Genome Editing in Bacteria (Part 1) E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Lebensstil
- Sprache: Englisch
- Veröffentlichungsjahr: 2024
This reference is a comprehensive review of genome editing in bacteria. The multi-part book meticulously consolidates research findings and insights on the applications of bacteria across several industries, including food processing and pharmaceutical development. The book covers four overarching themes for readers: a historical perspective of genome editing, genome editing in probiotics, applications of genome editing in agricultural microbiology and genetic engineering in environmental microbiology. The editors have also compiled chapters that provide an in-depth analysis of gene regulation and metabolic engineering through genome editing tools for specific bacteria.
Key topics in part 1:
- An Overview of advances in CRISPR-CAS research
- Applications of CRISPR/CAS9-based genome editing for industrial microorganisms
- Gut microbiome modulation to address gut dysbiosis
- Bifidobacterium genome editing for probiotic development and metabolic engineering.
- Insights into the use of lactic acid bacteria as starter cultures in the food
- Genome editing of vegetable-derived L. Plantarum
- Genome editing in Bacillus Licheniformis
Genome Editing in Bacteria is a definitive reference for scholars, researchers and industry professionals navigating the forefront of bacterial genomics.
Readership
Scholars and professionals interested in bacterial genomics.
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FOREWORD
The importance of Biotechnology and its applications in our life is well known, and with nearly four decades of experience at the University level, I am delighted and glad to write the foreword for the book “Genome Editing in Bacteria” edited by Dr. Prakash Halami and Dr. Aravind Sundararaman. I had known Dr. Halami since 2008, when I was nominated to the Institutional Biosafety Committee of CSIR-CFTRI, Mysuru, by the Dept. of Biotechnology, Govt. of India (New Delhi). I appreciate Dr. Halami abilities in planning, executing, monitoring research projects and dedication in mentoring many researchers in his lab.
Biotechnology is one of the most significant branches of biological sciences that is shaping the century and will continue to flourish and expand to newer frontiers in the present century too. Biotechnology draws parallel developments through research and applications in Microbiology, Genetics, Molecular biology, Bioinformatics, and Nanotechnology. Diverse branches of biotechnology have distinguished niches to well-defined dynamic research areas with both academic and industrial applications. Genome editing is a subject that has turned into a high science topic in our everyday vocabulary over a short period of time. Several positive and negative attributes have been associated with gene delivery techniques to develop transgenic microbes, animals and crops. The field has wide applications, and with every new development reported in leading peer-reviewed journals across the globe, the opportunities only become wider and the hopes brighter.
The editors have done an extraordinary job of bringing out a timely peer-reviewed volume titled "Genome Editing in Bacteria" with contributors spread across different continents. It is quite impressive to note that the editors have identified a wide range and dynamic topics that are organized in the form of a series of captivating articles highlighting different aspects of genetic engineering, both traditional and modern technologies in the field, protocols, advantages, new school of thoughts from around the world associated with some frontier development of biotechnological research. The attractive illustrations for presenting complex theoretical and experimental details and overall production design are sure to win the hearts of enthusiastic readers. The simplicity of the language and presentation style appealed to me a lot.
With years of experience in specific fields, several scientists and researchers have compiled and provided authentic and contemporary information. This compendium will be handy for faculty to update their lectures for the students pursuing higher education. Similarly, this book will be a ready reckoner for researchers working in specific areas to plan their future research.
It is a great pleasure on my part to pen a foreword for this prestigious, multi-authored, peer-reviewed, international publication on a topic that is very contemporary and close to my heart.
I wish the editors great success. I also extend my sincere greetings to all the contributors for their excellent effort in making this book a success.
PREFACE
Genetic engineering is essential in basic research and Industrial Biotechnology for metabolic and genomic manipulations to regulate microorganisms to produce valuable products. Genome editing is the cornerstone for scientists to interrogate the genetic basis of microorganisms for which the accessibility of the genome along with molecular tools is an essential factor. The classical genetic methods developed for genome editing in bacterial species include culture and transformation. The methods were highly laborious and required the introduction of at least one resistance marker cassette in the genome, which hampers the possibility of producing precise edits like single amino acid mutations. The breakthrough by the discovery of the CRISPR-Cas technology has shed light on the adaptive immune system of prokaryotes to explore tremendous opportunities for targeted genetic engineering approaches in prokaryotes. Here, we discuss the current state-of-the-art approach for gene editing in bacteria and different strategies used in this technology for prokaryotic organisms.
This book has eight chapters, including historical perspectives of genome editing and applications of probiotics and its metabolites. We attempted to update and collate information and research carried out on various applications of bacteria in different industries, such as the food and pharmaceutical industry and their gene regulation for metabolic engineering using genome editing tools. We are grateful to all contributing authors who accepted our invitation to contribute to this book. The contributing authors are well-recognized scientists and researchers with vast experience in the field of bacteriology and molecular biology. We are happy to bring them all together on the same platform to bring out this book. We are grateful to the Bentham Science Group for publishing this comprehensive book, and we hope it will be read by researchers, students, teachers, scientists and food entreprenuers who are interested in the metabolic engineering of bacteria for various health benefits. Although there are hundreds of research articles, review papers, and limited books on genome editing of prokaryotes, this book "Genome Editing in Bacteria" is the first of this kind, a compilation of various applications of bacteria across diverse fields of biotechnology.
We dedicate this book to the creators of the indigenous knowledge of molecular biology and genetic engineering for putting together both an ocean of knowledge and the basis for research to study in-depth genome editing techniques for bacteria.
List of Contributors
Recent Advances in CRISPR-Cas Genome Engineering: An Overview
Angelina Job Kolady1,Aritra Mukherjee1,Ranjith Kumavath1,2,*,Sarvepalli Vijay Kumar3,Pasupuleti Sreenivasa Rao4,5,*Abstract
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.
INTRODUCTION
Bacteria are single-celled prokaryotic microorganisms, all belonging to the kingdom of Monera in the system of classification of living organisms [1]. Bacteria are among the oldest living organisms as they were among the first life forms to appear on earth and are present in almost every habitat. We usually associate bacteria with an infectious disease. Nonetheless, every bacterium lives in parasitic relations with plants and animals. A significant number of bacterial species live in symbiotic associations with other living organisms. Bacteria are also prone to infection from specialized viruses called bacteriophages [2]. To evade conditions from bacteriophages, bacteria evolved to use a specialized tool in their genome and clustered regularly interspaced short palindromic repeats [3, 4].
GENOME EDITING
Genome editing is a process where specific changes can be made in the regions of interest with the help of explicit and engineered nucleases by introducing double-stranded breaks (DSB). These breaks can cause site-specific mutations, gene deletions, substitutions, or insertions, and later can be repaired by various mechanisms. Non-homologous end joining (NHEJ) is prone to error, and homology-directed repair (HDR) error-free is the repair mechanism used [5]. Genome editing is a powerful tool for understanding biological roles. It can treat genetic disorders by identifying 'molecular mistakes' and providing appropriate gene therapy. Restriction enzymes (RE) are natural genome editing tool, while transcription activator-like effector nucleases (TALENs) and Zinc Finger Nucleases (ZFNs) are artificial genome editing tools.
GENOME EDITING TOOLS AND THEIR LIMITATIONS
Restriction Enzymes (RE)
The discovery of restriction enzymes in early 1970 heralded a new age in molecular biology. Restriction enzymes or endonucleases are natural genome editing tools that recognize specific nucleotide sequences and cut the DNA at specific sites. The gene of interest could be inserted at a particular location.
The limitation of the restriction enzymes is the difficulty in predicting the location at which the gene of interest could be inserted. The primary reason behind it is that the recognition sequence of most of the restriction enzymes is base pairs long and often arises several times in a genome. The restriction specificity of endonucleases can depend on the environmental conditions.
In contrast, restriction enzymes are used for molecular cloning, DNA mapping, epigenome mapping, and constructing DNA libraries. These enzymes were modified to enhance the specificity of restriction endonucleases like the homing endonuclease systems. They could target specific sequences for genome editing. REs have long recognition sites and tolerate sequence degeneracy within their restriction site, unlike restriction enzymes [6]. One of the examples is meganucleases. It is designed to recognize long DNA sequences.
Zinc Finger Nucleases (ZNFs)
The artificial restriction enzymes consist of a subunit that recognizes desired DNA sequence and the DNA cutting part of restriction enzymes. They can be designed to identify specific DNA sequences and thereby enable targeted cleavage [7]. The hybrid restriction enzymes could be created using a zinc finger DNA binding domain fused to break up the naturally occurring FokI endonuclease domain. FokI, a naturally occurring IIS restriction enzyme, has played a pivotal role in the success of ZFNs. A lot of effort was required to produce a modified ZNF, which was a significant drawback. Thus, research has been done to customize ZNF.
Transcription Activator Like Effector Nucleases
The artificial restriction enzymes (ARE) consist of two components (i) restriction enzyme to cleave DNA and (ii) TAL, effector. TAL effectors comprise 33 repeat sequences, which helps them bind to long lines in the genome. TALENs are preferable over ZNF due to their ease of application. TALENs encode the FokI domain fused to the engineered DNA binding region, and when bound, dimerized FokI endonuclease could form a double-stranded break. The limitation of TALEN is TALE target search process is affected by genomic occlusions.
Discovery and History
The discovery of CRISPR revolutionized gene-editing technology (Fig. 1). CRISPR was initially discovered in 1987 from the E. coli genome, and its role in the adaptive immune system was elucidated in early 2000. In 2020, Prof. Emmanuelle Charpentier and Prof. Jennifer Doudna were honored with the Nobel Prize in chemistry for their discovery of CRISPR-Cas9 technology in Streptococcus pyogenes, which is considered an evolution in the fields of medicine, biotechnology, and agriculture [8]. This technique is favorable due to its precision in gene editing [9]. This helps to alter genes efficiently and rapidly. It is also widely used in treating genetic disorders [10].
Fig. (1)) Timeline of the discovery of CRISPR as the genome-editing tool. CRISPR was discovered in 1987 in E. coli and was later found in other species, from 2005 to 2011.Spacer Acquisition
Upon invasion of a prokaryote phage virus, the first stage of the immune response is to capture phage DNA and insert it into the CRISPR locus in a spacer. New spacers are usually added upstream of the CRISPR next to the leader sequence creating chronological order of the viral infections. Cas1 and Cas2 are found in both CRISPR-Cas immune systems, hinting that they are involved in spacer acquisition [11-15]. However, their crystal structures are similar, and purified Cas1 proteins are metalloenzymes acting as nucleases/integrates that bind to DNA sequence-independent [16]. Representative Cas2 proteins have been characterized and contain either ssRNA (single strand) or dsDNA specific endoribonuclease activity. The analysis of CRISPR-Cas systems showed PAMs as necessary for type I and type II but not for type III systems during spacer acquisition [17]. The conservation of the PAM sequences differs between CRISPR-Cas systems and appears to be evolutionarily linked to Cas1 and the leader sequence [17].
Biogenesis
Specialized Cas proteins to form crRNAs then cleave this transcript. However, the type I-E and type I-F systems, proteins Cas6e and Cas6f, respectively, recognize stem-loops created by pairing identical repeats that flank the crRNA [18]. The cleavage instead occurs by the longer transcript enveloping around the Cas6 to allow detachment just upstream of the repeat sequence [19]. Type-II systems lack the Cas6 gene and instead use RNaseIII for cleavage. Functional type II systems encode a specialized form of RNA known as a trans-activating crRNA (tracrRNA) [20]. Transcription of the tracrRNA and the immediate CRISPR transcript results in base pairing and dsRNA formation at the repeat sequence, which RNaseIII eventually targets to produce crRNAs. The crRNA only contains a truncated spacer at one end, unlike the other two systems. The type I-E Cascade requires five Cas proteins bound to a single crRNA [21].
Components of CRISPR-Cas
Cas9
It is an endonuclease enzyme used to cut DNA at the target sequence. Cas9 can recognize and bind to the target sequence in front of the adjacent protospacer motif (PAM) sequence to enhance the specificity. Cas9 cuts at the specific site and causes a double-stranded break (DSB), after which the DNA repairing mechanism such as non-homology end joining (NHEJ) and homology-directed repair (HDR) occurs. One such example is SpyCas9 cDNA is found in several plasmids, especially pX330.
sgRNA and How to Design sgRNAs
The complement the DNA sequences on either side of the cut and contain whatever arrangement is desired for insertion into the host genome [22]. They occur naturally, serving essential functions, but can also be designed to be used for targeted editing, such as with CRISPR-Cas9 [23]. Most prokaryotes, encompassing bacteria and archaea, use CRISPR with its associated Cas enzymes as their adaptive immune system. When prokaryotes are infected by phages and manage to fend off the attack, Cas enzymes will cut phage DNA or RNA and integrate parts between the repeats of the CRISPR sequence [22].
