Gene Editing with Artificial DNA Scissors.

Artificial metallo-nucleases (AMNs) are small molecule DNA cleavage agents, also known as DNA molecular scissors, and represent an important class of chemotherapeutic with high clinical potential. This review provides a primary level of exploration on the concepts key to this area including an introduction to DNA structure, function, recognition, along with damage and repair mechanisms. Building on this foundation, we describe hybrid molecules where AMNs are covalently attached to directing groups that provide molecular scissors with enhanced or sequence specific DNA damaging capabilities. As this research field continues to evolve, understanding the applications of AMNs along with synthetic conjugation strategies can provide the basis for future innovations, particularly for designing new artificial gene editing systems.

Genome Editing Approaches Using Zinc Finger Nucleases (ZFNs) for the Treatment of Motor Neuron Diseases.

Motor neuron disorders are diseases that can be passed through generations by heredity or they occur due to spontaneous mutations in the gene. These are the disorders that weaken the connection between motor neurons and the muscles, due to this the coordination between the neurons and muscles gets disturbed and thereby the actions become abnormal, every year millions of people around the world suffer from these different types of motor neuron disorders. Till now there is no proper known treatment for this type of disorder, there is active research work going on to treat these diseases permanently. Some gene therapy treatments are giving promising results in the treatment of these diseases, specifically, genetic modification techniques are the front liners, and many types of nucleases are doing their work to replace the mutated gene with a functional one. Zinc finger nucleases (ZFNs) are one of them with good disease treatment potential with accurate and desirable effects. In this review, we note the complete information about ZFNs and their drawbacks along with their future prospective in gene therapy and also shortly with other types of nucleases-mediated gene therapies. There also some factors that influence the gene therapy treatment are also noted along with some detailed information.

Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Development of Aptamers for RNase Inactivation in Xtract-Free™ Sample Collection and Transport Medium.

There is a significant need to develop new environmentally friendly, extraction-free sample collection mediums that can effectively preserve and protect genetic material for point-of-care and/or self-collection, home-collection, and mail-back testing. Systematic evolution of ligands by exponential enrichment (SELEX) was used to create anti-ribonuclease (RNase) deoxyribonucleic acid (DNA) aptamers against purified RNase A conjugated to paramagnetic carboxylated beads. Following eight rounds of SELEX carried out under various stringency conditions, e.g., selection using Xtract-Free™ (XF) specimen collection medium and elevated ambient temperature of 28 °C, a panel of five aptamers was chosen following bioinformatic analysis using next-generation sequencing. The efficacy of aptamer inactivation of RNase was assessed by monitoring ribonucleic acid (RNA) integrity via fluorometric and real-time RT-PCR analysis. Inclusion of aptamers in reaction incubations resulted in an 8800- to 11,200-fold reduction in RNase activity, i.e., digestion of viral RNA compared to control. Thus, anti-RNase aptamers integrated into XF collection medium as well as other commercial reagents and kits have great potential for ensuring quality intact RNA for subsequent genomic analyses.

Structure-specific nucleases in genome dynamics and strategies for targeting cancers.

Nucleases are a super family of enzymes that hydrolyze phosphodiester bonds present in genomes. They widely vary in substrates, causing differentiation in cleavage patterns and having a diversified role in maintaining genetic material. Through cellular evolution of prokaryotic to eukaryotic, nucleases become structure-specific in recognizing its own or foreign genomic DNA/RNA configurations as its substrates, including flaps, bubbles, and Holliday junctions. These special structural configurations are commonly found as intermediates in processes like DNA replication, repair, and recombination. The structure-specific nature and diversified functions make them essential to maintaining genome integrity and evolution in normal and cancer cells. In this article, we review their roles in various pathways, including Okazaki fragment maturation during DNA replication, end resection in homology-directed recombination repair of DNA double-strand breaks, DNA excision repair and apoptosis DNA fragmentation in response to exogeneous DNA damage, and HIV life cycle. As the nucleases serve as key points for the DNA dynamics, cellular apoptosis, and cancer cell survival pathways, we discuss the efforts in the field in developing the therapeutic regimens, taking advantage of recently available knowledge of their diversified structures and functions.

CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

All organisms, from animals to bacteria, are subject to genetic parasites, such as viruses and transposons. Genetic parasites are pieces of nucleic acids (DNA or RNA) that can use a cell's machinery to copy themselves at the expense of their hosts. This often leads to the host's demise, so organisms evolved many types of defense mechanisms. One of the most ancient and common forms of defense against viruses and transposons is the targeted restriction of nucleic acids, that is, deployment of host enzymes that can destroy or restrict nucleic acids containing specific sequence motifs or modifications. In bacteria, many of the restriction enzymes targeting parasitic genetic elements are formed by fusions of proteins from the so-called McrBC systems with a protein domain called EVE. EVE and other functionally similar domains are a part of proteins that recognize and bind modified bases in nucleic acids. Enzymes can use the ability of these specificity domains to bind modified bases to detect non-host nucleic acids. Bell et al. conducted a comprehensive computational search for McrBC systems and discovered a large and highly diverse branch of this family with unusual characteristic structural and functional domains. These features include regions that form long alpha-helices (coils) that coil with other alpha-helices (known as coiled-coils), as well as several distinct enzymatic domains that break down nucleic acids (known as nucleases). They call these systems CoCoNuTs (coiled-coiled nuclease tandems). All CoCoNuTs contain domains, including EVE-like ones, which are predicted to interact with components of the RNA-based systems responsible for producing proteins in the cell (translation), suggesting that the CoCoNuTs have an important impact on protein abundance and RNA metabolism. Bell et al.'s findings will be of interest to scientists working on prokaryotic immunity and virulence. Furthermore, similarities between CoCoNuTs and components of eukaryotic RNA-degrading systems suggest evolutionary connections between this diverse family of bacterial predicted RNA restriction systems and RNA regulatory pathways of eukaryotes. Further deciphering the mechanisms of CoCoNuTs could shed light on how certain pathways of RNA metabolism and regulation evolved, and how they may contribute to advances in biotechnology.

Engineering of Zinc Finger Nucleases Through Structural Modeling Improves Genome Editing Efficiency in Cells.

Genome Editing is widely used in biomedical research and medicine. Zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector (TALE) nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFN-encoding DNAs can be easily packaged into a viral vector with limited cargo space, such as adeno-associated virus (AAV) vectors, for in vivo and clinical applications. ZFNs have great potential for translational research and clinical use. However, constructing functional ZFNs and improving their genome editing efficiency is extremely difficult. Here, the efficient construction of functional ZFNs and the improvement of their genome editing efficiency using AlphaFold, Coot, and Rosetta are described. Plasmids encoding ZFNs consisting of six fingers using publicly available zinc-finger resources are assembled. Two functional ZFNs from the ten ZFNs tested are successfully obtained. Furthermore, the engineering of ZFNs using AlphaFold, Coot, or Rosetta increases the efficiency of genome editing by 5%, demonstrating the effectiveness of engineering ZFNs based on structural modeling.

Breeding rice for yield improvement through CRISPR/Cas9 genome editing method: current technologies and examples.

The impending climate change is threatening the rice productivity of the Asian subcontinent as instances of crop failures due to adverse abiotic and biotic stress factors are becoming common occurrences. CRISPR-Cas9 mediated genome editing offers a potential solution for improving rice yield as well as its stress adaptation. This technology allows modification of plant's genetic elements and is not dependent on foreign DNA/gene insertion for incorporating a particular trait. In this review, we have discussed various CRISPR-Cas9 mediated genome editing tools for gene knockout, gene knock-in, simultaneously disrupting multiple genes by multiplexing, base editing and prime editing the genes. The review here also presents how these genome editing technologies have been employed to improve rice productivity by directly targeting the yield related genes or by indirectly manipulating various abiotic and biotic stress responsive genes. Lately, many countries treat genome-edited crops as non-GMOs because of the absence of foreign DNA in the final product. Thus, genome edited rice plants with improved yield attributes and stress resilience are expected to be accepted by the public and solve food crisis of a major portion of the globe. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01423-y.

Recent Advances in Tomato Gene Editing.

The use of gene-editing tools, such as zinc finger nucleases, TALEN, and CRISPR/Cas, allows for the modification of physiological, morphological, and other characteristics in a wide range of crops to mitigate the negative effects of stress caused by anthropogenic climate change or biotic stresses. Importantly, these tools have the potential to improve crop resilience and increase yields in response to challenging environmental conditions. This review provides an overview of gene-editing techniques used in plants, focusing on the cultivated tomatoes. Several dozen genes that have been successfully edited with the CRISPR/Cas system were selected for inclusion to illustrate the possibilities of this technology in improving fruit yield and quality, tolerance to pathogens, or responses to drought and soil salinity, among other factors. Examples are also given of how the domestication of wild species can be accelerated using CRISPR/Cas to generate new crops that are better adapted to the new climatic situation or suited to use in indoor agriculture.

Interference Requirements of Type III CRISPR-Cas Systems from Thermus thermophilus.

Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes from Thermus thermophilus to interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that the T. thermophilus III-B system must elicit a more efficient cyclic oligoadenylate-dependent response to provide the immunity.

Improving DNA nanostructure stability: A review of the biomedical applications and approaches.

DNA's programmable, predictable, and precise self-assembly properties enable structural DNA nanotechnology. DNA nanostructures have a wide range of applications in drug delivery, bioimaging, biosensing, and theranostics. However, physiological conditions, including low cationic ions and the presence of nucleases in biological systems, can limit the efficacy of DNA nanostructures. Several strategies for stabilizing DNA nanostructures have been developed, including i) coating them with biomolecules or polymers, ii) chemical cross-linking of the DNA strands, and iii) modifications of the nucleotides and nucleic acids backbone. These methods significantly enhance the structural stability of DNA nanostructures and thus enable in vivo and in vitro applications. This study reviews the present perspective on the distinctive properties of the DNA molecule and explains various DNA nanostructures, their advantages, and their disadvantages. We provide a brief overview of the biomedical applications of DNA nanostructures and comprehensively discuss possible approaches to improve their biostability. Finally, the shortcomings and challenges of the current biostability approaches are examined.

Mitochondrial diseases and mtDNA editing.

Mitochondrial diseases are a heterogeneous group of inherited disorders characterized by mitochondrial dysfunction, and these diseases are often severe or even fatal. Mitochondrial diseases are often caused by mitochondrial DNA mutations. Currently, there is no curative treatment for patients with pathogenic mitochondrial DNA mutations. With the rapid development of traditional gene editing technologies, such as zinc finger nucleases and transcription activator-like effector nucleases methods, there has been a search for a mitochondrial gene editing technology that can edit mutated mitochondrial DNA; however, there are still some problems hindering the application of these methods. The discovery of the DddA-derived cytosine base editor has provided hope for mitochondrial gene editing. In this paper, we will review the progress in the research on several mitochondrial gene editing technologies with the hope that this review will be useful for further research on mitochondrial gene editing technologies to optimize the treatment of mitochondrial diseases in the future.

Type III CRISPR-Cas: beyond the Cas10 effector complex.

Type III CRISPR-Cas loci encode some of the most abundant, yet complex, immune systems of prokaryotes. They are composed of a Cas10 complex that uses an RNA guide to recognize transcripts from bacteriophage and plasmid invaders. Target recognition triggers three activities within this complex: ssDNA degradation, synthesis of cyclic oligoadenylates (cOA) that act as second messengers to activate CARF-domain effectors, and cleavage of target RNA. This review covers recent research in type III CRISPR-Cas systems that looked beyond the activity of the canonical Cas10 complexes towards: (i) ancillary nucleases and understanding how they provide defense by sensing cOA molecules; (ii) ring nucleases and their role in regulating cOA production; and (iii) CRISPR-associated proteases, including the function of the Craspase complex in a transcriptional response to phage infection.

Development of mitochondrial gene-editing strategies and their potential applications in mitochondrial hereditary diseases: a review.

Mitochondrial DNA (mtDNA) is a critical genome contained within the mitochondria of eukaryotic cells, with many copies present in each mitochondrion. Mutations in mtDNA often are inherited and can lead to severe health problems, including various inherited diseases and premature aging. The lack of efficient repair mechanisms and the susceptibility of mtDNA to damage exacerbate the threat to human health. Heteroplasmy, the presence of different mtDNA genotypes within a single cell, increases the complexity of these diseases and requires an effective editing method for correction. Recently, gene-editing techniques, including programmable nucleases such as restriction endonuclease, zinc finger nuclease, transcription activator-like effector nuclease, clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated 9 and base editors, have provided new tools for editing mtDNA in mammalian cells. Base editors are particularly promising because of their high efficiency and precision in correcting mtDNA mutations. In this review, we discuss the application of these techniques in mitochondrial gene editing and their limitations. We also explore the potential of base editors for mtDNA modification and discuss the opportunities and challenges associated with their application in mitochondrial gene editing. In conclusion, this review highlights the advancements, limitations and opportunities in current mitochondrial gene-editing technologies and approaches. Our insights aim to stimulate the development of new editing strategies that can ultimately alleviate the adverse effects of mitochondrial hereditary diseases.

Cellular Responses to Widespread DNA Replication Stress.

Replicative DNA polymerases are blocked by nearly all types of DNA damage. The resulting DNA replication stress threatens genome stability. DNA replication stress is also caused by depletion of nucleotide pools, DNA polymerase inhibitors, and DNA sequences or structures that are difficult to replicate. Replication stress triggers complex cellular responses that include cell cycle arrest, replication fork collapse to one-ended DNA double-strand breaks, induction of DNA repair, and programmed cell death after excessive damage. Replication stress caused by specific structures (e.g., G-rich sequences that form G-quadruplexes) is localized but occurs during the S phase of every cell division. This review focuses on cellular responses to widespread stress such as that caused by random DNA damage, DNA polymerase inhibition/nucleotide pool depletion, and R-loops. Another form of global replication stress is seen in cancer cells and is termed oncogenic stress, reflecting dysregulated replication origin firing and/or replication fork progression. Replication stress responses are often dysregulated in cancer cells, and this too contributes to ongoing genome instability that can drive cancer progression. Nucleases play critical roles in replication stress responses, including MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, FEN1, and TATDN2. Several of these nucleases cleave branched DNA structures at stressed replication forks to promote repair and restart of these forks. We recently defined roles for EEPD1 in restarting stressed replication forks after oxidative DNA damage, and for TATDN2 in mitigating replication stress caused by R-loop accumulation in BRCA1-defective cells. We also discuss how insights into biological responses to genome-wide replication stress can inform novel cancer treatment strategies that exploit synthetic lethal relationships among replication stress response factors.

Genome and transcriptome engineering by compact and versatile CRISPR-Cas systems.

Comparative genomics has enabled the discovery of tiny clustered regularly interspaced short palindromic repeat (CRISPR) bacterial immune system effectors with enormous potential for manipulating eukaryotic genomes. Recently, smaller Cas proteins, including miniature Cas9, Cas12, and Cas13 proteins, have been identified and validated as efficient genome editing and base editing tools in human cells. The compact size of these novel CRISPR effectors is highly desirable for generating CRISPR-based therapeutic approaches, mainly to overcome in vivo delivery constraints, providing a promising opportunity for editing pathogenic mutations of clinical relevance and knocking down RNAs in human cells without inducing chromosomal insertions or genome alterations. Thus, these tiny CRISPR-Cas systems represent new and highly programmable, specific, and efficient platforms, which expand the CRISPR toolkit for potential therapeutic opportunities.

Argonaute protein-based nucleic acid detection technology.

It is vital to diagnose pathogens quickly and effectively in the research and treatment of disease. Argonaute (Ago) proteins are recently discovered nucleases with nucleic acid shearing activity that exhibit specific recognition properties beyond CRISPR-Cas nucleases, which are highly researched but restricted PAM sequence recognition. Therefore, research on Ago protein-mediated nucleic acid detection technology has attracted significant attention from researchers in recent years. Using Ago proteins in developing nucleic acid detection platforms can enable efficient, convenient, and rapid nucleic acid detection and pathogen diagnosis, which is of great importance for human life and health and technological development. In this article, we introduce the structure and function of Argonaute proteins and discuss the latest advances in their use in nucleic acid detection.

Improved Combinatorial Assembly and Barcode Sequencing for Gene-Sized DNA Constructs.

Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.

Therapeutic-oligonucleotides activated by nucleases (TOUCAN): A nanocarrier system for the specific delivery of clinical nucleoside analogues.

Nucleoside analogues have been in clinical use since 1960s and they are still used as the first therapeutic option for several cancers and viral infections, due to their high therapeutic efficacy. However, their wide clinical acceptance has been limited due to their high toxicity and severe side effects to patients. Herein, we report on a nanocarrier system that delivers nucleosides analogues in a target-specific manner, making nucleoside-based therapeutics safer and with the possibility to be used in other human conditions. This system, named, Therapeutic OligonUCleotides Activated by Nucleases" (TOUCAN) combines: i) the recognition power of oligonucleotides as substrates, ii) the use of nucleases as enzymatic biomarkers and iii) the clinical efficacy of nucleoside analogues, in a single approach. As a proof-of-concept, we report on a TOUCAN that is activated by a specific nuclease produced by bacteria and releases a therapeutic nucleoside, floxuridine. We demonstrate, for the first time, that, by incorporating a therapeutic nucleoside analogue into oligonucleotide probes, we can specifically inhibit bacterial growth in cultures. In this study, Staphylococcus aureus was selected as the targeted bacteria and the TOUCAN strategy successfully inhibited its growth with minimal inhibitory concentration (MIC) values ranging from 0.62 to 40 mg/L across all tested strains. Moreover, our results indicate that the intravenous administration of TOUCANs at a dose of 20 mg/kg over a 24-h period is a highly effective method for treating bacterial infections in a mouse model of pyomyositis. Importantly, no signs of toxicity were observed in our in vitro and in vivo studies. This work can significantly impact the current management of bacterial infections, laying the grounds for the development of a different class of antibiotics. Furthermore, it can provide a safer delivery platform for clinical nucleoside therapeutics in any human conditions, such as cancer and viral infection, where specific nuclease activity has been reported.

Genome Editing and Improvement of Abiotic Stress Tolerance in Crop Plants.

Genome editing aims to revolutionise plant breeding and could assist in safeguarding the global food supply. The inclusion of a 12-40 bp recognition site makes mega nucleases the first tools utilized for genome editing and first generation gene-editing tools. Zinc finger nucleases (ZFNs) are the second gene-editing technique, and because they create double-stranded breaks, they are more dependable and effective. ZFNs were the original designed nuclease-based approach of genome editing. The Cys2-His2 zinc finger domain's discovery made this technique possible. Clustered regularly interspaced short palindromic repeats (CRISPR) are utilized to improve genetics, boost biomass production, increase nutrient usage efficiency, and develop disease resistance. Plant genomes can be effectively modified using genome-editing technologies to enhance characteristics without introducing foreign DNA into the genome. Next-generation plant breeding will soon be defined by these exact breeding methods. There is abroad promise that genome-edited crops will be essential in the years to come for improving the sustainability and climate-change resilience of food systems. This method also has great potential for enhancing crops' resistance to various abiotic stressors. In this review paper, we summarize the most recent findings about the mechanism of abiotic stress response in crop plants and the use of the CRISPR/Cas mediated gene-editing systems to improve tolerance to stresses including drought, salinity, cold, heat, and heavy metals.