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825900.9959Secondary Metabolite Transcriptomic Pipeline (SeMa-Trap), an expression-based exploration tool for increased secondary metabolite production in bacteria. For decades, natural products have been used as a primary resource in drug discovery pipelines to find new antibiotics, which are mainly produced as secondary metabolites by bacteria. The biosynthesis of these compounds is encoded in co-localized genes termed biosynthetic gene clusters (BGCs). However, BGCs are often not expressed under laboratory conditions. Several genetic manipulation strategies have been developed in order to activate or overexpress silent BGCs. Significant increases in production levels of secondary metabolites were indeed achieved by modifying the expression of genes encoding regulators and transporters, as well as genes involved in resistance or precursor biosynthesis. However, the abundance of genes encoding such functions within bacterial genomes requires prioritization of the most promising ones for genetic manipulation strategies. Here, we introduce the 'Secondary Metabolite Transcriptomic Pipeline' (SeMa-Trap), a user-friendly web-server, available at https://sema-trap.ziemertlab.com. SeMa-Trap facilitates RNA-Seq based transcriptome analyses, finds co-expression patterns between certain genes and BGCs of interest, and helps optimize the design of comparative transcriptomic analyses. Finally, SeMa-Trap provides interactive result pages for each BGC, allowing the easy exploration and comparison of expression patterns. In summary, SeMa-Trap allows a straightforward prioritization of genes that could be targeted via genetic engineering approaches to (over)express BGCs of interest.202235580059
919210.9958Antimicrobial peptides: Sustainable application informed by evolutionary constraints. The proliferation and global expansion of multidrug-resistant (MDR) bacteria have deepened the need to develop novel antimicrobials. Antimicrobial peptides (AMPs) are regarded as promising antibacterial agents because of their broad-spectrum antibacterial activity and multifaceted mechanisms of action with non-specific targets. However, if AMPs are to be applied sustainably, knowledge of how they induce resistance in pathogenic bacteria must be mastered to avoid repeating the traditional antibiotic resistance mistakes currently faced. Furthermore, the evolutionary constraints on the acquisition of AMP resistance by microorganisms in the natural environment, such as functional compatibility and fitness trade-offs, inform the translational application of AMPs. Consequently, the shortcut to achieve sustainable utilization of AMPs is to uncover the evolutionary constraints of bacteria on AMP resistance in nature and find the tricks to exploit these constraints, such as applying AMP cocktails to minimize the efficacy of selection for resistance or combining nanomaterials to maximize the costs of AMP resistance. Altogether, this review dissects the benefits, challenges, and opportunities of utilizing AMPs against disease-causing bacteria, and highlights the use of AMP cocktails or nanomaterials to proactively address potential AMP resistance crises in the future.202235752270
918220.9957Harnessing CRISPR/Cas9 in engineering biotic stress immunity in crops. There is significant potential for CRISPR/Cas9 to be used in developing crops that can adapt to biotic stresses such as fungal, bacterial, viral, and pest infections and weeds. The increasing global population and climate change present significant threats to food security by putting stress on plants, making them more vulnerable to diseases and productivity losses caused by pathogens, pests, and weeds. Traditional breeding methods are inadequate for the rapid development of new plant traits needed to counteract this decline in productivity. However, modern advances in genome-editing technologies, particularly CRISPR/Cas9, have transformed crop protection through precise and targeted modifications of plant genomes. This enables the creation of resilient crops with improved resistance to pathogens, pests, and weeds. This review examines various methods by which CRISPR/Cas9 can be utilized for crop protection. These methods include knocking out susceptibility genes, introducing resistance genes, and modulating defense genes. Potential applications of CRISPR/Cas9 in crop protection involve introducing genes that confer resistance to pathogens, disrupting insect genes responsible for survival and reproduction, and engineering crops that are resistant to herbicides. In conclusion, CRISPR/Cas9 holds great promise for advancing crop protection and ensuring food security in the face of environmental challenges and increasing population pressures. The most recent advancements in CRISPR technology for creating resistance to bacteria, fungi, viruses, and pests are covered here. We wrap up by outlining the most pressing issues and technological shortcomings, as well as unanswered questions for further study.202540663257
833530.9957Implementing Optogenetic-Controlled Bacterial Systems in Drosophila melanogaster for Alleviation of Heavy Metal Poisoning. Drosophila melanogaster (fruit fly) is an animal model chassis in biological and genetic research owing to its short life cycle, ease of cultivation, and acceptability to genetic modification. While the D. melanogaster chassis offers valuable insights into drug efficacy, toxicity, and mechanisms, several obvious challenges such as dosage control and drug resistance still limit its utility in pharmacological studies. Our research combines optogenetic control with engineered gut bacteria to facilitate the precise delivery of therapeutic substances in D. melanogaster for biomedical research. We have shown that the engineered bacteria can be orally administered to D. melanogaster to get a stable density of approximately 28,000 CFUs/per fly, leading to no detectable negative effects on the growth of D. melanogaster. In a model of D. melanogaster exposure to heavy metal, these orally administered bacteria uniformly express target genes under green light control to produce MtnB protein for binding and detoxifying lead, which significantly reduces the level of oxidative stress in the intestinal tract of Pb-treated flies. This pioneering study lays the groundwork for using optogenetic-controlled bacteria in the model chassis D. melanogaster to advance biomedical applications.202439312764
825640.9956Revolutionizing Tomato Cultivation: CRISPR/Cas9 Mediated Biotic Stress Resistance. Tomato (Solanum lycopersicon L.) is one of the most widely consumed and produced vegetable crops worldwide. It offers numerous health benefits due to its rich content of many therapeutic elements such as vitamins, carotenoids, and phenolic compounds. Biotic stressors such as bacteria, viruses, fungi, nematodes, and insects cause severe yield losses as well as decreasing fruit quality. Conventional breeding strategies have succeeded in developing resistant genotypes, but these approaches require significant time and effort. The advent of state-of-the-art genome editing technologies, particularly CRISPR/Cas9, provides a rapid and straightforward method for developing high-quality biotic stress-resistant tomato lines. The advantage of genome editing over other approaches is the ability to make precise, minute adjustments without leaving foreign DNA inside the transformed plant. The tomato genome has been precisely modified via CRISPR/Cas9 to induce resistance genes or knock out susceptibility genes, resulting in lines resistant to common bacterial, fungal, and viral diseases. This review provides the recent advances and application of CRISPR/Cas9 in developing tomato lines with resistance to biotic stress.202439204705
828350.9956Stress responses as determinants of antimicrobial resistance in Gram-negative bacteria. Bacteria encounter a myriad of potentially growth-compromising conditions in nature and in hosts of pathogenic bacteria. These 'stresses' typically elicit protective and/or adaptive responses that serve to enhance bacterial survivability. Because they impact upon many of the same cellular components and processes that are targeted by antimicrobials, adaptive stress responses can influence antimicrobial susceptibility. In targeting and interfering with key cellular processes, antimicrobials themselves are 'stressors' to which protective stress responses have also evolved. Cellular responses to nutrient limitation (nutrient stress), oxidative and nitrosative stress, cell envelope damage (envelope stress), antimicrobial exposure and other growth-compromising stresses, have all been linked to the development of antimicrobial resistance in Gram-negative bacteria - resulting from the stimulation of protective changes to cell physiology, activation of resistance mechanisms, promotion of resistant lifestyles (biofilms), and induction of resistance mutations.201222424589
826760.9955Why put up with immunity when there is resistance: an excursion into the population and evolutionary dynamics of restriction-modification and CRISPR-Cas. Bacteria can readily generate mutations that prevent bacteriophage (phage) adsorption and thus make bacteria resistant to infections with these viruses. Nevertheless, the majority of bacteria carry complex innate and/or adaptive immune systems: restriction-modification (RM) and CRISPR-Cas, respectively. Both RM and CRISPR-Cas are commonly assumed to have evolved and be maintained to protect bacteria from succumbing to infections with lytic phage. Using mathematical models and computer simulations, we explore the conditions under which selection mediated by lytic phage will favour such complex innate and adaptive immune systems, as opposed to simple envelope resistance. The results of our analysis suggest that when populations of bacteria are confronted with lytic phage: (i) In the absence of immunity, resistance to even multiple bacteriophage species with independent receptors can evolve readily. (ii) RM immunity can benefit bacteria by preventing phage from invading established bacterial populations and particularly so when there are multiple bacteriophage species adsorbing to different receptors. (iii) Whether CRISPR-Cas immunity will prevail over envelope resistance depends critically on the number of steps in the coevolutionary arms race between the bacteria-acquiring spacers and the phage-generating CRISPR-escape mutants. We discuss the implications of these results in the context of the evolution and maintenance of RM and CRISPR-Cas and highlight fundamental questions that remain unanswered. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.201930905282
826970.9955Molecular genetics of Rhizobium Meliloti symbiotic nitrogen fixation. The application of recombinant DNA techniques to the study of symbiotic nitrogen fixation has yielded a growing list of Rhizobium meliloti genes involved in the processes of nodulation, infection thread formation and nitrogenase activity in nodules on the roots of the host plant, Medicago sativa (alfalfa). Interaction with the plant is initiated by genes encoding sensing and motility systems by which the bacteria recognizes and approaches the root. Signal molecules, such as flavonoids, mediate a complex interplay of bacterial and plant nodulation genes leading to entry of the bacteria through a root hair. As the nodule develops, the bacteria proceed inward towards the cortex within infection threads, the formation of which depends on bacterial genes involved in polysaccharide synthesis. Within the cortex, the bacteria enter host cells and differentiate into forms known as bacteroids. Genes which encode and regulate nitrogenase enzyme are expressed in the mature nodule, together with other genes required for import and metabolism of carbon and energy sources offered by the plant.198914542173
877280.9954The role of drought response genes and plant growth promoting bacteria on plant growth promotion under sustainable agriculture: A review. Drought is a major stressor that poses significant challenges for agricultural practices. It becomes difficult to meet the global demand for food crops and fodder. Plant physiology, physico-chemistry and morphology changes in plants like decreased photosynthesis and transpiration rate, overproduction of reactive oxygen species, repressed shoot and root shoot growth and modified stress signalling pathways by drought, lead to detrimental impacts on plant development and output. Coping with drought stress requires a variety of adaptations and mitigation techniques. Crop yields could be effectively increased by employing plant growth-promoting rhizobacteria (PGPR), which operate through many mechanisms. These vital microbes colonise the rhizosphere of crops and promote drought resistance by producing exopolysaccharides (EPS), 1-aminocyclopropane-1-carboxylate (ACC) deaminase and phytohormones including volatile compounds. The upregulation or downregulation of stress-responsive genes causes changes in root architecture due to acquiring drought resistance. Further, PGPR induces osmolyte and antioxidant accumulation. Another key feature of microbial communities associated with crops includes induced systemic tolerance and the production of free radical-scavenging enzymes. This review is focused on detailing the role of PGPR in assisting plants to adapt to drought stress.202439002396
58990.9954Insulin Signaling and Insulin Resistance Facilitate Trained Immunity in Macrophages Through Metabolic and Epigenetic Changes. Adaptation of the innate immune system has been recently acknowledged, explaining sustained changes of innate immune responses. Such adaptation is termed trained immunity. Trained immunity is initiated by extracellular signals that trigger a cascade of events affecting cell metabolism and mediating chromatin changes on genes that control innate immune responses. Factors demonstrated to facilitate trained immunity are pathogenic signals (fungi, bacteria, viruses) as well non-pathogenic signals such as insulin, cytokines, adipokines or hormones. These signals initiate intracellular signaling cascades that include AKT kinases and mTOR as well as histone methylases and demethylases, resulting in metabolic changes and histone modifications. In the context of insulin resistance, AKT signaling is affected resulting in sustained activation of mTORC1 and enhanced glycolysis. In macrophages elevated glycolysis readily impacts responses to pathogens (bacteria, fungi) or danger signals (TLR-driven signals of tissue damage), partly explaining insulin resistance-related pathologies. Thus, macrophages lacking insulin signaling exhibit reduced responses to pathogens and altered metabolism, suggesting that insulin resistance is a state of trained immunity. Evidence from Insulin Receptor as well as IGF1Receptor deficient macrophages support the contribution of insulin signaling in macrophage responses. In addition, clinical evidence highlights altered macrophage responses to pathogens or metabolic products in patients with systemic insulin resistance, being in concert with cell culture and animal model studies. Herein, we review the current knowledge that supports the impact of insulin signaling and other insulin resistance related signals as modulators of trained immunity.201931244863
8633100.9954Bacterial interactions with arsenic: Metabolic pathways, resistance mechanisms, and bioremediation approaches. Arsenic contamination in natural waters is one of the biggest threats to human health, mainly due to its carcinogenic potential. Given its toxicity, nearly all organisms have evolved to develop an arsenic resistance mechanism. Conventional techniques of arsenic remediation suffer from various limitations of their applicability, cost and/or chemical intensive nature. In past few decades, bioremediation has emerged as a potential alternative to the conventional techniques. Microbial bioremediation, bacteria in particular, offers an eco-friendly and sustainable alternative, owing to its inherent metabolic capabilities to transform, immobilize or volatilize arsenic. Diverse biochemical pathways involving oxidation of As(III) to As(V), reduction of As(V) under anaerobic respiration or detoxification, methylation and demethylation, bioleaching and biomineralization into insoluble forms are essential mechanisms for arsenic remediation. These transformations, detoxification and resistance are regulated by specific genetic systems, including the ars operon, aio, arr and arsM, accessory genes such as arsR, arsB, acr3, arsC and arsP. The metabolic regulation of arsenic detoxification involves complex cofactor-dependent enzyme systems and environmental signal-responsive transcriptional control. Integrated approaches such as immobilization of bacteria on biochar or their encapsulation have also been known to enhance stability, reusability and stress tolerance. However, bioremediation is a very complex process due to the interrelationship of various influences such as, presence of specific microorganisms, nutrients and environmental factors. Therefore, it is of utmost importance to understand the bacterial interactions with arsenic for the development of bioremediation technologies. This review article tries to discuss the current status of arsenic bioremediation using bacteria, its field applications, challenges and future perspectives. It also includes the strengths, weaknesses, opportunities, threats (SWOT) analysis to assess the merits and demerits of using bacteria for bioremediation of arsenic.202541043264
8263110.9954CRISPR/Cas9: A Novel Weapon in the Arsenal to Combat Plant Diseases. Plant pathogens like virus, bacteria, and fungi incur a huge loss of global productivity. Targeting the dominant R gene resulted in the evolution of resistance in pathogens, which shifted plant pathologists' attention toward host susceptibility factors (or S genes). Herein, the application of sequence-specific nucleases (SSNs) for targeted genome editing are gaining more importance, which utilize the use of meganucleases (MN), zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN) with the latest one namely clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The first generation of genome editing technologies, due to their cumbersome nature, is becoming obsolete. Owing to its simple and inexpensive nature the use of CRISPR/Cas9 system has revolutionized targeted genome editing technology. CRISPR/Cas9 system has been exploited for developing resistance against virus, bacteria, and fungi. For resistance to DNA viruses (mainly single-stranded DNA viruses), different parts of the viral genome have been targeted transiently and by the development of transgenic plants. For RNA viruses, mainly the host susceptibility factors and very recently the viral RNA genome itself have been targeted. Fungal and bacterial resistance has been achieved mainly by targeting the host susceptibility genes through the development of transgenics. In spite of these successes CRISPR/Cas9 system suffers from off-targeting. This and other problems associated with this system are being tackled by the continuous discovery/evolution of new variants. Finally, the regulatory standpoint regarding CRISPR/Cas9 will determine the fate of using this versatile tool in developing pathogen resistance in crop plants.201830697226
8636120.9954Insights into the synthesis, engineering, and functions of microbial pigments in Deinococcus bacteria. The ability of Deinococcus bacteria to survive in harsh environments, such as high radiation, extreme temperature, and dryness, is mainly attributed to the generation of unique pigments, especially carotenoids. Although the limited number of natural pigments produced by these bacteria restricts their industrial potential, metabolic engineering and synthetic biology can significantly increase pigment yield and expand their application prospects. In this study, we review the properties, biosynthetic pathways, and functions of key enzymes and genes related to these pigments and explore strategies for improving pigment production through gene editing and optimization of culture conditions. Additionally, studies have highlighted the unique role of these pigments in antioxidant activity and radiation resistance, particularly emphasizing the critical functions of deinoxanthin in D. radiodurans. In the future, Deinococcus bacterial pigments will have broad application prospects in the food industry, drug production, and space exploration, where they can serve as radiation indicators and natural antioxidants to protect astronauts' health during long-term space flights.202439119139
8265130.9954Mathematical modelling of CRISPR-Cas system effects on biofilm formation. Clustered regularly interspaced short palindromic repeats (CRISPR), linked with CRISPR associated (Cas) genes, can confer adaptive immunity to bacteria, against bacteriophage infections. Thus from a therapeutic standpoint, CRISPR immunity increases biofilm resistance to phage therapy. Recently, however, CRISPR-Cas genes have been implicated in reducing biofilm formation in lysogenized cells. Thus CRISPR immunity can have complex effects on phage-host-lysogen interactions, particularly in a biofilm. In this contribution, we develop and analyse a series of dynamical systems to elucidate and disentangle these interactions. Two competition models are used to study the effects of lysogens (first model) and CRISPR-immune bacteria (second model) in the biofilm. In the third model, the effect of delivering lysogens to a CRISPR-immune biofilm is investigated. Using standard analyses of equilibria, stability and bifurcations, our models predict that lysogens may be able to displace CRISPR-immune bacteria in a biofilm, and thus suggest strategies to eliminate phage-resistant biofilms.201728426329
9060140.9954Targetable nano-delivery vehicles to deliver anti-bacterial small acid-soluble spore protein (SASP) genes. Interest in phage-based therapeutics is increasing, at least in part due to the need for new treatment options for infections caused by antibiotic-resistant bacteria. It is possible to use wild-type (WT) phages to treat bacterial infections, but it is also possible to modify WT phages to generate therapeutics with improved features. Here, we will discuss features of Phico Therapeutics' SASPject technology, which modifies phages for use as targetable nano-delivery vehicles (NDV), to introduce antibacterial Small Acid Soluble Spore Protein (SASP) genes into specific target bacteria.202134723318
8635150.9953Techniques for enhancing the tolerance of industrial microbes to abiotic stresses: A review. The diversity of stress responses and survival strategies evolved by microorganism enables them to survive and reproduce in a multitude of harsh environments, whereas the discovery of the underlying resistance genes or mechanisms laid the foundation for the directional enhancement of microbial tolerance to abiotic stresses encountered in industrial applications. Many biological techniques have been developed for improving the stress resistance of industrial microorganisms, which greatly benefited the bacteria on which industrial production is based. This review introduces the main techniques for enhancing the resistance of microorganisms to abiotic stresses, including evolutionary engineering, metabolic engineering, and process engineering, developed in recent years. In addition, we also discuss problems that are still present in this area and offer directions for future research.202031206805
8264160.9953Anti-CRISPR Phages Cooperate to Overcome CRISPR-Cas Immunity. Some phages encode anti-CRISPR (acr) genes, which antagonize bacterial CRISPR-Cas immune systems by binding components of its machinery, but it is less clear how deployment of these acr genes impacts phage replication and epidemiology. Here, we demonstrate that bacteria with CRISPR-Cas resistance are still partially immune to Acr-encoding phage. As a consequence, Acr-phages often need to cooperate in order to overcome CRISPR resistance, with a first phage blocking the host CRISPR-Cas immune system to allow a second Acr-phage to successfully replicate. This cooperation leads to epidemiological tipping points in which the initial density of Acr-phage tips the balance from phage extinction to a phage epidemic. Furthermore, both higher levels of CRISPR-Cas immunity and weaker Acr activities shift the tipping points toward higher initial phage densities. Collectively, these data help elucidate how interactions between phage-encoded immune suppressors and the CRISPR systems they target shape bacteria-phage population dynamics.201830033365
9179170.9953A detailed landscape of CRISPR-Cas-mediated plant disease and pest management. Genome editing technology has rapidly evolved to knock-out genes, create targeted genetic variation, install precise insertion/deletion and single nucleotide changes, and perform large-scale alteration. The flexible and multipurpose editing technologies have started playing a substantial role in the field of plant disease management. CRISPR-Cas has reduced many limitations of earlier technologies and emerged as a versatile toolbox for genome manipulation. This review summarizes the phenomenal progress of the use of the CRISPR toolkit in the field of plant pathology. CRISPR-Cas toolbox aids in the basic studies on host-pathogen interaction, in identifying virulence genes in pathogens, deciphering resistance and susceptibility factors in host plants, and engineering host genome for developing resistance. We extensively reviewed the successful genome editing applications for host plant resistance against a wide range of biotic factors, including viruses, fungi, oomycetes, bacteria, nematodes, insect pests, and parasitic plants. Recent use of CRISPR-Cas gene drive to suppress the population of pathogens and pests has also been discussed. Furthermore, we highlight exciting new uses of the CRISPR-Cas system as diagnostic tools, which rapidly detect pathogenic microorganism. This comprehensive yet concise review discusses innumerable strategies to reduce the burden of crop protection.202235835393
8297180.9953Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase. Gram-negative bacteria have effective methods of excluding toxic compounds, including a largely impermeable outer membrane (OM) and a range of efflux pumps. Furthermore, when cells become nutrient limited, RpoS enacts a global expression change providing cross-protection against many stresses. Here, we utilized sensitivity to an anionic detergent (sodium dodecyl sulfate [SDS]) to probe changes occurring to the cell's permeability barrier during nutrient limitation. Escherichia coli is resistant to SDS whether cells are actively growing, carbon limited, or nitrogen limited. In actively growing cells, this resistance depends on the AcrAB-TolC efflux pump; however, this pump is not necessary for protection under either carbon-limiting or nitrogen-limiting conditions, suggesting an alternative mechanism(s) of SDS resistance. In carbon-limited cells, RpoS-dependent pathways lessen the permeability of the OM, preventing the necessity for efflux. In nitrogen-limited but not carbon-limited cells, the loss of rpoS can be completely compensated for by the AcrAB-TolC efflux pump. We suggest that this difference simply reflects the fact that nitrogen-limited cells have access to a metabolizable energy (carbon) source that can efficiently power the efflux pump. Using a transposon mutant pool sequencing (Tn-Seq) approach, we identified three genes, sanA, dacA, and yhdP, that are necessary for RpoS-dependent SDS resistance in carbon-limited stationary phase. Using genetic analysis, we determined that these genes are involved in two different envelope-strengthening pathways. These genes have not previously been implicated in stationary-phase stress responses. A third novel RpoS-dependent pathway appears to strengthen the cell's permeability barrier in nitrogen-limited cells. Thus, though cells remain phenotypically SDS resistant, SDS resistance mechanisms differ significantly between growth states. IMPORTANCE: Gram-negative bacteria are intrinsically resistant to detergents and many antibiotics due to synergistic activities of a strong outer membrane (OM) permeability barrier and efflux pumps that capture and expel toxic molecules eluding the barrier. When the bacteria are depleted of an essential nutrient, a program of gene expression providing cross-protection against many stresses is induced. Whether this program alters the OM to further strengthen the barrier is unknown. Here, we identify novel pathways dependent on the master regulator of stationary phase that further strengthen the OM permeability barrier during nutrient limitation, circumventing the need for efflux pumps. Decreased permeability of nutrient-limited cells to toxic compounds has important implications for designing new antibiotics capable of targeting Gram-negative bacteria that may be in a growth-limited state.201727821607
8136190.9953Recent progress in CRISPR/Cas9-based genome editing for enhancing plant disease resistance. Nowadays, agricultural production is strongly affected by both climate change and pathogen attacks which seriously threaten global food security. For a long time, researchers have been waiting for a tool allowing DNA/RNA manipulation to tailor genes and their expression. Some earlier genetic manipulation methods such as meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) allowed site directed modification but their successful rate was limited due to lack of flexibility when targeting a 'site-specific nucleic acid'. The discovery of clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome editing domain in different living organisms during the past 9 years. Based on RNA-guided DNA/RNA recognition, CRISPR/Cas9 optimizations have offered an unrecorded scientific opportunity to engineer plants resistant to diverse pathogens. In this report, we describe the main characteristics of the primary reported-genome editing tools ((MNs, ZFNs, TALENs) and evaluate the different CRISPR/Cas9 methods and achievements in developing crop plants resistant to viruses, fungi and bacteria.202336871676