Figure - available from: Molecular Biology Reports
This content is subject to copyright. Terms and conditions apply.
CRISPR/Cas9 system is a monomeric protein consisting of a single Cas9 enzyme that makes a complex with a guide RNA (gRNA). The gRNA is itself composed of trans-activating crispr RNA (tracer RNA) and crispr RNA (crRNA). crRNA recognizes 20 – 22 bp host DNA. The protospacer adjacent motif (PAM) is a small sequence of 2 – 6 bp (NGG) and is required for Cas9 nuclease activity. It is situated 3 – 4 nucleotides downstream from the cut site. Seed sequence is 5 nucleotides upstream of PAM. For gRNA to bind to the target DNA and Cas9 to cut the DNA, it needs to have sufficient homology of seed sequence and PAM site. CRISPR/Cas9 can generate single stranded nicks and DSBs. The Cas9 protein and the gRNA can be delivered into the cells and as such cloning of the assembly is not necessary. The system has high target recognition efficiency, low mutation rate, and low cytotoxicity. It can be easily multiplexed and has high cost efficiency
Source publication
In order to meet the growing human food and nutrition demand a perpetual process of crop improvement is idealized. It has seen changing trends and varying concepts throughout human history; from simple selection to complex gene-editing. Among these techniques, random mutagenesis has been shown to be a promising technology to achieve desirable genet...
Similar publications
Genome editing technology is a technique for targeted genetic modifications, enabling the knockout and knock-in of specific DNA fragments in the selected genome. This technology has been widely used in various types of biomedical, clinical, and agricultural research. Three gene-editing techniques including Zinc-finger nucleases (ZFNs), Transcriptio...
Citations
... La mutagénesis consiste en la exposición de material biológico a agentes mutagénicos con el fin de incrementar, de forma artificial, la tasa de mutaciones respecto a la frecuencia de mutaciones espontáneas en condiciones naturales [34], [35]. De esta manera, la mutagénesis permite incrementar la variabilidad genética en plantas para obtener características de interés en la producción agrícola [36]. ...
... De esta manera, la mutagénesis permite incrementar la variabilidad genética en plantas para obtener características de interés en la producción agrícola [36]. La eficiencia de la inducción de mutaciones depende de factores como el tipo de mutágeno, el explante vegetal, la duración de la exposición y la concentración utilizada [34]. ...
... Estas herramientas emplean nucleasas dirigidas para generar cortes en un sitio específico en una o las dos hebras de la molécula de ADN [2], [13]. De acuerdo con Holme et al. [39] y Kashtwari et al. [34], el empleo de estos sistemas para la inducción de mutaciones resulta ser eficiente y preciso. ...
Las mutaciones y la variabilidad genética causada por estas desempeñan un rol fundamental en el mejoramiento genético de las plantas. La inducción de mutaciones o mutagénesis representa una opción rápida y efectiva para el desarrollo de nuevas variedades que conservan los niveles óptimos de productividad en entornos desafiantes para la agricultura. La inducción de mutaciones puede ser llevada a cabo mediante el uso de agentes físicos, agentes químicos o herramientas de edición génica. El empleo conjunto de la mutagénesis con técnicas de cultivo in vitro de tejidos permite establecer estrategias de selección eficientes y rentables. El presente artículo ofrece una breve recopilación sobre la inducción de mutaciones, las técnicas más empleadas para dicho fin y algunas mejoras obtenidas en diferentes cultivos, con relación al estrés biótico, al estrés abiótico y al rendimiento productivo. Finalmente, se examina el panorama histórico del mejoramiento genético mediante la inducción de mutaciones en Costa Rica; específicamente, en los cultivos de mayor relevancia económica y social para el país.
... CRISPR/Cas9 was first described in 2012 (Jinek et al. 2012) and earned Emmanuelle Charpentier and Jennifer A. Doudna the Nobel Prize in Chemistry in 2020 (Fernholm and Barnes 2020). Genome editing already existed when CRISPR/Cas9 debuted in the form of other techniques such as meganucleases, TALENS, Zinc-Finger-Nucleases (ZFNs), and Oligonucleotide-Directed Mutagenesis (ODM); CRISPR/Cas9, nonetheless, was more affordable, accessible to design, and highly precise, which hastened its adoption (Kashtwari et al. 2021;Songstad et al. 2017). The study of Cas9 properties, evolution, and technique improvement has given rise to new enzymes, such as the eukaryotic Caslike enzyme Fanzor and Omega (Badon et al. 2024;Saito et al. 2023), new variants with higher fidelity (Rabinowitz and Offen 2021), base editors (Kuscu et al. 2017;Li et al. 2020a, b;Marx 2018), prime editing (Li et al. 2020a, b;Lin et al. 2020;Xu et al. 2020), epigenome editing , and IP-free enzymes like MAD7 Cas12a/Cpf1 (Lin et al. 2021). ...
Genome editing, mainly CRISPR/Cas9, has been revolutionizing agricultural biotechnology through precise modifications to plant and animal genomes. This review highlights advancements in research and regulatory development of genome editing across Latin America. The region has seen substantial progress in building a regulatory framework to adopt genome editing technologies that enhance crop yield, nutritional content, and resistance to pests and diseases, as well as address critical challenges such as food insecurity and climate change. The article discusses the evolution of regulatory frameworks in various Latin American countries and their trends toward using foreign genetic material to determine regulatory oversight. Argentina pioneered this approach, followed by Brazil, Colombia, Chile, Paraguay, Honduras, and Guatemala. Recent updates from Costa Rica, El Salvador, Ecuador, Uruguay, and Colombia reflect a similar rationale to balance innovation with safety and compliance with international standards. Regional examples of genome editing demonstrate the potential to improve crop quality and sustainability of coffee, rice, cacao, beans, and potatoes. Public perception and acceptance of genome editing are also explored, with surveys indicating strong support for regulatory measures in Costa Rica. The article further explores public perception and acceptance of genome editing indicating strong support for regulation in Costa Rica. The review underscores the importance of continued research, public engagement, and international cooperation that foster responsible development of genome-editing technologies in Latin America.
... A particular advantage of using canes (cuttings) is that with this method, genetically identical plants and clones of the parent vine were obtained. Thus, it allows the maintenance of desirable traits of the Mother grapevine of interest as these traits are more likely to be fixed across generations thanks to the vegetative propagation (Kashtwari et al., 2022). On the contrary, seeds will lead to distinct grapevine varieties that result from crossings of two parent varieties, so there is a risk of losing some desirable traits. ...
The control of plant diseases represents a significant challenge in agriculture, particularly with the current emphasis on reducing pesticide use, necessitating alternative approaches. Among them, resistant plant breeding programs offer a promising alternative. Considering the stressful conditions in space, space conditions emerge as an innovative method for advancing crop improvement. This study explored the unique potential of the International Space Station (ISS) environment to induce beneficial modifications in grapevine, resulting in plants with reduced susceptibility to diseases. Canes of two Vitis vinifera L. cultivars, Cabernet Sauvignon (CS) and Merlot (Me), have undergone a 10-month space journey on-board the ISS, while others stayed on Earth (Ground Control, GC). We conducted a 2-year analysis of the response to downy mildew (Plasmopara viticola) of plants developed from ISS and GC canes, called Mother plants. The disease development of progenies produced from Merlot Mother plants was also evaluated. A higher proportion of individuals with low-susceptibility was noted in the ISS Mother group compared to the controls for both cultivars. This trend was also observed for the Merlot progenies. Finally, 11% of Merlot individuals (Mother and progenies, and for the 2 years of study) were distributed in the low susceptibility class. This study suggests that grapevine canes exposure to ISS conditions resulted in an enhanced percentage of plants showing reduction of susceptibility to downy mildew.
... Noteworthy examples of perennial crops that have been subject to this technique include sugar cane and banana. Surprisingly, despite these advancements, no mutant coffee varieties have yet emerged as a product of induced mutagenesis (Srivastava et al. 2011;Ali et al. 2014;Mago et al. 2017;Kashtwari et al. 2022). The type of mutagen, dosage and tissues used for mutagenesis affects the spectrum and density of heritable mutations. ...
... The most commonly employed chemical mutagens include ethyl methanesulfonate (EMS) and sodium azide, both of which induce DNA alkylation. These mutagens interact with DNA bases, either by reacting with them or substituting themselves as base analogs, resulting in various mutations, including insertions, deletions, transitions, and transversions (Kashtwari et al. 2022). ...
... Dendrogram of M1V1 mutant coffee plants groups and varietal controls, according to incidence, severity, incubation period, and latency period of the CLR fungus, under controlled conditions were used for the development of resistance against Fusarium oxysporum in banana(Xu et al. 2011;Kishor et al. 2017) Other investigations(Khan and Goyal 2009;Mago et al. 2017) report cases of crops treated with sodium azide, for alkylation of the DNA. In recent decades, several attempts for improvement in vegetatively propagated crop through chemical mutagens have been conducted(Bolívar-González et al. 2018;Kashtwari et al. 2022). EMS and sodium azide ...
Coffee leaf rust (CLR), caused by Hemileia vastatrix Berk. and Br, stands as a globally significant disease responsible for diminishing the grain yield and quality of Coffea arabica L. As such, the need arises for a practical and dependable method to quantify CLR disease severity and propose effective management strategies. The creation of a diagrammatic scale, featuring color photographs, proves instrumental in enhancing severity assessment and disease estimation. Mutagenesis serves as a potent tool for improving crop traits, notably resistance to pests and diseases, by inducing genetic variability, thus fostering phenotypic diversity. The primary objective of this study was to craft a novel diagrammatic scale for leaf discs, designed to gauge the severity of coffee leaf rust in C. arabica L. cv. Catuaí plants treated with sodium azide (NaN3) and ethyl methane sulfonate (EMS). The assessment encompassed a thorough examination of incidence, severity, incubation period, and latency in plant leaf discs inoculated with CLR isolates, all carried out under rigorously controlled laboratory conditions. The scale’s construction hinged on the frequency distribution of severity and Weber-Fechner’s visual stimulus law, which facilitated the determination of minimum and maximum limits, as well as intermediary levels. The resulting scale comprises seven distinct levels spanning a range from 0 to 52.15% of the afflicted leaf area. Employing conglomerate analysis (Wilks, p < 0.0001) unveiled four groups of progenies exhibiting significantly different (p < 0.05) resistance responses to CLR. Notably, Plant E154 displayed an incidence rate of merely 0.3% for CLR, while the remaining plants, including A123, E6, E64, A127, CES5, A107, A93, E29, E116, and A101, showcased an incidence rate of less than 10%. In conclusion, this scale represents a suitable, valuable, and dependable tool for visually estimating CLR severity in coffee leaf discs.
... CRISPR-Cas9 activation in the genome(Kashtwari et al. 2022) ...
In the early 21st century, gene therapy, specifically CRISPR-Cas9, was developed as a viable option to prevent heritable diseases and treat genetic disorders in somatic and germline cells. However, the advancement of this technology has led to misuse, as genetic “engineers” aim to alter the DNA of future generations to conform to an idealistic set of standards. The abuse of technology, coupled with the ability to reconstruct an individual’s genetic makeup, leads to the societal question about the best way to regulate this software. This paper addresses three key factors in preventing misuse while maximizing medical benefits: the public’s role, physician-patient relationships, and diversity in healthcare. This paper analyzes how the public should engage with national organizations and participate in formal discussions to express their views regarding CRISPR-Cas9 governance; without explicit expression of their views, their beliefs will likely be undermined by professionals. Furthermore, this paper emphasizes transparency and communication within physician-patient relationships: physicians must offer the comprehensive health benefits and drawbacks of gene editing to ensure patients considering gene editing operations are aware of its implications. Additionally, this paper asserts the need to bridge the socioeconomic gap within healthcare to extend access to gene editing to a larger group and also gain deeper insight into CRISPR-Cas9’s long-term impacts on people of various backgrounds. This paper states that the resolution of these three components is essential to reach a societal consensus of the appropriate regulations on gene editing, thereby halting the temporary ban on the technology.
... Global food security faces tremendous pressures from climate change and population growth. Moreover, crop diseases continue to exert a significant negative economic impact (Kashtwari et al. 2021). Fungal and bacterial pathogens are estimated to reduce crop yields by 15% on average, while viral pathogens lead to about 3% loss (Ristaino et al. 2021). ...
... Additionally, using gene editing tools, particularly CRISPR/Cas9 many alleles can be targeted simultaneously for modification or replacement in polyploids. To fulfill the requirements of plant systems, the system must be adjusted and customized (Kashtwari et al. 2021). Recent studies have suggested that Cas could be programmed to cut various DNA sites in vitro . ...
Sugarcane (Saccharum spp. hybrids) is among the significant industrial cash crops. It serves as an essential raw material to produce biofuels and the global sugar industry. Natural selection, conventional breeding, and more recently, the cutting-edge tools from genetic engineering and biotechnology, have been used to develop and improve the crop since the dawn of civilization. Heterozygous, polyploid, and complex genome of the sugarcane makes its improvement through breeding approaches a bit difficult. The vulnerability potential of sugarcane to abiotic/biotic stressors is further increased by their frequent occurrences. Due to this, efforts to improve sugarcane varietals using biotechnological approaches have increased. In this context, the implementation of genome editing technologies will result into yield promising output. The CRISPR/Cas9 approach was successful in targeting multiple alleles of the magnesium chelatase gene in sugarcane. The genome-edited lines of sugarcane with modified cell wall components for bioethanol production were developed in Brazil. As far as the regulatory system for genome editing is concerned, the site-directed nuclease 1 and 2 (SDN 1 and 2) are exempted. In the present paper, the applications of the CRISPR/Cas9 technology to combat biotic/abiotic stresses in sugarcane in order to improve its agricultural traits have been summarized.
... Many botanists are working to improve the breeding and cultivation techniques of C. sativus to increase production. It has been documented that C. sativus shows higher heterozygosity owing to its three homologous chromosomes, thus rendering efforts to alter its genome by traditional plant breeding, which follows Mendelian principles, a major challenge [18,36,37]. The breeding of polyploid plants requires the aggregation of each allele to achieve a state of polyploid homozygosity, but achieving this ideal with C. sativus would require considerable acreage, a great deal of manpower, and significant financial investment over a long period [37]. ...
... It has been documented that C. sativus shows higher heterozygosity owing to its three homologous chromosomes, thus rendering efforts to alter its genome by traditional plant breeding, which follows Mendelian principles, a major challenge [18,36,37]. The breeding of polyploid plants requires the aggregation of each allele to achieve a state of polyploid homozygosity, but achieving this ideal with C. sativus would require considerable acreage, a great deal of manpower, and significant financial investment over a long period [37]. Therefore, vegetative reproduction using bulb propagation is traditionally used, with its advantages of a loss of juvenility, rapid development, and a higher rate of growth, and it is conducive to obtaining a large number of propagules with standardized characteristics [38]. ...
Crocin is one of the most valuable components of the Chinese medicinal plant Crocus sativus and is widely used in the food, cosmetics, and pharmaceutical industries. Traditional planting of C. sativus is unable to fulfill the increasing demand for crocin in the global market, however, such that researchers have turned their attention to the heterologous production of crocin in a variety of hosts. At present, there are reports of successful heterologous production of crocin in Escherichia coli, Saccharomyces cerevisiae, microalgae, and plants that do not naturally produce crocin. Of these, the microalga Dunaliella salina, which produces high levels of β-carotene, the substrate for crocin biosynthesis, is worthy of attention. This article describes the biosynthesis of crocin, compares the features of each heterologous host, and clarifies the requirements for efficient production of crocin in microalgae.
... Physical mutagens consist in exposing parts of the plant to radiation, which causes the breakdown of the DNA chain, thus generating the loss of one or more nucleotides [63], causing modifications in DNA, cell membranes, lipids, enzymes, and other cellular constituents [64]. ...
... Alkylation causes the link-up of a hydrocarbon group with low molecular weight to the bases, which changes their pairing properties since true DNA replication depends on precise base mating. Base alkylation causes incorrect pairing and will therefore induce mutations [63]. ...
Biotechnological techniques provide a viable alternative to help improve and increase the production of plant species of agricultural and economic importance, which have been affected over the years by climate change, increasing their susceptibility to pests and/or diseases, generating losses in production as well as a decrease in their regenerative and genetic diversity. The application of biotechnological techniques such as in vitro mutagenesis offers a viable option for the generation of crops that are resistant to the different factors caused by abiotic and biotic stress. In vitro mutagenesis has been used in an efficient way to generate genetic changes in different plant species. However, these methods have not been studied thoroughly in crops of agro-industrial interest, such as agave, which represents an economic resource of national importance and are considered as endemic species of Mexico. Therefore, this literary review aimed to focus on the studies that have been used for the genetic improvement of this species via mutagenesis techniques in plants in the agave genus. Therefore, the objective was to set a precedent for future genetic studies that aim to obtain more productive regenerants for various industries, such as food and pharmaceutical. It is also of great interest to compile information from basic research that helps understand and elucidate a model of possible defense mechanisms that are activated in the Agave genus.
... Mutation breeding in strawberry has been carried out through conventional mutagenesis techniques in the past, by exploiting both gamma radiation and chemical mutagens [168]. Currently, point-mutagenized strawberry plants can be obtained in a more precise manner, by both stable transformation (mediated by Agrobacterium or direct) and transient expression to produce CRISPR/Cas9-edited plants [138,169]. ...
Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the ‘Made in Italy’ label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The ‘new genomic techniques’ (NGTs), renamed in Italy as ‘technologies for assisted evolution’ (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian contexts.
