1.
Genome editing in fruit, ornamental, and industrial crops.
Ramirez-Torres, F, Ghogare, R, Stowe, E, Cerdá-Bennasser, P, Lobato-Gómez, M, Williamson-Benavides, BA, Giron-Calva, PS, Hewitt, S, Christou, P, Dhingra, A
Transgenic research. 2021;(4):499-528
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Abstract
The advent of genome editing has opened new avenues for targeted trait enhancement in fruit, ornamental, industrial, and all specialty crops. In particular, CRISPR-based editing systems, derived from bacterial immune systems, have quickly become routinely used tools for research groups across the world seeking to edit plant genomes with a greater level of precision, higher efficiency, reduced off-target effects, and overall ease-of-use compared to ZFNs and TALENs. CRISPR systems have been applied successfully to a number of horticultural and industrial crops to enhance fruit ripening, increase stress tolerance, modify plant architecture, control the timing of flower development, and enhance the accumulation of desired metabolites, among other commercially-important traits. As editing technologies continue to advance, so too does the ability to generate improved crop varieties with non-transgenic modifications; in some crops, direct transgene-free edits have already been achieved, while in others, T-DNAs have successfully been segregated out through crossing. In addition to the potential to produce non-transgenic edited crops, and thereby circumvent regulatory impediments to the release of new, improved crop varieties, targeted gene editing can speed up trait improvement in crops with long juvenile phases, reducing inputs resulting in faster market introduction to the market. While many challenges remain regarding optimization of genome editing in ornamental, fruit, and industrial crops, the ongoing discovery of novel nucleases with niche specialties for engineering applications may form the basis for additional and potentially crop-specific editing strategies.
2.
A critical look on CRISPR-based genome editing in plants.
Ahmad, N, Rahman, MU, Mukhtar, Z, Zafar, Y, Zhang, B
Journal of cellular physiology. 2020;(2):666-682
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing, derived from prokaryotic immunity system, is rapidly emerging as an alternative platform for introducing targeted alterations in genomes. The CRISPR-based tools have been deployed for several other applications including gene expression studies, detection of mutation patterns in genomes, epigenetic regulation, chromatin imaging, etc. Unlike the traditional genetic engineering approaches, it is simple, cost-effective, and highly specific in inducing genetic variations. Despite its popularity, the technology has limitations such as off-targets, low mutagenesis efficiency, and its dependency on in-vitro regeneration protocols for the recovery of stable plant lines. Several other issues such as persisted CRISPR activity in subsequent generations, the potential for transferring to its wild type population, the risk of reversion of edited version to its original phenotype particularly in cross-pollinated plant species when released into the environment and the scarcity of validated targets have been overlooked. This article briefly highlights these undermined aspects, which may challenge the wider applications of this platform for improving crop genetics.
3.
CRISPR/Cas9; A robust technology for producing genetically engineered plants.
Farooq, R, Hussain, K, Nazir, S, Javed, MR, Masood, N
Cellular and molecular biology (Noisy-le-Grand, France). 2018;(14):31-38
Abstract
CRISPR/Cas9 is a technology evolved from modified type II immune system of bacteria and archaea. Exploitation of this bacterial immune system in all eukaryotes including plants may lead to site-specific targeted genome engineering. Genome engineering is objectively utilized to express/silence a trait harbouring gene in the plant genome. In this review, different genetic engineering techniques including classical breeding, RNAi and genetic transformation and synthetic sequence-specific nucleases (zinc finger nucleases; ZFNs and transcription activator-like effector nuclease; TALENs) techniques have been described and compared with advanced genome editing technique CRISPR/Cas9, on the basis of their merits and drawbacks. This revolutionary genome engineering technology has edge over all other approaches because of its simplicity, stability, specificity of the target and multiple genes can be engineered at a time. CRISPR/Cas9 requires only Cas9 endonuclease and single guide RNA, which are directly delivered into plant cells via either vector-mediated stable transformation or transient delivery of ribonucleoproteins (RNPs) and generate double-strand breaks (DSBs) at target site. These DSBs are further repaired by cell endogenous repairing pathways via HDR or NHEJ. The major advantage of CRISPR/Cas9 system is that engineered plants are considered Non-GM; can be achieved using in vitro expressed RNPs transient delivery. Different variants of Cas9 genes cloned in different plasmid vectors can be used to achieve different objectives of genome editing including double-stranded DNA break, single-stranded break, activate/repress the gene expression. Fusion of Cas9 with fluorescent protein can lead to visualize the expression of the CRISPR/Cas9 system. The applications of this technology in plant genome editing to improve different plant traits are comprehensively described.
4.
Boosting innate immunity to sustainably control diseases in crops.
Nicaise, V
Current opinion in virology. 2017;:112-119
Abstract
Viruses cause epidemics in all major crops, threatening global food security. The development of efficient and durable resistance able to withstand viral attacks represents a major challenge for agronomy, and relies greatly on the understanding of the molecular dialogue between viral pathogens and their hosts. Research over the last decades provided substantial advances in the field of plant-virus interactions. Remarkably, the advent of studies of plant innate immunity has recently offered new strategies exploitable in the field. This review summarizes the recent breakthroughs that define the mechanisms underlying antiviral innate immunity in plants, and emphasizes the importance of integrating that knowledge into crop improvement actions, particularly by exploiting the insights related to immune receptors.
5.
[Studies on protein-based identification method of genetically modified capsicum].
Liu, J, Deng, P, Fang, S, Zhao, J
Wei sheng yan jiu = Journal of hygiene research. 2003;(2):134-7
Abstract
The detection system based on protein is a method to evaluate the safety of genetically modified foods (GMF). Using cecropin BD gene in capsicum, a detecting method was set up. It is a system of evaluating the real expressive condition and safety of the foreign target protein of GMF. In this studies, with the preformative technic method, a satisfactory results by making use of hemolymph of immunized pupae of Antheraea pernyi as standard experimental material was achieved, comparing with the realities of the goal protein expressive condition of cecropin D gene in capsicum. The detecting steps were as following: the goal protein from material was extracted roughly, then with CM-Sepharose-FF ion-exchange chromatography twice, the goal protein was purified moderately. The purified product was identified by detecting the anti-bacterial activity, electrophoresis, biological auto-photography of the goal protein and MADDI-TOF mass spectrum. The results showed that the expressive foreign target protein in transgenic capsicum was in accordance with standard protein in the physical and chemical property, anti-bacterial activity and molecular weight. It indicated that expression of the target gene in capsicum is real, it corresponded to expected value. The separation, purification and identification methods of cecropin D were established in the study. By means of the comparative experiments about anti-bacterial activity and molecular weight of anti-bacterial peptide(ABP) from GM-capsicum and hemolymph of immunized pupae of Antheraea pernyi, the identification method of target protein from GM-capsicum was set up. The method is easy to be operated, fast and feasible.