Genome Editing in Bacteria (Part 1)

Bacteria is one of the most primitive organisms on earth. Its high susceptibility to bacteriophages has tailored them to use specific tools to edit their genome and evade the bacteriophages. This defense system has been developed to be the most specific genome editing technology of this current period. Previously, various other tools such as restriction enzymes (RE), zinc finger nucleases (ZNF), and transcription activator-like effector nucleases (TALENS) were utilized. Still, its major limitations led to exploiting the bacterial defense system to edit the genome. CRISPR technology can be applied in various microbiology, pathology, cancer biology, molecular biology, and industrial biotechnology, but its limitations, such as off-target effects due to unspecific alterations, are a major concern. In the future, this effective gene alteration technology will be developed to treat inherited rare genetic disorders. This chapter highlights the discovery, components, applications, limitations, and future prospects of CRISPR-Cas.

CRISPR-Cas technology has reshaped the field of microbiology. It has improved the microbial strains for better industrial and therapeutic utilization. In this chapter, we have tried to provide an overview of this technology with special reference to its associated applications in the various fields of interest. We have discussed the origin, classification, and different genome editing methods of CRISPR-Cas to understand its historical significance and the basic mechanism of action. Further, different applications in the area of agriculture, food industry, biotherapeutics, biofuel, and other valuable product synthesis were also explained to highlight the advancement of this system in industrial microbes. We have also tried to review some of the limitations offered by CRISPR and insights into its future perspective.

One of the components of the emerging lifestyle shows an exponential rise in the consumption of packaged or high-calorie food. This has caused an increase in the incidences of diseases which are considered to be a consequence of the changing lifestyle. It has been observed that these clinical conditions are linked with gut dysbiosis, and hence it has been proposed that by modulation of the composition of gut microbiota, the risk of such diseases can be lowered. Prebiotics and probiotics, in combination, possess tremendous potential for maintaining the homeostasis in individuals. In this chapter, a comparative assessment of CRISPR-mediated genome editing technique has been discussed with conventional omics tools and modelling approaches. These techniques substantially simplify the modification of target genome in complex microbial communities and could enhance their prebiotic and probiotic potential. The synthetic biology approach to microbiome therapies such as additive, subtractive, and modulatory therapies for curing gut dysbiosis are also discussed. The chapter is aimed at developing a better understanding about the role of CRISPR/Cas as a reliable technology that may be employed as a diagnostic tool for infectious disease diagnosis as well as its treatment. Although, the tool has already demonstrated its use in a wide range of genome editing and genetic engineering applications, additional study into its use in human genome editing and diagnostics is needed considering any potential side effects or ambiguities. 

Genome editing is a promising tool in the era of modern biotechnology that can alter the DNA of many organisms. It is now extensively used in various industries to obtain the well-desired and enhanced characteristics to improve the yield and nutritional quality of products. The positive health attributes of Bifidobacteria, such as prevention of diarrhoea, reduction of ulcerative colitis, prevention of necrotizing enterocolitis, etc., have shown promising reports in many clinical trials. The potential use of Bifidobacteria as starter or adjunct cultures has become popular. Currently, Bifidobacterium bifidum, B. adolescentis, B. breve, B. infantis, B. longum, and B. lactis find a significant role in the development of probiotic fermented dairy products. However, Bifidobacteria, one of the first colonizers of the human GI tract and an indicator of the health status of an individual, has opened new avenues for research and, thereby, its application. Besides this, the GRAS/QPS (Generally Regarded as Safe/Qualified Presumption of Safety) status of Bifidobacteria makes it safe for use. They belong to the subgroup (which are the fermentative types that are primarily found in the natural cavities of humans and animals) of Actinomycetes. B. lactis has been used industrially in fermented foods, such as yogurt, cheese, beverages, sausages, infant formulas, and cereals. In the present book chapter, the authors tried to explore the origin, health attributes, and various genetic engineering tools for genome editing of Bifidobacteria for the development of starter culture for dairy and non-dairy industrial applications as well as probiotics. 

The gut microbiome is significant in maintaining human health by facilitating absorption and digestion in the intestine. Probiotics have diverse and significant applications in the health sector, so probiotic strains require an understanding of the genome-level organizations. Probiotics elucidate various functional parameters that control their metabolic functions. Gut dysbiosis leads to inflammatory bowel disease and other neurological disorders. The application of probiotic bacteria to modulate the gut microbiota prevents diseases and has gained large interest. In a recent decade, the development of modern tools in molecular biology has led to the discovery of genome engineering. Synthetic biology approaches provide information about diverse biosynthetic pathways and also facilitate novel metabolic engineering approaches for probiotic strain improvement. The techniques enable engineering probiotics with the desired functionalities to benefit human health. This chapter describes the recent advances in probiotic strain improvement for diagnostic and therapeutic applications via CRISPR-Cas tools. Also, the application of probiotics, current challenges, and future perspectives in disease treatment are discussed.

Fermented food products are consumed by about 30% of the world's population due to their high nutritional value and health properties. The use of LAB in the fermentation process has resulted in a variety of fermented food products derived from both plant and animal sources. LAB have been used as starter cultures for food fermentation both traditionally and industrially, having certain specific characteristics such as rapid growth, product yield, higher biomass and also unique organoleptic properties, and are employed in food fermentation. The advancement of highthroughput genome sequencing methods has resulted in a tremendous improvement in our understanding of LAB physiology and has become more essential in the field of food microbiology. The complete genome sequence of Lactococcus lactis in 2001 resulted in a better understanding of metabolic properties and industrial applications of LAB. Genes associated with β-galactosidase, antimicrobial agents, bile salt hydrolase, exopolysaccharide, and GABA producing LAB have received a lot of attention in recent years. Genome editing techniques are required for the development of strains for novel applications and products. They can also play an important part as a research method for acquiring mechanistic insights and identifying new properties. The genome editing of lactic acid bacterial strains has a lot of potential applications for developing functional foods with a favourable influence on the food industries.

Lactobacillus plantarum is a widespread, versatile bacterium that plays a vital role in the preservation of innumerable fermented foods. These strains are commonly employed as silage additives and starter cultures of fermented goods. Genome editing could provide an added benefit by improving the fermentation profile and quality, as well as the accompanying therapeutic benefits.

Genome editing of various strains of L. plantarum can be used commercially to produce L-ribulose or succinic acid, direct lactic acid production, and increased ethanol production. L. plantarum strains or recombinant strains can help restore intestinal flora homeostasis, reduce the number of pathogenic organisms, and could even be employed as vaccine carriers. Food products such as raw and fermented vegetables, olives, and cereals inoculated with probiotic microbes have shown encouraging benefits as people now seek non-dairy based probiotics. L. plantarum of vegetable or plant origin, as well as applications of genome edited strains, are discussed in this book chapter.

Bacillus licheniformis has been regarded as an exceptional microbial cell factory for the production of biochemicals and enzymes. The complete genome sequencing and annotation of the genomes of industrially-relevant Bacillus species has uplifted our understanding of their properties and helped in the progress of genetic manipulations in other Bacillus species. The genome sequence analysis has given information on the different genes and their functional importance. Post-genomic studies require simple and highly efficient tools to enable genetic manipulation. With the developments of complete genome sequences and simple genetic manipulation tools, the metabolic pathways of B. licheniformis could be rewired for the efficient production of interest chemicals. However, gene editing (such as gene knockout) is laborious and time consuming using conventional methods. Recently, useful tools for the genetic engineering of Bacillus species have emerged from the fields of systems and synthetic biology. The recent progress in genetic engineering strategies as well as the available genetic tools that have been developed in Bacillus licheniformis species, has conveniently enabled multiple modifications in the genomes of Bacillus species and thereby improved its use in the industrial sector.