Despite abundant expression of DNAJB6 gene in the heart, its roles in cardiac diseases remain underexplored. We aimed to investigate the function of its zebrafish (Danio rerio) ortholog, the dnajb6b gene, in cardiomyopathy and heart failure.

Both loss-of-function mutation and gain-of-function transgenic approaches were employed in zebrafish. High frequency echocardiography was performed to evaluate cardiac function indices in adult zebrafish. 4-phenylbutyric acid (4-PBA) was used to pharmacologically inhibit sarcoplasmic reticulum (SR) stress in zebrafish. Western blot was carried out to determine expression of DNAJB6 isoforms in human patients' heart tissues.

Global loss-of-function mutations affecting both the sarcomere-localized short (Dnajb6b[S]) and nucleus-localized long (Dnajb6b[L]) isoforms appeared phenotypically normal. In contrast, cardiomyocyte-specific overexpression of a truncated, sarcomere-localized Dnajb6b(L) isoform (Dnajb6b[∆L]) led to severe cardiomyopathy and heart failure phenotypes. Mechanistically, Dnajb6b responded to sarcoplasmic reticulum (SR) stress and activation of Dnajb6b(∆L) resulted in elevated SR stress, accumulation of ubiquitinated protein aggregation, and aberrant activation of autophagy. 4-PBA treatment partially rescued cardiac dysfunction and extended the lifespan of zebrafish with cardiomyocyte-specific activation of Dnajb6b(∆L). Finally, elevated expression of both DNAJB6(S) and DNAJB6(L) isoforms was detected in failing human hearts, supporting their clinical relevance.

Gain-of-function mutation in Dnajb6b(∆L) isoform causes cardiomyopathy and heart failure, likely mediated by elevated SR stress. This study enhances our understanding of Dnajb6's role in cardiac proteostasis and highlights its potential as a therapeutic target for the treatment of cardiomyopathy and heart failure.

Genome editing technologies have emerged as the keystone of biotechnological research, enabling precise gene modification. The field has evolved rapidly through revolutionary advancements, transitioning from early explorations to the breakthrough of the CRISPR-Cas system. The emergence of the CRISPR-Cas system represents a huge leap in genome editing, prompting the development of advanced tools such as base and prime editors, thereby enhancing precise genomic engineering capabilities. The rapid integration of AI across disciplines is now driving another transformative phase in genome editing, streamlining workflows and enhancing precision. The application prospects of genome editing technology are extensive, particularly in plant breeding, where it has already presented unparalleled opportunities for improving plant traits. Here, we review early genome editing technologies, including meganucleases, ZFNs, TALENs, and CRISPR-Cas systems. We also provide a detailed introduction to next-generation editing tools-such as base editors and prime editors-and their latest applications in plants. At the same time, we summarize and prospect the cutting-edge developments and future trends of genome editing technologies in combination with the rapidly rising AI technology, including optimizing editing systems, predicting the efficiency of editing sites and designing editing strategies. We are convinced that as these technologies progress and their utilization expands, they will provide pioneering solutions to global challenges, ushering in an era of health, prosperity, and sustainability.

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.

The RUNX1/AML1 transcription factor is one of the key regulators of definitive hematopoietic development in mice. However, its role in early human hematopoiesis remains poorly investigated. In this study, we integrated a tdTomato reporter cassette into the RUNX1 locus of human pluripotent stem cells (hPSCs) to monitor and block the expression of the gene during hPSC differentiation. This approach demonstrated that expression of RUNX1 starts early in mesodermal specification focusing later on hemogenic endothelium (HE) and nascent hematopoietic cells. Lack of RUNX1 halted the development of CD43+ and CD235-CD45+ hematopoietic cells, preventing the production of clonogenic hematopoietic progenitors including the multilineage ones. The abrogation of RUNX1 resulted in the failure of definitive lineages, specifically T and NK cells. Remarkably, we instead observed the accumulation of RUNX1-null HE cells at the stage of blood cell generation. Moreover, the loss of the gene biased the development toward the lineage of CD43-CD146+CD90+CD73+ mesenchymal cells. RNA-seq analysis of RUNX1-null cells revealed the downregulation of top-level hematopoietic transcription factor genes and the reciprocal upregulation of genes associated with non-hematopoietic cells of mesodermal origin. Forced expression of RUNX1c in differentiating RUNX1-null hPSCs effectively rescued the development of CD45+ myeloid cells and megakaryocytes. Our data demonstrate that RUNX1 is a top hematopoietic inducer that simultaneously controls the expansion of non-hematopoietic lineages.

The skin is the largest organ of the body and forms and serves as the barrier for preventing external material from accessing and damaging internal organs. As the outward interface to the environment, it is accessible for the application of therapeutic agents and cellular and gene therapy represent attractive and promising options to treat severe genetic conditions for which palliation has long been the main stay. However, because of its barrier function, transit across and to the subdermal compartment can be challenging. This commentary examines the current approaches of cell and gene therapies for genetic skin disorders. We write this from a local and systemic "outside and inside." perspective. Delivery from the outside encompasses topical, intradermal, and transdermal strategies for cell and vector delivery and ex vivo cell expansion and grafting. The inside approach details systemic delivery via infusion of cells or agents toward providing benefit to the skin. We use recessive dystrophic epidermolysis bullosa (RDEB) as a representative and paradigmatic disease to showcase these approaches as a means to highlight potential broader applicability to other conditions.

The zinc finger transcription factor PRDM14, part of the PR domain containing protein family, is critical for mammalian primordial germ cell (PGC) specification, epigenetic reprogramming and maintaining naïve pluripotency in stem cells. However, PRDM14's role in other species is not well understood. In chicken, PRDM14 is broadly expressed in the early embryo, before becoming restricted to the forming neural plate, migratory PGCs, and later, in the adult testes. To investigate the role of PRDM14 we generated two independent targeted chicken lines and bred homozygous knockout embryos. Strikingly, we found that gastrulation was disrupted in PRDM14-/- embryos, which lacked a definitive primitive streak. Transcriptomic and in situ hybridisation analyses revealed a broad loss of anterior primitive streak marker genes, coupled with downregulation of the multifunctional antagonists CHRD and CER1, and expansion of the NODAL expression domain. Further analysis of PRDM14-/- embryos revealed PGCs were still specified but significantly reduced in number, and PRDM14-/- PGCs could not be propagated in vitro. Knockdown studies in vitro confirmed that PRDM14 is essential for PGC survival and antagonises FGF-induced somatic differentiation, similar to PRDM14's role in mammalian stem cells. Taken together, our results show that in chicken, PRDM14 plays a multifunctional and essential role during embryonic development.

Tracking of Insertions and DEletions (TIDE) analysis, which computationally deconvolves capillary sequencing data derived from the DNA of bulk or clonal cell populations to estimate the efficiency of targeted mutagenesis by programmable nucleases, has played a significant role in the field of genome editing. However, the detection range covered by conventional TIDE analysis is limited. Range extension for deconvolution is required to detect larger deletions and insertions (indels) derived from genome editing in TIDE analysis. However, extending the deconvolution range introduces uncertainty into the deconvolution process. Moreover, the accuracy and sensitivity of TIDE analysis tools for large deletions (> 50 bp) remain poorly understood.

In this study, we introduced a new software called PtWAVE that can detect a wide range of indel sizes, up to 200 bp. PtWAVE also offers options for variable selection and fitting algorithms to prevent uncertainties in the model. We evaluated the performance of PtWAVE by using in vitro capillary sequencing data that mimicked DNA sequencing, including large deletions. Furthermore, we confirmed that PtWAVE can stably analyze trace sequencing data derived from actual genome-edited samples.

PtWAVE demonstrated superior accuracy and sensitivity compared to the existing TIDE analysis tools for DNA samples, including large deletions. PtWAVE can accelerate genome editing applications in organisms and cell types in which large deletions often occur when programmable nucleases are applied.

As an emerging and innovative technology, gene-editing technology has been widely applied in crop breeding, human disease treatment, animal model research, drug and vaccine development, and microbial engineering. We mainly introduce the development of gene-editing technology, the application of clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) in Ganoderma lucidum breeding, the current challenges and optimization strategies in the use of gene-editing technology in Ganoderma breeding, as well as the current status of gene-editing technology in Ganoderma breeding. Finally, the future research directions and innovative strategies that gene editing may explore in Ganoderma breeding are prospects given the existing background, future research directions, and innovative strategies that gene editing may explore in Ganoderma breeding prospects.

Guide RNA (gRNA) arrays can enable targeting multiple genomic loci simultaneously using CRISPR-Cas9. In this study, we present a streamlined and efficient method to rapidly construct gRNA arrays with up to 10 gRNA units in a single day. We demonstrate that gRNA arrays maintain robust functional activity across all positions, and can incorporate libraries of gRNAs, combining scalability and multiplexing. Our approach will streamline combinatorial perturbation research by enabling the economical and rapid construction, testing, and iteration of gRNA arrays.

Malignant transformation of gene modified haematopoietic stem cells caused anxiety following adverse events in early clinical trials using gamma-retroviral vectors (γRV) to correct haematopoietic stem cells (HSC) in monogenic immune disorders. Adoption of HIV-derived lentiviral vectors (LV) with SIN (self-inactivating) configurations greatly reduced risks and subsequently hundreds of patients have been dosed with HSC gene therapy for blood, immune and metabolic conditions. Nevertheless, as experience builds, it's now well recognised that vector integration can drive clonal expansions and these may carry long term safety risks. Documented cases of haematological malignancy after SIN-LV gene therapy have recently emerged, in particular where heterologous retroviral promoters were employed and there are concerns around certain insulator elements and other possible contributors to clonal expansions. Similarly, tens of thousands of subjects have now received engineered T cell products, and longstanding dogma that mature T cells cannot be transformed is being questioned, with reports of a small number of malignant transformation events and wider concerns around secondary malignancies in some groups of patients. We summarize current clinical information and revisit genotoxicity risks following ex-vivo gene modification of HSC and T cells.

Fruit crops are rich source of important vitamins, minerals, and dietary fibres. They are essential for global agriculture with respect to nutritional security. Globally, there is a rapid decline in the genetic base of fruit crops warranting breeding strategies to overcome the challenge. Applied mutagenesis has emerged as a viable approach for the focused enhancement of fruit crops utilizing precise genetic alterations to increase a variety of desirable characteristics. However, traditional mutagenesis using physical and chemical mutagens are majorly random in nature. Directed mutagenesis with advancements in genetic engineering and molecular technology allows precise manipulation of genes, which facilitates the efficient and precise knockout of target genes and the targeted insertion or modification of specific DNA sequences within the genome via homologous recombination (HR)-mediated gene replacement. This review presents an in-depth exploration of several directed mutagenesis techniques including CRISPR-Cas9, TILLING, TALEN, MutMap, and MutMap + emphasizing their transformative applications in fruit crops. It also discusses about space mutagenesis. These advanced techniques empower researchers to precisely introduce specific mutations into the genome, skilfully altering gene expression and reshaping protein function with remarkable precision. This review highlights successful examples of directed mutagenesis in a variety of fruit crops such as apples, grapes, citrus, and strawberries and elucidates the impact of directed mutagenesis on traits such as fruit size, colour, flavour, shelf-life, and resistance to diseases and environmental stresses.

The CRISPR/Cas system is a sizable family that is currently a popular and efficient gene editing tool. Cas12i3, as a member of the Type V-I family, has the characteristics of recognizing T-rich PAM sequences and being guided by shorter crRNA and has higher gene editing efficiency than Cas9 in rice. However, as a potential tool in accelerating the breeding process, the application of Cas12i3 in mammalian embryos has not yet been reported. Our study systematically evaluated the feasibility of applying CRISPR/Cas12i3 to gene editing in mouse embryos, with the core pluripotency regulator gene Nanog as the target. We successfully constructed a Nanog loss-of-function mouse embryo model using CRISPR/Cas12i3. At the targeted Nanog locus, its editing efficiency exceeded that of the Cas9 system under matched experimental conditions; no off-target phenomenon was detected. Moreover, the Cas12i3 system exhibited no side effect on mouse embryo development and proliferation of blastocyst cells. Finally, we obtained healthy chimeric gene-edited offspring by optimizing the concentration of the Cas12i3 mixture. These results confirm the feasibility and safety of CRISPR/Cas12i3 for gene editing in mammals, which provides a reliable tool for one-step generation of gene-edited animals for applications in biology, medical research, and large livestock breeding.

Chromatin organization is a relevant layer of control of gene expression during plant development. Chromatin states strictly depend on associated features such as DNA methylation, histone modifications and histone variants. Thus, epigenome editing has become of primary interest to alter gene expression without disrupting genomic sequences. Different tools have been developed to address this challenge, starting with modular Zinc Finger Proteins (ZFPs) and Transcription Activator Like Effectors (TALEs). However, the discovery of CRISPR/Cas9 system and the adaptability of technologies based on enzymatically dead Cas9 (dCas9) have paved the way towards a reliable and adaptable epigenome editing in a great variety of organisms. In this review, we will focus on the application of targeted epigenome editing technologies in plants, summarizing the most updated advances in this field. The promising results obtained by altering the expression state of targets involved in flowering time and abiotic stress resistance are crucial not only for elucidating the molecular interactions that underly chromatin dynamics, but also for future applications in breeding programs as an alternative route to genetic manipulation towards the achievement of higher quality crops particularly in terms of nutritional properties, yield and tolerance.

Forkhead box L2 (FOXL2) is a transcription factor from the forkhead box family primarily expressed in the pituitary, ovaries, and eyelids. Human mutations in FOXL2 cause blepharophimosis, ptosis, epicanthus and inversus syndrome (BPES), which can be associated with primary ovarian insufficiency, and is indirectly linked with differences of sex development (DSD). Animal studies have shown the crucial role that FOXL2 plays in the development, function, and maintenance of the ovary as well as in sex determination. However, the specific role of FOXL2 in early human somatic cell ovarian development is largely unknown.

In this study, we utilised CRISPR/Cas9 genome activation and a previously published in-house 14-day gonadal differentiation protocol to study the role of FOXL2.

Our results demonstrate that FOXL2 downregulates coelomic epithelial markers GATA4 and LHX9, female gonadal markers RSPO1 and WNT4, and male gonadal markers SOX9, NR0B1 and DHH. The differentially expressed genes were mostly associated with Kyoto encyclopaedia of genes and genomes (KEGG) pathways relating to cell adhesion molecules and gene ontology (GO) pathways relating to extracellular matrix and junction formation. Furthermore, a comparative analysis with existing single cell RNA sequencing data from human in vivo-derived samples elucidated that FOXL2 initiates the downregulation of coelomic epithelial genes GATA4, LHX9 and UPK3B at day 6. By day 8, the genes ARX and GATA2 are transiently upregulated by FOXL2 induction and then downregulated as the genes LGR5, TSPAN8, OSR1 and TAC1 become upregulated.

These findings suggest that FOXL2 facilitates the exit of differentiating cells from the coelomic epithelium and initially drives them towards a transitional identity before progressing into early supporting gonadal-like cells. The findings of this study significantly advance our understanding of normal gonadal development which can be used as a basis to elucidate pathological gonadal development underlying BPES.

The brain ventricle system, composed of the ventricular cavities and the cerebral spinal fluid within, performs critical functions including circulation of nutrients, removal of wastes, and cushioning of neural tissues. Development of the hindbrain ventricle requires a series of factors that coordinate its initial formation and subsequent inflation. Previous work has demonstrated that the transcriptional co-activator Taz (also known as WW domain-containing transcription regulator protein 1, Wwtr1), a component of Hippo signalling, is active at hindbrain rhombomere boundaries where it is regulated by mechanotransduction and promotes proliferation. Here, we demonstrate that Taz is also a critical regulator of hindbrain ventricle development. Zebrafish embryos that lack Taz protein fail to undergo initial midline separation of the hindbrain ventricle. Furthermore, the ventricle phenotype is a result of disorganized cytoskeletal F-actin and apicobasal polarity components. In addition, we have demonstrated that the hindbrain rhombomere boundaries are a location of active Wnt-Hippo crosstalk. Through our work, we propose a model where Taz protein is stabilized at rhombomere boundaries and promotes proper cell polarity necessary for formation of the brain ventricle.

Pathogenic gene dysregulation can be attributed to chromatin state change that pre-transcriptionally regulates expression. Recent breakthroughs elucidating the rules governing this DNA control layer, an epigenetic code, unlock a modality in precision medicine to target gene dysregulation across myriad diseases. Here we present a modular platform to design programmable mRNA therapeutics, Epigenomic Controllers (EC), that control gene expression through directed epigenetic change. By leveraging natural mechanisms, ECs tune expression levels of one or multiple genes with durable effect of weeks-to-months in female mice following a single dose. We design and characterize ECs to multiple target genes and identify an EC that effectively inhibits the cancer- and inflammatory-disorder-associated multi-gene cluster CXCL1-8. With precision targeting of NF-kB signaling and identification of homologous murine surrogates, ECs significantly reduce neutrophil migration in vivo during acute lung inflammation in female mice. A platform approach to EC design for epigenomic modulation expands treatment frontiers for diverse gene targets, including those considered "undruggable."

Genome editing technologies have revolutionized biomedical sciences and biotechnology. However, their delivery in vivo remains one of the major obstacles for clinical translation. Here, we introduce various emerging genome editing systems and review different delivery systems have been developed to realize the promise of in vivo gene editing therapies. In particular, we focus on virus-like particles (VLPs), an emerging delivery platform and provide in depth analysis on recent advancements to improve VLPs delivery potential and highlight opportunities for future improvements. To this end, we also provide detail workflows for engineered VLP (eVLP) selection, production, and purification, along with methods for characterization and validation.

In rice bacterial blight, Xanthomonas oryzae pv. oryzae deploys transcription activator-like effectors (TALEs) that upregulate host susceptibility genes. Thirty-four amino acid repeats in TALEs each specify a base in the DNA target, via a repeat-variable diresidue (RVD; positions 12 and 13). Some aberrant-length repeats can disengage to accommodate single base deletions. Clade III SWEET genes SWEET11, -13 and -14 are major susceptibility targets of different TALEs. xa13 is a SWEET11 allele lacking the TALE binding site and thus confers resistance to some strains. It has been deployed widely in India. We report that an xa13-breaking Indian isolate, IX-221, harbours one SWEET14- and two SWEET13-activating TALEs, with one or two disengageable repeats. One, Tal7, orthologous to PthXo2B of Philippines strain PXO61 but with minor, non-RVD sequence differences, like PthXo2B upregulates SWEET13 in a japonica variety and no clade III SWEET in an indica, yet unlike PthXo2B renders both varieties susceptible. A designer TALE with distinct, minor differences also failed to render the indica susceptible. The results suggest that Tal7 activates an alternative susceptibility gene and that non-RVD polymorphism can affect TALE targeting. Moreover, IX-221 provides evidence that the deployment of xa13 in India resulted in strains super-equipped with TALEs that break it.

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.

Bacterial transcription activator-like effectors (TALEs) promote pathogenicity by activating host susceptibility (S) genes. To understand the pathogenicity and host adaptation of Xanthomonas citri pv. malvacearum (Xcm), we assemble the genome and the TALE repertoire of three recent Xcm Texas isolates. A newly evolved TALE, Tal7b, activates GhSWEET14a and GhSWEET14b, different from GhSWEET10 targeted by a TALE in an early Xcm isolate. Activation of GhSWEET14a and GhSWEET14b results in water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identify a pectin lyase gene as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9 gene editing supports the function of GhSWEETs in cotton bacterial blight and the promise of disrupting the TALE-binding site in S genes for disease management. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes to promote pathogenicity.

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.

Lipoprotein(a), or Lp(a), is encoded by the LPA gene and is a causal genetic risk factor for cardiovascular disease. Individuals with high Lp(a) are at risk for cardiovascular morbidity and are refractory to standard lipid-lowering agents. Lp(a)-lowering therapies currently in clinical development require repetitive dosing, while a gene editing approach presents an opportunity for a single-dose treatment. In this study, mRNAs encoding transcription activator-like effector nucleases (TALENs) were designed to target human LPA for gene disruption and permanent Lp(a) reduction. TALEN mRNAs were screened in vitro and found to cause on-target gene editing and target protein reduction with minimal off-target editing. TALEN mRNAs were then encapsulated with LUNAR, a proprietary lipid nanoparticle (LNP), and administered to transgenic mice that expressed a human LPA transgene. A single dose of TALEN mRNA-LNPs reduced plasma Lp(a) levels in mice by over 80%, which was sustained for at least 5 weeks. Moreover, both standard and long-read next-generation sequencing confirmed the presence of gene-inactivating deletions at LPA transgene loci. Overall, this study serves as a proof-of-concept for using TALEN-mediated gene editing to disrupt LPA in vivo, paving the way for the development of a feasible gene editing therapy for patients with high Lp(a).

Available treatment for chronic hepatitis B virus (HBV) infection offers modest functional curative efficacy. The viral replicative intermediate comprising covalently closed circular DNA (cccDNA) is responsible for persistent chronic HBV infection. Hence, current efforts have focused on developing therapies that disable cccDNA. Employing gene editing tools has emerged as an attractive strategy, with the end goal of establishing permanently inactivated cccDNA. Although anti-HBV designer nucleases are effective in vivo, none has yet progressed to clinical trial. Lack of safe and efficient delivery systems remains the limiting factor. Several vectors may be used to deliver anti-HBV gene editor-encoding sequences, with viral vectors being at the forefront. Despite the challenges associated with packaging large gene editor-encoding sequences into viral vectors, advancement in the field is overcoming such limitations. Translation of viral vector-mediated gene editing against HBV to clinical application is within reach. This review discusses the prospects of delivering HBV targeted designer nucleases using viral vectors.

The different spatiotemporal distribution of progenitor and neurogenic capacities permits that brain regions engage asynchronously in neurogenesis. In the hindbrain, rhombomere progenitor cells contribute to neurons during the first neurogenic phase, whereas boundary cells participate later. To analyze what maintains boundary cells as non-neurogenic progenitors, we addressed the role of Her9, a zebrafish Hes1-related protein. her9 expression is temporarily sustained in boundary cells independently of Notch at early embryonic stages, while they are non-neurogenic progenitors. Complementary functional approaches show that Her9 inhibits the onset of Notch signaling and the neurogenic program, keeping boundary cells as progenitors. Multicolor clonal analysis combined with genetic perturbations reveal that Her9 expands boundary progenitors by promoting symmetric proliferative and preventing neurogenic cell divisions. Her9 also regulates the proliferation of boundary cells by inhibiting the cell cycle arrest gene cdkn1ca and interplaying with Cyclin D1. Moreover, her9 is enriched in hindbrain radial glial cells at late embryonic stages independently of Notch. Together these data demonstrate that Her9 maintains the stemness properties of hindbrain boundary progenitors and late radial glial cells, ensuring the different temporal distribution of neurogenic capacities within the hindbrain.

Grasses, including turf and forage, cover most of the earth's surface; predominantly important for land, water, livestock feed, soil, and water conservation, as well as carbon sequestration. Improved production and quality of grasses by modern molecular breeding is gaining more research attention. Recent advances in genome-editing technologies are helping to revolutionize plant breeding and also offering smart and efficient acceleration on grass improvement. Here, we reviewed all recent researches using (CRISPR)/CRISPR-associated protein (Cas)-mediated genome editing tools to enhance the growth and quality of forage and turf grasses. Furthermore, we highlighted emerging approaches aimed at advancing grass breeding program. We assessed the CRISPR-Cas effectiveness, discussed the challenges associated with its application, and explored future perspectives primarily focusing on turf and forage grasses. Despite the promising potential of genome editing in grasses, its current efficiency remains limited due to several bottlenecks, such as the absence of comprehensive reference genomes, the lack of efficient gene delivery tools, unavailability of suitable vector and delivery for grass species, high polyploidization, and multiple homoeoalleles, etc. Despite these challenges, the CRISPR-Cas system holds great potential to fully harness its benefits in grass breeding and genetics, aiming to improve and sustain the quantity and quality of turf and forage grasses.

Japanese cedar or sugi (Cryptomeria japonica D. Don) is among the most important plantation conifers in Japan, occupying 12% of the total land area in the country. We have successfully established a CRISPR/Cas9-based genome editing system in C. japonica. However, in practical use, we encountered problems of low efficiency when generating biallelic mutations, i.e., target gene knockouts. As part of our efforts to improve efficiency, we codon-optimized the Cas9 gene, evaluated by the genome editing efficiency of CjChl I, a gene encoding a chlorophyll biosynthesis enzyme. As a result, our codon-optimized SpCas9, named CjSpCas9, performed the highest genome editing efficiency of two targets (t4, t1+t2). Specifically, the biallelic disruption efficiency of the CjChl I with CjSpCas9 was 1.8-fold higher than that of the SpCas9 gene optimized for Arabidopsis thaliana (AtSpCas9) and 2.0-fold higher than that of the SpCas9 gene optimized for Orysa sativa (OsSpCas9) for t4, respectively. For t1+t2, the efficiency was 4.9-fold higher than that of AtSpCas9 and 1.4-fold higher than that of OsSpCas9, respectively. Our western blotting analysis proved that the Cas9 protein accumulation increased upon codon frequency optimization. We concluded that the observed efficiency improvement was due to the increased Cas9 protein quantity. The efficient genome editing system we report here would accelerate molecular breeding in conifers.

The increasing prevalence of heart disease poses a significant threat to human survival and safety. However, the current treatments available for heart disease are quite limited. Therefore, it is important to utilize suitable animal models that can accurately simulate the physiological characteristics of heart disease. This would help improve our understanding of this disease and aid in the development of new treatment methods and drugs. Zebrafish heart not only exhibits similarities to mammalian hearts, but they also share ~70% of homologous genes with humans. Utilizing zebrafish as an alternative to expensive and time-consuming mammalian models offers numerous advantages. Zebrafish models can be easily established and maintained, and compound screening and genetic methods allow for the development of various economical and easily controlled zebrafish and zebrafish embryonic heart disease models in a short period of time. Consequently, zebrafish have become a powerful tool for exploring the pathological mechanisms of heart disease and identifying new effective genes. In this review, we summarize recent studies on different zebrafish models of heart disease. We also describe the techniques and protocols used to develop zebrafish models of myocardial infarction, heart failure, and congenital heart disease, including surgical procedures, forward and reverse genetics, and drug and combination screening. This review aims to promote the utilization of zebrafish models in investigating diverse pathological mechanisms of heart disease, enhancing our knowledge and comprehension of heart disease, and offering novel insights and objectives for exploring the prevention and treatment of heart disease.

Silk fiber is generally considered an excellent biological material due to its good biocompatibility, morphological plasticity and biodegradability. Previously, the construction of silkworm silk gland bioreactors based on the piggyBac transposon has been optimized. However, the inserted exogenous genes have problems such as position uncertainty, and expression is not strictly controlled. Here, we applied transcription activator-like effector nuclease (TALEN)-mediated homology-directed repair (HDR) to precisely insert histidine-rich cuticular protein (CP) into silkworm Sericin1 (Ser1) gene. The Ser1-CP fusion protein was successfully secreted into cocoon shell. Subsequently, based on the metal coordination ability of the histidine imidazole group, we crosslinked cocoon with metal ions in vitro. In this strategy, the mechanical properties of the fused silk fibers with crosslinked Zn2+ improved, and the maximum breaking stress of the crosslinked Zn2+-fused silk fibers was 23.5 % greater than that of the wild-type fibers. Analysis of the secondary structure of the silk protein showed that the fused silk fibers crosslinked with Zn2+ had more β-sheet structures. This study pioneered a method of improving the mechanical properties of silk fibers by crosslinking metal ions with fused exogenous proteins and expanded the application value of silk gland bioreactors in the development of novel biomaterials.

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.

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.

"GABA dysfunction" is a major hypothesis for the biological basis of schizophrenia with indirect supporting evidence from human post-mortem brain and genetic studies. Patient-derived induced pluripotent stem cells (iPSCs) have emerged as a valuable platform for modeling psychiatric disorders, and previous modeling has revealed glutamatergic synapse deficits. Whether GABAergic synapse properties are affected in patient-derived human neurons and how this impacts neuronal network activity remain poorly understood. Here we optimized a protocol to differentiate iPSCs into highly enriched ganglionic eminence-like neural progenitors and GABAergic neurons. Using a collection of iPSCs derived from patients of psychiatric disorders carrying a Disrupted-in-Schizophrenia 1 ( DISC1 ) mutation and their unaffected family member, together with respective isogenic lines, we identified mutation-dependent deficits in GABAergic synapse formation and function, a phenotype similar to that of mutant glutamatergic neurons. However, mutant glutamatergic and GABAergic neurons contribute differentially to neuronal network excitability and synchrony deficits. Finally, we showed that GABAergic synaptic transmission is also defective in neurons derived from several idiopathic schizophrenia patient iPSCs. Transcriptome analysis further showed some shared gene expression dysregulation, which is more prominent in DISC1 mutant neurons. Together, our study supports a functional GABAergic synaptic deficit in major psychiatric disorders.

Gene editing technologies have revolutionized the field of livestock breeding, offering unprecedented opportunities to enhance animal welfare, productivity, and sustainability. This paper provides a comprehensive review of recent innovations and applications of gene editing in livestock, exploring the diverse applications of gene editing in livestock breeding, as well as the regulatory and ethical considerations, and the current challenges and prospects of the technology in the industry. Overall, this review underscores the transformative potential of gene editing in livestock breeding and its pivotal role in shaping the future of agriculture and biomedicine.

Conventional gene therapy involving supplementation only treats loss-of-function diseases and is limited by viral packaging sizes, precluding therapy of large genes. The discovery of CRISPR/Cas has led to a paradigm shift in the field of genetic therapy, with the promise of precise gene editing, thus broadening the range of diseases that can be treated. The initial uses of CRISPR/Cas have focused mainly on gene editing or silencing of abnormal variants via utilising Cas endonuclease to trigger the target cell endogenous non-homologous end joining. Subsequently, the technology has evolved to modify the Cas enzyme and even its guide RNA, leading to more efficient editing tools in the form of base and prime editing. Further advancements of this CRISPR/Cas technology itself have expanded its functional repertoire from targeted editing to programmable transactivation, shifting the therapeutic focus to precise endogenous gene activation or upregulation with the potential for epigenetic modifications. In vivo experiments using this platform have demonstrated the potential of CRISPR-activators (CRISPRa) to treat various loss-of-function diseases, as well as in regenerative medicine, highlighting their versatility to overcome limitations associated with conventional strategies. This review summarises the molecular mechanisms of CRISPRa platforms, the current applications of this technology in vivo, and discusses potential solutions to translational hurdles for this therapy, with a focus on ophthalmic diseases.

Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.

Arrhythmias are disturbances in the normal rhythm of the heart and account for significant cardiovascular morbidity and mortality worldwide. Historically, preclinical research has been anchored in animal models, though physiological differences between these models and humans have limited their clinical translation. The discovery of human induced pluripotent stem cells (iPSC) and subsequent differentiation into cardiomyocyte has led to the development of new in vitro models of arrhythmias with the hope of a new pathway for both exploration of pathogenic variants and novel therapeutic discovery.

The authors describe the latest two-dimensional in vitro models of arrhythmias, several examples of the use of these models in drug development, and the role of gene editing when modeling diseases. They conclude by discussing the use of three-dimensional models in the study of arrythmias and the integration of computational technologies and machine learning with experimental technologies.

Human iPSC-derived cardiomyocytes models have significant potential to augment disease modeling, drug discovery, and toxicity studies in preclinical development. While there is initial success with modeling arrhythmias, the field is still in its nascency and requires advances in maturation, cellular diversity, and readouts to emulate arrhythmias more accurately.

CRISPR-Cas9 technology has revolutionized plant genome editing, providing precise and efficient methods for genetic modification. This study focuses on the advancements and delivery of CRISPR-Cas9 in plant gene editing.

A comprehensive search in scientific databases, including PubMed, ScienceDirect, and Google Scholar, was conducted to gather information on CRISPR-Cas9 gene editing and its delivery in precise gene modification in plants.

The evolving landscape of CRISPR nucleases has led to the development of innovative technologies, enhancing plant research. However, successful editing is contingent on efficient delivery of genome engineering reagents. CRISPR-based gene editing in plants utilizes diverse delivery methods: Agrobacterium-mediated transformation for bacterial transfer, biolistic transformation for physical gene insertion, electroporation for direct gene entry, expression of developmental regulators for gene expression modulation, and tobacco rattle virus as a viral vector, each offering distinct advantages for precise and efficient genetic modification in plants.

CRISPR-Cas9 gene editing stands as a pivotal advancement in plant genetics, offering precise gene manipulation with applications in agriculture and biotechnology. The continuous refinement of reagent delivery tools reinforces CRISPR-Cas9's transformative role in plant genome editing, with significant implications for broader scientific applications.

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.

Silkworm was the first domesticated insect and has important economic value. It has also become an ideal model organism with applications in genetic and expression studies. In recent years, the use of transgenic strategies has made the silkworm silk gland an attractive bioreactor for the production of recombinant proteins, in particular, piggyBac-mediated transgenes. However, owing to differences in regulatory elements such as promoters, the expression levels of exogenous proteins have not reached expectations. Here, we used targeted gene editing to achieve site-specific integration of exogenous genes on genomic DNA and established the fibroin light chain (FibL) in-fusion expression system by TALEN-mediated homology-directed recombination. First, the histidine-rich cuticular protein (CP) was successfully site-directed inserted into the native FibL, and the FibL-CP fusion gene was correctly transcribed and expressed in the posterior silk gland under the control of the endogenous FibL promoter, with a protein expression level comparable with that of the native FibL protein. Moreover, we showed based on molecular docking that the fusion of FibL with cuticular protein may have a negative effect on disulfide bond formation between the C-terminal domain of fibroin heavy chain (FibH) and FibL-CP, resulting in abnormal spinning and cocoon in homozygotes, indicating a significant role of FibL in silk protein formation and secretion. Our results demonstrate the feasibility of using the FibL fusion system to express exogenous proteins in silkworm. We expect that this bioreactor system will be used to produce more proteins of interest, expanding the application value of the silk gland bioreactor.

Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases. Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but, unexpectedly, recent studies have revealed that the Tie2 locus has been lost in many vertebrate species, whereas the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells, where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in endothelial cell migration and proliferation, and in lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show that Angpt1/Tie1 signaling constitutes an essential signaling pathway for lymphatic development in zebrafish.

Plastids (including chloroplasts) and mitochondria are remnants of endosymbiotic bacteria, yet they maintain their own genomes, which encode vital components for photosynthesis and respiration, respectively. Organellar genomes have distinctive features, such as being present as multicopies, being mostly inherited maternally, having characteristic genomic structures and undergoing frequent homologous recombination. To date, it has proven to be challenging to modify these genomes. For example, while CRISPR/Cas9 is a widely used system for editing nuclear genes, it has not yet been successfully applied to organellar genomes. Recently, however, precise gene-editing technologies have been successfully applied to organellar genomes. Protein-based enzymes, especially transcription activator-like effector nucleases (TALENs) and artificial enzymes utilizing DNA-binding domains of TALENs (TALEs), have been successfully used to modify these genomes by harnessing organellar-targeting signals. This short review introduces and discusses the use of targeted nucleases and base editors in organellar genomes, their effects and their potential applications in plant science and breeding.

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs are often deployed as pairs, with each arm comprised 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 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 constraint, 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 αDdCBEs that contain modified TALE proteins engineered to recognize all 5' bases. Notably, 5'-T-noncompliant, canonical DdCBEs efficiently edited mtDNA at diverse loci. However, DdCBEs were frequently outperformed by αDdCBEs, which consistently displayed significant improvements in activity and specificity, regardless of the 5'-most bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with DddA tox and its derivatives DddA6, and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes at a single target site. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.

The unique virus-cell interaction in Epstein-Barr virus (EBV)-associated malignancies implies targeting the viral latent-lytic switch is a promising therapeutic strategy. However, the lack of specific and efficient therapeutic agents to induce lytic cycle in these cancers is a major challenge facing clinical implementation. We develop a synthetic transcriptional activator that specifically activates endogenous BZLF1 and efficiently induces lytic reactivation in EBV-positive cancer cells. A lipid nanoparticle encapsulating nucleoside-modified mRNA which encodes a BZLF1-specific transcriptional activator (mTZ3-LNP) is synthesized for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more efficiently activates EBV lytic gene expression in EBV-associated epithelial cancers. Here we show the potency and safety of treatment with mTZ3-LNP to suppress tumor growth in EBV-positive cancer models. The combination of mTZ3-LNP and ganciclovir yields highly selective cytotoxic effects of mRNA-based lytic induction therapy against EBV-positive tumor cells, indicating the potential of mRNA nanomedicine in the treatment of EBV-associated epithelial cancers.