The poultry industry faces multifaceted challenges, including escalating demand for poultry products, climate change impacting feed availability, emergence of novel avian pathogens, and antimicrobial resistance. Traditional disease control measures are costly and not always effective, prompting the need for complementary methods. Gene editing (GE, also called genome editing) technologies, particularly CRISPR/Cas9, offer promising solutions. This article summarizes recent advancements in utilizing CRISPR/Cas GE to enhance infectious disease control in poultry. It begins with an overview of modern GE techniques, highlighting CRISPR/Cas9's advantages over other methods. The potential applications of CRISPR/Cas in poultry infectious disease prevention and control are explored, including the engineering of innovative vaccines, the generation of disease-resilient birds, and in vivo pathogen targeting. Additionally, insights are provided regarding regulatory frameworks and future perspectives in this rapidly evolving field.

Gene therapy development, re-engineering, and application to patients hold promise to revolutionize medicine, including therapies for disorders of the brain. Advances in delivery modalities, expression regulation, and improving safety profiles are of critical importance. Additionally, each inherited disorder has its own unique characteristics as to regions and cell types impacted and the temporal dynamics of that impact that are essential for the design of therapeutic design strategies. Here, we review the current state of the art in gene therapies for inherited brain disorders, summarizing key considerations for vector delivery, gene addition, gene silencing, gene editing, and epigenetic editing. We provide examples from animal models, human cell lines, and, where possible, clinical trials. This review also highlights the various tools available to researchers for basic research questions and discusses our views on the current limitations in the field.

Gene editing technologies, particularly CRISPR/Cas9, have emerged as transformative tools in genetic modification, significantly advancing the use of porcine embryos in biomedical and agricultural research. This review comprehensively examines the various methodologies for gene editing and delivery methods, such as somatic cell nuclear transfer (SCNT), microinjection, electroporation, and lipofection. This review, focuses on the advantages or limitations of using different biological sources (in vivo- vs. in vitro oocytes/embryos). Male germ cell manipulation using sperm-mediated gene transfer (SMGT) and testis-mediated gene transfer (TMGT) represent innovative approaches for producing genetically modified animals. Although these technologies have revolutionized the genetic engineering field, all these strategies face challenges, including high rates of off-target events and mosaicism. This review emphasizes the need to refine these methods, with a focus on reducing mosaicism and improving editing accuracy. Further advancements are essential to unlocking the full potential of gene editing for both agricultural applications and biomedical innovations.

The 1975 Asilomar conference contributed to the misperception that recombinant DNA (rDNA) technology is inherently risky to human health and the environment. It thus paved the way toward process-based regulation of genetically modified organisms (GMOs), such as in the EU. Initially, this regulatory approach obstructed technological uses of rDNA related to human health. However, regulators gradually softened the rules applicable to laboratories or industrial facilities. This encouraged R&D and production of pharmaceuticals derived from GMOs. Nevertheless, administering pharmaceuticals containing GMOs to patients may still be confronted with burdensome process-based GMO law on the deliberate release of GMOs into the environment. On the other hand, pharmaceutical law may need to be updated regarding, for example, novel gene therapies or xenotransplantation.

Genome editing techniques have potential to revolutionize the field of life sciences. Several limitations associated with traditional gene editing techniques have been resolved with the development of prime editors that precisely edit the DNA without double-strand breaks (DSBs). To further improve the efficiency, several modified versions of prime editing (PE) system have been introduced. Bi-directional PE (Bi-PE), for example, uses two PE guide RNAs enabling broad and improved editing efficiency. It has the potential to alter, delete, integrate, and replace larger genome sequences and edit multiple bases at the same time. This review aims to discuss the typical gene editing methods that offer DSB-mediated repair mechanisms, followed by the latest advances in genome editing technologies with non-DSB-mediated repair. The review specifically focuses on Bi-PE being an efficient tool to edit the human genome. In addition, the review discusses the applications, limitations, and future perspectives of Bi-PE for gene editing.

SNARE-dependent mast cell (MC) exocytosis causes the release of a wide variety of mediators with important physiological/pathological consequences. Unlike synaptic transmission in the brain, which relies primarily on one set of exocytic SNAREs (i.e., Syntaxin1, SNAP-25, and VAMP2), MCs produce a multitude of exocytic SNAREs that can form a minimum of 8 distinct sets of fusogenic trans-SNARE complexes. Here we describe the genetic approaches we have developed to dissect the specific roles of these SNAREs in RBL-2H3 cells, a widely utilized model for studying MC signaling and exocytosis.

Sub-Saharan Africa's agricultural sector faces a multifaceted challenge due to climate change consisting of high temperatures, changing precipitation trends, alongside intensified pest and disease outbreaks. Conventional plant breeding methods have historically contributed to yield gains in Africa, and the intensifying demand for food security outpaces these improvements due to a confluence of factors, including rising urbanization, improved living standards, and population growth. To address escalating food demands amidst urbanization, rising living standards, and population growth, a paradigm shift toward more sustainable and innovative crop improvement strategies is imperative. Genome editing technologies offer a promising avenue for achieving sustained yield increases while bolstering resilience against escalating biotic and abiotic stresses associated with climate change. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) is unique due to its ubiquity, efficacy, alongside precision, making it a pivotal tool for Sub-Saharan African crop improvement. This review highlights the challenges and explores the prospect of gene editing to secure the region's future foods.

Inflammation-related disorders, such as autoimmune diseases and cancer, impose a significant global health burden. Zinc finger proteins (ZFs) are ubiquitous metalloproteins which regulate inflammation and many biological signaling pathways related to growth, development, and immune function. Numerous ZFs are involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway, associating them with inflammation-related diseases that feature chronically elevated pro-inflammatory cytokines. This review highlights the predominance of ZFs in NFκB-related signaling and summarizes the breadth of functions that these proteins perform. The cysteine-specific post-translational modification (PTM) of persulfidation is also discussed in the context of these cysteine-rich ZFs, including what is known from the few available reports on the functional implications of ZF persulfidation. Persulfidation, mediated by endogenously produced hydrogen sulfide (H2S), has a recently established role in signaling inflammation. This work will summarize the known connections between ZFs and persulfidation and has the potential to inform on the development of related therapies.

Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the Pcsk9 gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.

DNA-PKcs is a crucial protein target involved in DNA repair and response pathways, with its abnormal activity closely associated with the occurrence and progression of various cancers. In this study, we employed a deep learning-based screening and molecular dynamics (MD) simulation-based pipeline, identifying eight candidates for DNA-PKcs targets. Subsequent experiments revealed the effective inhibition of DNA-PKcs-mediated cell proliferation by three small molecules (5025-0002, M769-1095, and V008-1080). These molecules exhibited anticancer activity with IC50 (inhibitory concentration at 50%) values of 152.6 μM, 30.71 μM, and 74.84 μM, respectively. Notably, V008-1080 enhanced homology-directed repair (HDR) mediated by CRISPR/Cas9 while inhibiting non-homologous end joining (NHEJ) efficiency. Further investigations into the structure-activity relationships unveiled the binding sites and critical interactions between these small molecules and DNA-PKcs. This is the first application of DeepBindGCN_RG in a real drug screening task, and the successful discovery of a novel DNA-PKcs inhibitor demonstrates its efficiency as a core component in the screening pipeline. Moreover, this study provides important insights for exploring novel anticancer therapeutics and advancing the development of gene editing techniques by targeting DNA-PKcs.

Liver cancer, which is well-known to us as one of human most prevalent malignancies across the globe, poses a significant risk to live condition and life safety of individuals in every region of the planet. It has been shown that immune checkpoint treatment may enhance survival benefits and make a significant contribution to patient prognosis, which makes it a promising and popular therapeutic option for treating liver cancer at the current time. However, there are only a very few numbers of patients who can benefit from the treatment and there also exist adverse events such as toxic effects and so on, which is still required further research and discussion. Fortunately, the clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) provides a potential strategy for immunotherapy and immune checkpoint therapy of liver cancer. In this review, we focus on elucidating the fundamentals of the recently developed CRISPR/Cas9 technology as well as the present-day landscape of immune checkpoint treatment which pertains to liver cancer. What's more, we aim to explore the molecular mechanism of immune checkpoint treatment in liver cancer based on CRISPR/Cas9 technology. At last, its encouraging and powerful potential in the future application of the clinic is discussed, along with the issues that already exist and the difficulties that must be overcome. To sum up, our ultimate goal is to create a fresh knowledge that we can utilize this new CRISPR/Cas9 technology for the current popular immune checkpoint therapy to overcome the treatment issues of liver cancer.

Gene editing technology has emerged as a powerful tool in all aspects of health research and continues to advance our understanding of critical and essential elements in disease pathophysiology. The clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology has been used with precision to generate gene knockouts, alter genes, and identify genes that cause disease. The full spectrum of allergic/atopic diseases, in part because of shared pathophysiology, is ripe for studies with this technology. In this way, novel culprit genes are being identified and allow for manipulation of triggering allergens to reduce allergenicity and disease. Notwithstanding current limitations on precision and potential off-target effects, newer approaches are rapidly being introduced to more fully understand specific gene functions as well as the consequences of genetic manipulation. In this review, we examine the impact of editing technologies of novel genes relevant to peanut allergy and asthma as well as how gene modification of common allergens may lead to the deletion of allergenic proteins.

Our ability to edit genomes lags behind our capacity to sequence them, but the growing understanding of CRISPR biology and its application to genome, epigenome and transcriptome engineering is narrowing this gap. In this Review, we discuss recent developments of various CRISPR-based systems that can transiently or permanently modify the genome and the transcriptome. The discovery of further CRISPR enzymes and systems through functional metagenomics has meaningfully broadened the applicability of CRISPR-based editing. Engineered Cas variants offer diverse capabilities such as base editing, prime editing, gene insertion and gene regulation, thereby providing a panoply of tools for the scientific community. We highlight the strengths and weaknesses of current CRISPR tools, considering their efficiency, precision, specificity, reliance on cellular DNA repair mechanisms and their applications in both fundamental biology and therapeutics. Finally, we discuss ongoing clinical trials that illustrate the potential impact of CRISPR systems on human health.

Genome editing involves precise modification of specific nucleotides in the genome using nucleases like CRISPR/Cas, ZFN, or TALEN, leading to increased efficiency of homologous recombination (HR) for gene editing, and it can result in gene disruption events via non-homologous end joining (NHEJ) or homology-driven repair (HDR). Genome editing, particularly CRISPR-Cas9, revolutionizes vaccine development by enabling precise modifications of pathogen genomes, leading to enhanced vaccine efficacy and safety. It allows for tailored antigen optimization, improved vector design, and deeper insights into host genes' impact on vaccine responses, ultimately enhancing vaccine development and manufacturing processes. This review highlights different types of genome editing methods, their associated risks, approaches to overcome the shortcomings, and the diverse roles of genome editing.

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.

In recent years, a growing number of clinical trials have been initiated to evaluate gene therapy approaches for the treatment of patients with transfusion-dependent β-thalassemia and sickle cell disease (SCD). Therapeutic modalities being assessed in these trials utilize different molecular techniques, including lentiviral vectors to add functional copies of the gene encoding the hemoglobin β subunit in defective cells and CRISPR-Cas9, transcription activator-like effector protein nuclease, and zinc finger nuclease gene editing strategies to either directly address the underlying genetic cause of disease or induce fetal hemoglobin production by gene disruption. Here, we review the mechanisms of action of these various gene addition and gene editing approaches and describe the status of clinical trials designed to evaluate the potentially for these approaches to provide one-time functional cures to patients with transfusion-dependent β-thalassemia and SCD.

The CRISPR/Cas9 system is a simpler and more versatile method compared to other engineered nucleases such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), and since its discovery, the efficiency of CRISPR-based genome editing has increased to the point that multiple and different types of edits can be made simultaneously. These advances in gene editing have revolutionized biotechnology by enabling precise genome editing with greater simplicity and efficacy than ever before. This tool has been successfully applied to a wide range of animal species, including cattle, pigs, dogs, and other small animals. Engineered nucleases cut the genome at specific target positions, triggering the cell's mechanisms to repair the damage and introduce a mutation to a specific genomic site. This review discusses novel genome-based CRISPR/Cas9 editing tools, methods developed to improve efficiency and specificity, the use of gene-editing on animal models and translational medicine, and the main challenges and limitations of CRISPR-based gene-editing approaches.

Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates. Using a traffic light reporter in mouse embryonic stem cells and a fluorescence conversion reporter in human-induced pluripotent stem cells, we confirm that a transient cold shock leads to an increase in the rate of HDR, with a corresponding decrease in the rate of NHEJ repair. We then investigated whether a similar cold shock could lead to an increase in the rate of HDR in the mouse embryo. By analyzing the efficiency of gene editing using single nucleotide polymorphism changes and loxP insertion at three different genetic loci, we found that a transient reduction in temperature after zygote electroporation of CRISPR-Cas9 ribonucleoprotein with a single-stranded oligodeoxynucleotide repair template did indeed increase knockin efficiency, without affecting embryonic development. The efficiency of gene editing with and without the cold shock was first assessed by genotyping blastocysts. As a proof of concept, we then confirmed that the modified embryo culture conditions were compatible with live births by targeting the coat color gene tyrosinase and observing the repair of the albino mutation. Taken together, our data suggest that a transient cold shock could offer a simple and robust way to improve knockin outcomes in both stem cells and zygotes.

The neuroscience and neurobiology of gene editing to enhance learning and memory is of paramount interest to the scientific community. The advancements of CRISPR system have created avenues to treat neurological disorders by means of versatile modalities varying from expression to suppression of genes and proteins. Neurodegenerative disorders have also been attributed to non-canonical DNA secondary structures by affecting neuron activity through controlling gene expression, nucleosome shape, transcription, translation, replication, and recombination. Changing DNA regulatory elements which could contribute to the fate and function of neurons are thoroughly discussed in this review. This study presents the ability of CRISPR system to boost learning power and memory, treat or cure genetically-based neurological disorders, and alleviate psychiatric diseases by altering the activity and the irritability of the neurons at the synaptic cleft through DNA manipulation, and also, epigenetic modifications using Cas9. We explore and examine how each different OMIC techniques can come useful when altering DNA sequences. Such insight into the underlying relationship between OMICs and cellular behaviors leads us to better neurological and psychiatric therapeutics by intelligently designing and utilizing the CRISPR/Cas9 technology.

The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route.

We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods.

Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9-4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.

Precision genome editing is a rapidly evolving field in gene therapy, allowing for the precise modification of genetic material. The CRISPR and Cas systems, particularly the CRISPRCas9 system, have revolutionized genetic research and therapeutic development by enabling precise changes like single-nucleotide substitutions, insertions, and deletions. This technology has the potential to correct disease-causing mutations at their source, allowing for the treatment of various genetic diseases. Programmable nucleases like CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs) can be used to restore normal gene function, paving the way for novel therapeutic interventions. However, challenges, such as off-target effects, unintended modifications, and ethical concerns surrounding germline editing, require careful consideration and mitigation strategies. Researchers are exploring innovative solutions, such as enhanced nucleases, refined delivery methods, and improved bioinformatics tools for predicting and minimizing off-target effects. The prospects of precision genome editing in gene therapy are promising, with continued research and innovation expected to refine existing techniques and uncover new therapeutic applications.

Anti-nutrients are substances either found naturally or are of synthetic origin, which leads to the inactivation of nutrients and limits their utilization in metabolic processes. Phytic acid is classified as an anti-nutrient, as it has a strong binding affinity with most minerals like Fe, Zn, Mg, Ca, Mn, and Cd and impairs their proper metabolism. Removing anti-nutrients from cereal grains may enable the bioavailability of both macro- and micronutrients which is the desired goal of genetic engineering tools for the betterment of agronomic traits. Several strategies have been adopted to minimize phytic acid content in plants. Pursuing the molecular strategies, there are several studies, which result in the decrement of the total phytic acid content in grains of major as well as minor crops. Biosynthesis of phytic acid mainly takes place in the seed comprising lipid-dependent and lipid-independent pathways, involving various enzymes. Furthermore, some studies show that interruption of these enzymes may involve the pleiotropic effect. However, using modern biotechnological approaches, undesirable agronomic traits can be removed. This review presents an overview of different genes encoding the various enzymes involved in the biosynthetic pathway of phytic acid which is being targeted for its reduction. It also, highlights and enumerates the variety of potential applications of genome editing tools such as TALEN, ZFN, and CRISPR/Cas9 to knock out the desired genes, and RNAi for their silencing.

Alzheimer's disease (AD) is a chronic illness marked by increasing cognitive decline and nervous system deterioration. At this time, there is no known medication that will stop the course of Alzheimer's disease; instead, most symptoms are treated. Clinical trial failure rates for new drugs remain high, highlighting the urgent need for improved AD modeling for improving understanding of the underlying pathophysiology of disease and improving drug development. The development of induced pluripotent stem cells (iPSCs) has made it possible to model neurological diseases like AD, giving access to an infinite number of patient-derived cells capable of differentiating neuronal fates. This advance will accelerate Alzheimer's disease research and provide an opportunity to create more accurate patient-specific models of Alzheimer's disease to support pathophysiological research, drug development, and the potential application of stem cell-based therapeutics. This review article provides a complete summary of research done to date on the potential use of iPSCs from AD patients for disease modeling, drug discovery, and cell-based therapeutics. Current technological developments in AD research including 3D modeling, genome editing, gene therapy for AD, and research on familial (FAD) and sporadic (SAD) forms of the disease are discussed. Finally, we outline the issues that need to be elucidated and future directions for iPSC modeling in AD.

CRISPR RNA-guided endonucleases have enabled precise editing of DNA. However, options for editing RNA remain limited. Here, we combine sequence-specific RNA cleavage by CRISPR ribonucleases with programmable RNA repair to make precise deletions and insertions in RNA. This work establishes a recombinant RNA technology with immediate applications for the facile engineering of RNA viruses.

Dangers confronting forest ecosystems are many and the strength of these biological systems is deteriorating, thus substantially affecting tree physiology, phenology, and growth. The establishment of genetically engineered trees into degraded woodlands, which would be adaptive to changing climate, could help in subsiding ecological threats and bring new prospects. This should not be resisted due to the apprehension of transgene dispersal in forests. Consequently, it is important to have a deep insight into the genetic structure and phenotypic limits of the reproductive capability of tree stands/population(s) to endure tolerance and survival. Importantly, for a better understanding of genes and their functional mechanisms, gene editing (GeEd) technology is an excellent molecular tool to unravel adaptation progressions. Therefore, GeEd could be harnessed for resolving the allelic interactions for the creation of gene diversity, and transgene dispersal may be alleviated among the population or species in different bioclimatic zones around the globe. This review highlights the potential of the CRISPR/Cas tools in genomic, transcriptomic, and epigenomic-based assorted and programmable alterations of genes in trees that might be able to fix the trait-specific gene function. Also, we have discussed the application of diverse forms of GeEd to genetically improve several traits, such as wood density, phytochemical constituents, biotic and abiotic stress tolerance, and photosynthetic efficiency in trees. We believe that the technology encourages fundamental research in the forestry sector besides addressing key aspects, which might fasten tree breeding and germplasm improvement programs worldwide.

The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9-mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV-infected human hepatoma cell line HepG2.2.15 with HBV-specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene-editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off-target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA/Cas9 RNP-based gene-editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.

The clinical outcome for children and adolescents with homozygous familial hypercholesterolaemia (HoFH) can be devastating, and treatment options are limited in the presence of a null variant. In HoFH, atherosclerotic risk accumulates from birth. Gene therapy is an appealing treatment option as restoration of low-density lipoprotein receptor (LDLR) gene function could provide a cure for HoFH. A clinical trial using a recombinant adeno-associated vector (rAAV) to deliver LDLR DNA to adult patients with HoFH was recently completed; results have not yet been reported. However, this treatment strategy may face challenges when translating to the paediatric population. The paediatric liver undergoes substantial growth which is significant as rAAV vector DNA persists primarily as episomes (extra-chromosomal DNA) and are not replicated during cell division. Therefore, rAAV-based gene addition treatment administered in childhood would likely only have a transient effect. With over 2,000 unique variants in LDLR, a goal of genomic editing-based therapy development would be to treat most (if not all) mutations with a single set of reagents. For a robust, durable effect, LDLR must be repaired in the genome of hepatocytes, which could be achieved using genomic editing technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and a DNA repair strategy such as homology-independent targeted integration. This review discusses this issue in the context of the paediatric patient group with severe compound heterozygous or homozygous null variants which are associated with aggressive early-onset atherosclerosis and myocardial infarction, together with the important pre-clinical studies that use genomic editing strategies to treat HoFH in place of apheresis and liver transplantation.

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.

CRISPR RNA-guided endonucleases have enabled precise editing of DNA. However, options for editing RNA remain limited. Here, we combine sequence-specific RNA cleavage by CRISPR ribonucleases with programmable RNA repair to make precise deletions and insertions in RNA. This work establishes a new recombinant RNA technology with immediate applications for the facile engineering of RNA viruses.

Programmable CRISPR RNA-guided ribonucleases enable recombinant RNA technology.

CRISPR technologies have progressed by leaps and bounds over the past decade, not only having a transformative effect on biomedical research but also yielding new therapies that are poised to enter the clinic. In this review, I give an overview of (i) the various CRISPR DNA-editing technologies, including standard nuclease gene editing, base editing, prime editing, and epigenome editing, (ii) their impact on cardiovascular basic science research, including animal models, human pluripotent stem cell models, and functional screens, and (iii) emerging therapeutic applications for patients with cardiovascular diseases, focusing on the examples of hypercholesterolaemia, transthyretin amyloidosis, and Duchenne muscular dystrophy.

The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.

Genetically modified mouse models have shaped our understanding of biological systems in both physiological and pathological conditions. For decades, mouse genome engineering has relied on transgenesis and spontaneous gene replacement in embryonic stem (ES) cells. While these technologies provided a wealth of knowledge, they remain imprecise and expensive to use. Recent advances in genome editing technologies such as the development of targetable nucleases, the improvement of delivery systems, and the simplification of targeting strategies now allow for the rapid, precise manipulation of the mouse genome. In this review article, we discuss novel methods and targeting strategies for the generation of mouse models for the study of bone and skeletal muscle biology.

Sickle cell disease (SCD) results from a single base pair change in the sixth codon of the β-globin chain of hemoglobin, which promotes aggregation of deoxyhemoglobin, increasing rigidity of red blood cells and causing vaso-occlusive and hemolytic complications. Allogeneic transplant of hematopoietic stem cells (HSCs) can eliminate SCD manifestations but is limited by absence of well-matched donors and immune complications. Gene therapy with transplantation of autologous HSCs that are gene-modified may provide similar benefits without the immune complications. Much progress has been made, and patients are realizing significant clinical improvements in multiple trials using different approaches with lentiviral vector-mediated gene addition to inhibit hemoglobin aggregation. Gene editing approaches are under development to provide additional therapeutic opportunities. Gene therapy for SCD has advanced from an attractive concept to clinical reality.

Our ability to modify DNA molecules and to introduce them into mammalian cells or embryos almost appears in parallel, starting from the 1970s of the last century. Genetic engineering techniques rapidly developed between 1970 and 1980. In contrast, robust procedures to microinject or introduce DNA constructs into individuals did not take off until 1980 and evolved during the following two decades. For some years, it was only possible to add transgenes, de novo, of different formats, including artificial chromosomes, in a variety of vertebrate species or to introduce specific mutations essentially in mice, thanks to the gene-targeting methods by homologous recombination approaches using mouse embryonic stem (ES) cells. Eventually, genome-editing tools brought the possibility to add or inactivate DNA sequences, at specific sites, at will, irrespective of the animal species involved. Together with a variety of additional techniques, this chapter will summarize the milestones in the transgenesis and genome engineering fields from the 1970s to date.

CRSPR/Cas9-mediated base editing introduces point mutations in cellular DNA by exploiting target-specific single guide RNA (sgRNA) along with a genetically modified Cas9. Existing plasmid vectors for sgRNA expression in base editing contain either a fluorescent marker or an antibiotic resistance cassette but not both, preventing simultaneous monitoring and enrichment of transfected host cells. In this study, we introduced a fluorescent marker into pGL3-U6-sgRNA-PGK-puromycin, a popular sgRNA expression vector available at Addgene. Specifically, the cDNAs of mRFP and a T2A linker were inserted in between the hPGK promoter and the puromycin resistance gene (PuroR). After correct insertion was verified by DNA sequencing, this new plasmid, pGL3-U6-sgRNA-PGK-mRFP-T2A-PuroR, was utilized to generate a stop codon in the second exon of the Munc13-1 gene in RBL-2H3 cells. Both the mRFP fluorescent marker and the puromycin resistance marker functioned accordingly in the process. This new sgRNA vector therefore represents a useful addition to the CRISPR tool kit.

Parkinson's disease (PD) is primarily idiopathic and a highly heterogenous neurodegenerative disease with patients experiencing a wide array of motor and non-motor symptoms. A major challenge for understanding susceptibility to PD is to determine the genetic and environmental factors that influence the mechanisms underlying the variations in disease-associated traits. The pathological hallmark of PD is the degeneration of dopaminergic neurons in the substantia nigra pars compacta region of the brain and post-mortem Lewy pathology, which leads to the loss of projecting axons innervating the striatum and to impaired motor and cognitive functions. While the cause of PD is still largely unknown, genome-wide association studies provide evidence that numerous polymorphic variants in various genes contribute to sporadic PD, and 10 to 15% of all cases are linked to some form of hereditary mutations, either autosomal dominant or recessive. Among the most common mutations observed in PD patients are in the genes LRRK2, SNCA, GBA1, PINK1, PRKN, and PARK7/DJ-1. In this review, we cover these PD-related mutations, the use of induced pluripotent stem cells as a disease in a dish model, and genetic animal models to better understand the diversity in the pathogenesis and long-term outcomes seen in PD patients.

Common wheat is a major source of nutrition around the globe, but unlike maize and rice hybrids, no breakthrough has been made to enhance wheat yield since Green Revolution. With the availability of reference genome sequence of wheat and advancement of allied genomics technologies, understanding of genes involved in grain yield components and disease resistance/susceptibility has opened new avenues for crop improvement. Wheat has a huge hexaploidy genome of approximately 17 GB with 85% repetition, and it is a daunting task to induce any mutation across three homeologues that can be helpful for the enhancement of agronomic traits. The CRISPR-Cas9 system provides a promising platform for genome editing in a site-specific manner. In wheat, CRISPR-Cas9 is being used in the improvement of yield, grain quality, biofortification, resistance against diseases, and tolerance against abiotic factors. The promising outcomes of the CRISPR-based multiplexing approach circumvent the constraint of targeting merely one gene at a time. Deployment of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) 9 endonuclease (CRISPR-Cas9) and Cas9 variant systems such as cytidine base editing, adenosine base editing, and prime editing in wheat has been used to induce point mutations more precisely. Scientists have acquired major events such as induction of male sterility, fertility restoration, and alteration of seed dormancy through Cas9 in wheat that can facilitate breeding programs for elite variety development. Furthermore, a recent discovery in tissue culturing enables scientists to significantly enhance regeneration efficiency in wheat by transforming the GRF4-GIF1 cassette. Rapid generation advancement by speed breeding technology provides the opportunity for the generation advancement of the desired plants to segregate out unwanted transgenes and allows rapid integration of gene-edited wheat into the breeding pipeline. The combination of these novel technologies addresses some of the most important limiting factors for sustainable and climate-smart wheat that should lead to the second "Green Revolution" for global food security.

Plant gene editing has become an important molecular tool to revolutionize modern breeding of crops. Over the past years, remarkable advancement has been made in developing robust and efficient editing methods for plants. Despite a variety of available genome editing methods, the discovery of most recent system of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) has been one of the biggest advancement in this path, with being the most efficient approach for genome manipulation. Until recently, genetic manipulations were confined to methods, like Agrobacterium-mediated transformations, zinc-finger nucleases, and TAL effector nucleases. However this technology supersedes all other methods for genetic modification. This RNA-guided CRISPR-Cas system is being rapidly developed with enhanced functionalities for better use and greater possibilities in biological research. In this review, we discuss and sum up the application of this simple yet powerful tool of CRISPR-Cas system for crop improvement with recent advancement in this technology.

The CRISPR-Cas genome editing system is an intrinsic property of a bacteria-based immune system. This employs a guide RNA to detect and cleave the PAM-associated target DNA or RNA in subsequent infections, by the invasion of a similar bacteriophage. The discovery of Cas systems has paved the way to overcome the limitations of existing genome editing tools. In this review, we focus on Cas proteins that are available for gene modifications among which Cas9, Cas12a, and Cas13 have been widely used in the areas of medicine, research, and diagnostics. Since CRISPR has been already proven for its potential research applications, the next milestone for CRISPR will be proving its efficacy and safety. In this connection, we systematically review recent advances in exploring multiple variants of Cas proteins and their modifications for therapeutic applications.

Domestication spanning over thousands of years led to the evolution of crops that are being cultivated in recent times. Later, selective breeding methods were practiced by human to produce improved cultivars/germplasm. Classical breeding was further transformed into molecular- and genomics-assisted breeding strategies, however, these approaches are labor-intensive and time-consuming. The advent of omics technologies has facilitated the identification of genes and genetic determinants that regulate particular traits allowing the direct manipulation of target genes and genomic regions to achieve desirable phenotype. Recently, genome editing technologies such as meganucleases (MN), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-Associated protein 9 (Cas9) have gained popularity for precise editing of genes to develop crop varieties with superior agronomic, physiological, climate-resilient, and nutritional traits. Owing to the efficiency and precision, genome editing approaches have been widely used to design the crops that can survive the challenges posed by changing climate, and also cater the food and nutritional requirements for ever-growing population. Here, we briefly review different genome editing technologies deployed for crop improvement, and the fundamental differences between GE technology and transgene-based approach. We also summarize the recent advances in genome editing and how this radical expansion can complement the previously established technologies along with breeding for creating designer crops.

Choanoflagellates, the closest living relatives of animals, have the potential to reveal the genetic and cell biological foundations of complex multicellular development in animals. Here we describe the history of research on the choanoflagellate Salpingoeca rosetta. From its original isolation in 2000 to the establishment of CRISPR-mediated genome editing in 2020, S. rosetta provides an instructive case study in the establishment of a new model organism.

Genetically modified (GM) rabbits are outstanding animal models for studying human genetic and acquired diseases. As such, GM rabbits that express human genes have been extensively used as models of cardiovascular disease. Rabbits are genetically modified via prokaryotic microinjection. Through this process, genes are randomly integrated into the rabbit genome. Moreover, gene targeting in embryonic stem (ES) cells is a powerful tool for understanding gene function. However, rabbits lack stable ES cell lines. Therefore, ES-dependent gene targeting is not possible in rabbits. Nevertheless, the RNA interference technique is rapidly becoming a useful experimental tool that enables researchers to knock down specific gene expression, which leads to the genetic modification of rabbits. Recently, with the emergence of new genetic technology, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated protein 9 (CRISPR/Cas9), major breakthroughs have been made in rabbit gene targeting. Using these novel genetic techniques, researchers have successfully modified knockout (KO) rabbit models. In this paper, we aimed to review the recent advances in GM technology in rabbits and highlight their application as models for cardiovascular medicine.