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The mutant project : inside the global race to genetically modify humans
As scientists elsewhere start to catch up with China's vast genetic research programme, gene editing is fuelling an innovation economy that threatens to widen racial and economic inequality. Fundamental questions about science, health and social justice are at stake. Who gets access to gene-editing technologies? As countries loosen regulations around the globe, can we shape research agendas to promote an ethical and fair society? Professor Eben Kirksey takes us on a groundbreaking journey to meet the key scientists, lobbyists and entrepreneurs who are bringing cutting-edge genetic modification tools like CRISPR to your local clinic.
Book
The application of a heat‐inducible CRISPR/Cas12b (C2c1) genome editing system in tetraploid cotton (G. hirsutum) plants
Summary The CRISPR/Cas9 and Cas12a (Cpf1) tools have been used on a large scale for genome editing. A new effector with a single nuclease domain, a relatively small size, low‐frequency off‐target effects and cleavage capability under high temperature has been recently established and designated CRISPR/Cas12b (C2c1). Cas12b has also shown temperature inducibility in mammalian systems. Therefore, this system is potentially valuable for editing the genomes of plant species, such as cotton, that are resistant to high temperatures. Using this new system, mutants of upland cotton were successfully generated following Agrobacterium‐mediated genetic transformation under a range of temperatures. Transformants (explants infected by Agrobacterium) exposed to 45 °C for 4 days showed the highest editing efficiency. No off‐target mutation was detected by whole‐genome sequencing. Genome edits by AacCas12b in T0 generation were faithfully passed to the T1 generation. Taken together, CRISPR/Cas12b is therefore an efficient and precise tool for genome editing in cotton plants.
Journal Article
Challenges Facing CRISPR/Cas9-Based Genome Editing in Plants
The development of plant varieties with desired traits is imperative to ensure future food security. The revolution of genome editing technologies based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has ushered in a new era in plant breeding. Cas9 and the single-guide RNA (sgRNA) form an effective targeting complex on a locus or loci of interest, enabling genome editing in all plants with high accuracy and efficiency. Therefore, CRISPR/Cas9 can save both time and labor relative to what is typically associated with traditional breeding methods. However, despite improvements in gene editing, several challenges remain that limit the application of CRISPR/Cas9-based genome editing in plants. Here, we focus on four issues relevant to plant genome editing: (1) plant organelle genome editing; (2) transgene-free genome editing; (3) virus-induced genome editing; and (4) editing of recalcitrant elite crop inbred lines. This review provides an up-to-date summary on the state of CRISPR/Cas9-mediated genome editing in plants that will push this technique forward.
Journal Article
Prevention of Enzymatic Browning by Natural Extracts and Genome-Editing: A Review on Recent Progress
Fresh fruits and vegetable products are easily perishable during postharvest handling due to enzymatic browning reactions. This phenomenon has contributed to a significant loss of food quality and appearance. Thus, a safe and effective alternative method from natural sources is needed to tackle enzymatic browning prevention. The capabilities of natural anti-browning agents derived from plant- and animal-based resources in inhibiting enzymatic activity have been demonstrated in the literature. Some also possess strong antioxidants properties. This review aims to summarize a recent investigation regarding the use of natural anti-browning extracts from different sources for controlling the browning. The potential applications of genome-editing in preventing browning activity and improving postharvest quality is also discussed. Moreover, the patents on the anti-browning extract from natural sources is also presented in this review. The information reviewed here could provide new insights, contributing to the development of natural anti-browning extracts and genome-editing techniques for the prevention of food browning.
Journal Article
Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox
Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.
Journal Article
Development and optimization of a Barley stripe mosaic virus‐mediated gene editing system to improve Fusarium head blight resistance in wheat
Unfortunately, most wheat genotypes have extremely low callus induction and regeneration efficiency, which limits the application of genome editing in wheat breeding. [...]a new gRNA delivery system that bypasses the tissue culture is critical to the successful use of gene editing in wheat breeding. Lanes 4 and 6 are the vector control and two mutants with TaHRC-specific cleavage bands (768 and 298 bp). (d) and (e) Edited In/del sequences at TaPDS and TaHRC target sites of the mutants. (f) Seedlings of a TaPDS albino mutant (left) and TaHRC mutant. (g) Sequence of edited and non-edited TaHRC in Bobwhite showing insertions in Mut01 (57 bp) and Mut02 (3 bp). (h) and (i) FHB symptoms and percentages of symptomatic spikelets (PSS) between the mutants and control. (j) Sequences of edited and non-edited TaHRC in Everest. (k) and (l) PSS and infected spikes of mutants and control. (m) Recovery rates of TaHRC mutants generated using TaHRC_sgRNA, TaHRC_sgRNA-mTaFT and TaHRC_sgRNA-tRNAIleu vectors. To expand the utility of this editing system in other wheat cultivars with low transformation efficiency, we transferred the Cas9 gene into a locally adapted winter wheat cultivar 'Everest' by crossing Everest to a Cas9-OE Bobwhite plant and selecting Cas9-OE Everest F2 progeny that carried homozygous Cas9 and the target gene.
Journal Article
Advancing genome editing with artificial intelligence: opportunities, challenges, and future directions
Clustered regularly interspaced short palindromic repeat (CRISPR)-based genome editing (GED) technologies have unlocked exciting possibilities for understanding genes and improving medical treatments. On the other hand, Artificial intelligence (AI) helps genome editing achieve more precision, efficiency, and affordability in tackling various diseases, like Sickle cell anemia or Thalassemia. AI models have been in use for designing guide RNAs (gRNAs) for CRISPR-Cas systems. Tools like DeepCRISPR, CRISTA, and DeepHF have the capability to predict optimal guide RNAs (gRNAs) for a specified target sequence. These predictions take into account multiple factors, including genomic context, Cas protein type, desired mutation type, on-target/off-target scores, potential off-target sites, and the potential impacts of genome editing on gene function and cell phenotype. These models aid in optimizing different genome editing technologies, such as base, prime, and epigenome editing, which are advanced techniques to introduce precise and programmable changes to DNA sequences without relying on the homology-directed repair pathway or donor DNA templates. Furthermore, AI, in collaboration with genome editing and precision medicine, enables personalized treatments based on genetic profiles. AI analyzes patients’ genomic data to identify mutations, variations, and biomarkers associated with different diseases like Cancer, Diabetes, Alzheimer’s, etc. However, several challenges persist, including high costs, off-target editing, suitable delivery methods for CRISPR cargoes, improving editing efficiency, and ensuring safety in clinical applications. This review explores AI’s contribution to improving CRISPR-based genome editing technologies and addresses existing challenges. It also discusses potential areas for future research in AI-driven CRISPR-based genome editing technologies. The integration of AI and genome editing opens up new possibilities for genetics, biomedicine, and healthcare, with significant implications for human health.
Journal Article
Plant and Fungal Genome Editing to Enhance Plant Disease Resistance Using the CRISPR/Cas9 System
Crop production has been substantially reduced by devastating fungal and oomycete pathogens, and these pathogens continue to threaten global food security. Although chemical and cultural controls have been used for crop protection, these involve continuous costs and time and fungicide resistance among plant pathogens has been increasingly reported. The most efficient way to protect crops from plant pathogens is cultivation of disease-resistant cultivars. However, traditional breeding approaches are laborious and time intensive. Recently, the CRISPR/Cas9 system has been utilized to enhance disease resistance among different crops such as rice, cacao, wheat, tomato, and grape. This system allows for precise genome editing of various organisms via RNA-guided DNA endonuclease activity. Beyond genome editing in crops, editing the genomes of fungal and oomycete pathogens can also provide new strategies for plant disease management. This review focuses on the recent studies of plant disease resistance against fungal and oomycete pathogens using the CRISPR/Cas9 system. For long-term plant disease management, the targeting of multiple plant disease resistance mechanisms with CRISPR/Cas9 and insights gained by probing fungal and oomycete genomes with this system will be powerful approaches.
Journal Article
Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives
CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.
Journal Article
Genome Editing: Targeting Susceptibility Genes for Plant Disease Resistance
Plant pathogens pose a major threat to crop productivity. Typically, phytopathogens exploit plants’ susceptibility (S) genes to facilitate their proliferation. Disrupting these S genes may interfere with the compatibility between the host and the pathogens and consequently provide broad-spectrum and durable disease resistance. In the past, genetic manipulation of such S genes has been shown to confer disease resistance in various economically important crops. Recent studies have accomplished this task in a transgene-free system using new genome editing tools, including clustered regularly interspaced palindromic repeats (CRISPR). In this Opinion article, we focus on the use of genome editing to target S genes for the development of transgene-free and durable disease-resistant crop varieties.
CRISPR has emerged as a revolutionary tool for plant genome editing. Although developed recently, it has been established in several important plant species, including rice, wheat, and maize, to introduce agronomically important traits such as heat/cold tolerance, disease resistance, herbicide tolerance, and yield improvement.
Transgene-free methods are being introduced in CRISPR-mediated plant genome editing, such as segregating out transgenes, delivering the ribonucleoprotein complex of Cas9 and gRNA through particle bombardment or using a protoplast system, and using viral vectors for editing germline cells.
Targeting susceptibility (S) genes using CRISPR methodologies offers new frontiers to break molecular plant–microbe compatibility and introducing durable pathogen resistance.
Journal Article