Genome Editing in Bacteria (Part 2)

Now and in the future, meeting the global demand for healthy food for the ever-increasing population is a crucial challenge. In the last seven decades, agricultural practices have shifted to the use of synthetic fertilizers and pesticides to achieve higher yields. Despite the huge contribution of synthetic fertilizers in agronomy, their adverse effects on the environment, natural microbial habitat, and human health cannot be underrated. Besides, synthetic fertilizers are manufactured from non-renewable sources such as earth mining or rock exploitation. In this context, understanding and exploiting soil microbiota appears promising to enhance crop production without jeopardizing the environment and human health. This chapter reviews the historical as well as current research efforts made in identifying the interaction between soil microbes and root exudates for crop improvement. First, microbial consortium viz. bacteria, algae, fungi, and protozoa are briefly discussed. Then, the application of bio-stimulants followed by genome editing of microbes for crop improvement is summarized. Finally, the perspectives and opportunities to produce bioenergy and bio-fertilizers are analyzed. 

Meeting the crucial demand for sustainable agriculture is an upcoming challenge worldwide, leading to global food security concerns. Approximately 50% of agricultural loss is caused by both biotic and abiotic stresses. As per the estimation of Agrios, 42% of crop loss is characterized by biotic stress alone. Bacteria are the second largest contributor in terms of economic losses caused by various plant diseases. Hence, there is a need to develop elite cultivars in amalgamation with readily available sequenced plant database and progressive genome editing. This has proved to be a groundbreaking/milestone in the field of plant breeding for any desired trait. Until now, many new plant breeding techniques (NPBTs) have been introduced for crop improvement. These techniques include site-specific mutagenesis, cisgenesis, intragenesis, breeding with transgenic inducer lines, etc. This book chapter provides a comparative understanding of enrichment in plant genome editing approach about bacterial pathogens aiming for sustainable agriculture development. This chapter also brings a broad aspect of the application, advantages, unsighted aspects of genome editing, and future challenges.

Clustered Regularly Interspaced Short Palindromic Repeats, abbreviated as CRISPR, is a genome-editing technology that permits the creation of precise knock-out mutants by aiding the modification of gene sequences devoid of the steps involving the insertion of foreign DNA into pathogenic microorganisms. The microorganisms are ubiquitous in nature and harbor in the complex ecosystem of the human being. Cas (acronym for CRISPR-associated) genes are present in many microbial genomes. The variable nature of the microbial genome has been utilized as an integral typing tool in epidemiologic, diagnostic, and evolutionary analyses of the prokaryotic species. The past decade has seen an accumulating growth in the development of gene-editing tools utilizing the CRISPR-Cas system, which essentially is a part of the prokaryotic immune system. The development of these unique gene-editing techniques has empowered researchers to alter and investigate organisms with ease and efficiency as never before. This editing tool can efficiently be programmed and delivered into the bacterial populations to explicitly eliminate members of a targeted micro biome. Manipulation of the gene expression and regulation of the synthesis of metabolites and proteins can be achieved by utilizing an engineered CRISPR-Cas system. Put together, these tools present with the exhilarating opportunity to explore the complex interaction between the individual species of the microbiome and the host organism and thereby reveal novel avenues for the generation of drugs to selectively target the microbiome. CRISPR-Cas technology has been employed to cope with antibiotic resistance in intracellular and extracellular pathogens. The widespread use of antibiotics and the escalation of multidrug-resistant (MDR) bacteria boost the prospect of a post-antibiotic era, which emphasizes the need for novel strategies to target MDR pathogens. The development of permissive synthetic biology techniques offers favorable solutions to carry through safe and efficient antibacterial therapies.

Plant-associated microbes, referred to as plant microbiomes, are an integral part of the plant system. The multifaceted role of plant microbiota in combating both abiotic and biotic stresses is well documented in different crop species. However, understanding the co-evolution of plant growth- promoting microbes (PGPM) and PGP traits at genetic and molecular levels requires robust molecular tools to unravel the functional gene orthologues involved in plant-microbe interaction. The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 (CRISPRassociated protein 9) is of paramount importance in deciphering the plant-microbe interaction and addressing the challenges of unraveling endophytic microbes and their benefits thereof. Our knowledge of plant microbiome composition, signaling cues, secondary metabolites, microbial volatiles, and other driving factors in plant microbiome has been enlightened. In recent years, scientists have focused more on below-ground dialogue in recruiting efficient microbiome/engineered rhizosphere. More recently, base editing techniques using endo-nucleolytic ally deactivated dCas9 protein and sgRNAs (CRISPR interference or CRISPRi) have emerged as a useful approach to study the gene functions and have potential merits in exploring plantmicrobe interactions and the signaling cues involved. A systemic understanding of the signaling events and the respective metabolic pathways will enable the application of genome editing tools to enhance the capacity of microbes to produce more targeted metabolites that will enhance microbial colonization.

Further, it will be exciting to employ CRISPR technologies for editing plant-microbe interactions to discover novel metabolic pathways and their modulation for plant immunity and fitness against abiotic as well as biotic stresses. Such metabolites possess tremendous scope in tailoring newer smart nano-based bio-formulations, besides formulating beneficial microbiomes or cocktails, which is the best alternative for climate resilient farming. The present review sheds light upon the deployment of CRISPR/Cas techniques to comprehend plant-microbe interactions, microbe-mediated abiotic and biotic stress resistance, genes edited for the development of fungal, bacterial, and viral disease resistance, nodulation process, PGP activity, CRISPR interference-based gene repression in the PGPM, metabolic pathway editing and their future implications in sustainable agriculture.

Excessive utilization of chemicals based substances such as pesticides, pharmaceuticals, fertilizers, inappropriate dumping of industrial materials and local wastes, etc., into the environment is leading towards deliverance of high amounts of contaminants such as chlorinated hydrocarbons, dyes, toxins, petroleum and diesel spills into the soil. The mingling of these materials with soil and water is becoming one of the supreme complications associated with the environment, as these contaminants are a potential menace to human health. Bioremediation is a process that has the ability to destroy harmful contaminants and transform them into less toxic forms using living organisms such as bacteria, fungi, plants, etc. It is the most up-to-date nature-friendly approach to lower the extent of pollutants in the environment. With continuous developments in the scientific area, researchers are focussing on improving the process of bioremediation by using genome editing technologies. The gene editing techniques have the potential to significantly improve bioremediation processes such as xenobiotic removal, conversion of toxic compounds to less toxic compounds and pesticide degradation to simple components. The main gene editing techniques, CRISPR-Cas, ZFN and TALEN, have the potential to meet the aforementioned goals. This chapter focuses on the various gene editing tools and different genomic strategies such as gene editing, gene circuit, etc., for the alteration or editing of the genome so that their potential value or applications can be seen in various areas.

Genetic engineering involves the manipulation of DNA to either improve, enhance or repair a function by using recombinant DNA technology, which has contributed greatly to the fields of medicine and agriculture. In recent times, the CRISPR-Cas system of gene editing has come to the forefront of genome engineering, transforming disease treatment strategies and the production of modified crops. Industrial activities cause environmental pollution by releasing heavy metal-containing xenobiotic compounds into the environment and affect animal health by causing organ dysfunction and even cancer. Although plants utilize heavy metals from soil in small quantities for their growth, excessive exposure leads to disruption of plant cell machinery and reduces productivity. Similarly, heavy metals degrade soil health by interfering with microbial processes that contribute to soil fertility. Apart from existing methods available for the remediation of contaminated sites, bioremediation is emerging as a potent technique due to its high efficacy, cost-effectiveness and ecofriendly nature. Microbes possess a number of physiological and biochemical properties that have been exploited for the removal and detoxification of metal pollutants. This chapter elaborates on the approaches of gene editing and the development of genetically engineered bacteria to modify the expression of specific genes coding for enzymes that take part in the degradative or detoxification pathway of metals and xenobiotic compounds. It is crucial to address the scope as well as limitations involved in the use of genetically engineered microbes to ensure a safe and cost-effective method for the bioremediation of heavy metal contaminants.

The ubiquity of the CRISPR gene system in bacteria and archaea is characterized by the Cas9 protein, which functions in the repression and activation of several genes. This inherent function of the CRISPR system can find application in bioprocess optimization in environmental and health research. Owing to the complex and dynamic nature of microbial communities catalysing the bioremediation of urban and industrial toxic waste effluents in wastewater treatment plant (WWTP)/common effluent treatment plant (CETP), such sites represent a relatively untapped area for applying the CRISPR technique. DNA editing using CRISPR can enable the sitespecific enhancement in process efficiency of bacterial remediation, which under normal conditions is hampered by its non-selectivity and saturation of binding sites with multiple non-targeted pollutants. Similarly, under the second generation biorefinery concept, CRISPR can serve as a powerful tool in strengthening and improving the anaerobic bio-processes by genome editing in microbes for the heterogeneous expression of various genes associated with anaerobic digestion. Not only has the CRISPR system been used to insert desired genes in the host genome but also to regulate the expression of the host-specific genes. The role of methanotrophic and nitrogen metabolizing bacteria in shaping the atmospheric gaseous composition can also be monitored via CRISPR aided manipulation so as to regulate the nutritional exchange between the atmosphere and the soil. Additionally, genome editing of targeted organisms and crops has found extensive applications in various areas ranging from the nutrigenomics, food and pharmaceutical industry, diagnostics and therapeutics, health and disease prevention.

Methanotrophs use methane gas as their carbon and energy source, but their industrial use has not yet fully been realized due to undiscovered genetic engineering methods that could amend their slow growth rate and economically inefficient product yield. This chapter informs upon genetic engineering approaches taken on methanotrophs so far to enable their widespread use in industry, as well as the reasoning behind these interests. Specific examples of successful engineering performed so far, including conjugation and electroporation methods, CRISPR, genome-scale metabolic modeling, and specific vectors reported as successful, are presented. In addition, the reading provides insights into existing knowledge gaps in the field of methanotrophic engineering and future prospects for optimizing growth and product yield from methanotrophs.

Cyanobacteria are potential organisms being exploited for a wide range of biotechnological applications. They are photosynthetic bacteria and grow in a carbonfree medium and become attractive hosts for biotechnology industries. Cyanobacteria can utilize solar energy and atmospheric CO2 for the growth and synthesis of biomolecules. It is used in many large-scale preparations of various bioproducts such as pharmaceuticals, biofuels, etc. Cyanobacteria become target organisms for the next generation of biofactories for producing desired products with a low-cost technology. The problem in the metabolic engineering of Cyanobacteria is due to ploidy. It has multiple copies of chromosomes ranging from 3-218 copies. There are 12 copies of the genome in Synechocystis PCC 6803 and 3 copies in Synechococcus PCC 7942. Segregation analysis in the conventional genetic approaches of Cyanobacteria becomes laborious due to its polyploidy. Modern genome editing tools such as CRISPR-Cas9 and 12 are available to perform genome editing. CRISPR-Cas9 has been used in a wide range of Cyanobacteria such as Synechococcus elongates UTEX 2973, Synechocystis sp. PCC 6803. To avoid toxic effects caused by Cas-9, a low-level expression system is adopted in Cyanobacteria. Cas-9 base genome editing was applied in Synechococcus and produced succinate 11-fold higher than the normal. Cas-9 is used to cure plasmids in Synechocystis sp. PCC 6803 to develop a shuttle vector for heterologous expression. Another variant of genome editing tool is CRISPR-Cas12a, which is successfully used in Synechocystis sp. 

Streptomyces are Gram-positive, filamentous bacteria belonging to the group actinomycetes. This bacterium is important to the modern industrial world because of the presence of 20-50 biosynthetic gene clusters (BGCs). BGCs contain the genes for the production of industrially important natural products (NP), which includes antibiotics, anti-tumor drugs, anti-depressants, etc., naturally originated from this microorganism. Strain improvement is required to enhance the production of these NP in Streptomyces. Different methods have been used to enhance NP production and strain improvement. In this chapter, we will be discussing strain improvement of Streptomyces species by different genome editing tools. The information, which is put together, includes the basic techniques used for genome editing to the most advanced CRISPR/Cas system associated genome editing in Streptomyces (PCR targeting system, Cre-loxP recombination system, I SceI meganuclease promoted recombination system and CRISPR/Cas system). The authors have discussed about multiplex automated genome editing (MAGE) tool associated with CRISPR/Cas system.