the knockdown agents used. Nevertheless, given the recent evidence of some knockdown phenotypes being recapitulated in knockout mutants lacking the morpholino or RNAi target, we stress that results of knockdown experiments need to be interpreted with caution. We ultimately argue that knockdown experiments should not be discontinued if proper control experiments are performed, and that with careful interpretation, knockdown approaches remain useful to complement the limitations of knockout studies (e.g. lethality of knockout and compensatory responses).

Creating Targeted Gene Knockouts in Brassica oleracea Using CRISPR/Cas9.

While public and political views on genetic modification (inserting "foreign" genes to elicit new traits) have resulted in limited exploitation of the technology in some parts of the world, the new era of genome editing (to edit existing genes to gain new traits/genetic variation) has the potential to change the biotech landscape. Genome editing offers a faster and simpler approach to gene knockout in both single and multiple genetic locations, within a single or small number of generations, in a way that has not been possible through alternative breeding methods. Here we describe an Agrobacterium-mediated delivery approach to deliver Cas9 and dual sgRNAs into 4-day-old cotyledonary petioles of Brassica oleracea. Mutations are detected in approximately 10% of primary transgenic plants and go on in subsequent T1 and T2 generations to segregate away from the T-DNA. This enables the recovery of non-transgenic, genome-edited plants carrying a variety of mutations at the target locus.