Search Results (256)

Microalgae, with their unparalleled capabilities for sunlight-driven growth, CO₂ fixation, and synthesis of diverse high-value compounds, represent sustainable cell factories for a circular bioeconomy. However, industrial deployment has been hindered by biological constraints and the inadequacy of conventional genetic tools. The advent of CRISPR-Cas systems initially provided precise gene editing via targeted DNA cleavage. This review argues that the true transformative potential lies in moving decisively beyond cutting to harness CRISPR as a versatile synthetic biology “Swiss Army Knife”. We synthesize the rapid evolution of CRISPR-derived tools—including transcriptional modulators (CRISPRa/i), epigenome editors, base/prime editors, multiplexed systems, and biosensor-integrated logic gates—and their revolutionary applications in microalgal engineering. These tools enable tunable gene expression, stable epigenetic reprogramming, DSB-free nucleotide-level precision editing, coordinated rewiring of complex metabolic networks, and dynamic, autonomous control in response to environmental cues. We critically evaluate their deployment to enhance photosynthesis, boost lipid/biofuel production, engineer high-value compound pathways (carotenoids, PUFAs, proteins), improve stress resilience, and optimize carbon utilization. Persistent challenges—species-specific tool optimization, delivery efficiency, genetic stability, scalability, and biosafety—are analyzed, alongside emerging solutions and future directions integrating AI, automation, and multi-omics. The strategic integration of this CRISPR toolkit unlocks the potential to engineer robust, high-productivity microalgal cell factories, finally realizing their promise as sustainable platforms for next-generation biomanufacturing. Full article

The recent discovery of TIGR-Tas (Tandem Interspaced Guide RNA-Targeting Systems) marks a major advance in the field of genome editing, introducing a new class of compact, programmable DNA-targeting systems that function independently of traditional CRISPR-Cas pathways. TIGR-Tas effectors use a novel dual-spacer guide RNA (tigRNA) to recognize both strands of target DNA without requiring a protospacer adjacent motif (PAM). These Tas proteins introduce double-stranded DNA cuts with characteristic 8-nucleotide 3′ overhangs and are significantly smaller than Cas9, offering delivery advantages for in vivo editing. Structural analyses reveal homology to box C/D snoRNP proteins, suggesting a previously unrecognized evolutionary lineage of RNA-guided nucleases. This review positions TIGR-Tas at the forefront of a new wave of RNA-programmable genome-editing technologies. In parallel, I provide comparative insight into the diverse and increasingly modular CRISPR-Cas systems, including Cas9, Cas12, Cas13, and emerging effectors like Cas3, Cas10, CasΦ, and Cas14. While the CRISPR-Cas universe has revolutionized molecular biology, TIGR-Tas systems open a complementary and potentially more versatile path for programmable genome manipulation. I discuss mechanistic distinctions, evolutionary implications, and potential applications in human cells, synthetic biology, and therapeutic genome engineering. Full article

Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription–translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology. Full article

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering. Full article

RNA viruses pose significant threats to global health, causing diseases such as COVID-19, HIV/AIDS, influenza, and dengue. These viruses are characterized by high mutation rates, rapid evolution, and the ability to evade traditional antiviral therapies, making effective treatment and prevention particularly challenging. In recent years, CRISPR/Cas13 has emerged as a promising antiviral tool due to its ability to specifically target and degrade viral RNA. Unlike conventional antiviral strategies, Cas13 functions at the RNA level, providing a broad-spectrum and programmable approach to combating RNA viruses. Its flexibility allows for rapid adaptation of guide RNAs to counteract emerging viral variants, making it particularly suitable for highly diverse viruses such as SARS-CoV-2 and HIV. This review discusses up-to-date applications of Cas13 in targeting a wide range of RNA viruses, including SARS-CoV-2, HIV, dengue, influenza, and other RNA viruses, focusing on its therapeutic potential. Preclinical studies have demonstrated Cas13’s efficacy in degrading viral RNA and inhibiting replication, with applications spanning prophylactic interventions to post-infection treatments. However, challenges such as collateral cleavage, inefficient delivery, potential immunogenicity, and the development of an appropriate ethical framework must be addressed before clinical translation. Future research should focus on optimizing crRNA design, improving delivery systems, and conducting rigorous preclinical evaluations to enhance specificity, safety, and therapeutic efficacy. With continued advancements, Cas13 holds great promise as a revolutionary antiviral strategy, offering novel solutions to combat some of the world’s most persistent viral threats. Full article

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9)-mediated genome editing has emerged as a transformative tool in medicine, offering significant potential for cancer therapy because of its capacity to precisely target and alter the genetic modifications associated with the disease. However, a major challenge for its clinical translation is the safe and efficient in vivo delivery of CRISPR/Cas9 components to target cells. Nanotechnology is a promising solution to this problem. Nanocarriers, owing to their tunable physicochemical properties, can encapsulate and protect CRISPR/Cas9 components, enabling targeted delivery and enhanced cellular uptake. This review provides a comprehensive examination of the synergistic potential of CRISPR/Cas9 and nanotechnology in cancer therapy and explores their integrated therapeutic applications in gene editing and immunotherapy. A critical aspect of in vivo CRISPR/Cas9 application is to achieve effective localization at the tumor site while minimizing off-target effects. Nanocarriers can be engineered to overcome biological barriers, thereby augmenting tumor-specific delivery and facilitating intracellular uptake. Furthermore, their design allows for controlled release of the therapeutic payload, ensuring sustained efficacy and reduced systemic toxicity. The optimization of nanocarrier attributes, including size, shape, surface charge, and composition, is crucial for improving the cellular internalization, endosomal escape, and nuclear localization of CRISPR/Cas9. Moreover, surface functionalization with targeting ligands can enhance the specificity of cancer cells, leading to improved gene-editing accuracy. This review thoroughly discusses the challenges associated with in vivo CRISPR/Cas9 delivery and the innovative nanotechnological strategies employed to overcome them, highlighting their combined potential for advancing cancer treatment for clinical application. Full article

CRISPR technology, which is derived from the bacterial adaptive immune system, has transformed traditional genetic engineering techniques, made strain engineering significantly easier, and become a very versatile genome editing system that allows for precise, programmable modifications to a wide range of microbial genomes. The economies of fermentation-based manufacturing are changing because of its quick acceptance in both academic and industry labs. CRISPR processes have been used to modify industrially significant bacteria, including the lactic acid producers, Clostridium spp., Escherichia coli, and Corynebacterium glutamicum, in order to increase the yields of bioethanol, butanol, succinic acid, acetone, and polyhydroxyalkanoate precursors. CRISPR-mediated promoter engineering and single-step multiplex editing have improved inhibitor tolerance, raised ethanol titers, and allowed for the de novo synthesis of terpenoids, flavonoids, and recombinant vaccines in yeasts, especially Saccharomyces cerevisiae and emerging non-conventional species. While enzyme and biopharmaceutical manufacturing use CRISPR for quick strain optimization and glyco-engineering, food and beverage fermentations benefit from starter-culture customization for aroma, texture, and probiotic functionality. Off-target effects, cytotoxicity linked to Cas9, inefficient delivery in specific microorganisms, and regulatory ambiguities in commercial fermentation settings are some of the main challenges. This review provides an industry-specific summary of CRISPR–Cas9 applications in microbial fermentation and highlights technical developments, persisting challenges, and industrial advancements. Full article

Bacterial structures formed from the outer membrane and the periplasm components carry biomolecules to expel cellular material and interact with other cells. These outer membrane vesicles (OMVs) can encapsulate bioactive content, which confers OMVs with high potential as alternative drug delivery vehicles or as a platform for novel vaccine development. Single-gene mutants derived from Escherichia coli JC8031 were engineered to further enhance OMV production based on metabolic network modelling and in silico gene knockout design (ΔpoxB, ΔsgbE, ΔgmhA, and ΔallD). Mutants were experimentally obtained by genome editing using CRISPR-Cas9 and tested for OMVs recovery observing an enhanced OMV production in all of them. Lipidomic analysis through LC-ESI-QTOF-MS was performed for OMVs obtained from each engineered strain and compared to the wild-type E. coli JC8031 strain. The lipid profile of OMVs from the wild-type E. coli JC8031 did not change significantly confirmed by multivariate statistical analysis when compared to the mutant strains. The obtained results suggest that the vesicle production can be further improved while the obtained vesicles are not altered in their composition, allowing further study for stability and integrity for use in therapeutic settings. Full article

The type V CRISPR/Cas12f system, with its broad PAM recognition range, small size, and ease of delivery, has significantly contributed to the gene editing toolbox. In this study, enOsCas12f1 activity was detected during transient expression in rice protoplasts. The results showed that enOsCas12f1 exhibited DNA cleavage activity when it recognized TTN PAMs. Subsequently, we examined the gene editing efficiency of enOsCas12f1 in stably transformed rice plants, and the results showed that enOsCas12f1 could identify the TTT and TTC PAM sequences of the OsPDS gene, resulting in gene mutations and an albino phenotype. The editing efficiencies of TTT and TTC PAMs were 6.21% and 44.21%, respectively. Furthermore, all mutations were base deletions, ranging in size from 7 to 29 base pairs. Then, we used enOsCas12f1 to edit the promoter and 5′ UTR of the OsDREB1C gene, demonstrating that enOsCas12f1 could stably produce base deletion, mutant rice plants. Additionally, we fused the transcriptional activation domain TV with the dead enOsCas12f1 to enhance the expression of the target gene OsIPA1. Our study demonstrates that enOsCas12f1 can be utilized for rice gene modification, thereby expanding the toolbox for rice gene editing. Full article

Epigenome engineering, particularly utilizing CRISPR/dCas-based systems, is a powerful strategy to modulate gene expression and genome functioning without altering the DNA sequence. In this review we summarized current achievements and prospects in dCas-mediated epigenome editing, primarily focusing on its applications in biomedicine, but also providing a wider context for its applications in biotechnology. The diversity of CRISPR/dCas architectures is outlined, recent innovations in the design of epigenetic editors and delivery methods are highlighted, and the therapeutic potential across a wide range of diseases, including hereditary, neurodegenerative, and metabolic disorders, is examined. Opportunities for the application of dCas-based tools in animal, agricultural, and industrial biotechnology are also discussed. Despite substantial progress, challenges, such as delivery efficiency, specificity, stability of induced epigenetic modifications, and clinical translation, are emphasized. Future directions aimed at enhancing the efficacy, safety, and practical applicability of epigenome engineering technologies are proposed. Full article

Background: Human herpesviruses are double-stranded DNA viruses of which eight types have been identified at present. Herpesvirus infection comprises an active lytic phase and a lifelong latency phase with the possibility of reactivation. These infections are highly prevalent worldwide and can lead to a broad spectrum of clinical manifestations, ranging from mild symptoms to severe disease, particularly in immunocompromised individuals. Clustered regularly interspaced palindromic repeats (CRISPR)-based therapy is an interesting alternative to current antiviral drugs, which fail to cure latent infections and are increasingly challenged by viral resistance. Objective: This scoping review aimed to summarize the current state of CRISPR-based antiviral strategies against herpesvirus infections, highlighting the underlying mechanisms, study design and outcomes, and challenges for clinical implementation. Design: A literature search was conducted in the databases PubMed and Web of Science, using both a general and an individual approach for each herpesvirus. Results: This scoping review identified five main mechanisms of CRISPR-based antiviral therapy against herpesvirus infections in vitro and/or in vivo. First, CRISPR systems can inhibit the active lytic replication cycle upon targeting viral lytic genes or host genes. Second, CRISPR technologies can remove latent viral genomes from infected cells by targeting viral genes essential for latency maintenance or destabilizing the viral genome. Third, reactivation of multiple latent herpesvirus infections can be inhibited by CRISPR-Cas-mediated editing of lytic viral genes, preventing a flare-up of clinical symptoms and reducing the risk of viral transmission. Fourth, CRISPR systems can purposefully induce viral reactivation to enhance recognition by the host immune system or improve the efficacy of existing antiviral therapies. Fifth, CRISPR technology can be applied to develop or enhance the efficiency of cellular immunotherapy. Conclusions: Multiple studies demonstrate the potential of CRISPR-based antiviral strategies to target herpesvirus infections through various mechanisms in vitro and in vivo. However, aspects regarding the delivery and biosafety of CRISPR systems, along with the time window for treatment, require further investigation before broad clinical implementation can be realized. Full article

HIV-1 Tat acts as a central molecular switch governing the transition between viral latency and active replication, making it a pivotal target for HIV-1 functional cure strategies. By binding to the viral long terminal repeat (LTR) and hijacking host transcriptional machinery, Tat dynamically regulates RNA polymerase II processivity to alter viral transcription states. Recent studies reveal its context-dependent variability: while Tat recruits chromatin modifiers and scaffolds non-coding RNAs to stabilize epigenetic silencing in latently infected cells, it also triggers rapid transcriptional amplification upon cellular activation. This review systematically analyzes the bistable regulatory mechanism of Tat and investigates advanced technologies for reprogramming this switch to eliminateviral reservoirs and achieve functional cures. Conventional approaches targeting Tat are limited by compensatory viral evolution and poor bioavailability. Next-generation interventions will employ precision-engineered tools, such as AI-optimized small molecules blocking Tat-P-TEFb interfaces and CRISPR-dCas9/Tat chimeric systems, for locus-specific LTR silencing or reactivation (“block and lock” or “shock and kill”). Advanced delivery platforms, including brain-penetrant lipid nanoparticles (LNPs), enable the targeted delivery of Tat-editing mRNA or base editors to microglial reservoirs. Single-cell multiomics elucidates Tat-mediated clonal heterogeneity, identifying “switchable” subpopulations for timed interventions. By integrating systems-level Tat interactomics, epigenetic engineering, and spatiotemporally controlled delivery, this review proposes a roadmap to disrupt HIV-1 persistence by hijacking the Tat switch, ultimately bridging mechanistic insights to clinical applications. Full article

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterized by progressive photoreceptor degeneration and vision loss. While current management is largely supportive—relying on visual aids, orientation training, and nutritional supplementation—these interventions offer only symptomatic relief and do not halt disease progression. Advances in molecular genetics have led to the development of targeted treatments, including gene replacement therapy, RNA-based therapies, and CRISPR/Cas9 gene editing, offering promising strategies for disease modification. The approval of voretigene neparvovec for RPE65-associated RP marked a milestone in gene therapy, while ongoing trials targeting mutations in RPGR, USH2A, and CEP290 are expanding therapeutic options. Optogenetic therapy and stem cell transplantation represent additional strategies, particularly for patients with advanced disease. Challenges persist in delivery efficiency, immune responses, and treating large or dominant-negative mutations. Non-viral vectors, nanoparticle systems, and artificial intelligence-guided diagnostics are being explored to address these limitations and support personalized care. This review summarizes the current and emerging therapeutic landscape for RP, highlighting the shift toward precision medicine and the need for continued innovation to overcome genetic and phenotypic variability. Full article

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. One of its major genetic causes is C9ORF72, where mutations lead to hexanucleotide repeat expansions in the C9ORF72 gene. These expansions drive disease progression through mechanisms, including the formation of toxic RNAs and the accumulation of damaged proteins such as dipeptide repeats (DPRs). This review highlights these pathogenic mechanisms, focusing on RNA foci formation and the accumulation of toxic DPRs, which contribute to neuronal damage. It also discusses promising targeted therapies, including small molecules and biological drugs, designed to counteract these specific molecular events. Small molecules such as G-quadruplex stabilizers, proteasome and autophagy modulators, and RNase-targeting chimeras show potential in reducing RNA foci and DPR accumulation. Furthermore, targeting enzymes involved in repeat-associated non-AUG (RAN) translation and nucleocytoplasmic transport, which are crucial for disease pathogenesis, opens new therapeutic avenues. Even some anti-viral drugs show encouraging results in preclinical studies. Biological drugs, such as antisense oligonucleotides and gene-editing technologies like CRISPR-Cas, were explored for their potential to specifically target C9ORF72 mutations and modify the disease’s molecular foundations. While preclinical and early clinical data show promise, challenges remain in optimizing delivery methods, ensuring long-term safety, and improving efficacy. This review concludes by emphasizing the importance of continued research and the potential for these therapies to alter the disease trajectory and improve patient outcomes. Full article

Epstein–Barr virus (EBV), a double-stranded DNA virus, is implicated in nasopharyngeal carcinoma (NPC), with particularly high incidence in regions such as southern China and Hong Kong. Although NPC is typically treated with radio- and chemotherapy, outcomes remain poor for advanced-stage diagnoses, highlighting the need for targeted therapies. This study explores the potential of CRISPR/CRISPR-associated protein 13 (Cas13) technology to target essential EBV RNA in NPC cells. Previous research demonstrated that CRISPR/Cas9 could partially reduce EBV load, but suppression was incomplete. Here, the combination of CRISPR/Cas13 with CRISPR/Cas9 shows enhanced viral clearance. Long-term EBNA1 suppression via CRISPR/Cas13 reduced the EBV genome, improved CRISPR/Cas9 effectiveness, and identified suitable AAV serotypes for delivery. Furthermore, cotreatment increased NPC cell sensitivity to 5-fluorouracil and cisplatin. These findings underscore the potential of CRISPR/Cas13 as an anti-EBV therapeutic approach, effectively targeting latent EBV transcripts and complementing existing treatments. The study suggests a promising new direction for developing anti-EBV strategies, potentially benefiting therapies for NPC and other EBV-associated malignancies. Full article