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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 18 Feb 2025 at 01:45 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: ( "CRISPR.CAS" OR "crispr/cas" ) NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

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RevDate: 2025-02-17
CmpDate: 2025-02-17

Stewart C, Manriquez Roman C, SS Kenderian (2025)

Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells.

Journal of visualized experiments : JoVE.

Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, the durable response remains limited. Recent studies have shown that the epigenetic landscape of preinfusion CART cell products can influence response to therapy, and gene editing has been proposed as a solution. However, more work needs to be done to determine the optimal gene editing strategy. Genome-wide CRISPR screens have become popular tools to both investigate mechanisms of resistance and optimize gene editing strategies. Yet their application to primary cells presents many challenges. Here we describe a method to complete a genome-wide CRISPR knockout screen in CART cells from healthy donors. As a proof-of-concept model, we designed this method to investigate the development of exhaustion in CART cells targeting the CD19 antigen. However, we believe that this approach can be used to address a variety of mechanisms of resistance to therapy in different CAR constructs and tumor models.

RevDate: 2025-02-17
CmpDate: 2025-02-17

Meng H, Nan M, Li Y, et al (2025)

PD-L1 knockout or ZG16 overexpression inhibits PDAC progression and modulates TAM polarization.

Frontiers in immunology, 16:1510179.

CRISPR/Cas9-mediated genome editing has the potential to delete PD-L1 both on the cell surface and inside the cell, thereby inhibiting tumor growth and migration and overcoming immunosuppression. ZG16, with its lectin structure, can reduce PD-L1 expression on the cell surface. However, direct comparison of two approaches on PD-L1 expression in Pancreatic ductal adenocarcinoma (PDAC) cells has not yet been investigated. In this study, we established two Panc-1 cell line: one with PD-L1 knockout and another with ZG16 overexpression. Both methods promoted the polarization of tumor-associated macrophages (TAMs) to the M1 phenotype, as indicated by increased levels of the M1 marker CD11c+ in vitro and in vivo. Meanwhile, we observed a reduction in the M2 marker CD206+, upregulation of immune activation-related cytokines/chemokines, and a decrease in immunosuppressive cytokines and tumor angiogenesis factors. In summary, both PD-L1 knockout and ZG16 overexpression represent promising approaches for PDAC treatment.

RevDate: 2025-02-16
CmpDate: 2025-02-17

Izzo M, Battistini J, Golini E, et al (2025)

Muscle-specific gene editing improves molecular and phenotypic defects in a mouse model of myotonic dystrophy type 1.

Clinical and translational medicine, 15(2):e70227.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is a genetic multisystemic disease, characterised by pleiotropic symptoms that exhibit notable variability in severity, nature and age of onset. The genetic cause of DM1 is the expansion of unstable CTG-repeats in the 3' untranslated region (UTR) of the DMPK gene, resulting in the accumulation of toxic CUG-transcripts that sequester RNA-binding proteins and form nuclear foci in DM1 affected tissues and, consequently, alter various cellular processes. Therapeutic gene editing for treatment of monogenic diseases is a powerful technology that could in principle remove definitively the disease-causing genetic defect. The precision and efficiency of the molecular mechanisms are still under investigation in view of a possible use in clinical practice.

METHODS: Here, we describe the application of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) strategy to remove the CTG-expansion in the DMPK gene in a mouse model carrying the human transgene from a DM1 patient. To optimise the editing efficiency in vivo, we identified new tools that allowed to improve the expression levels and the activity of the CRISPR/Cas9 machinery. Newly designed guide RNA pairs were tested in DM1-patient derived cells before in vivo application. Edited cells were analysed to assess the occurrence of off-target and the accuracy of on-target genomic events. Gene editing-dependent and -independent mechanisms leading to decreased accumulation of the mutated DMPK transcripts were also evaluated.

RESULTS AND CONCLUSION: Systemic delivery of CRISPR/Cas9 components in DM1 mice, through myotropic adeno-associated viral vectors, led to significant improvement of molecular alterations in the heart and skeletal muscle. Importantly, a persistent increase of body weight, improvement of muscle strength and body composition parameters were observed in treated animals. Accurate evaluation of CRISPR/Cas9-mediated-phenotypic recovery in vivo is a crucial preclinical step for the development of a gene therapy for DM1 patients.

KEY POINTS: In vivo application of a therapeutic gene editing strategy for permanent deletion of the pathogenetic CTG-repeat amplification in the DMPK gene that causes myotonic dystrophy type 1. Following treatment, diseased mice show a significant improvement of both molecular and phenotypic defects.

RevDate: 2025-02-16
CmpDate: 2025-02-16

Breusegem SY, Houghton J, Romero-Bueno R, et al (2025)

A multiparametric anti-aging CRISPR screen uncovers a role for BAF in protein synthesis regulation.

Nature communications, 16(1):1681.

Progeria syndromes are very rare, incurable premature aging conditions recapitulating most aging features. Here, we report a whole genome, multiparametric CRISPR screen, identifying 43 genes that can rescue multiple cellular phenotypes associated with progeria. We implement the screen in fibroblasts from Néstor-Guillermo Progeria Syndrome male patients, carrying a homozygous A12T mutation in BAF. The hits are enriched for genes involved in protein synthesis, protein and RNA transport and osteoclast formation and are validated in a whole-organism Caenorhabditis elegans model. We further confirm that BAF A12T can disrupt protein synthesis rate and fidelity, which could contribute to premature aging in patients. This work highlights the power of multiparametric genome-wide suppressor screens to identify genes enhancing cellular resilience in premature aging and provide insights into the biology underlying progeria-associated cellular dysfunction.

RevDate: 2025-02-16
CmpDate: 2025-02-16

Florez-Vargas O, Ho M, Hogshead MH, et al (2025)

Genetic regulation of TERT splicing affects cancer risk by altering cellular longevity and replicative potential.

Nature communications, 16(1):1676.

The chromosome 5p15.33 region, which encodes telomerase reverse transcriptase (TERT), harbors multiple germline variants identified by genome-wide association studies (GWAS) as risk for some cancers but protective for others. Here, we characterize a variable number tandem repeat within TERT intron 6, VNTR6-1 (38-bp repeat unit), and detect a strong link between VNTR6-1 alleles (Short: 24-27 repeats, Long: 40.5-66.5 repeats) and GWAS signals rs2242652 and rs10069690 within TERT intron 4. Bioinformatics analyses reveal that rs10069690-T allele increases intron 4 retention while VNTR6-1-Long allele expands a polymorphic G-quadruplex (G4, 35-113 copies) within intron 6, with both variants contributing to variable TERT expression through alternative splicing and nonsense-mediated decay. In two cell lines, CRISPR/Cas9 deletion of VNTR6-1 increases the ratio of TERT-full-length (FL) to the alternative TERT-β isoform, promoting apoptosis and reducing cell proliferation. In contrast, treatment with G4-stabilizing ligands shifts splicing from TERT-FL to TERT-β isoform, implicating VNTR6-1 as a splicing switch. We associate the functional variants VNTR6-1, rs10069690, and their haplotypes with multi-cancer risk and age-related telomere shortening. By regulating TERT splicing, these variants may contribute to fine-tuning cellular longevity and replicative potential in the context of stress due to tissue-specific endogenous and exogenous exposures, thereby influencing the cancer risk conferred by this locus.

RevDate: 2025-02-16

Sun J, Jiang X, Xu F, et al (2025)

Constructing pyrG marker by CRISPR/Cas facilities the highly-efficient precise genome editing on industrial Aspergillus niger strain.

Bioprocess and biosystems engineering [Epub ahead of print].

To prevent the unique difficulty of hygromycin-based gene editing on industrial A. niger strain and increase the working efficiency, the local pyrG marker was removed by well-designed dual sgRNAs and repair template through Cas9-ribonucleoprotein (RNP) strategy in this study. The positive rate of the desired pyrG auxotroph construction was 100%, while no transformant was observed using the traditional methods. The complementation strain showed similar fermentation character as the starting strain. Moreover, an efficient and seamless knock out plasmid-based strategy was established, achieving positive rate at 90% and 50% for challenging Δku70 and Δku80 respectively. Further, combined hygromycin markers and miniaturization cultivation were conducted to select the poor growth strain. Finally, skillfully designed sgRNA and amdS counter-selection repair template were used to obtain ERG3[Tyr185] mutation. A highly-efficient precise strategy was established for A. niger through a diagnostic PCR method, with nearly 100% positive rate. Highly- precise desired point mutation was achieved by the developed gene toolbox.

RevDate: 2025-02-17
CmpDate: 2025-02-17

Masarwy R, Breier D, Stotsky-Oterin L, et al (2025)

Targeted CRISPR/Cas9 Lipid Nanoparticles Elicits Therapeutic Genome Editing in Head and Neck Cancer.

Advanced science (Weinheim, Baden-Wurttemberg, Germany), 12(7):e2411032.

Squamous cell carcinomas of the head and neck (HNSCC) originate in the upper aerodigestive tract, including the oral cavity, pharynx, and larynx. Current treatments of locally advanced HNSCC often lead to high treatment failure, and disease recurrence, resulting in poor survival rates. Advances in mRNA technologies and lipid nanoparticle (LNP) delivery systems led to several clinical trials involving LNP-CRISPR-Cas9 mRNA-based therapeutics. Despite these advances, achieving cell-type-specific extrahepatic mRNA delivery is still challenging. This study introduces a safe and effective intratumoral EGFR-targeted CRISPR-LNP delivery strategy for knocking out SOX2, which is a cancer-specific gene. To assess their therapeutic potential, it is shown that LNPs made from ionizable lipids with helper lipids co-encapsulating Cas9 mRNA and sgRNA targeting SOX2 (sgSOX2), lead to a ≈60% reduction in HNSCC cell viability in vitro. Next, using a xenograft HNSCC mouse model, targeted delivery of 𝜶EGFR- CRISPR-sgSOX2-LNPs to HNSCC cells resulted in a 90% inhibition of tumor growth and a 90% increase in survival for > 84 days, with tumor disappearance observed in 50% of the mice. These findings emphasize the potential of targeted mRNA-Cas9-LNPs in clinically accessible solid tumors, specifically in reaching tumor cells and inducing persistent therapeutic responses in tumors with high-recurrence rates like HNSCC.

RevDate: 2025-02-17
CmpDate: 2025-02-17

Huang W, Wang J, Wang C, et al (2025)

Expanding Cas12a Activity Control with an RNA G-Quadruplex at the 5' end of CRISPR RNA.

Advanced science (Weinheim, Baden-Wurttemberg, Germany), 12(7):e2411305.

Precise control of Cas12a activity is essential for the improvement of the detection limit of clinical diagnostics and the minimization of errors. This study addresses the challenge of controlling Cas12a activity, especially in the context of nucleic acid detection where the inherent incompatibility between isothermal amplification and CRISPR reactions complicates accurate diagnostics. An RNA G-quadruplex (RG4) structure at the 5' end of crRNA is introduced to modulate Cas12a activity accurately without the need for chemical modifications. The results indicate that the presence of RG4 does not significantly impact Cas12a's cleavage activity but can be controlled by RG4 stabilizers, enabling the suppression and subsequent restoration of Cas12a activity with potential for precise activity control. Moreover, the use of RG4 is expanded by incorporating it into split crRNA, introducing RG4 directly at the 5' end of the direct repeat (DR) region, enabling tailored activity regulation for different targets by matching with various Spacer regions. Additionally, a light-controlled one-pot method for activating Cas12a is developed, thereby enhancing the accuracy and sensitivity of clinical samples. This study showcases the pioneering use of RG4 in manipulating Cas12a activity, streamlining diagnostics, and paving the way for advances in clinical nucleic acid testing.

RevDate: 2025-02-17
CmpDate: 2025-02-17

Fracassi G, Lorenzin F, Orlando F, et al (2024)

CRISPR/Cas9 screens identify LIG1 as a sensitizer of PARP inhibitors in castration-resistant prostate cancer.

The Journal of clinical investigation, 135(4):.

PARP inhibitors (PARPi) have received regulatory approval for the treatment of several tumors, including prostate cancer (PCa), and demonstrate remarkable results in the treatment of castration-resistant prostate cancer (CRPC) patients characterized by defects in homologous recombination repair (HRR) genes. Preclinical studies showed that DNA repair genes (DRG) other than HRR genes may have therapeutic value in the context of PARPi. To this end, we performed multiple CRISPR/Cas9 screens in PCa cell lines using a custom sgRNA library targeting DRG combined with PARPi treatment. We identified DNA ligase 1 (LIG1), essential meiotic structure-specific endonuclease 1 (EME1), and Fanconi anemia core complex associated protein 24 (FAAP24) losses as PARPi sensitizers and assessed their frequencies from 3% to 6% among CRPC patients. We showed that concomitant inactivation of LIG1 and PARP induced replication stress and DNA double-strand breaks, ultimately leading to apoptosis. This synthetic lethality (SL) is conserved across multiple tumor types (e.g., lung, breast, and colorectal), and its applicability might be extended to LIG1-functional tumors through a pharmacological combinatorial approach. Importantly, the sensitivity of LIG1-deficient cells to PARPi was confirmed in vivo. Altogether, our results argue for the relevance of determining the status of LIG1 and potentially other non-HRR DRG for CRPC patient stratification and provide evidence to expand their therapeutic options.

RevDate: 2025-02-17
CmpDate: 2025-02-17

Fu Z, Huang Z, Xu H, et al (2024)

IL-2-inducible T cell kinase deficiency sustains chimeric antigen receptor T cell therapy against tumor cells.

The Journal of clinical investigation, 135(4):.

Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several key challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of T cell receptor signaling, while its role in CAR signaling is unknown. By electroporation of CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing analyses revealed downregulation of exhaustion and upregulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T cell products for clinical use.

RevDate: 2025-02-15
CmpDate: 2025-02-15

Zheng W, Li H, Liu M, et al (2025)

Molecular insights and rational engineering of a compact CRISPR-Cas effector Cas12h1 with a broad-spectrum PAM.

Signal transduction and targeted therapy, 10(1):66.

Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif (PAM) distinct from that of other type V Cas effectors. Here, we report the nickase preference of Cas12h1, which predominantly cleaves the nontarget strand (NTS) of a double-stranded DNA (dsDNA) substrate. In addition, Cas12h1 acts as a nickase in human cells. We further determined the cryo-EM structures of Cas12h1 in the surveillance, R-loop formation, and interference states, revealing the molecular mechanisms involved in the crRNA maturation, target recognition, R-loop formation, nuclease activation and target degradation. Cas12h1 notably recognizes a broad 5'-DHR-3' PAM (D is A, G, or T; H is A, C, or T; R is A or G) both in vitro and in human cells. In addition, Cas12h1 utilizes a distinct activation mechanism that the lid motif undergoes a "flexible to stable" transition to expose the catalytic site to the substrate. A high-fidelity nucleic acid detector, Cas12h1[hf], was developed through rational engineering, which distinguishes single-base mismatches and retains comparable on-target activities. Our results shed light on the molecular mechanisms underlying Cas12h1 nickase, improve the understanding of type V Cas effectors, and expand the CRISPR toolbox for genome editing and molecular diagnosis.

RevDate: 2025-02-15

Vilar LC, Rego ACS, Miguel MAL, et al (2025)

Staphylococcus spp. and methicillin-resistance gene mecA dispersion in seawater: A case study of Guanabara Bay's recreational and touristic waters.

Comparative immunology, microbiology and infectious diseases, 118:102326 pii:S0147-9571(25)00034-7 [Epub ahead of print].

Environmental Staphylococci, particularly coagulase-negative Staphylococci (CoNS), are known reservoirs of antimicrobial resistance genes and human-animal opportunistic pathogens, yet their role within the One Health framework remains underexplored. In this study, we isolated 12 species of CoNS from two sites 10 km apart in Guanabara Bay, Rio de Janeiro, with the most frequent species being the opportunistic Staphylococcus saprophyticus (30.3 %), Staphylococcus warneri (25.7 %), and Staphylococcus epidermidis (16.7 %). GTG5-PCR fingerprinting revealed significant genetic diversity, yet identical profiles persisted across both sites throughout the year, indicating strain dispersion and persistence. Among the 66 strains analyzed, 42 exhibited resistance to clinically significant antimicrobials, including methicillin-resistant strains harboring the mecA gene. Remarkably, 22.7 % of the strains carried CRISPR-Cas systems, a frequency unusually high for Staphylococcus spp., suggesting that bacteriophage pressure in the seawater environment may drive this increase. The presence of antimicrobial-resistant CoNS in Guanabara Bay, a popular recreational area, represents a potential public health risk.

RevDate: 2025-02-14
CmpDate: 2025-02-14

Chambial P, Thakur N, Bhukya PL, et al (2025)

Frontiers in superbug management: innovating approaches to combat antimicrobial resistance.

Archives of microbiology, 207(3):60.

Anti-microbial resistance (AMR) is a global health issue causing significant mortality and economic burden. Pharmaceutical companies' discontinuation of research hinders new agents, while MDR pathogens or "superbugs" worsen the problem. Superbugs pose a threat to common infections and medical procedures, exacerbated by limited antibiotic development and rapid antibiotic resistance. The rising tide of antimicrobial resistance threatens to undermine progress in controlling infectious diseases. This review examines the global proliferation of AMR, its underlying mechanisms, and contributing factors. The study explores various methodologies, emphasizing the significance of precise and timely identification of resistant strains. We discuss recent advancements in CRISPR/Cas9, nanoparticle technology, light-based techniques, and AI-powered antibiogram analysis for combating AMR. Traditional methods often fail to effectively combat multidrug-resistant bacteria, as CRISPR-Cas9 technology offers a more effective approach by cutting specific DNA sequences, precision targeting and genome editing. AI-based smartphone applications for antibiogram analysis in resource-limited settings face challenges like internet connectivity, device compatibility, data quality, energy consumption, and algorithmic limitations. Additionally, light-based antimicrobial techniques are increasingly being used to effectively kill antibiotic-resistant microbial species and treat localized infections. This review provides an in-depth overview of AMR covering epidemiology, evolution, mechanisms, infection prevention, control measures, antibiotic access, stewardship, surveillance, challenges and emerging non-antibiotic therapeutic approaches.

RevDate: 2025-02-16
CmpDate: 2025-02-16

Chen X, Zheng M, Lin S, et al (2025)

The application of CRISPR/Cas9-based genome-wide screening to disease research.

Molecular and cellular probes, 79:102004.

High-throughput genetic screening serves as an indispensable approach for deciphering gene functions and the intricate relationships between phenotypes and genotypes. The CRISPR/Cas9 system, with its ability to precisely edit genomes on a large scale, has revolutionized the field by enabling the construction of comprehensive genomic libraries. This technology has become a cornerstone for genome-wide screenings in disease research. This review offers a comprehensive examination of how CRISPR/Cas9-based genetic screening has been leveraged to uncover genes that play a role in disease mechanisms, focusing on areas such as cancer development and viral replication processes. The insights presented in this review hold promise for the development of novel therapeutic strategies and precision medicine approaches.

RevDate: 2025-02-16
CmpDate: 2025-02-16

Guo F, Li J, Ma P, et al (2025)

A magnetic bead-based dual-aptamer sandwich assay for quantitative detection of ciprofloxacin using CRISPR/Cas12a.

Molecular and cellular probes, 79:101998.

Ciprofloxacin (CIP) is a broad-spectrum fluoroquinolone antibiotic, and its excessive residues in food and water sources pose potential risks to human health. Therefore, there is a need for a rapid and convenient method for its accurate quantification. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system has gained extensive application in signal detection and amplification due to the trans-cleavage activity of Cas12a. In this study, we devised a novel magnetic bead-based dual sandwich aptamer coupled with a CRISPR/Cas12a system for the precise quantification of CIP in milk, river water, and honey. Through the incorporation of a magnetic bead-based dual aptamer sandwich approach, the concentration of CIP in the samples was pre-enriched. Additionally, by optimizing the Fluorescence-Quencher (F-Q) probe concentration, detection aptamer (APTd) concentration, and assay duration, the limit of blank (LOB) of the system was determined as 362 nM, while the limit of detection (LOD) was determined as 403 nM. This enabled the accurate quantification of CIP within the linear range of 0.5 μM to 0.2 mM with high specificity. Moreover, the performance of this detection method was comparable to that of high-performance liquid chromatography (HPLC) in river water, milk, and honey samples.

RevDate: 2025-02-16
CmpDate: 2025-02-16

Wang J, Liu H, Yang Y, et al (2023)

Genome-scale CRISPR screen identifies TRIM2 and SLC35A1 associated with porcine epidemic diarrhoea virus infection.

International journal of biological macromolecules, 250:125962.

Porcine epidemic diarrhoea (PED) caused by the porcine epidemic diarrhoea virus (PEDV) is the most devastating disease in the global pig industry due to its high mortality rate in piglets. The host factors critical for PEDV replication are poorly understood. Here, we designed a pooled African green monkey genome-scale CRISPR/Cas9 knockout (VeroCKO) library containing 75,608 single guide RNAs targeting 18,993 protein-coding genes. Subsequently, we use the VeroCKO library to identify key host factors facilitating PEDV infection in Vero E6 cells. Several previously unreported genes associated with PEDV infection are highly enriched post-PEDV selection. We discovered that knocking out the tripartite motif 2 (TRIM2) and the solute carrier family 35 member A1 (SLC35A1) inhibited PEDV replication. Virtual screening and molecular docking approaches showed that chem-80,048,685 (M2) s ignificantly inhibited PEDV attachment and late replication by impeding SLC35A1. Furthermore, we found that knocking out SLC35A1 in Vero E6 cells upregulated a disintegrin and metalloprotease protein-17 (ADAM17) by splicing porcine aminopeptidase N (pAPN) and angiotensin-converting enzyme 2 (ACE2) ectodomains to reduce PEDV-infection in a CMP-Sialic Acid (CMP-SA) cell entry-independent manner. These findings provide a new perspective for a better understanding of host-pathogen interactions and new therapeutic targets for PEDV infection.

RevDate: 2025-02-14
CmpDate: 2025-02-14

El Kassem G, Hillmer J, M Boettcher (2025)

Evaluation of Cas13d as a tool for genetic interaction mapping.

Nature communications, 16(1):1631.

Mapping genetic interactions (GIs) is crucial for understanding genetic network complexity. In this study, we investigate the utility of Cas13d, a CRISPR system targeting RNA, for GI mapping and compare it to Cas9 and Cas12a, two DNA nucleases commonly used for GI mapping. We find that Cas13d induces faster target gene perturbation and generates more uniform cell populations with double perturbations than Cas9 or Cas12a. We then encounter Cas13d gRNA-gRNA interference when concatenating gRNAs targeting different genes into one gRNA array, which we overcome by a dual promoter gRNA expression strategy. Moreover, by concatenating three gRNAs targeting the same gene into one array, we are able to maximize the Cas13d-mediated knockdown effects. Combining these strategies enhances proliferation phenotypes while reducing library size and facilitates reproducible quantification of GIs in oncogenic signaling pathways. Our study highlights the potential of Cas13d for GI mapping, promising advancements in understanding therapeutically relevant drug response pathways.

RevDate: 2025-02-14
CmpDate: 2025-02-14

Fu Y, Yang X, Li S, et al (2025)

Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation.

Nature communications, 16(1):1640.

CRISPR activation (CRISPRa) is a powerful tool for endogenous gene activation, yet the mechanisms underlying its optimal transcriptional activation remain unclear. By monitoring real-time transcriptional bursts, we find that CRISPRa modulates both burst duration and amplitude. Our quantitative imaging reveals that CRISPR-SunTag activators, with three tandem VP64-p65-Rta (VPR), form liquid-like transcriptional condensates and exhibit high activation potency. Although visible CRISPRa condensates are associated with some RNA bursts, the overall levels of phase separation do not correlate with transcriptional bursting or activation strength in individual cells. When the number of SunTag scaffolds is increased to 10 or more, solid-like condensates form, sequestering co-activators such as p300 and MED1. These condensates display low dynamicity and liquidity, resulting in ineffective gene activation. Overall, our studies characterize various phase-separated CRISPRa systems for gene activation, highlighting the foundational principles for engineering CRISPR-based programmable synthetic condensates with appropriate properties to effectively modulate gene expression.

RevDate: 2025-02-14

Alhabsi A, Ling Y, Crespi M, et al (2025)

Alternative Splicing Dynamics in Plant Adaptive Responses to Stress.

Annual review of plant biology [Epub ahead of print].

Plants thrive in dynamic environments by activating sophisticated molecular networks that fine-tune their responses to stress. A key component of these networks is gene regulation at multiple levels, including precursor messenger RNA (pre-mRNA) splicing, which shapes the transcriptome and proteome landscapes. Through the precise action of the spliceosome complex, noncoding introns are removed and coding exons are joined to produce spliced RNA transcripts. While constitutive splicing always generates the same messenger RNA (mRNA), alternative splicing (AS) produces multiple mRNA isoforms from a single pre-mRNA, enriching proteome diversity. Remarkably, 80% of multiexon genes in plants generate multiple isoforms, underscoring the importance of AS in shaping plant development and responses to abiotic and biotic stresses. Recent advances in CRISPR-Cas genome and transcriptome editing technologies offer revolutionary tools to dissect AS regulation at molecular levels, unveiling the functional significance of specific isoforms. In this review, we explore the intricate mechanisms of pre-mRNA splicing and AS in plants, with a focus on stress responses. Additionally, we examine how leveraging AS insights can unlock new opportunities to engineer stress-resilient crops, paving the way for sustainable agriculture in the face of global environmental challenges.

RevDate: 2025-02-14

Ahsan M, Pindi C, G Palermo (2025)

Emerging Mechanisms of Metal-Catalyzed RNA and DNA Modifications.

Annual review of physical chemistry [Epub ahead of print].

Metal ions play a critical role in various chemical, biological, and environmental processes. This review reports on emerging chemical mechanisms in the catalysis of DNA and RNA. We provide an overview of the metal-dependent mechanisms of DNA cleavage in CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems that are transforming life sciences through genome editing technologies, and showcase intriguing metal-dependent mechanisms of RNA cleavages. We show that newly discovered CRISPR-Cas complexes operate as protein-assisted ribozymes, highlighting RNA's versatility and the enhancement of CRISPR-Cas functions through strategic metal ion use. We demonstrate the power of computer simulations in observing chemical processes as they unfold and in advancing structural biology through innovative approaches for refining cryo-electron microscopy maps. Understanding metal ion involvement in nucleic acid catalysis is crucial for advancing genome editing, aiding therapeutic interventions for genetic disorders, and improving the editing tools' specificity and efficiency.

RevDate: 2025-02-14

Contiliani DF, Sretenovic S, Dailey M, et al (2025)

Harnessing novel cytidine deaminases from the animal kingdom for robust multiplexed base editing in rice.

Plant biotechnology journal [Epub ahead of print].

CRISPR-Cas-based cytosine base editors (CBEs) are prominent tools that perform site-specific and precise C-to-T conversions catalysed by cytidine deaminases. However, their use is often constrained by stringent editing preferences for genomic contexts, off-target effects and restricted editing windows. To expand the repertoire of CBEs, we systematically screened 66 novel cytidine deaminases sourced from various organisms, predominantly from the animal kingdom and benchmarked them in rice protoplasts using the nCas9-BE3 configuration. After selecting candidates in rice protoplasts and further validation in transgenic rice lines, we unveiled a few cytidine deaminases exhibiting high editing efficiencies and wide editing windows. CBEs based on these cytidine deaminases also displayed minimal frequencies of indels and C-to-R (R = A/G) conversions, suggesting high purity in C-to-T base editing. Furthermore, we highlight the highly efficient cytidine deaminase OoA3GX2 derived from Orca (killer whale) for its comparable activity across GC/CC/TC/AC sites, thus broadening the targeting scope of CBEs for robust multiplexed base editing. Finally, the whole-genome sequencing analyses revealed very few sgRNA-dependent and -independent off-target effects in independent T0 lines. This study expands the cytosine base-editing toolkit with many cytidine deaminases sourced from mammals, providing better-performing CBEs that can be further leveraged for sophisticated genome engineering strategies in rice and likely in other plant species.

RevDate: 2025-02-15
CmpDate: 2025-02-14

Goncharov NV, Baklanov IN, Gulaia VS, et al (2025)

Therapy enhancing chromosome instability may be advantageous for IDH1 [R132H/WT] gliomas.

NAR cancer, 7(1):zcaf003.

Recently revised brain tumor classification suggested a glioma treatment strategy that takes into consideration molecular variants in IDH1 and TP53 marker genes. While pathogenic variants of IDH1 and TP53 can be accompanied by chromosomal instability (CIN), the impact of IDH1 and TP53 mutations on genome stability remains unstudied. Elevated CIN might provide therapeutic targets, based on synergistic effects of chemotherapy with CIN-inducing drugs. Using an assay based on human artificial chromosomes, we investigated the impact of common glioma missense mutations in IDH1 and TP53 on chromosome transmission and demonstrated that IDH1R132H and TP53R248Q variants elevate CIN. We next found enhanced CIN levels and the sensitivity of IDH1 [R132H/WT] and TP53 [R248Q/R248Q] genotypes, introduced into U87 MG glioma cells by CRISPR/Cas9, to different drugs, including conventional temozolomide. It was found that U87 MG cells carrying IDH1 [R132H/WT] exhibit dramatic sensitivity to paclitaxel, which was independently confirmed on cell cultures derived from patients with naturally occurring IDH1 [R132H/WT]. Overall, our results suggest that the development of CIN-enhancing therapy for glioma tumors with the IDH1 [R132H/WT] genotype could be advantageous for adjuvant treatment.

RevDate: 2025-02-14
CmpDate: 2025-02-14

Bhardwaj P, Dhangur P, Kalichamy A, et al (2025)

RT-RPA Assisted CRISPR/Cas12a Based One-Pot Rapid and Visual Detection of the Pan-Dengue Virus.

Journal of medical virology, 97(2):e70219.

Globally ≤ 4 billion of the population are at potential risk of contracting dengue virus (DENV) infection. Seasonal outbreaks of dengue are frequently reported causing a high healthcare burden. Undiagnosed DENV can lead to severe morbidity and mortality. Early diagnosis of DENV relies on molecular methods, which are impractical in resource-constrained settings (RCSs). Dengue can be caused by any of the four distinct DENV serotypes. Therefore, a simple method for rapid diagnosis of Pan-DENV serotypes is of utmost importance at RCSs. A fluorescence detection platform for Pan-DENV using RT-RPA and CRISPR/Cas12a was developed targeting nonstructural 1 (NS1) gene for DENV-1, 2, and 3, and envelope (E) gene for DENV-2. Further, crRNA specific to DENV serotypes were designed to facilitate CRISPR/Cas12a detection. Analytical sensitivity was determined using synthetic RNA and DENV serotypes genome. Clinical validation of the assay was performed using RNA extracted from AES/AFI clinical samples. The developed CRISPR/Cas12a-based detection platform can detect all four serotypes of DENV viz 1-4 in a single pot using fluorescence detection. This assay showed the limit of detection ≥ 781 zg reaction[-] [1], ≥ 1.81 ag reaction[-1], ≥ 62.5 fg reaction[-1], and ≥ 2.5 pg reaction[-1] for synthetic DENV-1, DENV-2, DENV-3, and DENV-4 template, respectively. Our assay demonstrated the analytic sensitivity of ≥ 10 ng reaction[-1] for DENV-1 and DENV-4, and ≥ 0.5 ng reaction[-1] for DENV-3 and DENV-4 genomes. This assay showed no cross-reactivity with other related etiologies tested causing AFI/AES. With 76 clinical samples (DENV PCR positive = 16, DENV PCR negative = 60), the assay demonstrated 93.7% sensitivity and 100% specificity with an overall accuracy of 98.7% for detection of the Pan-DENV serotypes. Our assay displayed comparable results to that of RT-PCR. The ease of interpretation and rapid detection of the Pan-DENV, represents the potential of the developed assay as an ideal point-of-care test. This assay upon field-deployment could help in reducing healthcare burden, provide differential diagnosis and support initiating early and prompt treatment to patients at RCS.

RevDate: 2025-02-14
CmpDate: 2025-02-14

Li Y, Xu R, Quan F, et al (2025)

Topologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution.

EBioMedicine, 112:105564.

BACKGROUND: The widespread and evolution of RNA viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the importance of fast identification of virus subtypes, particularly in non-laboratory settings. Rapid and inexpensive at-home testing of viral nucleic acids with single-base resolution remains a challenge.

METHODS: Topologically constrained DNA ring is engineered as substrates for the trans-cleavage of Cas13a to yield an accelerated post isothermal amplification. The capacity of CRISPR/Cas13a for discriminating single nucleotide variant (SNV) in viral genome is leveraged by designing synthetic mismatches and hairpin structure in CRISPR RNA (crRNA), enabling robust discrimination of different SARS-CoV-2 variants. Via optimisation of CasTDR3pot to be one-pot assay, CasTDR1pot can detect Omicron and its subvariants, with only a few copies in clinical samples in less than 30 min without pre-amplification.

FINDINGS: The detection system boasts high sensitivity (0.1 aM), single-base specificity, and the advantage of a rapid "sample-to-answer" process, which takes only 30 min. In the detection of SARS-CoV-2 clinical samples and their variant strains, CasTDR1pot has achieved 100% accuracy. Furthermore, the design of a portable signal-reading device facilitates user-friendly result interpretation. For the detection needs of different RNA viruses, the system can be adapted simply by designing the corresponding crRNA.

INTERPRETATION: Our study provides a rapid and accurate molecular diagnostic tool for point-of-care testing, epidemiological screening, and the detection of diseases associated with other RNA biomarkers with excellent single nucleotide differentiation, high sensitivity, and simplicity.

FUNDING: National Key Research and Development Program of China (No. 2023YFB3208302), National Natural Science Foundation of China (No. 22377110, 22034004, 82402749, 82073787, 22122409), National Key Research and Development Program of China (No. 2021YFA1200104), Henan Province Fund for Cultivating Advantageous Disciplines (No. 222301420019).

RevDate: 2025-02-14
CmpDate: 2025-02-14

Dahl-Jessen M, Terkelsen T, Bak RO, et al (2025)

Characterization of the role of spatial proximity of DNA double-strand breaks in the formation of CRISPR-Cas9-induced large structural variations.

Genome research, 35(2):231-241 pii:gr.278575.123.

Structural variations (SVs) play important roles in genetic diversity, evolution, and carcinogenesis and are, as such, important for human health. However, it remains unclear how spatial proximity of double-strand breaks (DSBs) affects the formation of SVs. To investigate if spatial proximity between two DSBs affects DNA repair, we used data from 3C experiments (Hi-C, ChIA-PET, and ChIP-seq) to identify highly interacting loci on six different chromosomes. The target regions correlate with the borders of megabase-sized topologically associated domains (TADs), and we used CRISPR-Cas9 nuclease and pairs of single guide RNAs (sgRNAs) against these targets to generate DSBs in both K562 cells and H9 human embryonic stem cells (hESCs). Droplet digital PCR (ddPCR) was used to quantify the resulting recombination events, and high-throughput sequencing was used to analyze the chimeric junctions created between the two DSBs. We observe a significantly higher formation frequency of deletions and inversions with DSBs in proximity compared with deletions and inversions with DSBs not in proximity in K562 cells. Additionally, our results suggest that DSB proximity may affect the ligation of chimeric deletion junctions. Taken together, spatial proximity between DSBs is a significant predictor of large-scale deletion and inversion frequency induced by CRISPR-Cas9 in K562 cells. This finding has implications for understanding SVs in the human genome and for the future application of CRISPR-Cas9 in gene editing and the modeling of rare SVs.

RevDate: 2025-02-15
CmpDate: 2025-02-15

Hildebrandt F, N Matias A, M Treeck (2025)

A CRISPR view on genetic screens in Toxoplasma gondii.

Current opinion in microbiology, 83:102577.

Genome editing technologies, such as CRISPR-Cas9, have revolutionised the study of genes in a variety of organisms, including unicellular parasites. Today, the CRISPR-Cas9 technology is vastly applied in high-throughput screens to investigate interactions between the Apicomplexan parasite Toxoplasma gondii and its hosts. In vitro and in vivo T. gondii screens performed in naive and restrictive conditions have led to the discovery of essential and fitness-conferring T. gondii genes, as well as factors important for virulence and dissemination. Recent studies have adapted the CRISPR-Cas9 screening technology to study T. gondii genes based on phenotypes unrelated to parasite survival. These advances were achieved by using conditional systems coupled with imaging, as well as single-cell RNA sequencing and phenotypic selection. Here, we review the state-of-the-art of CRISPR-Cas9 screening technologies with a focus on T. gondii, highlighting strengths, current limitations and future avenues for its development, including its application to other Apicomplexan species.

RevDate: 2025-02-15
CmpDate: 2025-02-15

Wang T, Chen T, Li D, et al (2025)

Core-shell vector-mediated co-delivery of CRISPR/Cas9 system and hydrophobic drugs against triple-negative breast cancer stem cells.

Journal of controlled release : official journal of the Controlled Release Society, 378:1080-1091.

Cancer stem cells (CSCs) play an important role in the development of triple-negative breast cancer (TNBC), including metastasis, invasion, tumorigenicity, and drug resistance. Moreover, non-CSCs can spontaneously transform into CSCs in special tumor microenvironments, thereby leading to poor prognosis or even failed treatments. Therefore, reversing CSCs into normal tumor cells in a sustained-acting manner is a promising strategy. It has been reported that down-regulation of FBXO44 protein expression inhibits tumor cell stemness. Moreover, CRISPR/Cas9 technology, a well-known precise gene editing tool, was adopted to permanently block FBXO44 within the genome upon its successful implementation. Given this, a core-shell nanoparticle (NP) consisting of amphiphilic polymer core and crosslinked-hyaluronic acid shell (nDOX-PL/pFBXO44 NPs) is developed in this work to concurrently deliver FBXO44-targeted CRISPR/Cas9 plasmids (pFBXO44) and doxorubicin (DOX) for combinational CSC reprogramming and chemotherapy of TNBC, which exhibits tumor cell targeting, endosomal escape, and reduction responsiveness to release DOX and plasmids in the cytoplasma. CRISPR/Cas9-mediated downregulation of FBXO44 expression could convert CSC into normal tumor cells, and effectively inhibit tumor growth without obvious side effects in vivo after combining with chemotherapy. In summary, we developed an intelligent system to co-deliver genetic and hydrophobic drugs, achieving effective cancer stemness reversal and synergistic suppression of contractable TNBC.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Mazzaferro E, Mujica E, Zhang H, et al (2025)

Functionally characterizing obesity-susceptibility genes using CRISPR/Cas9, in vivo imaging and deep learning.

Scientific reports, 15(1):5408.

Hundreds of loci have been robustly associated with obesity-related traits, but functional characterization of candidate genes remains a bottleneck. Aiming to systematically characterize candidate genes for a role in accumulation of lipids in adipocytes and other cardiometabolic traits, we developed a pipeline using CRISPR/Cas9, non-invasive, semi-automated fluorescence imaging and deep learning-based image analysis in live zebrafish larvae. Results from a dietary intervention show that 5 days of overfeeding is sufficient to increase the odds of lipid accumulation in adipocytes by 10 days post-fertilization (dpf, n = 275). However, subsequent experiments show that across 12 to 16 established obesity genes, 10 dpf is too early to detect an effect of CRISPR/Cas9-induced mutations on lipid accumulation in adipocytes (n = 1014), and effects on food intake at 8 dpf (n = 1127) are inconsistent with earlier results from mammals. Despite this, we observe effects of CRISPR/Cas9-induced mutations on ectopic accumulation of lipids in the vasculature (sh2b1 and sim1b) and liver (bdnf); as well as on body size (pcsk1, pomca, irs1); whole-body LDLc and/or total cholesterol content (irs2b and sh2b1); and pancreatic beta cell traits and/or glucose content (pcsk1, pomca, and sim1a). Taken together, our results illustrate that CRISPR/Cas9- and image-based experiments in zebrafish larvae can highlight direct effects of obesity genes on cardiometabolic traits, unconfounded by their - not yet apparent - effect on excess adiposity.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Caragine CM, Le VT, Mustafa M, et al (2025)

Comprehensive dissection of cis-regulatory elements in a 2.8 Mb topologically associated domain in six human cancers.

Nature communications, 16(1):1611.

Cis-regulatory elements (CREs), such as enhancers and promoters, are fundamental regulators of gene expression and, across different cell types, the MYC locus utilizes a diverse regulatory architecture driven by multiple CREs. To better understand differences in CRE function, we perform pooled CRISPR inhibition (CRISPRi) screens to comprehensively probe the 2.8 Mb topologically-associated domain containing MYC in 6 human cancer cell lines with nucleotide resolution. We map 32 CREs where inhibition leads to changes in cell growth, including 8 that overlap previously identified enhancers. Targeting specific CREs decreases MYC expression by as much as 60%, and cell growth by as much as 50%. Using 3-D enhancer contact mapping, we find that these CREs almost always contact MYC but less than 10% of total MYC contacts impact growth when silenced, highlighting the utility of our approach to identify phenotypically-relevant CREs. We also detect an enrichment of lineage-specific transcription factors (TFs) at MYC CREs and, for some of these TFs, find a strong, tumor-specific correlation between TF and MYC expression not found in normal tissue. Taken together, these CREs represent systematically identified, functional regulatory regions and demonstrate how the same region of the human genome can give rise to complex, tissue-specific gene regulation.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Liu Y, Zhang L, Lei W, et al (2025)

Development of a rapid and sensitive RPA-CRISPR/Cas12a assay for non-invasive pre-implantation genetic testing.

Analytica chimica acta, 1343:343687.

BACKGROUND: Pre-implantation genetic testing (PGT) is served as the primary technology for diagnosing genetic disorders. However, invasive operation may affect embryonic development, which indicates non-invasive methods might have important clinical value. Free DNA in blastocoele fluid provides the possibility for non-invasive diagnosis. The combination of RPA and CRISPR/Cas12a technology is expected to achieve analysis of free DNA in blastocoele fluid and develop an instant diagnostic platform for non-invasive PGT.

RESULTS: In this study, we collected 65 samples of day 6/7 blastocysts formed through intracytoplasmic sperm injection, and blastocysts hatched from the zona pellucida, with the corresponding blastocoele fluid, from the Center of Reproductive Medicine at the Second Affiliated Hospital of Zhengzhou University. The TSPY1 and TBC1D3 genes were analyzed using the RPA-CRISPR/Cas12a system to investigate the diagnostic potential of free DNA in the blastocoele fluid. A single-tube dual-gene assay for blastocoele fluid was successfully constructed using the RPA-CRISPR/Cas12a technology achieving specific detection of the Y chromosome and fluorescence visualization. Interpretatable results could be completed within 1h. By detecting the TSPY1 and TBC1D3 genes in 65 pairs of blastocysts, the accuracy of the Y chromosome in the interpretable results reached 95.4 %.

SIGNIFICANCE: Free DNA in the blastocoele fluid could serve as a genetic information source for non-invasive PGT. We first established a single-tube dual-gene RPA-CRISPR/Cas12a assay to detect free DNA in blastocoele fluid and achieved rapid amplification and detection with the advantages of easy operation and fluorescence visualization, providing a rapid detection platform for the diagnosis of sex-linked disorders.

RevDate: 2025-02-14
CmpDate: 2025-02-14

Varghese M, Kumar R, Sharma A, et al (2025)

Isopropylmalate synthase regulatory domain removal abolishes feedback regulation at the expense of leucine homeostasis in plants.

Plant physiology, 197(2):.

In the leucine (Leu) biosynthesis pathway, homeostasis is achieved through a feedback regulatory mechanism facilitated by the binding of the end product Leu at the C-terminal regulatory domain of the first committed enzyme, isopropylmalate synthase (IPMS). In vitro studies have shown that removing the regulatory domain abolishes the feedback regulation on plant IPMS while retaining its catalytic activity. However, the physiological consequences and underlying molecular regulation of Leu flux upon removing the IPMS regulatory domain remain to be explored in plants. Here, we removed the IPMS C-terminal regulatory domain using a CRISPR/Cas9-based gene editing system and studied the resulting impact on the Leu biosynthesis pathway under in planta conditions. Absence of the IPMS regulatory domain unexpectedly reduced the formation of the end product Leu but increased the levels of Leu pathway intermediates in mustard (Brassica juncea). Additionally, delayed growth was observed when IPMS devoid of the regulatory domain was introduced into IPMS-null mutants of Escherichia coli and Arabidopsis thaliana. Further, a detailed biochemical analysis showed that in the absence of the C-terminal regulatory domain, a Leu pathway intermediate (α-ketoisocaproate) could compete with the native IPMS substrate (2-oxoisovalerate) for the active site. Combining these metabolomic, biochemical, and in planta analyses, we demonstrate that the C-terminal regulatory domain of IPMS is critical for maintaining Leu-Val homeostasis in plants.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Rohm D, Black JB, McCutcheon SR, et al (2025)

Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing.

Cell genomics, 5(2):100770.

Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi syndrome (PWS) results from loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control the expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Kumar K, Crobu L, Thiam R, et al (2025)

Apoptotic proteins in Leishmania donovani: in silico screening, modeling, and validation by knock-out and gene expression analysis.

Parasite (Paris, France), 32:9.

Visceral leishmaniasis, a life-threatening vector-borne illness that disproportionately affects children and elderly immunocompromised people, is a primary tropical neglected disease. No apoptotic partner proteins have yet been reported in Leishmania donovani, while their identification could contribute to knowledge on parasite cell death and the establishment of alternative therapeutics. We searched for mammalian Bcl-2 family protein orthologs and found one anti-apoptotic and two pro-apoptotic orthologs in L. donovani. A pro-death aquaporin protein, due to its characteristic BH3 domain known to interact with pro-apoptotic proteins in mammalian Bcl-2 family proteins, was also included in this study. Molecular docking and molecular dynamics simulations were conducted to assess protein-protein interactions between the identified apoptotic proteins and mimic mammalian intrinsic apoptotic pathways. The results showed that both pro-apoptotic proteins interacted with the hydrophobic pocket of the anti-apoptotic ortholog, forming a stable complex. This interaction may represent a critical event in an apoptotic pathway in L. donovani. To further characterise it, we used CRISPR-Cas9 approaches to target the identified proteins. Pure knocked population mutants, and episomal over-expressing mutant cells were exposed to apoptotic stimuli. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and quantitative expression profiling suggested that these proteins are involved in the parasite's apoptosis and could play a role in its survival.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Fagerberg E, Attanasio J, Dien C, et al (2025)

KLF2 maintains lineage fidelity and suppresses CD8 T cell exhaustion during acute LCMV infection.

Science (New York, N.Y.), 387(6735):eadn2337.

Naïve CD8 T cells have the potential to differentiate into a spectrum of functional states during an immune response. How these developmental decisions are made and what mechanisms exist to suppress differentiation toward alternative fates remains unclear. We employed in vivo CRISPR-Cas9-based perturbation sequencing to assess the role of ~40 transcription factors (TFs) and epigenetic modulators in T cell fate decisions. Unexpectedly, we found that knockout of the TF Klf2 resulted in aberrant differentiation to exhausted-like CD8 T cells during acute infection. KLF2 was required to suppress the exhaustion-promoting TF TOX and to enable the TF TBET to drive effector differentiation. KLF2 was also necessary to maintain a polyfunctional tumor-specific progenitor state. Thus, KLF2 provides effector CD8 T cell lineage fidelity and suppresses the exhaustion program.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Deaver JW, Ryan PJ, O'Reilly CL, et al (2025)

MCF7 breast cancer anabolic capacity reduced with CRISPR/Cas9-mediated stable overexpression of DEPTOR.

American journal of physiology. Cell physiology, 328(2):C670-C678.

The hyperactivation of mTOR is a significant contributor to the development and progression of a number of human diseases, including a majority of human cancers. Although there have been many scientific and clinical efforts to reduce the impact of mTOR hyperactivation on downstream cellular metabolism, we aimed to mitigate this hyperactivation through a novel targeted gene edit of the intrinsic mTOR inhibitor, DEP domain containing MTOR interacting protein (DEPTOR), in MCF7 human breast cancer cells. Using publicly available bioinformatics tools, we demonstrate that DEPTOR gene expression is low in breast cancers compared with healthy tissues and that DEPTOR expression predicts overall survival, recurrence-free survival, and distant metastasis-free survival in breast cancer patients. We show that a directed overexpression of DEPTOR protein leads to significant alteration of downstream mTORC1 targets and subsequently reduces overall rates of protein synthesis. In addition, treatment of DEPTOR overexpressing cells with small-molecule DEPTOR inhibitor NSC126405 leads to a reversal of this effect, indicating a direct causal mechanism between DEPTOR protein levels and mTORC1 activation.NEW & NOTEWORTHY We identify DEPTOR as a predictor of mortality in breast cancer and show that precision gene editing to restore DEPTOR expression in breast cancer slows cell growth by inhibiting mTOR activity.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Lee H, Rho WY, Kim YH, et al (2025)

CRISPR-Cas9 Gene Therapy: Non-Viral Delivery and Stimuli-Responsive Nanoformulations.

Molecules (Basel, Switzerland), 30(3): pii:molecules30030542.

The CRISPR-Cas9 technology, one of the groundbreaking genome editing methods for addressing genetic disorders, has emerged as a powerful, precise, and efficient tool. However, its clinical translation remains hindered by challenges in delivery efficiency and targeting specificity. This review provides a comprehensive analysis of the structural features, advantages, and potential applications of various non-viral and stimuli-responsive systems, examining recent progress to emphasize the potential to address these limitations and advance CRISPR-Cas9 therapeutics. We describe how recent reports emphasize that nonviral vectors, including lipid-based nanoparticles, extracellular vesicles, polymeric nanoparticles, gold nanoparticles, and mesoporous silica nanoparticles, can offer diverse advantages to enhance stability, cellular uptake, and biocompatibility, based on their structures and physio-chemical stability. We also summarize recent progress on stimuli-responsive nanoformulations, a type of non-viral vector, to introduce precision and control in CRISPR-Cas9 delivery. Stimuli-responsive nanoformulations are designed to respond to pH, redox states, and external triggers, facilitate controlled and targeted delivery, and minimize off-target effects. The insights in our review suggest future challenges for clinical applications of gene therapy technologies and highlight the potential of delivery systems to enhance CRISPR-Cas9's clinical efficacy, positioning them as pivotal tools for future gene-editing therapies.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Zhou S, Tian D, Liu H, et al (2025)

Editing the RR-TZF Gene Subfamily in Rice Uncovers Potential Risks of CRISPR/Cas9 for Targeted Genetic Modification.

International journal of molecular sciences, 26(3): pii:ijms26031354.

The CRISPR/Cas9 system offers a powerful tool for gene editing to enhance rice productivity. In this study, we successfully edited eight RR-TZF genes in japonica rice Nipponbare using CRISPR-Cas9 technology, achieving a high editing efficiency of 73.8%. Sequencing revealed predominantly short insertions or deletions near the PAM sequence, along with multi-base deletions often flanked by identical bases. Off-target analysis identified 5 out of 31 predicted sites, suggesting the potential for off-target effects, which can be mitigated by designing gRNAs with more than three base mismatches. Notably, new mutations emerged in the progeny of several gene-edited mutants, indicating inheritable genetic mutagenicity. Phenotypic analysis of homozygous mutants revealed varied agronomic traits, even within the same gene, highlighting the complexity of gene-editing outcomes. These findings underscore the importance of backcrossing to minimize off-target and inheritable mutagenicity effects, ensuring more accurate trait evaluation. This study offers insights into CRISPR/Cas9 mechanisms and uncertain factors and may inform future strategies for rice improvement, prompting further research into CRISPR/Cas9's precision and long-term impacts.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Qi S, Wang Y, Liu Z, et al (2025)

Construction of a TAT-Cas9-EGFP Site-Specific Integration Eukaryotic Cell Line Using Efficient PEG10 Modification.

International journal of molecular sciences, 26(3): pii:ijms26031331.

The CRISPR/Cas9 system enables precise and efficient modification of eukaryotic genomes. Among its various applications, homology-directed repair (HDR) mediated knock-in (KI) is crucial for creating human disease models, gene therapy, and agricultural genetic enhancements. Despite its potential, HDR-mediated knock-in efficiency remains relatively low. This study investigated the impact of 5' end PEG10 modification on site-specific integration of the target gene. The HEK293 cell line is considered a highly attractive expression system for the production of recombinant proteins, with the construction of site-specific integration cell lines at the AAVS1 locus enabling stable protein expression. This study investigated the impact of the 5' end PEG10 modification on the site-specific integration of the target gene at the AAVS1 locus in the 293T cell line. Utilizing this 5' end PEG10 modification resulted in a 1.9-fold increase in knock-in efficiency for a 1.8 kb target fragment, improving efficiency from 26% to 49%. An optimized system was utilized to successfully establish a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP, providing a reliable resource of seed cells for subsequent protein production.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Liu Y, Elshan M, Li G, et al (2025)

Perspectives of Genome Editing Mediated Haploid Inducer Systems in Legumes.

International journal of molecular sciences, 26(3): pii:ijms26031154.

Genome editing-mediated haploid inducer systems (HISs) present a promising strategy for enhancing breeding efficiency in legume crops, which are vital for sustainable agriculture due to their nutritional benefits and ability to fix nitrogen. Traditional legume breeding is often slow and complicated by the complexity of legumes' genomes and the challenges associated with tissue culture. Recent advancements have broadened the applicability of HISs in legume crops, facilitating a reduction in the duration of the breeding cycle. By integrating genome editing technology with haploid breeding systems, researchers can achieve precise genetic modifications and rapidly produce homozygous lines, thereby significantly accelerating the development of desired traits. This review explores the current status and future prospects of genome editing-mediated HISs in legumes, emphasizing the mechanisms of haploid induction; recent breakthroughs; and existing technical challenges. Furthermore, we highlight the necessity for additional research to optimize these systems across various legume species, which has the potential to greatly enhance breeding efficiency and contribute to the sustainability of legume production.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Wang H, Yang H, Li T, et al (2025)

Optimization of CRISPR/Cas9 Gene Editing System in Sheep (Ovis aries) Oocytes via Microinjection.

International journal of molecular sciences, 26(3): pii:ijms26031065.

The CRISPR/Cas9 system has become a powerful tool for molecular design breeding in livestock such as sheep. However, the efficiency of the Cas9 system combined with zygote microinjection remains suboptimal. In this study, mature sheep oocytes were used for microinjection to assess the impact of various factors on Cas9 editing efficiency. We found that the in vitro maturation efficiency of oocytes is related to environmental factors such as air temperature, pressure, and humidity. Our results indicate that high-efficiency gene editing can be achieved when targeting the SOCS2, DYA, and TBXT, using a microinjection mixture with a concentration of 10 ng/μL Cas9 and sgRNA. By optimizing the injection capillary, we significantly reduced the oocyte invalidation rate post-microinjection to 3.1-5.3%. Furthermore, we observed that using either Cas9 protein or mRNA in the microinjection process resulted in different genotypes in the edited oocytes. Importantly, parthenogenetic activation did not appear to affect the editing efficiency. Using this high-efficiency system, we successfully generated SOCS2 or DYA gene-edited sheep, with all lambs confirmed to be genetically modified. This study presents a highly efficient method for producing gene-edited sheep, potentially enabling more precise and effective strategies for livestock breeding.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Danso Ofori A, Zheng T, Titriku JK, et al (2025)

The Role of Genetic Resistance in Rice Disease Management.

International journal of molecular sciences, 26(3): pii:ijms26030956.

Rice (Oryza sativa) is a crucial staple crop for global food security, particularly in Asia. However, rice production faces significant challenges from various diseases that can cause substantial yield losses. This review explores the role of genetic resistance in rice disease management, focusing on the molecular mechanisms underlying plant-pathogen interactions and strategies for developing resistant varieties. The paper discusses qualitative and quantitative resistance, emphasizing the importance of resistance (R) genes, defense-regulator genes, and quantitative trait loci (QTLs) in conferring broad-spectrum disease resistance. Gene-for-gene relationships in rice-pathogen interactions are examined, particularly for Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae. The review also covers recent advancements in breeding techniques, including marker-assisted selection, genetic engineering, and genome editing technologies like CRISPR-Cas. These approaches offer promising avenues for enhancing disease resistance in rice while maintaining yield potential. Understanding and exploiting genetic resistance mechanisms is crucial for developing durable and broad-spectrum disease-resistant rice varieties, essential for ensuring sustainable rice production and global food security in the face of evolving pathogen threats and changing environmental conditions.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Sosnovtseva AO, Le TH, Karpov DS, et al (2025)

Establishment of a Panel of Human Cell Lines to Identify Cellular Receptors Used by Enteroviruses to Infect Cells.

International journal of molecular sciences, 26(3): pii:ijms26030923.

Non-pathogenic natural and recombinant strains of human Enteroviruses are the subject of ongoing study with some strains having been approved for use as anticancer agents. The efficacy of oncolytic virotherapy depends upon identifying the receptor utilized by a specific strain for cell entry, and the presence of this receptor on the surface of cancer cells. Accordingly, a rapid and straightforward approach to determining the enteroviral receptors is necessary for developing an effective patient-specific, virus-based cancer therapy. To this end, we created a panel of seven lines with double knockouts on the background of the HEK293T cell line, which lacks the IFNAR1 gene. In these lines, the main viral receptor genes, including PVR, CXADR, CD55, ITGA2, SCARB2, ICAM1, and FCGRT, were knocked out using the CRISPR/Cas9 system. The panel of lines was validated on twelve different Enteroviruses types, providing a basis for studying the molecular mechanisms of enterovirus entry into cells, and for developing new therapeutic strains.

RevDate: 2025-02-12
CmpDate: 2025-02-13

Chey YCJ, Gierus L, Lushington C, et al (2025)

Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA transcript levels through scaffold poly-T tract reduction.

BMC genomics, 26(1):138.

Ensuring sufficient gRNA transcript levels is critical for obtaining optimal CRISPR-Cas9 gene editing efficiency. The standard gRNA scaffold contains a sequence of four thymine nucleotides (4T), which is known to inhibit transcription from Pol III promoters such as the U6 promoter. Our study showed that using standard plasmid transfection protocols, the presence of these 4Ts did not significantly affect editing efficiency, as most of the gRNAs tested (55 gRNAs) achieved near-perfect editing outcomes. We observed that gRNAs with lower activity were T-rich and had reduced gRNA transcript levels. However, this issue can be effectively resolved by increasing transcript levels, which can be readily achieved by shortening the 4T sequences. In this study, we demonstrated this by modifying the sequences to 3TC. Although the 3TC scaffold modification did not improve editing efficiency for already efficient gRNAs when high vector quantities were available, it proved highly beneficial under conditions of limited vector availability, where the 3TC scaffold yielded higher editing efficiency. Additionally, we demonstrated that the 3TC scaffold is compatible with SpCas9 high-fidelity variants and ABEmax base editing, enhancing their editing efficiency. Another commonly used natural Cas9 variant, SaCas9, also benefited from the 3TC scaffold sequence modification, which increased gRNA transcription and subsequently improved editing activity. This modification was applied to the EDIT-101 therapeutic strategy, where it demonstrated marked improvements in performance. This study highlights the importance of shortening the 4T sequences in the gRNA scaffold to optimize gRNA transcript expression for enhanced CRISPR-Cas9 gene editing efficiency. This optimization is particularly important for therapeutic applications, where the quantity of vector is often limited, ensuring more effective and optimal outcomes.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Xiong Y, Su Y, He R, et al (2025)

EXPERT expands prime editing efficiency and range of large fragment edits.

Nature communications, 16(1):1592.

Prime editing systems (PEs) hold great promise in modern biotechnology. However, their editing range is limited as PEs can only modify the downstream sequences of the pegRNA nick. Here, we report the development of the extended prime editor system (EXPERT) to overcome this limitation by using an extended pegRNA (ext-pegRNA) with modified 3' extension, and an additional sgRNA (ups-sgRNA) targeting the upstream region of the ext-pegRNA. We demonstrate that EXPERT can efficiently perform editing on both sides of the ext-pegRNA nick, a task that is unattainable by canonical PEs. EXPERT exhibits prominent capacity in replacing sequences up to 88 base pairs and inserting sequences up to 100 base pairs within the upstream region of the ext-pegRNA nick. Compared to canonical PEs such as PE2, the utilization of the EXPERT strategy significantly enhances the editing efficiency for large fragment edits with an average improvement of 3.12-fold, up to 122.1 times higher. Safety wise, the use of ups-sgRNA does not increase the rates of undesirable insertions and deletions (indels), as the two nicks are on the same strand. Moreover, we do not observe increased off-target editing rates genome-wide. Our work introduces EXPERT as a PE tool with significant potential in life sciences.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Ren LM, Qi YH, Cao FY, et al (2025)

Study on the framework of ATP energy cycle system in Escherichia coli.

Applied microbiology and biotechnology, 109(1):42.

The high mortality rate associated with single-use CRISPR-Cas9 in Escherichia coli limits its application. Recently, new CRISPR-based techniques for E.coli gene editing have emerged. Research aims to develop a system for rapid, marker-free, multi-site, and multi-copy genome editing in E.coli to advance synthetic biology. ATP, essential for energy in living organisms, plays a crucial role in various metabolic processes. To reduce the cost of ATP-requiring reactions, it is crucial to identify and efficiently express genes in ATP synthesis pathway. This study identified a single ppk gene (No.8) capable of completing the cyclic reaction. Using MUCICAT technology, the ppk gene (No.8) was inserted into various positions and copy numbers in the E.coli genome, resulting in different activity levels. The findings suggest that the difficulty of inserting the ppk gene (No.8) into the genome follows this order: IS186 < 8array < IS186 + 8array < IS1. A single genome insertion can mimic plasmid expression level. This study explores promoter competition and offers solutions, inspiring researchers in constructing the AMP-ATP cycle system in E.coli. KEY POINTS: • The single ppk gene (No.8) can regenerate the AMP-ATP cycle, crucial for ATP-dependent reactions. • Inserting the ppk gene (No.8) into the cr5 site of the E.coli genome achieves expression levels comparable to the pET29a plasmid. • The expression level of the ppk gene (No.8) is not significantly affected by its copy number in the E.coli genome.

RevDate: 2025-02-13
CmpDate: 2025-02-12

Choi J, Chen W, Liao H, et al (2025)

A molecular proximity sensor based on an engineered, dual-component guide RNA.

eLife, 13:.

One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here, we demonstrate a strategy termed 'P3 editing', which links protein-protein proximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells.

RevDate: 2025-02-12

Wang Z, Zhu X, Jiang T, et al (2025)

Alkaline Phosphatase-Regulated DNAzyme Cleavage Coupled with CRISPR/Cas12a for Quantitative Detection of Deoxynivalenol in Agricultural Crops.

Journal of agricultural and food chemistry [Epub ahead of print].

Sensitive and simplified detection of a mycotoxin such as deoxynivalenol (DON) is crucial for food safety. In recent years, the CRISPR/Cas technology has demonstrated significant potential in detecting non-nucleic acids. Herein, we present a triple enzyme-assisted fluorescence immunoassay (TEFIA) that integrates alkaline phosphatase (ALP)-regulated DNAzyme cleavage with the CRISPR/Cas12a assay for the accurate detection of mycotoxin. By employing this method for detecting DON, we exhibit a low detection limit of 0.05 ng/mL and a satisfactory linear response between 0.1 and 10 ng/mL. This performance exceeds the conventional sensitivity levels found in traditional methods. TEFIA also demonstrates a good correlation with ic-ELISA for testing DON in real samples. Thus, it offers a robust and efficient detection platform for DON in complex matrices. Furthermore, TEFIA can be employed to identify various targets of interest by merely altering the antibody-antigen pairs, indicating its great potential in a wide range of applications.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Liu S, Hamilton MC, Cowart T, et al (2025)

Characterization and bioinformatic filtering of ambient gRNAs in single-cell CRISPR screens using CLEANSER.

Cell genomics, 5(2):100766.

Single-cell RNA sequencing CRISPR (perturb-seq) screens enable high-throughput investigation of the genome, allowing for characterization of thousands of genomic perturbations on gene expression. Ambient gRNAs, which are contaminating gRNAs, are a major source of noise in perturb-seq experiments because they result in an excess of false-positive gRNA assignments. Here, we utilize CRISPR barnyard assays to characterize ambient gRNAs in perturb-seq screens. We use these datasets to develop CRISPR Library Evaluation and Ambient Noise Suppression for Enhanced single-cell RNA-seq (CLEANSER), a mixture model that filters ambient gRNAs. CLEANSER includes both gRNA and cell-specific normalization parameters, correcting for confounding technical factors that affect individual gRNAs and cells. The output of CLEANSER is the probability that a gRNA-cell assignment is in the native distribution over the ambient distribution. We find that ambient gRNA filtering methods impact differential gene expression analysis outcomes and that CLEANSER outperforms alternate approaches by increasing gRNA-cell assignment accuracy across multiple screen formats.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Zhang Y, Su R, Zhang Z, et al (2025)

An ultrasensitive one-pot Cas13a-based microfluidic assay for rapid multiplexed detection of microRNAs.

Biosensors & bioelectronics, 274:117212.

Aberrant microRNA expression is associated with tumor progression in various organs. Detecting microRNAs as clinical cancer biomarkers can facilitate early cancer diagnosis and monitoring. However, the rapid and accurate quantification of microRNAs from biological samples remains a significant challenge. Here we developed a one-pot isothermal assay utilizing a molecular circuit with CRISPR/Cas13a (CRISPR-circuit) to rapidly convert, amplify and report different microRNAs within 15 min at the attomolar (aM) level. Then the full process was performed on an active centrifugal microfluidic chip and its corresponding portable equipment for parallel detection of multiple microRNAs, including miR-21, miR-141, miR-196a, and miR-1246. We also demonstrated its application for identifying cell lines and clinical samples of cancer patients with varying microRNA levels, which showed a strong correlation with the RT-qPCR. The assay can be easily adapted for the detection of any microRNA by simply modifying the converter primer, thereby holding significant potential for accurate disease detection and clinical diagnosis.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Shu T, Yin X, Xiong Q, et al (2025)

Lift-CM: An integrated lift-heater centrifugal microfluidic platform for point-of-care pathogen nucleic acid detection using isothermal amplification and CRISPR/Cas12a.

Biosensors & bioelectronics, 274:117178.

Pathogen nucleic acid detection technology based on isothermal amplification and CRISPR/Cas12a system offers advantages in terms of high sensitivity, high specificity, and rapidity. However, this method has not been widely applied because of its shortcomings in utilizing conventional instruments, which cannot satisfy the requirements for Point of Care Testing (POCT), such as integration, convenience, and miniaturization. In this study, we developed an integrated lift-heater centrifugal microfluidic platform (Lift-CM) to automate the processes of isothermal amplification and CRISPR/Cas12a detection. A spatially encoded centrifugal microfluidic disc (SEC-disc) was employed to physically separate the amplification and detection processes while expanding the number of targets. The design of the dual-temperature and lift-heating centrifugal mechanism of the Lift-CM platform ensures that there is no manual intervention during amplification and detection processes. A smartphone-based app enables the setting of key parameters and monitoring of the experimental process, presenting results through a generated report that includes real-time fluorescence curves. We analyzed the plasmids of the Crimean-Congo hemorrhagic fever virus, Ebola virus, and five influenza viruses using different amplification methods (RPA/LAMP) to demonstrate the good compatibility of the Lift-CM platform with various amplification schemes. In clinical validation, the detection of H3N2-positive samples was completed within 30 min, and the results were highly consistent with qPCR results. This portable and compact platform offers a novel alternative solution for both clinical and at-home pathogen nucleic acid detection in the future.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Zhu F, Q Zhao (2025)

A CRISPR/Cas12a-based competitive aptasensor for ochratoxin A detection.

Analytical methods : advancing methods and applications, 17(7):1487-1492.

The serious contamination of ochratoxin A (OTA) in agricultural products has promoted the development of rapid, sensitive, and selective analytical methods for OTA monitoring. We demonstrated a competitive aptasensor for OTA detection using CRISPR/Cas12a as an effective signal amplifier. OTA competes with complementary DNA of the aptamer on the microplate to bind to the aptamer. Streptavidin bridges the biotinylated aptamer and biotinylated activator to convert the OTA input into Cas12a activation, which cleaves fluorescent DNA reporters. Under optimized experimental conditions, the aptasensor was demonstrated to work well for sensitive detection of OTA, with a linear range from 0.5 nM to 62.5 nM and a detection limit of 0.5 nM. Moreover, our method not only exhibits high selectivity, but also has satisfactory anti-interference ability against complex sample matrices. Taken together, the CRISPR/Cas12a-based competitive aptasensor offers a simple and sensitive platform for OTA detection, and it holds great promise for food security monitoring.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Zhao Y, Xie J, Yu S, et al (2025)

A novel method of species-specific molecular target mining and accurate discrimination of Bacillus cereus sensu lato.

International journal of food microbiology, 431:111068.

Bacillus cereus, a member of the Bacillus cereus sensu lato (B. cereus s.l.), is widely distributed in nature and can contaminate a variety of foods, leading to foodborne illnesses and substantial losses in the food industry. Although culture-based methods remain the gold standard for identifying B. cereus due to their high sensitivity under specific conditions, they are often complex and labor-intensive to implement. Furthermore, the high genetic similarity among certain members of the B. cereus s.l. makes it challenging to identify species-specific molecular targets, hindering the rapid and accurate differentiation of these bacteria. In this study, we introduce a novel method, comparative analysis based on whole genome slices (CAWGS), combined with the Basic Local Alignment Search Tool (BLAST) for efficient molecular target mining. Using CAWGS-BLAST and pan-genome analysis, we successfully identified new molecular targets for B. cereus, Bacillus thuringiensis, emetic B. cereus, Bacillus anthracis, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus megaterium. Based on these newly discovered targets, we developed a PCR-CRISPR/Cas12a method for detecting B. cereus s.l. and related species. Our research not only provides a rapid and accurate approach for discriminating B. cereus s.l. and related species, but also offers a universal and valuable reference for detecting foodborne pathogens, especially those with highly similar phenotypic and genetic characteristics.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Hashimoto T, Suenaga H, K Shin-Ya (2025)

Application of Cas9-Based Gene Editing to Engineering of Nonribosomal Peptide Synthetases.

Chembiochem : a European journal of chemical biology, 26(3):e202400765.

Engineering of nonribosomal peptide synthetases (NRPSs) could transform the production of bioactive natural product derivatives. A number of recent reports have described the engineering of NRPSs without marked reductions in yield. Comparative analysis of evolutionarily related NRPSs can provide insights regarding permissive fusion sites for engineering where recombination may occur during evolutionary processes. Studies involving engineering of NRPSs using these recombination sites showed that they have great potential. Moreover, we highlight recent advances in engineering of NRPSs using CRISPR-associated protein 9 (Cas9)-based gene editing technology. The use of Cas9 facilitates the editing of even larger biosynthetic gene clusters (BGCs) close to or over 100 kb in size by precisely targeting and digesting DNA sequences at specific sites. This technology combined with growing understanding of potential fusion sites from large-scale informatics analyses will accelerate the scalable exploration of engineered NRPS assembly lines to obtain bioactive natural product derivatives in high yields.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Balaraman AK, Babu MA, Moglad E, et al (2025)

Exosome-mediated delivery of CRISPR-Cas9: A revolutionary approach to cancer gene editing.

Pathology, research and practice, 266:155785.

Several molecular strategies based on targeted gene delivery systems have been developed in recent years; however, the CRISPR-Cas9 technology introduced a new era of targeted gene editing, precisely modifying oncogenes, tumor suppressor genes, and other regulatory genes involved in carcinogenesis. However, efficiently and safely delivering CRISPR-Cas9 to cancer cells across the cell membrane and the nucleus is still challenging. Using viral vectors and nanoparticles presents issues of immunogenicity, off-target effects, and low targeting affinity. Naturally, extracellular vesicles called exosomes have garnered the most attention as delivery vehicles in oncology-related CRISPR-Cas9 calls due to their biocompatibility, loading capacity, and inherent targeting features. The following review discusses the current progress in using exosomes to deliver CRISPR-Cas9 components, the approaches to load the CRISPR components into exosomes, and the modification of exosomes to increase stability and tumor-targeted delivery. We discuss the latest strategies in targeting recently accomplished in the exosome field, including modifying the surface of exosomes to enhance their internalization by cancer cells, as well as the measures taken to overcome the impacts of TME on delivery efficiency. Focusing on in vitro and in vivo experimentation, this review shows that exosome-mediated CRISPR-Cas9 can potentially treat cancer types, including pancreatic, lymphoma, and leukemia, for given gene targets. This paper compares exosome-mediated delivery and conventional vectors regarding safety, immune response, and targeting ability. Last but not least, we present the major drawbacks and potential development of the seemingly promising field of exosome engineering in gene editing, with references to CRISPR technologies and applications that may help make the target exosomes therapeutic in oncology.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Choudhery MS, Arif T, R Mahmood (2025)

Bidirectional Prime Editing: Combining Precision with Versatility for Genome Editing.

Cellular reprogramming, 27(1):10-23.

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

RevDate: 2025-02-13
CmpDate: 2025-02-13

Dong Y, Xu X, Qian L, et al (2025)

Genome-wide identification of yellow gene family in Hermetia illucens and functional analysis of yellow-y by CRISPR/Cas9.

Insect science, 32(1):115-126.

The yellow gene family plays a crucial role in insect pigmentation. It has potential for use as a visible marker gene in genetic manipulation and transgenic engineering in several model and non-model insects. Sadly, yellow genes have rarely been identified in Stratiomyidae species and the functions of yellow genes are relatively unknown. In the present study, we first manually annotated and curated 10 yellow genes in the black soldier fly (BSF), Hermetia illucens (Stratiomyidae). Then, the conserved amino acids in the major royal jelly proteins (MRJPs) domain, structural architecture and phylogenetic relationship of yellow genes in BSF were analyzed. We found that the BSF yellow-y, yellow-c and yellow-f genes are expressed at all developmental stages, especially in the prepupal stage. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we successfully disrupted yellow-y, yellow-c and yellow-f in the BSF. Consequently, the mutation of yellow-y clearly resulted in a pale-yellow body color in prepupae, pupae and adults, instead of the typical black body color of the wild type. However, the mutation of yellow-c or yellow-f genes did not result in any change in color of the insects, when compared with the wild type. Our study indicates that the BSF yellow-y gene plays a role in body pigmentation, providing an optimal marker gene for the genetic manipulation of BSF.

RevDate: 2025-02-13
CmpDate: 2025-02-13

Suchy FP, Karigane D, Nakauchi Y, et al (2025)

Genome engineering with Cas9 and AAV repair templates generates frequent concatemeric insertions of viral vectors.

Nature biotechnology, 43(2):204-213.

CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Liu C, Tan X, Wang J, et al (2025)

Upgrading of the genetic engineering toolkit accelerated the discovery process of the virulence effect of PsGH7d on Phytophthora sojae invasion.

Physiologia plantarum, 177(1):e70083.

The genus of Phytophthora includes numerous phytopathogens that have devastating impacts on agricultural production. However, the limited availability of selection markers for numerous pathogenicity pathogens of the genus Phytophthora genetic transformation hinders further research on their pathogenic functional genes. Here we report a gene of NAT I, which serves as a novel selection marker for the Phytophthora sojae transformation. Additionally, we developed a new genetic manipulation toolkit based on vectors containing NAT I, which facilitates gene editing in P. sojae. With the toolkit, the gene PsGH7d of P. sojae, which encodes a glycosyl hydrolase, was edited consecutively via the CRISPR/Cas9 system to obtain gene knockout and enzymatic active site mutation strains. The pathogenicity analysis of these transformants revealed that PsGH7d is a virulence factor dependent on its bifunctional glucanase-xylanase activities. This study develops an updated toolkit for the genus Phytophthora genetic transformation and provides initial insights into the virulence of the bifunctional enzyme PsGH7d.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Ghaznavi G, Vosough P, Ghasemian A, et al (2025)

Engineering bacteriophages for targeted superbug eradication.

Molecular biology reports, 52(1):221.

The rise of antibiotic-resistant bacteria, termed "superbugs," presents a formidable challenge to global health. These pathogens, often responsible for persistent nosocomial infections, threaten the effectiveness of conventional antibiotic therapies. This review delves into the potential of bacteriophages, viruses specifically targeting bacteria, as a powerful tool to combat superbugs. We examined the latest developments in genetic engineering that improve the efficacy of bacteriophages, focusing on modifications in host range, lysis mechanisms, and their ability to overcome bacterial defense systems. This review article highlights the CRISPR-Cas system as a promising method for precisely manipulating phage genomes, enabling the development of novel phage therapies with enhanced efficacy and specificity. Furthermore, we discussed developing novel phage-based strategies, such as phage cocktails and phage-antibiotic combinations. We also analyzed the challenges and ethical considerations associated with phage engineering, emphasizing the need for responsible and rigorous research to ensure this technology's safe and effective deployment to combat the growing threat of antibiotic resistance.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Li J, Liu S, Kim S, et al (2025)

Biomolecular condensation of human IDRs initiates endogenous transcription via intrachromosomal looping or high-density promoter localization.

Nucleic acids research, 53(4):.

Protein intrinsically disordered regions (IDRs) are critical gene-regulatory components and aberrant fusions between IDRs and DNA-binding/chromatin-associating domains cause diverse human cancers. Despite this importance, how IDRs influence gene expression, and how aberrant IDR fusion proteins provoke oncogenesis, remains incompletely understood. Here we develop a series of synthetic dCas9-IDR fusions to establish that locus-specific recruitment of IDRs can be sufficient to stimulate endogenous gene expression. Using dCas9 fused to the paradigmatic leukemogenic NUP98 IDR, we also demonstrate that IDRs can activate transcription via localized biomolecular condensation and in a manner that is dependent upon overall IDR concentration, local binding density, and amino acid composition. To better clarify the oncogenic role of IDRs, we construct clinically observed NUP98 IDR fusions and show that, while generally non-overlapping, oncogenic NUP98-IDR fusions convergently drive a core leukemogenic gene expression program in donor-derived human hematopoietic stem cells. Interestingly, we find that this leukemic program arises through differing mechanistic routes based upon IDR fusion partner; either distributed intragenic binding and intrachromosomal looping, or dense binding at promoters. Altogether, our studies clarify the gene-regulatory roles of IDRs and, for the NUP98 IDR, connect this capacity to pathological cellular programs, creating potential opportunities for generalized and mechanistically tailored therapies.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Muhar MF, Farnung J, Cernakova M, et al (2025)

C-terminal amides mark proteins for degradation via SCF-FBXO31.

Nature, 638(8050):519-527.

During normal cellular homeostasis, unfolded and mislocalized proteins are recognized and removed, preventing the build-up of toxic byproducts[1]. When protein homeostasis is perturbed during ageing, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites[2,3]. Such modifications have been proposed to trigger the selective removal of chemically marked proteins[3-6]; however, identifying modifications that are sufficient to induce protein degradation has remained challenging. Here, using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified FBXO31 as a reader of C-terminal amides. FBXO31 is a substrate receptor for the SKP1-CUL1-F-box protein (SCF) ubiquitin ligase SCF-FBXO31, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind to almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed after oxidative stress. A dominant human mutation found in neurodevelopmental disorders reverses CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Mouro Pinto R, Murtha R, Azevedo A, et al (2025)

In vivo CRISPR-Cas9 genome editing in mice identifies genetic modifiers of somatic CAG repeat instability in Huntington's disease.

Nature genetics, 57(2):314-322.

Huntington's disease, one of more than 50 inherited repeat expansion disorders[1], is a dominantly inherited neurodegenerative disease caused by a CAG expansion in HTT[2]. Inherited CAG repeat length is the primary determinant of age of onset, with human genetic studies underscoring that the disease is driven by the CAG length-dependent propensity of the repeat to further expand in the brain[3-9]. Routes to slowing somatic CAG expansion, therefore, hold promise for disease-modifying therapies. Several DNA repair genes, notably in the mismatch repair pathway, modify somatic expansion in Huntington's disease mouse models[10]. To identify novel modifiers of somatic expansion, we used CRISPR-Cas9 editing in Huntington's disease knock-in mice to enable in vivo screening of expansion-modifier candidates at scale. This included testing of Huntington's disease onset modifier genes emerging from human genome-wide association studies as well as interactions between modifier genes, providing insight into pathways underlying CAG expansion and potential therapeutic targets.

RevDate: 2025-02-12

Almotiri A, Abogosh A, Abdelfattah A, et al (2025)

Treating genetic blood disorders in the era of CRISPR-mediated genome editing.

Molecular therapy : the journal of the American Society of Gene Therapy pii:S1525-0016(25)00035-8 [Epub ahead of print].

In the setting of monogenic disease, advances made in genome editing technologies can, in principle, be deployed as a therapeutic strategy to precisely correct a specific gene mutation in an affected cell type and restore functionality. Using the β-hemoglobinopathies and hemophilia as exemplars, we review recent experimental breakthroughs using CRISPR-derived genome editing technology that have translated to significant improvements in the management of inherited hematologic disorders. Yet there are also challenges facing the use of CRISPR-mediated genome editing in these patients; we discuss possible ways to obviate those issues for furtherance of clinical benefit.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Pollitt SL, Levy AD, Anderson MC, et al (2025)

Large Donor CRISPR for Whole-Coding Sequence Replacement of Cell Adhesion Molecule LRRTM2.

The Journal of neuroscience : the official journal of the Society for Neuroscience, 45(7): pii:JNEUROSCI.1461-24.2024.

The cell adhesion molecule leucine-rich repeat transmembrane neuronal protein 2 (LRRTM2) is crucial for synapse development and function. However, our understanding of its endogenous trafficking has been limited due to difficulties in manipulating its coding sequence (CDS) using standard genome editing techniques. Instead, we replaced the entire LRRTM2 CDS by adapting a two-guide CRISPR knock-in method, enabling complete control of LRRTM2. In primary rat hippocampal cultures dissociated from embryos of both sexes, N-terminally tagged, endogenous LRRTM2 was found in 80% of synapses, and synaptic LRRTM2 content correlated with PSD-95 and AMPAR levels. LRRTM2 was also enriched with AMPARs outside synapses, demonstrating the sensitivity of this method to detect relevant new biology. Finally, we leveraged total genomic control to increase the synaptic levels of LRRTM2 via simultaneous mutation of its C-terminal domain, which did not correspondingly increase AMPAR enrichment. The coding region of thousands of genes span lengths suitable for whole-CDS replacement, suggesting this simple approach will enable straightforward structure-function analysis in neurons.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Libby ARG, Rito T, Radley A, et al (2025)

An in vivo CRISPR screen in chick embryos reveals a role for MLLT3 in specification of neural cells from the caudal epiblast.

Development (Cambridge, England), 152(3):.

Tissue development relies on the coordinated differentiation of stem cells in dynamically changing environments. The formation of the vertebrate neural tube from stem cells in the caudal lateral epiblast is a well-characterized example. Despite an understanding of the signalling pathways involved, the gene regulatory mechanisms remain poorly defined. To address this, we developed a multiplexed in vivo CRISPR screening approach in chick embryos targeting genes expressed in the caudal epiblast and neural tube. This revealed a role for MLLT3, a component of the super elongation complex, in the specification of neural fate. Perturbation of MLLT3 disrupted neural tube morphology and reduced neural fate acquisition. Mutant forms of retinoic acid receptor A lacking the MLLT3 binding domain similarly reduced neural fate acquisition. Together, these findings validate an in vivo CRISPR screen strategy in chick embryos and identify a previously unreported role for MLLT3 in caudal neural tissue specification.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Huang H, Hu C, Na J, et al (2025)

Functional evaluation and clinical classification of BRCA2 variants.

Nature, 638(8050):528-537.

Germline BRCA2 loss-of function variants, which can be identified through clinical genetic testing, predispose to several cancers[1-5]. However, variants of uncertain significance limit the clinical utility of test results. Thus, there is a need for functional characterization and clinical classification of all BRCA2 variants to facilitate the clinical management of individuals with these variants. Here we analysed all possible single-nucleotide variants from exons 15 to 26 that encode the BRCA2 DNA-binding domain hotspot for pathogenic missense variants. To enable this, we used saturation genome editing CRISPR-Cas9-based knock-in endogenous targeting of human haploid HAP1 cells[6]. The assay was calibrated relative to nonsense and silent variants and was validated using pathogenic and benign standards from ClinVar and results from a homology-directed repair functional assay[7]. Variants (6,959 out of 6,960 evaluated) were assigned to seven categories of pathogenicity based on a VarCall Bayesian model[8]. Single-nucleotide variants that encode loss-of-function missense variants were associated with increased risks of breast cancer and ovarian cancer. The functional assay results were integrated into models from ClinGen, the American College of Medical Genetics and Genomics, and the Association for Molecular Pathology[9] for clinical classification of BRCA2 variants. Using this approach, 91% were classified as pathogenic or likely pathogenic or as benign or likely benign. These classified variants can be used to improve clinical management of individuals with a BRCA2 variant.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Sahu S, Galloux M, Southon E, et al (2025)

Saturation genome editing-based clinical classification of BRCA2 variants.

Nature, 638(8050):538-545.

Sequencing-based genetic tests have uncovered a vast array of BRCA2 sequence variants[1]. Owing to limited clinical, familial and epidemiological data, thousands of variants are considered to be variants of uncertain significance[2-4] (VUS). Here we have utilized CRISPR-Cas9-based saturation genome editing in a humanized mouse embryonic stem cell line to determine the functional effect of VUS. We have categorized nearly all possible single nucleotide variants (SNVs) in the region that encodes the carboxylate-terminal DNA-binding domain of BRCA2. We have generated function scores for 6,551 SNVs, covering 96.4% of possible SNVs in exons 15-26 spanning BRCA2 residues 2479-3216. These variants include 1,282 SNVs that are categorized as missense VUS in the clinical variant database ClinVar, with 77.2% of these classified as benign and 20.4% classified as pathogenic using our functional score. Our assay provides evidence that 3,384 of the SNVs in the region are benign and 776 are pathogenic. Our classification aligns closely with pathogenicity data from ClinVar, orthogonal functional assays and computational meta predictors. We have integrated our embryonic stem cell-based BRCA2-saturation genome editing dataset with other available evidence and utilized the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines for clinical classification of all possible SNVs. This classification is available as a sequence-function map and serves as a valuable resource for interpreting unidentified variants in the population and for physicians and genetic counsellors to assess BRCA2 VUS in patients.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Cao XX, Yuan JJ, Bai ZY, et al (2025)

Effect of CHO cell line constructed with CMAH gene-directed integration on the recombinant protein expression.

International journal of biological macromolecules, 292:139274.

Chinese hamster ovary (CHO) cells are the most widely used platform for recombinant therapeutic protein (RTP) production. Traditionally, the development of CHO cell lines has mainly depended on random integration of transgenes into the genome, which is not conducive to stable long-term expression. Cytidine monophosphate N-acetylneuraminic acid hydroxylase (CMAH) is expressed in CHO cells and produces N-hydroxyacetylneuraminic acid, which may cause a human immune response. However, the effects of transgene integration at the CMAH site on RTP expression in CHO cells remain unclear. In this study, we selected CMAH gene, which is lacking in humans, as the target site to construct recombinant CHO cell line using the CRISPR/Cas9 technique. Erythropoietin (EPO) and EGFP integration at the CMAH site resulted in more stable expression levels and lower heterogeneity than random integration. In addition, the proportion of N-glycosylation levels in the EPO glycoside of CMAH integration site also changed. In conclusion, CMAH site integration improved the stability of RTP expression in CHO cells.

RevDate: 2025-02-12
CmpDate: 2025-02-12

Liu X, Zhao Z, Li W, et al (2025)

Rationally Engineering Pro-Proteins and Membrane-Penetrating α‑Helical Polypeptides for Genome Editing Toward Choroidal Neovascularization Treatment.

Advanced materials (Deerfield Beach, Fla.), 37(6):e2412366.

Ribonucleoprotein (RNP)-based CRISPR/Cas9 genome editing holds great potential for the treatment of choroidal neovascularization (CNV), which however, is challenged by the lack of efficient cytosolic protein delivery tools. Herein, reversibly-phosphorylated pro-proteins (P-proteins) with conjugated adenosine triphosphate (ATP) tags are engineered and coupled with a membrane-penetrating, guanidine-enriched, α-helical polypeptide (LGP) to mediate robust and universal cytosolic delivery. LGP forms salt-stable nanocomplexes (NCs) with P-proteins via electrostatic interaction and salt bridging, and the helix-assisted, strong membrane activities of LGP enabled efficient cellular internalization and endolysosomal escape of NCs. Therefore, this approach allows efficient cytosolic delivery of a wide range of protein cargoes and maintains their bioactivities due to endolysosomal acidity-triggered traceless restoration of P-proteins. Notably, intravitreally delivered LGP/P-RNP NCs targeting hypoxia-inducible factor-1α (HIF-1α) induce pronounced gene disruption to downregulate pro-angiogenic factors and alleviate subretinal fibrosis, ultimately provoking robust therapeutic efficacy in CNV mice. Such a facile and versatile platform provides a powerful tool for cytosolic protein delivery and genome editing, and it holds promising potential for the treatment of CNV-associated diseases, such as age-related macular degeneration.

RevDate: 2025-02-11

Paauw M, Schravesande WEW, Taks NW, et al (2025)

Evolution of a vascular plant pathogen is associated with the loss of CRISPR-Cas and an increase in genome plasticity and virulence genes.

Current biology : CB pii:S0960-9822(25)00003-X [Epub ahead of print].

A major question in infectious disease research is how bacteria have evolved into highly niche-adapted pathogens with efficient host infection strategies. The plant pathogenic bacterium Xanthomonas campestris is subdivided into pathovars that occupy two distinct niches of the same plant leaf: the vasculature and the mesophyll tissue. Using a pangenome comparison of 94 X. campestris isolates, we discovered that the vasculature-infecting pathovar emerged in one monophyletic clade, has lost its CRISPR-Cas system, and showed an increase in both genomic plasticity and acquisition of virulence factors, such as type III effector proteins, compared with the ancestral pathovar. In addition, we show that the CRISPR spacers of isolates belonging to the ancestral pathovar map to plasmids that circulate in Xanthomonas populations and encode high numbers of transposons and virulence factors, suggesting that CRISPR-Cas restricts gene flow toward this pathovar. Indeed, we demonstrate experimentally reduced plasmid uptake in a CRISPR-Cas-encoding isolate. Based on our data, we propose that the loss of the CRISPR-Cas system was a pivotal step in X. campestris evolution by facilitating increased genome dynamics and the emergence of the vasculature-adapted X. campestris pathovar campestris, a major pathogen of Brassica crops.

RevDate: 2025-02-11

Singh MA, Chang MM, Wang Q, et al (2025)

Rapid Enzymatic Assay for Antiretroviral Drug Monitoring Using CRISPR-Cas12a-Enabled Readout.

ACS synthetic biology [Epub ahead of print].

Maintaining the efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse transcriptase ACTivity crispR (REACTR) for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to the synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR's sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Arshad S, Qadir ML, Hussain N, et al (2025)

Advances in CRISPR/Cas9 technology: shaping the future of photosynthetic microorganisms for biofuel production.

Functional plant biology : FPB, 52:.

Use of fossil fuels causes environmental issues due to its inefficiency and and imminent depletion. This has led to interest in identifying alternative and renewable energy sources such as biofuel generation from photosynthetic organisms. A wide variety of prokaryotic and eukaryotic microorganisms, known as microalgae, have the potential to be economical and ecologically sustainable in the manufacture of biofuels such as bio-hydrogen, biodiesel, bio-oils, and bio-syngas. By using contemporary bioengineering techniques, the innate potential of algae to produce biomass of superior quality may be enhanced. In algal biotechnology, directed genome modification via RNA-guided endonucleases is a new approach. CRISPR/Cas systems have recently been frequently used to modify the genetic makeup of several aquatic and freshwater microalgae. The majority of research has used the Cas9-driven Type II system, one of two classes and six unique kinds of CRISPR systems, to specifically target desired genes in algae, and knock them out and down, or both. Using CRISPR technology to modify its genetic makeup, microalgae has produced more biomass and increased in lipid content. This review highlights the attempts made so far to target microalgae genome modification, discusses the prospects for developing the CRISPR platform for large-scale genome modification of microalgae, and identifies the opportunities and challenges in the development and distribution of CRISPR/Cas9 components.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Mohamed AA, Wang PY, Bartel DP, et al (2025)

The structural basis for RNA slicing by human Argonaute2.

Cell reports, 44(1):115166.

Argonaute (AGO) proteins associate with guide RNAs to form complexes that slice transcripts that pair to the guide. This slicing drives post-transcriptional gene silencing through RNA interference (RNAi), which is essential for many eukaryotes and the basis for new clinical therapies. Despite this importance, structural information on eukaryotic AGOs in a fully paired, slicing-competent conformation-hypothesized to be intrinsically unstable-has been lacking. Here, we present the cryogenic electron microscopy structure of a human AGO-guide complex bound to a fully paired target, revealing structural rearrangements that enable this conformation. Critically, the N domain of AGO rotates to allow the RNA full access to the central channel and forms contacts that license rapid slicing. Moreover, a conserved loop in the PIWI domain secures the RNA near the active site to enhance slicing rate and specificity. These results explain how AGO accommodates targets possessing pairing specificity typically observed in biological and clinical slicing substrates.

RevDate: 2025-02-11

Huq TB, Anil Kumar Jeeja P, Dam SK, et al (2025)

Recent Applications of Mesoporous Silica Nanoparticles in Gene Therapy.

Advanced healthcare materials [Epub ahead of print].

Gene therapy offers transformative potential for treating genetic disorders by directly addressing the molecular root causes of diseases. However, the primary challenges of gene therapy involve the efficient delivery of therapeutic genetic material to target cells, crossing biological barriers, managing toxicity and immune responses. Mesoporous silica nanoparticles (MSNs), due to their unique structural features have emerged as a promising platform to overcome these challenges. In recent years, MSNs have gained significant attention as potential nanocarriers for the efficient delivery of various nucleic acids. This review comprehensively examines the role of MSNs in gene therapy, focusing on their capabilities in the targeted delivery of siRNA, DNA, CRISPR-Cas systems, and other genetic therapeutics. This work explores the modern advancements in MSNs synthesis and functionalization strategies and the impact of structural modifications on their stability, cellular uptake, and controlled release under physiological conditions. Additionally, the review highlights the use of MSNs to develop theranostic systems, where gene delivery is combined with diagnostic imaging for real-time monitoring and personalized treatment strategies. Finally, this work discusses the future perspectives of MSNs in gene delivery, addressing regulatory challenges, enhancing clinical translation, and expanding their application for treating various genetic disorders and cancers.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Léger-Charnay E, Slembrouck-Brec A, O Goureau (2025)

Engineering Specific Human iPS Reporter Cell Lines to Generate Optogenetically Modified Photoreceptors.

Advances in experimental medicine and biology, 1468:409-414.

Cell therapy, by transplantation of photoreceptors derived from induced pluripotent stem cells (iPSCs), has been proposed as a promising therapeutic approach for photoreceptor degenerative diseases. A remaining obstacle is that such transplanted cells have to develop into functional light-sensitive photoreceptors, which require outer segment formation and interaction with the underlying retinal pigmented epithelium (RPE). To overcome this limitation, a combination of cell therapy and optogenetics allows to confer light sensitivity to the donor cells thanks to the expression of a microbial opsin and therefore independently of the formation of mature outer segment or RPE contact. To ensure stable and homogenous expression of the microbial opsin in photoreceptors, we inserted the coding sequence of the red-light sensitive chloride pump Jaws under specific photoreceptor promoter into the iPSC genome, using the CRISPR/Cas9 system at the safe AAVS1 locus. We successfully generated a knock-in Jaws-EGFP iPSC line and validated its stemness and pluripotency status. These engineered iPSCs will be used to produce photoreceptors expressing Jaws that will be grafted to assess their ability to restore vision in blind animal models.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Xiao H, Marshall R, Saxena MT, et al (2025)

The Power of Zebrafish in Disease Modeling and Therapy Discovery for Inherited Retinal Degeneration.

Advances in experimental medicine and biology, 1468:229-233.

In the research of inherited retinal degeneration (IRD), zebrafish have emerged as a powerful model system, offering profound insights into disease mechanisms and opening new therapeutic avenues. This mini-review discusses the distinctive advantages that zebrafish provide for investigating retinal degeneration. It outlines contemporary genetic tools, with a specific focus on advanced CRISPR/Cas9 gene targeting technology, utilized for genome manipulation and disease modeling in zebrafish. By emphasizing the pivotal role of zebrafish in large-scale high-throughput drug discovery and the exploration of innovative gene therapy strategies, this succinct review underscores the adaptability and significance of the zebrafish model in advancing IRD research. It establishes a robust foundation for future studies and therapeutic developments in the field.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Xia X, Liang Z, Xu G, et al (2025)

Split crRNA Precisely Assisted Cas12a Expansion Strategy for Simultaneous, Discriminative, and Low-Threshold Determination of Two miRNAs Associated with Multiple Sclerosis.

Analytical chemistry, 97(5):2873-2882.

Multiple sclerosis (MS) can proceed into secondary progressive MS accompanied by persistent neurological deterioration; therefore, accurate diagnosis of MS is of vital significance. Irregularities of microRNAs (miRNAs) expression have been observed in MS, so miRNAs have been evaluated as novel biomarkers and therapeutic targets. Herein, a new strategy named split crRNA precisely assisted Cas12a expansion (SPACE) was developed for simultaneous, discriminative, and low-threshold determination of two MS-related miRNAs: miRNA-155 and miRNA-326. On the one hand, owing to the property that split crRNA could activate Cas12a, miRNAs were designed as the spacers of crRNA to combine with scaffold. These integrated crRNAs then recognized the activators, activating Cas12a and enabling RNA target identification. On the other hand, the SPACE strategy dexterously integrated the activator with reporter probe, and utilized Cas12a's cis-cleavage to achieve simultaneous detection and differential signal output for miRNA-155 and miRNA-326. Moreover, trans-cleavage with ultra-high efficiency was assembled in the SPACE strategy to achieve sensitive quantification of total miRNAs in blood samples at low thresholds. Overall, the diversified and integrated design of the SPACE strategy enabled simultaneous, discriminative, and low-threshold detection of dual MS-related miRNAs in one pot and one step, providing a reliable and accurate Cas12a detection tool for clinical low-threshold diagnosis.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Huang D, He Y, Xu C, et al (2025)

DNAzyme-Triggered Equilibrium Transfer with Self-Activated CRISPR-Cas12a Biosensor Enables One-Pot Diagnosis of Nucleic Acids.

Analytical chemistry, 97(5):3026-3035.

Integrating recombinase-polymerase amplification (RPA) with CRISPR-Cas12a holds significant potential to simplify and improve nucleic acid diagnostic procedures. However, current strategies face limitations, such as complexity, reduced efficiency, and potential compromises in Cas12a activity. In response, we developed a DNAzyme-triggered equilibrium transfer with a self-activated CRISPR-Cas12a biosensor (DESCRIBER) for integrated nucleic acid detection. This platform features varying balance points to minimize interference between RPA and Cas12a in one pot and maximize their activity at different stages. Initially, the reaction focused on RPA, while Cas12a was silenced by circular-crRNA (C-crRNA). Then, DNAzyme, the activator, was generated during the RPA process, which linearizes C-crRNA to activate Cas12a and transfer the equilibrium toward signal readout. Meanwhile, activated Cas12a can further linearize C-crRNA to promote self-activation and accelerate equilibrium transfer. According to this principle, highly sensitive detection of the HIV-1 genome, as low as 500 CPs/mL, was achieved within 1 h while maintaining universality in detecting common subtypes and specificity against opportunistic infectious pathogens. Compared with qRT-PCR, it also exhibited good accuracy in detecting 35 spiked samples. Overall, we believe that the proposed strategy will enhance existing CRISPR systems to promote their practical applications in clinical diagnosis.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Jones JD, TD Maloney (2025)

sgRNA Single-Nucleotide Resolution by Ion-Pairing Reversed-Phase Chromatography.

Analytical chemistry, 97(5):2837-2844.

Single-stranded guide RNAs (sgRNAs) are important therapeutic modalities that facilitate selective genome editing by the CRISPR/Cas9 system. While these therapeutic modalities are synthesized through solid phase oligonucleotide synthesis similar to small interfering RNA (siRNAs) and antisense oligonucleotide (ASOs) therapeutics, their sequence length and complex secondary and tertiary structure hinder analytical characterization. The resulting current sgRNA methodologies have limited chromatographic selectivity near the FLP and limited MS compatibility. Here, we perform a systematic evaluation of chromatographic parameters to develop a highly selective ion-pairing reversed-phase chromatography separation for sgRNAs. We identify that stronger and more hydrophobic ion-pairing reagents promote the selectivity for long truncation impurities. Further, reduced flow rates and temperatures promote selectivity near the FLP. Together, this study reports the first single-nucleotide resolution chromatography method for an sgRNA modality while employing a highly MS-compatible mobile phase. This work provides further evidence that the sgRNA chromatographic selectivity is highly independent of on-column mass transfer. This methodology will enable the high-resolution characterization of sgRNA therapeutics, providing further insights into impurity profiles to facilitate toxicological studies and process development of these genetic medicines.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Hu C, Xiang H, Yin Y, et al (2025)

Electrochemiluminescence Resonance Energy Transfer Biosensor for the Human-Associated Clade of Streptococcus suis Based on Prereduction-Enhanced Yttrium MOFs.

Analytical chemistry, 97(5):3153-3160.

Streptococcus suis, a significant zoonotic pathogen, annually caused substantial economic losses in the swine industry and had intensified threat to public health due to the recent emergence of human-associated clade. In this study, we discovered that the rare-earth metal-based metal-organic frameworks (Y-BTC) possessed excellent ECL capabilities. After prereduction at high voltage, its ECL intensity was enhanced by two times. Subsequently, we developed an efficient CRISPR/Cas12a-mediated electrochemiluminescence resonance energy transfer (ECL-RET) biosensor utilizing Y-BTC for the detection of the human-associated S. suis clade. Y-BTC was employed as the ECL-RET donor and ECL emitter, and the spherical nucleic acid Au NP was utilized as the ECL-RET receptor. In the presence of the target, isothermal amplification was triggered to generate a large number of amplicons, which subsequently activated the trans-cleavage activity of Cas12a. Cas12a cleaved the nucleic acid shell on the surface of Au NPs, reducing the spatial distance between Au NPs and Y-BTC due to electrostatic adsorption, thereby quenching the ECL of Y-BTC via ECL-RET. Consequently, the presence of targets can be observed by a reduced ECL signal. The sensor exhibited a detection range of 25 pM to 50 nM, with a detection limit as low as 17 pM. The practical utility was verified through actual sample testing. Our proposed ECL-RET sensing strategy provides a new avenue for the sensitive detection of S. suis. The universality has also been demonstrated using Fusobacterium nucleatum, Salmonella pullorum, and Listeria monocytogenes, holding great promise in the field of food safety and public health.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Zhang X, Wang F, Guo X, et al (2025)

Developing a RecT-assisted endogenous CRISPR/SzCas9 system for precise genome editing in Streptococcus zooepidemicus.

International journal of biological macromolecules, 291:138758.

Streptococcus zooepidemicus is the most commonly utilized strain for industrial-scale hyaluronic acid (HA) production due to its capacity to produce high-quality HA. However, the lack of efficient genetic manipulation approaches has significantly hindered the industrial application of this species. In this study, we developed a RecT-assisted endogenous CRISPR/SzCas9 system to enable markerless gene deletion, gene substitution, stop codon insertion, and iterative editing in the industrially significant strain S. zooepidemicus for hyaluronic acid production. A variety of editing outcomes were achieved, including a 3186-bp deletion of the hylb gene, replacement of VHb, and insertion of stop codons into the sagA gene. Ultimately, we successfully developed an engineered strain, designated SD-3, which exhibited reduced toxicity and non-hemolytic properties. The hyaluronic acid production of SD-3 increased by 39.7%, reaching 10.8 g/L in a 5-L fermenter. This achievement represents the highest titer of high molecular weight hyaluronic acid to date, characterized by a uniform and high molecular weight of up to 2.8 × 10[6] Da.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Zou J, Meng X, Hong Z, et al (2025)

Cas9-PE: a robust multiplex gene editing tool for simultaneous precise editing and site-specific random mutation in rice.

Trends in biotechnology, 43(2):433-446.

In molecular design breeding, the simultaneous introduction of desired functional genes through specific nucleotide modifications and the elimination of genes regulating undesired phenotypic traits or agronomic components require advanced gene editing tools. Due to limited editing efficiency, even with the use of highly precise editing tools, such as prime editing (PE), simultaneous editing of multiple mutation types poses a challenge. Here, we replaced Cas9 nickase (nCas9) with Cas9 to construct a Cas9-mediated PE (Cas9-PE) system in rice. This system not only enables precise editing, but also allows for site-specific random mutation. Moreover, leveraging the precision of Cas9-PE, we established a transgene-free multiplex gene editing system using a co-editing strategy. This strategy involved the Agrobacterium-mediated transient expression of the precise editing rice endogenous acetolactate synthase gene ALS[S627I] to confer herbicide bispyribac-sodium (BS) resistance as a selection marker. This study provides a versatile and efficient multiplex gene editing tool for molecular design breeding.

RevDate: 2025-02-11
CmpDate: 2025-02-11

Carter JL, Halmai JANM, Waldo JJ, et al (2025)

A de novo missense mutation in PPP2R5D alters dopamine pathways and morphology of iPSC-derived midbrain neurons.

Stem cells (Dayton, Ohio), 43(1):.

Induced pluripotent stem cell (iPSC) models of neurodevelopmental disorders (NDDs) have promoted an understanding of commonalities and differences within or across patient populations by revealing the underlying molecular and cellular mechanisms contributing to disease pathology. Here, we focus on developing a human model for PPP2R5D-related NDD, called Jordan syndrome, which has been linked to Early-Onset Parkinson's Disease (EOPD). Here we sought to understand the underlying molecular and cellular phenotypes across multiple cell states and neuronal subtypes in order to gain insight into Jordan syndrome pathology. Our work revealed that iPSC-derived midbrain neurons from Jordan syndrome patients display significant differences in dopamine-associated pathways and neuronal architecture. We then evaluated a CRISPR-based approach for editing heterozygous dominant G-to-A mutations at the transcript level in patient-derived neural stem cells. Our findings show that site-directed RNA editing is influenced by sgRNA length and cell type. These studies support the potential for a CRISPR RNA editor system to selectively edit mutant transcripts harboring G-to-A mutations in neural stem cells while providing an alternative editing technology for those suffering from NDDs.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Ahmadian M, Okan ICT, Uyanik G, et al (2025)

Precise Gene Editing Technologies in Retinal Applications.

Advances in experimental medicine and biology, 1468:119-123.

Gene therapy is emerging as a promising treatment for inherited retinal diseases (IRDs). One of the first successful applications of gene therapy for IRDs was the gene replacement therapy for the RPE65 mutation. This therapy delivers a functional copy of the RPE65 gene to patients via AAV vectors, rather than targeting the mutation itself. Gene editing technologies have advanced significantly in recent years, allowing it to make precise in vivo modifications to the genetic code. After the discovery of CRISPR-Cas9, other gene editing technologies such as base editing and prime editing have been developed by modifying and combining the original CRISPR-Cas9 technology with other methods. Moreover, recently discovered CRISPR-Cas systems allow RNA editing to correct mutations at the posttranscriptional level. These technologies have potential applications in various fields, including inherited retinal diseases. This mini-review evaluates and summarizes the most current advancements in genome editing methods, including prime editing, base editing, and RNA editing, and their applications on retinal diseases.

RevDate: 2025-02-10
CmpDate: 2025-02-10

da Costa BL, Pincay J, Brodie SE, et al (2025)

Prime Editing Strategy to Install the Mfrp Retinal Degeneration 6 Mutation.

Advances in experimental medicine and biology, 1468:113-118.

Mutations in the MFRP (membrane-type frizzled-related protein) gene are associated with a spectrum of ocular diseases. Here, we report on a patient with MFRP-linked autosomal recessive retinitis pigmentosa (arRP) with nanophthalmos who exhibited yellow deposits circumferentially along with far temporal intraretinal pigment migration. In future studies, we plan to explore the amelioration of MFRP-associated phenotypes in patient-specific induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium and in vivo using the classical Mfrp[rd6] mouse model of RP. To effectively screen gene editing correction approaches for the Mfrp[rd6] mouse model, we require a strategy to install the desired mutation in the Neuro-2a (N2a) mouse neuroblastoma cell line. In this study, we developed a prime editing strategy for the installation of the Mfrp[rd6] c.445+3_6AAGTdel mutation.

RevDate: 2025-02-10
CmpDate: 2025-02-10

da Costa BL, Knudsen AS, Alves CH, et al (2025)

Megabase Deletion of the Human EYS Locus Using CRISPR/Cas9.

Advances in experimental medicine and biology, 1468:107-111.

Mutations in the Eyes Shut Homolog (EYS) gene are associated with autosomal recessive retinitis pigmentosa (arRP). To date, four retinal isoforms of EYS have been identified. However, the precise retinal function of EYS is not fully understood, but it has apparent roles in retinal morphogenesis, architecture, and ciliary transport. Clustered-regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) nuclease-mediated approaches are powerful tools for genome engineering in mammalian cells. The use of paired CRISPR/Cas9-induced double-strand breaks (DSBs) using dual single guide RNAs (sgRNA) can lead to precise genomic deletions. In this study, we developed a dual sgRNA strategy to facilitate CRISPR/Cas9-mediated deletion of 1,988,210 bp of the EYS locus, removing the four currently identified human retinal EYS isoforms. This approach can be used to produce EYSdel induced pluripotent stem cell (iPSC) lines to explore the function of EYS in human iPSC-derived retinal organoids.

RevDate: 2025-02-10
CmpDate: 2025-02-11

da Costa BL, Caruso SM, Tsai YT, et al (2025)

Prime Editing Strategy to Install the RPE65 c.1430A>G Dominant Mutation.

Advances in experimental medicine and biology, 1468:101-106.

The retinal pigment epithelium 65-kDa protein (RPE65) is a retinal isomerase that is an essential component of the visual cycle. Mutations in RPE65 are typically associated with autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. Here, we report on a patient with RPE65-mediated autosomal dominant retinitis pigmentosa (adRP) who has widespread chorioretinal atrophy with significant macular involvement and only small areas of retinal preservation. In future studies, we plan to model the pathobiology of RPE65-mediated adRP using induced pluripotent stem cell (iPSC)-derived RPE. To effectively model rare mutations using iPSC-derived RPE and screen gene editing correction approaches, we require a strategy to install the desired mutation in wild-type iPSC and HEK293T. In this study, we developed a prime editing strategy for the installation of the pathogenic RPE65 c.1430A>G mutation underlying our patient's disease.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Mittas DM, Gavrilov Z, Ucambarlic E, et al (2025)

CRISPR/Cas-Mediated Gene Activation as a Versatile Tool for Treatment of Inherited Retinal Dystrophies.

Advances in experimental medicine and biology, 1468:95-99.

CRISPR/Cas-mediated genome editing is an effective and attractive tool for the treatment of diseases or genes that cannot be adequately covered by gene replacement strategies. The first FDA-approved AAV-vector- and CRISPR/Cas-based clinical trials were each designed for the treatment of a subtype of an inherited retinal dystrophy, underscoring the importance of ophthalmic diseases in the field of gene (editing) therapies. This and most other pre-clinical therapeutic CRISPR/Cas approaches are typically designed for the treatment of single mutations. For the treatment of larger patient cohorts, however, mutation- or ideally gene-independent approaches appear to be more suitable. CRISPR/Cas-mediated transcriptional activation of genes (CRISPRa) is a promising approach to achieve these ambitious goals. In this minireview, focusing on inherited retinal dystrophies, we will discuss recent developments, advantages and limitations, and future prospects of CRISPRa as a therapeutic tool.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Allen A, Cooper BH, Singh J, et al (2025)

PAM-adjacent DNA flexibility tunes CRISPR-Cas12a off-target binding.

Scientific reports, 15(1):4930.

Cas12a is a class 2 type V CRISPR-associated nuclease that uses an effector complex comprised of a single protein activated by a CRISPR-encoded small RNA to cleave double-stranded DNA at specific sites. Cas12a processes unique features as compared to other CRISPR effector nucleases such as Cas9, and has been demonstrated as an effective tool for manipulating complex genomes. Prior studies have indicated that DNA flexibility at the region adjacent to the protospacer-adjacent-motif (PAM) contributes to Cas12a target recognition. Here, we adapted a SELEX-seq approach to further examine the connection between PAM-adjacent DNA flexibility and off-target binding by Cas12a. A DNA library containing DNA-DNA mismatches at PAM + 1 to + 6 positions was generated and subjected to binding in vitro with FnCas12a in the absence of pairing between the RNA guide and DNA target. The bound and unbound populations were sequenced to determine the propensity for off-target binding for each of the individual sequences. Analyzing the position and nucleotide dependency of the DNA-DNA mismatches showed that PAM-dependent Cas12a off-target binding requires unpairing of the protospacer at PAM + 1 and increases with unpairing at PAM + 2 and + 3. This revealed that PAM-adjacent DNA flexibility can tune Cas12a off-target binding. The work adds support to the notion that physical properties of the DNA modulate Cas12a target discrimination, and has implications on Cas12a-based applications.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Li S, Yu J, Shen Y, et al (2025)

Transdermal microneedle-assisted ultrasound-enhanced CRISPRa system to enable sono-gene therapy for obesity.

Nature communications, 16(1):1499.

Obesity, a surging global health challenge, necessitates effective, accessible and innovative therapeutic models. Here we develop a spatiotemporally controllable microneedle (MN) drug delivery platform for sono-gene therapy to fight obesity. The platform delivers the methoxy polyethylene glycol-polyethyleneimine (mPEG-PEI) modified metal-organic frameworks (MOFs) sonosensitizer and the clustered regularly interspaced short palindromic repeats-activating (CRISPRa)/CRISPRa-uncoupling protein 1 (UCP1) system intradermally to adipocytes. Overall, this therapy platform is capable of achieving two major strategies of "annihilation" and "countermeasure": one is to kill redundant white adipocytes by sonodynamic therapy, and the other is to promote the browning of white adipocytes through the controllable release of CRISPRa-UCP1 system and sonodynamic effect. Obese male mice treated with this sono-gene therapy shows significant ameliorate in glucose tolerance and insulin sensitivity, successfully achieves weight loss and restrains weight rebound. This study may enable a standard treatment paradigm for sono-gene therapy of obesity and other metabolic diseases.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Hossain KA, Nierzwicki L, Orozco M, et al (2025)

Flexibility in PAM recognition expands DNA targeting in xCas9.

eLife, 13: pii:102538.

xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognising a series of alternative protospacer adjacent motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Jin X, Zhang B, Sun Y, et al (2025)

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish.

Journal of visualized experiments : JoVE.

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Fry LE, Major L, Salman A, et al (2025)

Comparison of CRISPR-Cas13b RNA base editing approaches for USH2A-associated inherited retinal degeneration.

Communications biology, 8(1):200.

CRISPR-Cas13 systems have therapeutic promise for the precise correction of point mutations in RNA. Using adenosine deaminase acting on RNA (ADAR) effectors, A-I base conversions can be targeted using guide RNAs (gRNAs). We compare the Cas13 effectors PspCas13b and Cas13bt3 for the repair of the gene USH2A, a common cause of inherited retinal disease and Usher syndrome. In cultured cells, we demonstrate up to 80% efficiency for the repair of the common c.11864 G > A and its murine equivalent c.11840 G > A, across different gRNAs and promoters. We develop and characterize a mouse model of Usher syndrome carrying the c.11840 G > A mutation designed for the evaluation of base editors for inherited retinal disease. Finally, we compare Cas13 effectors delivered via AAV for the repair of Ush2a in photoreceptors. Mean RNA editing rates in photoreceptors across different constructs ranged from 0.32% to 2.04%, with greater efficiency in those injected with PspCas13b compared to Cas13bt3 constructs. In mice injected with PspCas13b constructs, usherin protein was successfully restored and correctly localized to the connecting cilium following RNA editing. These results support the development of transcriptome targeting gene editing therapies for retinal disease.

RevDate: 2025-02-08
CmpDate: 2025-02-08

Harris DT, CH Jan (2025)

CRISPuRe-seq: pooled screening of barcoded ribonucleoprotein reporters reveals regulation of RNA polymerase III transcription by the integrated stress response via mTOR.

Nucleic acids research, 53(4):.

Genetic screens using CRISPR (Clustered Regularly Interspaced Palindromic Repeats) provide valuable information about gene function. Nearly all pooled screening technologies rely on the cell to link genotype to phenotype, making it challenging to assay mechanistically informative, biochemically defined phenotypes. Here, we present CRISPuRe-seq (CRISPR PuRification), a novel pooled screening strategy that expands the universe of accessible phenotypes through the purification of ribonucleoprotein complexes that link genotypes to expressed RNA barcodes. While screening for regulators of the integrated stress response (ISR), we serendipitously discovered that the ISR represses transfer RNA (tRNA) production under conditions of reduced protein synthesis. This regulation is mediated through inhibition of mTORC1 and corresponding activation of the RNA polymerase III inhibitor MAF1. These data demonstrate that coherent downregulation of tRNA expression and protein synthesis is achieved through cross-talk between the ISR and mTOR, two master integrators of cell state.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Ju C, Li X, Wang D, et al (2025)

Ultrasensitive detection of microRNAs based on cascade amplification strategy of RCA-PER and Cas12a.

The Analyst, 150(4):692-699.

Since microRNAs (miRNAs) serve as markers for early cancer diagnosis, it is crucial to develop a novel biosensor to detect miRNAs quickly, sensitively and selectively. Hence, we developed a fluorescence biosensor based on target miRNA-initiated rolling circle amplification (RCA) to generate RCA products with multiple tandem catalytic hairpin DNA templates that trigger primer exchange reactions (PER) which extend short single-strand DNA (ssDNA) primers into long ssDNA. Subsequently, the long ssDNA activates the trans-cleavage activity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system to cleave a fluorescent reporter chain, enabling ultrasensitive detection of miRNAs through the output fluorescence signal. The biosensor could quantify miRNA-141 concentrations from 10[0] to 10[5] pM, with a detection limit of 94 fM. Therefore, the biosensing strategy proposed in this study offers a robust technique for the clinical diagnosis of miRNA-141.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Gong Y, Zhang J, Lu Z, et al (2025)

Dual signal amplification in ECL biosensors: A novel approach for argonaute2 detection using SAHARA CRISPR-Cas12a technology.

Bioelectrochemistry (Amsterdam, Netherlands), 163:108896.

Argonaute 2 (Ago2) is a crucial enzyme in the RNA interference (RNAi) pathway, essential for gene silencing via the cleavage of target messenger RNA (mRNA) mediated by microRNA (miRNA) or small interfering RNA (siRNA). The activity of Ago2 is a significant biomarker for various diseases, including cancer and viral infections, necessitating precise monitoring techniques. Traditional methods for detecting Ago2 activity are often cumbersome and lack the necessary sensitivity and specificity for low-abundance targets in complex samples. This study presents an innovative biosensor utilizing electrochemiluminescence (ECL) technology combined with the SAHARA (Split Activator for Highly Accessible RNA Analysis) CRISPR-Cas12a system to detect Ago2 activity with high sensitivity and specificity. The introduction of Blocker RNA in the activation mechanism enhances the specificity of CRISPR-Cas12a, ensuring accurate signal generation. The dual signal amplification strategy, combining RISC-assisted and CRISPR-Cas12a-mediated cleavage, enhances the biosensor's sensitivity. The developed ECL biosensor demonstrated a remarkable limit of detection (LOD) of 0.145 aM, along with excellent precision, stability, and specificity. These attributes make it a powerful tool for detecting Ago2 activity in clinical diagnostics and research settings.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Sang N, Ma B, Liu H, et al (2025)

CRISPR/Cas9-mediated GhFT-targeted mutagenesis prolongs indeterminate growth and alters plant architecture in cotton.

Plant science : an international journal of experimental plant biology, 352:112374.

The shift from vegetative to reproductive growth is an important developmental transition that affects flowering and maturation, architecture, and ecological adaptability in plants. The florigen-antiflorigen system universally controls flowering and plant architecture, and changes to the ratio of these components alter this transition and disrupt growth. The genes FT (FLOWERING LOCUS T), encoding the florigen protein FT, and CETS [CENTRORADIALIS (CEN)/TERMINAL FLOWER1 (TFL1)/SELF-PRUNING (SP)], encoding antiflorigen proteins, have opposing roles. Upland cotton (Gossypium hirsutum) is one of the world's most widely cultivated cotton varieties, and its complex allotetraploid genome contains only one homoeologous pair of FT genes (GhFT-A and GhFT-D). The functionally conserved gene GhFT promotes flowering and plays a role in plant architecture, although the molecular regulation of flowering and plant architecture in cotton remains unclear. In this study, CRISPR/Cas9 technology was used to induce mutations in the first and second exons of GhFT, respectively. G. hirsutum cv. YZ-1 was transformed with a CRISPR/Cas9-GhFT vector using Agrobacterium tumefaciens, and a diverse set of mutations was identified at the editing site. Compared with the wild type, mutant plants could not transition between vegetative and reproductive growth, and significant alterations to plant architecture were observed. Quantitative RT-PCR revealed downregulation of the homologous floral meristem identity genes APETALA1 (GhAP1) and OVEREXPRESSION OF CONSTANS 1 (GhSOC1) and upregulation of the TFL1 homologs GhTFL1-1 and GhTFL1-2. These results suggested that GhFT played a significant role in flowering time and plant architecture and that the ratio of florigen-antiflorigen components was critical to producing improved cotton varieties. This study provided a basis for future investigations of molecular breeding in cotton and guidance for the agricultural production of this crop.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Peña-Gutiérrez I, Olalla-Sastre B, Río P, et al (2025)

Beyond precision: evaluation of off-target clustered regularly interspaced short palindromic repeats/Cas9-mediated genome editing.

Cytotherapy, 27(3):279-286.

The gene editing field has advanced rapidly since the development of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system because of its applicability in precisely modifying the genome. Among its multiple applications, the correction of genetic diseases has emerged as a potential curative treatment for many disorders that have eluded a cure to date. Despite its efficiency in achieving therapeutic levels of correction, the unexpected adverse effects of editing due to CRISPR/Cas9 nuclease activity are a major concern when translating these new strategies to the clinic. Multiple in silico tools and empirical methods have been developed to evaluate these off-target edits as well as other adverse alterations of the genome, including rearrangements, not only in ex vivo experiments but also in in vivo experiments. In this review, we summarize the available methods for the assessment of off-target effects of CRISPR/Cas9 systems, highlighting their advantages and limitations. Special attention will be paid to their application in pre-clinical studies and clinical trials, both in the manufacturing product and in the long-term follow-up of patients.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Kaul R, Thangaraj A, Sharda S, et al (2025)

Optimization of tissue culture and Cas9 transgene expression in tomato: A step towards CRISPR/Cas9-based genetic improvement.

Plant science : an international journal of experimental plant biology, 352:112324.

Tomato (Solanum lycopersicum L.) is an essential source of antioxidants and a prime candidate for bioengineering experiments. Many studies have aimed to improve tomatoes using CRISPR/Cas9 technology; however, the success rate is limited due to the lack of efficient regeneration and genetic modification techniques. Here, we report an efficient regeneration and transformation procedure focused on developing efficient Cas9 gene transgenic tomato plants using the Agrobacterium tumefaciens strain LBA4404 harbouring pCRISPR/Cas9TK2-NIC binary vector. We optimized the concentrations and combinations of growth hormones to promote direct shoot and root regeneration via hypocotyl explants. We found that MS medium 2.0 mg/l Zeatin (Zn) + 1.5 mg/l Indole -3- acetic acid (IAA) + 0.3 mg/l Benzyl amino purine (BAP) was preeminent for shoot regeneration medium, and 0.5 mg/l BAP+ 0.1 mg/l IAA was appropriate for root regeneration. Cas9 transgenes in the tomato genome of putative tomato plants were validated using various methods, including polymerase chain reaction (PCR), and confirmed via Southern blotting. The developed protocol showed improved regeneration and transformation efficiencies in tomatoes of 88 % and 54 %, respectively. In this study, we successfully established a gene delivery platform for tomatoes using the CRISPR/Cas9 system.

RevDate: 2025-02-10
CmpDate: 2025-02-10

Feng J, Ma Y, Zhang D, et al (2025)

High-Efficiency Genome Editing in Naturally Isolated Aeromonas hydrophila and Edwardsiella Piscicida Using the CRISPR-Cas9 System.

Biotechnology and bioengineering, 122(3):606-614.

Aeromonas hydrophila and Edwardsiella piscicida are significant bacterial pathogens in aquaculture, causing severe diseases and tremendous economic losses worldwide. Additionally, both of them can act as opportunistic pathogens in humans, leading to severe infections. Efficient genome editing tools for these pathogens are essential for understanding their pathogenic mechanisms and physiological behaviors, enabling the development of targeted strategies to control and mitigate their effects. In this study, we adapted the CRISPR-Cas9 system for high-efficiency, marker-less genome editing in multiple naturally isolated strains of these two aquaculture pathogens. We developed a streamlined procedure that successfully generated deletion mutants of the aerA gene (encoding for aerolysin, a pore-forming toxin that plays a critical role in the pathogenicity) and the gfp insertion mutants in three naturally isolated A. hydrophila strains. Additionally, we deleted five putative hemolysin-encoding genes in both A. hydrophila ML10-51K and its ∆aerA derivative. The same system was also applied to the naturally isolated E. piscicida S11-285 strain, successfully deleting the ssaV gene (a component of the Type III Secretion System-a critical virulence mechanism in many pathogenic bacteria). The methodologies developed herein could be broadly applied to other pathogenic strains from natural environments, providing valuable tools for studying bacterial pathogenesis and aiding in the development of effective control strategies.

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ESP Quick Facts

ESP Origins

In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

ESP Support

In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

ESP Rationale

Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

ESP Goal

In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

ESP Usage

Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

ESP Content

When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

ESP Help

Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

ESP Plans

With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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CRISPR-Cas

By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

Electronic Scholarly Publishing
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Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin and even a collection of poetry — Chicago Poems by Carl Sandburg.

Timelines

ESP now offers a large collection of user-selected side-by-side timelines (e.g., all science vs. all other categories, or arts and culture vs. world history), designed to provide a comparative context for appreciating world events.

Biographies

Biographical information about many key scientists (e.g., Walter Sutton).

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 28 JUL 2024 )