Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has shown a high incidence rate in rice fields in recent years. Rice resistance breeding is considered as the most effective method for achieving economical and sustainable management of BLB disease. The essential basis for resistance breeding is rooted in the exploration of rice resistance genes and the clarification of the molecular mechanisms that underlie Xoo resistance. In our previous research, we showed that Xanthomonas outer protein XopZ and rice oxysterol-binding related protein ORP1C collaboratively regulate the compatible interaction between Xoo strain PXO99 and Nipponbare rice, but the deeper regulatory mechanisms remain unknown. In this study, we successfully constructed ORP1C overexpression rice using the plant binary expression vector pCAMBIA1301. Through a series of virulence and effector translocation detections in Xoo-rice interactions, we revealed that overexpression of the ORP1C gene largely increases rice resistance to multiple Xoo strains from different countries and regions. Mechanistically, ORP1C plays a Xoo resistant role through negatively regulating transcription activator-like effectors (TALEs) translocation, ORP1C has become a potential candidate gene resource for disease-resistant breeding in rice. Further studies also indicated that XopZ and ORP1C collaboratively regulate the compatible interaction of PXO99-Nipponbare by modulating TALEs translocation.

Patient-derived induced pluripotent stem cells (iPSCs) are a useful pathological model for debilitating diseases caused by mitochondrial DNA (mtDNA) mutations. We established iPSCs derived from mitochondrial disease patients, heteroplasmic for the m.3243A>G mutation. The proportion of a selected mtDNA can be reduced by delivering a programmable nuclease into the mitochondria, and we developed various mtDNA-targeted Platinum TALENs (mpTALENs) to modify m.3243A>G-iPSC heteroplasmy levels in either wild-type or mutant direction. For TALEN optimization, the use of non-conventional repeat-variable di-residues (ncRVD)-LK/WK or NM-enhanced cleavage activity and specificity, and the replacement of conventional with obligate heterodimeric FokI nuclease domains increased target specificity and protected mtDNA from copy number depletion. In vitro, depending on whether wild-type or mutant mtDNA was targeted, we could obtain m.3243A>G-iPSCs with a higher or lower mutation load, while the cells retained their ability to differentiate into three germ layers. These results demonstrate that our mpTALEN optimization created a useful tool for altering heteroplasmy levels in m.3243A>G-iPSCs, improving the potential for studying mutation pathology. The enhanced efficiency also holds promise for using m.3243G(MUT)-mpTALEN as a therapeutic strategy for treating patients suffering from m.3243A>G mitochondrial diseases.

With the global aging trend, neurodegenerative diseases (NDs) have emerged as a significant public health concern in the 21st century, imposing substantial economic burdens on families and society. NDs are characterized by cognitive and motor decline, resulting from a combination of genetic and environmental factors. Currently, there is no cure for NDs. Gene and RNA editing therapies offer new possibilities for addressing NDs. Gene editing involves modifying mutant genes associated with NDs, while RNA editing can directly modify RNA molecules to regulate the protein translation process, potentially influencing the expression of genes related to NDs. In this review, we examined the historical evolution, mechanisms of action, applications in NDs, advantages and disadvantages, as well as ethical and safety considerations of gene and RNA editing. While gene and RNA editing technologies hold promise for treating NDs, further research and development are needed to address safety, efficacy, and treatment timing issues, ultimately offering improved treatment options for ND patients. Our review provides valuable insights for future gene and RNA editing applications in ND treatment.

Rice, a major global food staple, is threatened by viral infections that hinder its growth and yield. We have recently shown that the virulence protein P3 of rice grassy stunt virus promotes pathogenesis by inducing proteasome-controlled degradation of the rice RNA polymerase IV (RNA Pol IV) protein NRPD1a controlled by the P3-interacting E3 ubiquitin ligase P3IP1. However, the underlying mechanisms remain elusive. In this study, we show that P3 acts as a virus-encoded transcription activator-like effector to upregulate transcription of somatic embryogenesis receptor kinase 4 (SERK4) by directly binding to its promoter. SERK4 phosphorylates P3IP1 and enhances RNA Pol IVa (NRPD1a) degradation following P3IP1-controlled ubiquitination, leading to attenuated antiviral defense in rice. Thus, our study finds a critical viral virulence strategy by encoding a transcription factor-like protein that activates a host kinase to promote proteasome-controlled degradation of NRPD1a, thereby disarming RNA-directed DNA methylation (RdDM) antiviral defense.

The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.

To address the increasing demand for high-quality pork protein, it is essential to implement strategies that enhance diets and produce pigs with excellent production traits. Selective breeding and crossbreeding are the primary methods used for genetic improvement in modern agriculture. However, these methods face challenges due to long breeding cycles and the necessity for beneficial genetic variation associated with high-quality traits within the population. This limitation restricts the transfer of desirable alleles across different genera and species. This article systematically reviews past and current research advancements in porcine molecular breeding. It discusses the screening of clustered regularly interspaced short palindromic repeats (CRISPR) to identify resistance loci in swine and the challenges and future applications of genetically modified pigs.

The emergence of transgenic and gene editing technologies has prompted researchers to apply these methods to pig breeding. These advancements allow for alterations in the pig genome through various techniques, ranging from random integration into the genome to site-specific insertion and from target gene knockout (KO) to precise base and prime editing. As a result, numerous desirable traits, such as disease resistance, high meat yield, improved feed efficiency, reduced fat deposition, and lower environmental waste, can be achieved easily and effectively by genetic modification. These traits can serve as valuable resources to enhance swine breeding programmes.

In the era of genome editing, molecular breeding of pigs is critical to the future of agriculture. Long-term and multidomain analyses of genetically modified pigs by researchers, related policy development by regulatory agencies, and public awareness and acceptance of their safety are the keys to realizing the transition of genetically modified products from the laboratory to the market.

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.

Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multiscale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution and the visualization of macromolecular complexes in situ, especially if these molecules are expressed with appropriately engineered and easily detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our system comprises a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion. As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express six fluorescently tagged proteins and simultaneously visualize five different subcellular compartments. Our protocol leads from the in silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.

Rice CC-BED NLR Xa1 recognizes TAL effectors through the interaction between ERF101 and TAL effectors. The rice Xa1 gene encodes a nucleotide-binding leucine-rich repeat receptor with an N-terminal coiled coil-zinc finger BED (CC-BED) domain. Xa1 recognizes the transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo) in the nucleus, triggering a number of immune responses, including hypersensitive cell death. We previously discovered that the rice transcription factor ERF101 directly interacts with Xa1, and functions as a positive regulator of Xa1-dependent immunity. However, the involvement of ERF101 in Xa1-induced immunity remains unclear. We herein demonstrated that the expression of the CC-BED domain in rice protoplasts inhibited Xa1-induced cell death. However, the CC-BEDC165A,C168A domain which has mutations of cysteine residues conserved in the zinc-finger motifs of BED domains and is essential for forming tetrahedral coordination geometry, failed to inhibit cell death or interact with ERF101. Therefore, Xa1-induced cell death appears to depend on the interaction between the BED domain and ERF101. In addition, we generated transgenic plants overexpressing N-terminal or C-terminal FLAG-tagged ERF101. FLAG-ERF101 transgenic plants exhibited reduced levels of Xa1-mediated immunity against Xoo, even though the overexpression of ERF101-FLAG or non-tagged ERF101 enhanced immunity. This result was consistent with the CC-BED domain interacting with C-terminal tagged ERF101, but not N-terminal tagged ERF101, whereas N-terminal and C-terminal tagged ERF101 both interacted with TAL effectors. Therefore, the interaction between the BED domain and ERF101 appears to be essential for the recognition of TAL effectors by Xa1.

The rapid increase in global population poses a significant challenge to food security, compounded by the adverse effects of climate change, which limit crop productivity through both biotic and abiotic stressors. Despite decades of progress in plant breeding and genetic engineering, the development of new crop varieties with desirable agronomic traits remains a time-consuming process. Traditional breeding methods often fall short of addressing the urgent need for improved crop varieties. Genome editing technologies, which enable precise modifications at specific genomic loci, have emerged as powerful tools for enhancing crop traits. These technologies, including RNA interference, Meganucleases, ZFNs, TALENs, and CRISPR/Cas systems, allow for the targeted insertion, deletion, or alteration of DNA fragments, facilitating improvements in traits such as herbicide and insect resistance, nutritional quality, and stress tolerance. Among these, CRISPR/Cas9 stands out for its simplicity, efficiency, and ability to reduce off-target effects, making it a valuable tool in both agricultural biotechnology and plant functional genomics. This review examines the functional mechanisms and applications of various genome editing technologies for crop improvement, highlighting their advantages and limitations. It also explores the ethical considerations associated with genome editing in agriculture and discusses the potential of these technologies to contribute to sustainable food production in the face of growing global challenges.

Base editors are precise editing tools that employ deaminases to modify target DNA bases. The DYW-family of cytosine deaminases is structurally and phylogenetically distinct and might be harnessed for genome editing tools. We report a novel CRISPR/Cas9-cytosine base editor using SsdA, a DYW-like deaminase and bacterial toxin. A G103S mutation in SsdA enhances C-to-T editing efficiency while reducing its toxicity. Truncations result in an extraordinarily small enzyme. The SsdA-base editor efficiently converts C-to-T in rice and barley protoplasts and induces mutations in rice plants and mammalian cells. The engineered SsdA is a highly efficient genome editing tool.

Traditional pig breeding has improved production traits but faces limitations in genetic diversity, disease resistance, and environmental adaptation. Gene editing technologies, such as CRISPR/Cas9, base editing, and prime editing, enable precise genetic modifications, overcoming these limitations and expanding applications to biomedical research. Here, we reviewed the advancements in gene editing technologies in pigs and explored pathways toward optimized swine genetics for a resilient and adaptive livestock industry. This review synthesizes recent research on gene editing tools applied to pigs, focusing on CRISPR/Cas9 and its derivatives. It examines their impact on critical swine production traits and their role as human disease models. Significant advancements have been made in targeting genes for disease resistance, such as those conferring immunity to porcine reproductive and respiratory syndrome viruses. Additionally, gene-edited pigs are increasingly used as models for human diseases, demonstrating the technology's broader applications. However, challenges such as off-target effects, ethical concerns, and varying regulatory frameworks remain. Gene editing holds substantial potential for sustainable and productive livestock production by enhancing key traits and supporting biomedical applications. Addressing technical and ethical challenges through integrated approaches will be essential to realize its full potential, ensuring a resilient, ethical, and productive livestock sector for future generations.

Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimize a bacterial CRISPR-hybrid system coupled with FACS, and identified high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allows effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.

Common bacterial blight (CBB) is a devastating seed-transmitted disease of common bean (Phaseolus vulgaris L.), caused by Xanthomonas phaseoli pv. phaseoli and Xanthomonas citri pv. fuscans. The genes responsible for CBB resistance are largely unknown. Moreover, the lack of a reproducible and universal transformation protocol limits the study of genetic traits in common bean. We produced X. phaseoli pv. phaseoli strains expressing artificially designed transcription-activator like effectors (dTALEs) to target 14 candidate genes for resistance to CBB based on previous transcriptomic data. In planta assays in a susceptible common bean genotype showed that induction of PvOFP7, PvAP2-ERF71, or PvExpansinA17 expression by dTALEs resulted in CBB symptom reduction. After PvOFP7 induction, in planta bacterial growth was reduced at early colonization stages, and RNA-seq analysis revealed up-regulation of cell wall formation and primary metabolism, together with major down-regulation of heat shock proteins. Our results demonstrated that PvOFP7 contributes to CBB resistance, and underlined the usefulness of dTALEs for functional validation of genes whose induction impacts Xanthomonas-plant interactions.

Brain disorders, especially neurodegenerative diseases, affect millions of people worldwide. There is no causal treatment available; therefore, there is an unmet clinical need for finding therapeutic options for these diseases. Epigenetic research has resulted in identification of various genomic loci with differential disease-specific epigenetic modifications, mainly DNA methylation. These biomarkers, although not yet translated into clinically approved options, offer therapeutic targets as epigenetic modifications are reversible. Indeed, clinical trials are designed to inhibit epigenetic writers, erasers, or readers using epigenetic drugs to interfere with epigenetic dysregulation in brain disorders. However, since such drugs elicit genome-wide effects and potentially cause toxicity, the recent developments in the field of epigenetic editing are gaining widespread attention. In this review, we provide examples of epigenetic biomarkers and epi-drugs, while describing efforts in the field of epigenetic editing, to eventually make a difference for the currently incurable brain disorders.

With the advent of gene editing technologies like CRISPR/Cas9, it has become possible to edit genomic regions of interest for research and therapeutic purposes. These technologies have also been adapted to alter gene expression without changing their DNA sequence, allowing epigenetic edits. While genetic editors make edits by cutting the genome at specified regions, epigenetic editors leverage the same targeting mechanism but act based on the epigenetic modifier fused to them, such as a methyltransferase. Here, we discuss two recently employed epigenetic editors (epi-editors) that silenced target genes involved in disease to mitigate their effects. Neumann et al. reported the construction of an epigenetic editor called CHARM that could methylate and silence the prion gene in mouse brains and subsequently switch itself off. Additionally, Capelluti et al. developed an epi-editor called EvoETR that knocked down Pcsk9 in the murine liver to reduce LDL levels. We aim to highlight the design principles underlying the design of these epi-editors to inform future editor designs.

Cotton (Gossypium hirsutum L.), a vital global cash crop, significantly impacts both the agricultural and industrial sectors, providing essential fiber for textiles and valuable byproducts such as cottonseed oil and animal feed. The cultivation of cotton supports millions of livelihoods worldwide, particularly in developing regions, making it a cornerstone of rural economies. Despite its importance, cotton production faces numerous challenges, including biotic stresses from pests and diseases, and abiotic stresses like drought, salinity, and extreme temperatures. These challenges necessitate innovative solutions to ensure sustainable production. Genome editing technologies, particularly CRISPR/Cas9, have revolutionized cotton breeding by enabling precise genetic modifications. These advancements hold promise for developing cotton varieties with enhanced resistance to pests, diseases, and environmental stresses. Early genome editing tools like ZFNs and TALENs paved the way for more precise modifications but were limited by complexity and cost. The introduction of CRISPR/Cas-based technology with its simplicity and efficiency, has dramatically transformed the field, making it the preferred tool for genome editing in crops. Improved version of the technology like CRISPR/Cas12a, CRISPR/Cas13, base and prime editing, developed from CRISPR/Cas systems, provide additional tools with distinct mechanisms, further expanding their potential applications in crop improvement. This comprehensive review explores the impact of genome editing on cotton breeding and production. It discusses the technical challenges, including off-target effects and delivery methods for genome editing components, and highlights ongoing research efforts to overcome these hurdles. The review underscores the potential of genome editing technologies to revolutionize cotton cultivation, enhancing yield, quality, and resilience, ultimately contributing to a sustainable future for the cotton industry.

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.

Recently a cytidine deaminase-based method for highly efficient C-to-T targeted base editing was developed and has been used with CRISPR-mediated systems. It is a powerful method for genome engineering, although it is prone to off-target effects and has a limited targeting scope. Transcription activator-like effector (TALE)-based tools which allow longer recognition sequences than do CRISPR/Cas9 systems, can also be used for targeted C-to-T base editing. Here, we describe a method that efficiently achieved targeted C-to-T substitutions in Arabidopsis nuclear genes using cytidine deaminase fused to a TALE DNA-binding domain. We used a single pair of TALEs with a novel TALE-repeat unit that can recognize all four DNA bases, especially to allow for variations in the third base of codons in homologous genes. This targeting strategy makes it possible to simultaneously base edit almost identical sites in multiple isoforms of a gene while suppressing off-target substitutions.

Genome editing technologies hold significant potential for targeted mutagenesis in crop development, aligning with evolving agricultural needs. Point mutations, or single nucleotide polymorphisms (SNPs), define key agronomic traits in various crop species and play a pivotal role. The implementation of single nucleotide variations through genome editing-based base editing offers substantial promise in expediting crop improvement by inducing advantageous trait variations. Among many genome editing techniques, base editing stands out as an advanced next-generation technology, evolved from the CRISPR/Cas9 system.Base editing, a recent advancement in genome editing, enables precise DNA modification without the risks associated with double-strand breaks. Base editors, designed as precise genome editing tools, enable the direct and irreversible conversion of specific target bases. Base editors consist of catalytically active CRISPR-Cas9 domains, including Cas9 variants, fused with domains like cytidine deaminase, adenine deaminase, or reverse transcriptase. These fusion proteins enable the introduction of specific point mutations in target genomic regions. Currently developed are cytidine base editors (CBEs), mutating C to T; adenine base editors (ABEs), changing A to G; and prime editors (PEs), enabling arbitrary base conversions, precise insertions, and deletions. In this review, the research, development, and progress of various base editing systems, along with their potential applications in crop improvement, were intended to be summarized. The limitations of this technology will also be discussed. Finally, an outlook on the future of base editors will be provided.

Epigenetic modifications are chemical groups in our DNA (and chromatin) that determine which genes are active and which are shut off. Importantly, they integrate environmental signals to direct cellular function. Upon chronic environmental exposures, the epigenetic signature of lung cells gets altered, triggering aberrant gene expression programs that can lead to the development of chronic lung diseases. In addition to driving disease, epigenetic marks can serve as attractive lung disease biomarkers, due to early onset, disease specificity, and stability, warranting the need for more epigenetic research in the lung field. Despite substantial progress in mapping epigenetic alterations (mostly DNA methylation) in chronic lung diseases, the molecular mechanisms leading to their establishment are largely unknown. This review is meant as a guide for clinicians and lung researchers interested in epigenetic regulation with a focus on DNA methylation. It provides a short introduction to the main epigenetic mechanisms (DNA methylation, histone modifications and non-coding RNA) and the machinery responsible for their establishment and removal. It presents examples of epigenetic dysregulation across a spectrum of chronic lung diseases and discusses the current state of epigenetic therapies. Finally, it introduces the concept of epigenetic editing, an exciting novel approach to dissecting the functional role of epigenetic modifications. The promise of this emerging technology for the functional study of epigenetic mechanisms in cells and its potential future use in the clinic is further discussed.

The pathogen Verticillium dahliae secreted effector V. dahliae Aspf2-like protein (VDAL) has been found to cause leaf wilting in cotton, but the ectopic expression of VDAL-encoding gene enhances the resistance to V. dahliae in cotton and Arabidopsis. The application of the VDAL protein powder with optimal dosage promotes the growth and yield in multiple crop species, such as rice and wheat. However, the promotive effects of VDAL on these aspects are sporadically reported in asexually propagated species, including potato, while the molecular regulatory network involved in the process remains unclear. In this study, we observed that VDAL promotes sprouting of the potato pre-basic seed (PBS) tubers and enhances the development of both above-ground and below-ground tissues. Strikingly, VDAL increases the tuber yield in both greenhouse and field trials by up to 18.97%. The time-course transcriptomic analysis and the endogenous phytohormone detection revealed that cytokinin may play an important role in response to VDAL-promoted growth. Interestingly, VDAL-treated PBS tubers show higher resistance to cold temperature (late-spring cold), a phenomenon that is diminished when the lovastatin, a cytokinin inhibitor is applied, indicating that the VDAL-promoted potato growth, particularly under low temperature, is associated with cytokinin.

Patients suffering from an inherited severe liver disorder require lifelong treatment to prevent premature death. Until recently, the only curative treatment option was liver transplantation, which requires lifelong immune suppression. Now, liver-directed gene therapy, which is a much less invasive procedure, has become a market-approved treatment for hemophilia A and B. This may pave the way for it to become the treatment of choice for many other recessive inherited liver disorders with loss-of-function mutations. Inherited liver disease with toxic-gain-of-function or intrinsic hepatocyte damage may require alternative applications, such as integrating vectors or genome editing technologies, that can provide permanent or specific modification of the genome. We present an overview of currently available gene therapy strategies, i.e., gene supplementation, gene editing, and gene repair investigated in preclinical and clinical studies to treat inherited severe liver disorders. The advantages and limitations of these gene therapy applications are discussed in relation to the underlying disease mechanism.

Genome-wide CRISPR library screening technology is a gene function research tool developed based on the CRISPR/Cas9 gene-editing system. The clustered regularly interspaced short palindromic repeats/CRISPR-associated genes (CRISPR/Cas) system, considered the third generation of gene editing after zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), is widely used for screening various viral host factors. CRISPR libraries are classified into three main categories based on the different functions of Cas9 enzymes: CRISPR knockout (CRISPR KO) library screening, CRISPR transcriptional activation (CRISPRa) library screening, and CRISPR transcriptional interference (CRISPRi) library screening. Recently, genome-wide CRISPR library screening technology has been used to identify host factors that interact with viruses at various stages, including adsorption, endocytosis, and replication. By specifically modulating the expression of these host factors, it becomes possible to cultivate disease-resistant varieties, establish disease models, and design and develop vaccines, among other applications. This review provides an overview of the development and technical processes of genome-wide CRISPR library screening, as well as its applications in identifying viral host factors in livestock and poultry.

Lepidopteran insects are a major threat to global agriculture, causing significant crop losses and economic damage. Traditional pest control methods are becoming less effective due to the rapid evolution of insecticide resistance. This study explores the current status and genomic characteristics of 1315 Lepidopteran records, alongside an overview of relevant research, utilizing advanced functional genomics techniques, including RNA-seq and CRISPR/Cas9 gene-editing technologies to uncover the molecular mechanisms underlying insecticide resistance. Our genomic analysis revealed significant variability in genome size, assembly quality, and chromosome number, which may influence species' biology and resistance mechanisms. We identified key resistance-associated genes and pathways, including detoxification and metabolic pathways, which help these insects evade chemical control. By employing CRISPR/Cas9 gene-editing techniques, we directly manipulated resistance-associated genes to confirm their roles in resistance, demonstrating their potential for targeted interventions in pest management. These findings emphasize the value of integrating genomic data into the development of effective and sustainable pest control strategies, reducing reliance on chemical insecticides and promoting environmentally friendly integrated pest management (IPM) approaches. Our study highlights the critical role of functional genomics in IPM and its potential to provide long-term solutions to the growing challenge of Lepidopteran resistance.

Recent times have witnessed remarkable progress in cancer immunotherapy, drastically changing the cancer treatment landscape. Among the various immunotherapeutic approaches, adoptive cell therapy (ACT), particularly chimeric antigen receptor (CAR) T cell therapy, has emerged as a promising strategy to tackle cancer. CAR-T cells are genetically engineered T cells with synthetic receptors capable of recognising and targeting tumour-specific or tumour-associated antigens. By leveraging the intrinsic cytotoxicity of T cells and enhancing their tumour-targeting specificity, CAR-T cell therapy holds immense potential in achieving long-term remission for cancer patients. However, challenges such as antigen escape and cytokine release syndrome underscore the need for the continued optimisation and refinement of CAR-T cell therapy. Here, we report on the challenges of CAR-T cell therapies and on the efforts focused on innovative CAR design, on diverse therapeutic strategies, and on future directions for this emerging and fast-growing field. The review highlights the significant advances and changes in CAR-T cell therapy, focusing on the design and function of CAR constructs, systematically categorising the different CARs based on their structures and concepts to guide researchers interested in ACT through an ever-changing and complex scenario. UNIVERSAL CARs, engineered to recognise multiple tumour antigens simultaneously, DUAL CARs, and SUPRA CARs are some of the most advanced instances. Non-molecular variant categories including CARs capable of secreting enzymes, such as catalase to reduce oxidative stress in situ, and heparanase to promote infiltration by degrading the extracellular matrix, are also explained. Additionally, we report on CARs influenced or activated by external stimuli like light, heat, oxygen, or nanomaterials. Those strategies and improved CAR constructs in combination with further genetic engineering through CRISPR/Cas9- and TALEN-based approaches for genome editing will pave the way for successful clinical applications that today are just starting to scratch the surface. The frontier lies in bringing those approaches into clinical assessment, aiming for more regulated, safer, and effective CAR-T therapies for cancer patients.

Chloroplasts are organelles that are derived from a photosynthetic bacterium and have their own genome. Genome editing is a recently developing technology that allows for specific modifications of target sequences. The first successful application of genome editing in chloroplasts was reported in 2021, and since then, this research field has been expanding. Although the chloroplast genome of several dicot species can be stably modified by a conventional method, which involves inserting foreign DNAs into the chloroplast genome via homologous recombination, genome editing offers several advantages over this method. In this review, we introduce genome editing methods targeting the chloroplast genome and describe their advantages and limitations. So far, CRISPR/Cas systems are inapplicable for editing the chloroplast genome because guide RNAs, unlike proteins, cannot be efficiently delivered into chloroplasts. Therefore, protein-based enzymes are used to edit the chloroplast genome. These enzymes contain a chloroplast-transit peptide, the DNA-binding domain of transcription activator-like effector nuclease (TALEN), or a catalytic domain that induces DNA modifications. To date, genome editing methods can cause DNA double-strand break or introduce C:G-to-T:A and A:T-to-G:C base edits at or near the target sequence. These methods are expected to contribute to basic research on the chloroplast genome in many species and to be fundamental methods of plant breeding utilizing the chloroplast genome.

Chromatin organization plays a key role in gene regulation throughout the cell cycle. Understanding the dynamics governing the accessibility of chromatin is crucial for insight into mechanisms of gene regulation, DNA replication, and cell division. Extensive research has been done to track chromatin dynamics to explain how cells function and how diseases develop, in the hope of this knowledge leading to future therapeutics utilizing proteins or drugs that modify the accessibility or expression of disease-related genes. Traditional methods for studying the movement of chromatin throughout the cell relied on cross-linking spatially adjacent sections or hybridizing fluorescent probes to chromosomal loci and then constructing dynamic models from the static data collected at different time points. While these traditional methods are fruitful in understanding fundamental aspects of chromatin organization, they are limited by their invasive sample preparation protocols and diffraction-limited microscope resolution. These limitations have been challenged by modern methods based on high- or super-resolution microscopy and specific labeling techniques derived from gene targeting tools. These modern methods are more sensitive and less invasive than traditional methods, therefore allowing researchers to track chromosomal organization, compactness, and even the distance or rate of chromatin domain movement in detail and real time. This review highlights a selection of recently developed methods of chromatin tracking and their applications in fixed and live cells.

DNA lies at the heart of the central dogma of life. Altering DNA can modify the flow of information in fundamental cellular processes such as transcription and translation. The ability to precisely manipulate DNA has led to remarkable advances in treating incurable human genetic ailments and has changed the landscape of biological research. Genome editors such as CRISPR-Cas nucleases and TALENs have become ubiquitous tools in basic and applied biological research and have been translated to the clinic to treat patients. The specificity and modularity of these genome editors have made it possible to efficiently engineer genomic DNA; however, underlying principles governing editing outcomes in eukaryotes are still being uncovered. Editing efficiency can vary from cell type to cell type for the same DNA target sequence, necessitating de novo design and validation efforts. Chromatin structure and epigenetic modifications have been shown to affect the activity of genome editors because of the role they play in hierarchical organization of the underlying DNA. Understanding the nuclear search mechanism of genome editors and their molecular interactions with higher order chromatin will lead to improved models for predicting precise genome editing outcomes. Insights from such studies will unlock the entire genome to be engineered for the creation of novel therapies to treat critical illnesses.

A considerable fraction of population in the world suffers from rare diseases. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its related Cas proteins offer a modern form of curative gene therapy for treating the rare diseases. Hereditary transthyretin amyloidosis, hereditary angioedema, duchenne muscular dystrophy and Rett syndrome are a few examples of such rare diseases. CRISPR/Cas9, for example, has been used in the treatment of β-thalassemia and sickle cell disease (Frangoul et al., 2021; Pavani et al., 2021) [1,2]. Neurological diseases such as Huntington's have also been focused in some studies involving CRISPR/Cas (Yang et al., 2017; Yan et al., 2023) [3,4]. Delivery of these biologicals via vector and non vector mediated methods depends on the type of target cells, characteristics of expression, time duration of expression, size of foreign genetic material etc. For instance, retroviruses find their applicability in case of ex vivo delivery in somatic cells due to their ability to integrate in the host genome. These have been successfully used in gene therapy involving X-SCID patients although, incidence of inappropriate activation has been reported. On the other hand, ex vivo gene therapy for β-thalassemia involved use of BB305 lentiviral vector for high level expression of CRISPR biological in HSCs. The efficacy and safety of these biologicals will decide their future application as efficient genome editing tools as they go forward in further stages of human clinical trials. This review focuses on CRISPR/Cas based therapies which are at various stages of clinical trials for treatment of rare diseases and the constraints and ethical issues associated with them.

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5' bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5'-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5' bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.

Genome editing technologies have become widely used research tools. To assess the rate of growth with respect to federal funding of gene editing projects, we analyzed publicly available data retrieved from the NIH RePORTER and Clinicaltrials.gov databases. We identified 6,111 awards between 1977 and 2023, the majority being extramural, investigator-driven R (noneducational) awards (66.7%). There was an average growth rate of 40% between 2008 and 2022, and the biggest increase in awards was observed between 2017 and 2018 (doubling from 140 to 280). Five administering institutes/centers accounted for more than 60% of awards with the highest number of awards from the National Cancer Institute (20.0%). The majority of clinical trials involving some type of genome editing (75%) started in or after 2020. This analysis illuminates the rapid and widespread growth of gene editing research across disciplines and the eventual launch of clinical trials using gene editing tools.

In photosynthetic cells, plants convert carbon dioxide to sugars that can be moved between cellular compartments by transporters before being subsequently metabolized to support plant growth and development. Most pathogens cannot synthesize sugars directly but have evolved mechanisms to obtain plant-derived sugars as C resource for successful infection and colonization. The availability of sugars to pathogens can determine resistance or susceptibility. Here, we summarize current progress on the roles of sugar transporters in plant-pathogen interactions. We highlight how transporters are manipulated antagonistically by both host and pathogens in competing for sugars. We examine the potential application of this target in resistance breeding and discuss opportunities and challenges for the future.

Tandem repeat proteins (TRPs) are widely distributed and bind to a wide variety of ligands. DNA-binding TRPs such as zinc finger (ZNF) and transcription activator-like effector (TALE) play important roles in biology and biotechnology. In this study, we first conducted an extensive analysis of TRPs in public databases, and found that the enormous diversity of TRPs is largely unexplored. We then focused our efforts on identifying novel TRPs possessing DNA-binding capabilities. We established a protein language model for DNA-binding protein prediction (PLM-DBPPred), and predicted a large number of DNA-binding TRPs. A subset was then selected for experimental screening, leading to the identification of 11 novel DNA-binding TRPs, with six showing sequence specificity. Notably, members of the STAR (Short TALE-like Repeat proteins) family can be programmed to target specific 9 bp DNA sequences with high affinity. Leveraging this property, we generated artificial transcription factors using reprogrammed STAR proteins and achieved targeted activation of endogenous gene sets. Furthermore, the members of novel families such as MOON (Marine Organism-Originated DNA binding protein) and pTERF (prokaryotic mTERF-like protein) exhibit unique features and distinct DNA-binding characteristics, revealing interesting biological clues. Our study expands the diversity of DNA-binding TRPs, and demonstrates that a systematic approach greatly enhances the discovery of new biological insights and tools.

Covering: up to the end of 2023Type I CRISPR-Cas systems are widely distributed, found in over 40% of bacteria and 80% of archaea. Among genome-sequenced actinomycetes (particularly Streptomyces spp.), 45.54% possess type I CRISPR-Cas systems. In comparison to widely used CRISPR systems like Cas9 or Cas12a, these endogenous CRISPR-Cas systems have significant advantages, including better compatibility, wide distribution, and ease of operation (since no exogenous Cas gene delivery is needed). Furthermore, type I CRISPR-Cas systems can simultaneously edit and regulate genes by adjusting the crRNA spacer length. Meanwhile, most actinomycetes are recalcitrant to genetic manipulation, hindering the discovery and engineering of natural products (NPs). The endogenous type I CRISPR-Cas systems in actinomycetes may offer a promising alternative to overcome these barriers. This review summarizes the challenges and recent advances in CRISPR-based genome engineering technologies for actinomycetes. It also presents and discusses how to establish and develop genome editing tools based on type I CRISPR-Cas systems in actinomycetes, with the aim of their future application in gene editing and the discovery of NPs in actinomycetes.

Patients with gain-of-function mutations of Dyrk1b have higher fasting blood glucose (FBG) levels. However, the role of Dyrk1b in glucose metabolism is not fully elucidated. Herein, we found that hepatic Dyrk1b overexpression in mice impaired systemic glucose tolerance and hepatic insulin signaling. Dyrk1b overexpression in vitro attenuated insulin signaling in a kinase activity-dependent manner, and its kinase activity was required for its effect on systemic glucose homeostasis and hepatic insulin signaling in vivo. Dyrk1b ablation improved systemic glucose tolerance and hepatic insulin signaling in mice. Quantitative proteomic analyses showed that Dyrk1b downregulated WW domain-binding protein 2 (Wbp2) protein abundance. Mechanistically, Dyrk1b enhanced Wbp2 ubiquitylation and proteasomal degradation. Restoration of hepatic Wbp2 partially rescued the impaired glucose homeostasis in Dyrk1b overexpression mice. In addition, Dyrk1b inhibition with AZ191 moderately improved systemic glucose homeostasis. Our study uncovers that hepatic Dyrk1b impairs systemic glucose homeostasis via its modulation of Wbp2 expression in a kinase activity-dependent manner.

A tool for precise, target-specific, efficient, and affordable genome editing is a dream for many researchers, from those who conduct basic research to those who use it for applied research. Since 2012, we have tool that almost fulfils such requirements; it is based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight key factors affecting its mutagenesis outcomes: (i) factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state, or Cas9 variant, and how long it remains in place after cleavage; and (ii) factors affecting the follow-up DNA repair mechanisms including mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts, and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.

Xanthomonas species infect many important crops and cause huge yield loss. These pathogens deliver transcription activator-like (TAL) effectors into the cytoplasm of plant cells. TAL effectors move to host nuclei, directly bind to the promoters of host susceptible genes, and activate their transcription. However, the molecular mechanisms by which TAL effectors induce host transcription remain unclear. We herein demonstrated that TAL effectors interacted with the SIMILAR TO RCD ONE (SRO) family proteins OsSRO1a and OsSRO1b in nuclei. A transactivation assay using rice protoplasts indicated that OsSRO1a and OsSRO1b enhanced the activation of the OsSWEET14 promoter by the TAL effector AvrXa7. The AvrXa7-mediated expression of OsSWEET14 was significantly reduced in ossro1a mutants. However, the overexpression of OsSRO1a increased disease resistance by up-regulating the expression of defense-related genes, such as WRKY62 and PBZ1. This was attributed to OsSRO1a and OsSRO1b also enhancing the transcriptional activity of WRKY45, a direct regulator of WRKY62 expression. Therefore, OsSRO1a and OsSRO1b appear to positively contribute to transcription mediated by bacterial TAL effectors and rice transcription factors.

N6-methyladenosine (6mA) plays a pivotal role in diverse biological processes, including cancer, bacterial toxin secretion, and bacterial drug resistance. However, to date there has not been a selective, sensitive, and simple method for quantitative detection of 6mA at single base resolution. Herein, we present a series piezoelectric quartz crystal (SPQC) sensor based on the specific recognition of transcription-activator-like effectors (TALEs) for locus-specific detection of 6mA. Detection sensitivity is enhanced through the use of a hybridization chain reaction (HCR) in conjunction with silver staining. The limit of detection (LOD) of the sensor was 0.63 pM and can distinguish single base mismatches. We demonstrate the applicability of the sensor platform by quantitating 6mA DNA at a specific site in biological matrix. The SPQC sensor presented herein offers a promising platform for in-depth study of cancer, bacterial toxin secretion, and bacterial drug resistance.

Mitochondria, mainly known as energy factories of eukaryotic cells, also exert several additional signaling and metabolic functions and are today recognized as major cellular biosynthetic and signaling hubs. Mitochondria possess their own genome (mitochondrial DNA-mtDNA), that encodes proteins essential for oxidative phosphorylation, and mutations in it are an important contributor to human disease. The mtDNA mutations often exist in heteroplasmic conditions, with both healthy and mutant versions of the mtDNA residing in patients' cells and the level of mutant mtDNA may vary between different tissues and organs and affect the clinical outcome of the disease. Thus, shifting the ratio between healthy and mutant mtDNA in patients' cells provides an intriguing therapeutic option for mtDNA diseases. In this review we describe current strategies for modulating mitochondrial heteroplasmy levels with engineered endonucleases including mitochondrially targeted TALENs and Zinc finger nucleases (ZFNs) and discuss their therapeutic potential. These gene therapy tools could in the future provide therapeutic help both for patients with mitochondrial disease as well as in preventing the transfer of pathogenic mtDNA mutations from a mother to her offspring.