@article {pmid41785636,
year = {2026},
author = {Xue, S and Sun, H and Hou, X and Li, N and Xue, L and Dai, E and Wan, J},
title = {An off-target exonuclease activity in AsCpf1 undermines CRISPR diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {303},
number = {},
pages = {118578},
doi = {10.1016/j.bios.2026.118578},
pmid = {41785636},
issn = {1873-4235},
abstract = {The extensive utilization of CRISPR-Cas systems in molecular diagnostics stems from their crRNA-guided trans-cleavage capabilities. However, AsCpf1-based detection systems frequently exhibit unexplained sensitive variations. This research reveals that AsCpf1 maintains a crRNA-independent function, similar to exonuclease I, when utilized in standard buffers containing Mg[2+]. From a structural perspective, this exonuclease activity is independent of the RuvC domain-mediated canonical trans-cleavage activity. It is predicted by structural modeling to be potentially localized within the WED-PI domain. In the context of diagnostics, the effective target concentration is diminished by AsCpf1-mediated degradation of the free 3' ends of target DNA, which impairs detection sensitivity. To mitigate this interference, we demonstrate that 3' end capping effectively restores detection performance. This approach was validated in a CRISPR-EXPAR-based microRNA biosensor, which exhibited approximately 10-fold improvement in sensitivity following 3' end capping. Overall, this investigation characterizes a previously unidentified exonuclease activity within the AsCpf1 system and establishes practical design criteria to improve the robustness and accuracy of CRISPR-based diagnostic tools.},
}

@article {pmid41785320,
year = {2026},
author = {Zhang, X and Richart, D and McFarlin, S and Cheng, F and Park, SY and Zhang-Chen, A and McFarlane, R and Xiao, C and Yan, Z and Qiu, J},
title = {Identification of CD164 as an essential entry receptor for divergent adeno-associated viruses.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2525865123},
doi = {10.1073/pnas.2525865123},
pmid = {41785320},
issn = {1091-6490},
support = {AI166293//HHS | National Institutes of Health (NIH)/ ; AI180416//HHS | National Institutes of Health (NIH)/ ; HL174593//HHS | National Institutes of Health (NIH)/ ; AI182645//HHS | National Institutes of Health (NIH)/ ; HL174593//HHS | National Institutes of Health (NIH)/ ; AI182645//HHS | National Institutes of Health (NIH)/ ; GM129525//HHS | National Institutes of Health (NIH)/ ; YAN23G0//Cystic Fibrosis Foundation (CFF)/ ; AH-2126-20220331//Welch Foundation (The Welch Foundation)/ ; },
mesh = {*Dependovirus/genetics/physiology/metabolism ; Animals ; Mice ; Humans ; Mice, Knockout ; *Virus Internalization ; *Receptors, Cell Surface/metabolism/genetics ; HEK293 Cells ; *Antigens, CD/metabolism/genetics ; Genetic Vectors/genetics ; Transduction, Genetic ; CRISPR-Cas Systems ; *Receptors, Virus/metabolism/genetics ; Capsid/metabolism ; },
abstract = {Recombinant adeno-associated viruses (rAAVs) are widely used for in vivo gene delivery. While KIAA0319L, known as AAV receptor (AAVR), is essential for the transduction of multiserotype AAVs, it is dispensable for AAV4-related (Clade G) AAVs. We conducted a genome-wide CRISPR/Cas9 screen and identified CD164, a type I transmembrane sialomucin, as an essential entry receptor for Clade G AAVs. Ablation of CD164 expression substantially impaired both entry and transduction of Clade G AAVs. CD164-targeting antibodies and soluble CD164 ectodomain effectively blocked transduction. AAV4 capsids colocalized with CD164 at the plasma membrane and in endosomal compartments. In vitro, CD164 interacted with AAV4 or AAVrh32.33 capsids at high affinity. Importantly, systemic administration of rAAV4 or rAAVrh32.33 in CD164 knockout (KO) mice resulted in nearly complete loss of transgene expression. These findings establish CD164 as an essential entry receptor for Clade G AAV vectors and uncover a distinct AAVR-independent mechanism of AAV tropism.},
}

*Dependovirus/genetics/physiology/metabolism
Animals
Mice
Humans
Mice, Knockout
*Virus Internalization
*Receptors, Cell Surface/metabolism/genetics
HEK293 Cells
*Antigens, CD/metabolism/genetics
Genetic Vectors/genetics
Transduction, Genetic
CRISPR-Cas Systems
*Receptors, Virus/metabolism/genetics
Capsid/metabolism

@article {pmid41785318,
year = {2026},
author = {Weber, LI and Timpen, LE and Egger-Hörschinger, AS and Schöpf, P and Ayhan, ND and Demmel, D and Hotze, M and Zhang, Y and Mehrabi, M and Puglisi, K and Stefan, E and Ghaffari-Tabrizi-Wizsy, N and Ramos-Pittol, JM and Kwiatkowski, M and Hartl, M},
title = {Reactivation of the silenced BASP1 gene suppresses oncogenic WNT signaling in human colorectal cancer cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2524159123},
doi = {10.1073/pnas.2524159123},
pmid = {41785318},
issn = {1091-6490},
support = {P33662//Austrian Science Fund (FWF)/ ; },
mesh = {Humans ; *Wnt Signaling Pathway/genetics ; *Colorectal Neoplasms/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; *Nerve Tissue Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; beta Catenin/metabolism/genetics ; Promoter Regions, Genetic ; Gene Silencing ; Cell Movement ; },
abstract = {Starting from human colon cancer cells showing aberrant WNT/β-catenin/TCF signaling, hyperactivated MYC, and silenced BASP1, we generated stable cell lines overexpressing BASP1, either ectopically, or by reactivating the dormant BASP1 promoter using a lentiviral CRISPR-based system. BASP1 encodes a neuronal signaling protein and transcriptional corepressor, from which tumor-suppressive functions have been described in avian cell systems and in multiple human cancer cell types. Proteome and transcriptome analyses revealed activation of several tumor and metastasis suppressors in BASP1-expressing cells, which also show strong repression of the transformed phenotype in terms of contact inhibition, anchorage-independent growth, and tumor formation. Cells with reactivated BASP1 display a flat and differentiated morphology with enhanced migratory potential, accompanied by expression of multiple genes implicated in actin polymerization, focal adhesion, and neuronal migration. Furthermore, MYC protein expression is substantially repressed due to BASP1-mediated transcriptional MYC downregulation involving BASP1 interaction with β-catenin and binding to the MYC promoter. Upon BASP1 activation, multiple key proteins of the canonical WNT signaling pathway become suppressed. One of these BASP1 targets is the protein kinase TNIK catalyzing phosphorylation of TCF7L2, the latter required for transcriptional MYC activation. Results obtained with a preclinical TNIK inhibitor in human colorectal cancer cells show efficient abrogation of MYC expression and consequently impaired dimerization with its interaction partner MAX. The antagonistic BASP1 effect on MYC and the MYC dependency on TNIK could enhance the development of strategies to interfere with oncogenic functions of the cancer driver MYC.},
}

Humans
*Wnt Signaling Pathway/genetics
*Colorectal Neoplasms/genetics/metabolism/pathology
Gene Expression Regulation, Neoplastic
Cell Line, Tumor
Proto-Oncogene Proteins c-myc/genetics/metabolism
*Nerve Tissue Proteins/genetics/metabolism
*Membrane Proteins/genetics/metabolism
beta Catenin/metabolism/genetics
Promoter Regions, Genetic
Gene Silencing
Cell Movement

@article {pmid41784822,
year = {2026},
author = {Eweda, MA and Li, J and Hassan, U and Jalil, S and Jin, X},
title = {Peroxisomal acyl-CoA oxidase OsACX4 negatively regulates salt and drought stress tolerance by modulating cellular redox homeostasis in rice.},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {},
pmid = {41784822},
issn = {1432-203X},
support = {517000-N12502ZJ//National Natural Science Foundation of China/ ; 2025ZDXT01-4//The Major Agricultural Technology Collaborative Extension Project/ ; LZ25C130006//Natural Science Foundation of Zhejiang Province/ ; 2021C02063-6//Science and Technology Department of Zhejiang Province/ ; },
mesh = {*Oryza/genetics/physiology/enzymology/metabolism ; Homeostasis ; Oxidation-Reduction ; *Plant Proteins/metabolism/genetics ; Droughts ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Peroxisomes/metabolism/enzymology ; *Acyl-CoA Oxidase/metabolism/genetics ; Reactive Oxygen Species/metabolism ; Stress, Physiological/genetics ; *Salt Tolerance/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Oxidative Stress ; },
abstract = {OsACX4 knockout reduces peroxisomal oxidative stress, enhancing rice drought and salt tolerance through metabolic-redox rebalancing for climate-resilient breeding. Climate change is intensifying the frequency and severity of abiotic stress, such as salt and drought stresses, which severely limit rice productivity worldwide, necessitating the identification of molecular targets for crop improvement. This study provides the first comprehensive functional characterization of the peroxisomal acyl-CoA oxidase OsACX4 in rice (Oryza sativa L.) drought and salinity tolerance, revealing its unexpected role as a negative regulator of stress tolerance through modulation of cellular redox homeostasis. Through genome editing using CRISPR/Cas9-mediated knockout and overexpression approaches, we generated transgenic lines to investigate the function of OsACX4 under salt and drought stress. Knockout lines exhibited superior stress tolerance compared to the wild-type (WT) and overexpression lines, demonstrating significantly higher survival rates under severe stress conditions. Enhanced tolerance correlated with coordinated upregulation of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities. Knockout lines accumulated substantially higher proline (Pro) levels while maintaining markedly reduced reactive oxygen species (ROS) compared to overexpression lines. Transcriptional analysis revealed that OsACX4 disruption triggered upregulation of stress-responsive genes, including OsSOD1, OsDREB2A, OsDREB1B, and OsAPX1 under severe stress. Subcellular localization confirmed peroxisomal targeting of OsACX4, where its β-oxidation activity generates hydrogen peroxide (H2O2) as a metabolic by-product. Our results reveal a metabolic trade-off whereby stress-induced OsACX4 expression mobilizes energy reserves but compromises cellular redox homeostasis. The superior performance of knockout lines under both stresses demonstrates that strategic OsACX4 disruption enhances plant resilience, identifying this gene as a promising breeding target for developing climate-resilient rice varieties through precision genome editing.},
}

*Oryza/genetics/physiology/enzymology/metabolism
Homeostasis
Oxidation-Reduction
*Plant Proteins/metabolism/genetics
Droughts
Gene Expression Regulation, Plant
Plants, Genetically Modified
Peroxisomes/metabolism/enzymology
*Acyl-CoA Oxidase/metabolism/genetics
Reactive Oxygen Species/metabolism
Stress, Physiological/genetics
*Salt Tolerance/genetics
Gene Knockout Techniques
CRISPR-Cas Systems
Oxidative Stress

@article {pmid41784340,
year = {2026},
author = {Zhou, C and Cheng, T and Zhou, J and Zhang, B and Liu, L and Jiang, G and Li, W and Wang, C},
title = {Synthetic Biofilms for Green Membranes: Engineering Low-Energy Filtration Systems.},
journal = {Environmental science & technology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.est.5c15661},
pmid = {41784340},
issn = {1520-5851},
abstract = {Membrane filtration is a key technology to modern water purification, yet its sustainability is compromised by biofouling, which increases energy consumption and ecological impacts. Conventional control strategies often struggle to balance efficacy and environmental footprint. In this study, an inducible, engineered quorum-quenching (QQ) bacterium was constructed via a genomic integration strategy, thereby achieving control over the biofilm structure in membrane filtration biofouling layers. By using the clustered regularly interspaced short palindromic repeat (CRISPR-Cas) targeted gene editing technology, the engineered bacteria that were constructed to express aiiO under l-ribose induction have achieved the regulation of biofilms. Validation using a gravity-driven membrane ultrafiltration system (UF-GDM) model showed that the engineered bacterium effectively reduced extracellular polymeric substances (EPS) components, increased the hydrophilic porosity of the residual biofilm, and decreased its stickiness. This approach reduced transmembrane pressure by 64.5%, increased total organic carbon (TOC) removal by 13.2%, and extended membrane lifespan by 16.1%. A technical-economic analysis indicates that the 100,000 m[3]/day treatment plant achieves an annual net profit increase of 31.52%, reaching 1.55 × 10[7] CNY, while reducing its net carbon footprint by 27.43%, with an annual net reduction of 2.96 × 10[5] kg CO2eq. This study provides a novel solution strategy for achieving biofouling resistance and sustainable, low-energy operation in membrane filtration processes, which contributes to the broader application and adoption of this technology.},
}

@article {pmid41784267,
year = {2026},
author = {Wang, Z and Wu, Y and Wang, Z and Zhang, S and Liu, H and Nie, Y and Chen, K and Huang, Y and Zhou, Y and Cao, Y and Sun, L and Hao, R},
title = {crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41784267},
issn = {1362-4962},
support = {//Prevention and Control of Emerging/ ; 2025ZD01903403//National Science and Technology Major Project/ ; L234051//Beijing Municipal Natural Science Foundation/ ; L246011//Beijing Municipal Natural Science Foundation/ ; 2024-03-18//Training Plan for High-Level Public Health Technical Talents of Beijing Municipal Health Commission/ ; 2025ZD01903403//Prevention and Control of Emerging and Major Infectious Diseases-National Science/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Mycobacterium tuberculosis/genetics/isolation & purification ; Klebsiella pneumoniae/genetics/isolation & purification ; Nucleic Acid Conformation ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Endodeoxyribonucleases/metabolism/genetics/chemistry
Mycobacterium tuberculosis/genetics/isolation & purification
Klebsiella pneumoniae/genetics/isolation & purification
Nucleic Acid Conformation
*Bacterial Proteins/genetics/metabolism

@article {pmid41783940,
year = {2026},
author = {Berti, M and Ceriotti, S and Santi, L and Alberti, G and Beretta, S and Degl'Innocenti, S and Ruatti, C and Savoia, EO and Jofra-Hernandez, R and De Ponti, G and Bolamperti, S and Villa, I and Galeotti, F and Romano, A and Visigalli, I and Norata, R and Rocchi, M and Cristofori, P and Cossutta, M and Consiglieri, G and Tucci, F and Santorelli, L and Grumati, P and Ronfani, L and D'Adamo, P and Giustina, A and Angelozzi, M and Settembre, C and Mortellaro, A and Scala, S and Sanvito, F and Volpi, N and Aiuti, A and Bernardo, ME and Crippa, S},
title = {Development and characterization of a model of mucopolysaccharidosis type IVA for evaluating therapies targeting bone disease.},
journal = {Disease models & mechanisms},
volume = {19},
number = {2},
pages = {},
doi = {10.1242/dmm.052540},
pmid = {41783940},
issn = {1754-8411},
support = {20228H9T82//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; P20223MF7X_001//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; CN_00000041 - CUP G83C22000270001//NextGenerationEU/ ; TTAGTXEKFA//Else Kröner-Fresenius-Zentrum für Ernährungsmedizin/ ; TELE-MB//Fondazione Telethon/ ; TELE-AA//Fondazione Telethon/ ; //Ospedale San Raffaele/ ; },
mesh = {Animals ; Disease Models, Animal ; *Mucopolysaccharidosis IV/therapy/pathology/blood/complications ; *Bone Diseases/therapy/pathology/complications ; Chondroitinsulfatases/metabolism/deficiency ; Glycosaminoglycans/metabolism ; Keratan Sulfate/metabolism/urine ; Chondroitin Sulfates/metabolism/urine ; Mice ; Humans ; Genetic Therapy ; Mice, Knockout ; Bone and Bones/pathology ; CRISPR-Cas Systems/genetics ; },
abstract = {Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disease (LSD) caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which causes the accumulation of keratan sulphate (KS) and chondroitin sulphate (CS). Patients with MPSIVA typically present with severe skeletal and joint disorders, which are not addressed by conventional therapies. Currently, no animal model accurately replicates the human disease, hindering the development of novel therapeutic interventions. To overcome this limitation, we established, by CRISPR-Cas9 technology, a Galns-/- mouse model that expresses a non-functional enzyme and accumulates CS and KS in the urine, plasma and distinct tissues, and glycosaminoglycans in the spleen. The mice exhibit shortened long bones, trabecular bone alterations and skeletal abnormalities in the growth plate. Additionally, we observed increased levels of inflammatory and oxidative markers in visceral organs and plasma. Our newly developed model of MPSIVA demonstrates clear and quantifiable signs of skeletal alterations, providing novel means of assessment of the safety and efficacy of innovative therapies, including hematopoietic stem and progenitor cell gene therapy, which has recently been shown to provide a beneficial effect on skeletal alterations in Hurler syndrome.},
}

Animals
Disease Models, Animal
*Mucopolysaccharidosis IV/therapy/pathology/blood/complications
*Bone Diseases/therapy/pathology/complications
Chondroitinsulfatases/metabolism/deficiency
Glycosaminoglycans/metabolism
Keratan Sulfate/metabolism/urine
Chondroitin Sulfates/metabolism/urine
Mice
Humans
Genetic Therapy
Mice, Knockout
Bone and Bones/pathology
CRISPR-Cas Systems/genetics

@article {pmid41781627,
year = {2026},
author = {Hoffmann, FT and Wiegand, T and Palmieri, AI and Glass-Klaiber, J and Xiao, R and Tang, S and Le, HC and Meers, C and Lampe, GD and Chang, L and Sternberg, SH},
title = {Exapted CRISPR-Cas12f homologues drive RNA-guided transcription.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41781627},
issn = {1476-4687},
abstract = {Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP)[1]. Although the seven σ factors in Escherichia coli have been extensively characterized[2], Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ[E]) whose precise roles are unknown, pointing to additional layers of regulatory potential[3]. Here we uncover a mechanism of RNA-guided gene activation involving the coordinated action of σ[E] factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and σ[E] homologues in E. coli using RNA and chromatin immunoprecipitation experiments, revealing systems that exhibit robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif. Recruitment of σ[E] was dependent on dCas12f and guide RNA, suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ[E] ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ[E] complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription that embodies natural features reminiscent of CRISPR activation (CRISPRa) technology[4,5].},
}

@article {pmid41781609,
year = {2026},
author = {Xiao, R and Hoffmann, FT and Xie, D and Wiegand, T and Palmieri, AI and Sternberg, SH and Chang, L},
title = {Structural basis of RNA-guided transcription by a dCas12f-σ[E]-RNAP complex.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41781609},
issn = {1476-4687},
abstract = {In both natural and engineered biological systems, RNA-guided proteins have emerged as critical transcriptional regulators by modulating RNA polymerase (RNAP) and its associated factors[1-3]. In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity[1,4,5]. A distinct class of nuclease-dead Cas12f homologues (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ[E])[6], although the molecular basis has remained elusive. Here we reveal a new mode of RNA-guided transcription initiation by determining the cryo-electron microscopy structures of the dCas12f-σ[E] system from Flagellimonas taeanensis. We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ[E]-RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ[E]-RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ[E]-dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, whereas the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.},
}

@article {pmid41747766,
year = {2026},
author = {Cai, Y and Zhuang, L and Wang, Z and He, L and Li, X and Liu, BF and Li, T and Zhang, G and Zhou, H and Huang, X and Li, Y},
title = {Gravity-Driven Formation of Water-in-Wax Spheres for Efficient One-Pot CRISPR Diagnostics.},
journal = {ACS nano},
volume = {20},
number = {9},
pages = {8055-8067},
doi = {10.1021/acsnano.6c01232},
pmid = {41747766},
issn = {1936-086X},
mesh = {*Gravitation ; *Water/chemistry ; Humans ; *Waxes/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microspheres ; CRISPR-Cas Systems ; Human papillomavirus 16/genetics/isolation & purification ; },
abstract = {Rapid, decentralized molecular diagnostics are urgently needed for effective infectious disease control. Here, we present "Wax-Sphere CRISPR" (WS-CRISPR), a paradigm-shifting platform for CRISPR-based diagnostics centered on a gravity-driven, interfacial phase-change self-encapsulation mechanism. This system fundamentally decouples bioreagent engineering from specific reaction vessels, transforming conventional, labor-intensive manual encapsulation into a standardized, physics-driven assembly process that generates discrete wax microspheres. Guided by fluid mechanics and interfacial thermodynamics, aqueous CRISPR droplets spontaneously traverse air/molten wax/ethanol to self-encapsulate and solidify, enabling standardized, high-throughput fabrication without manual wax handling. Upon temperature modulation, the wax phase change triggers sequential recombinase polymerase amplification (RPA) and CRISPR detection within a sealed, one-pot vessel. As a clinically oriented demonstration, WS-CRISPR enables multiplexed detection and risk stratification of all 14 high-risk HPV genotypes (HPV16/18 vs others) with a detection limit of 1 × 10[-18] M in under 30 min. Integrated with widely accessible devices─including a thermocycler, hand-held fluorescence reader, and microfluidic platform─the system demonstrated 97.4% sensitivity and 100% specificity across 70 clinical samples. By solving the engineering bottlenecks of scalability and universality, WS-CRISPR offers a robust tool for decentralized, large-scale pathogen surveillance.},
}

*Gravitation
*Water/chemistry
Humans
*Waxes/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Microspheres
CRISPR-Cas Systems
Human papillomavirus 16/genetics/isolation & purification

@article {pmid41742365,
year = {2026},
author = {Yao, W and Li, Y and Du, Y and Gao, R and Tan, J and Fan, J and Liu, Y},
title = {Aptamer-Mediated Protein Corona In Situ Multiplex Microfluidic Detection for Cancer Diagnosis.},
journal = {Analytical chemistry},
volume = {98},
number = {9},
pages = {7123-7135},
doi = {10.1021/acs.analchem.6c00170},
pmid = {41742365},
issn = {1520-6882},
mesh = {Humans ; *Aptamers, Nucleotide/chemistry ; *Neoplasms/diagnosis/blood ; *Protein Corona/chemistry ; *Microfluidic Analytical Techniques ; *Biomarkers, Tumor/blood ; *Lab-On-A-Chip Devices ; CRISPR-Cas Systems ; Silicon Dioxide/chemistry ; },
abstract = {Liquid biopsy faces critical limitations in detecting low-abundance protein biomarkers due to serum complexity, single-target constraints, and insufficient sensitivity. Here, we present an integrated microfluidic protein aptamer corona-CRISPR (PACC) platform that synergizes nanoparticle protein corona (PC) enrichment, multiplexed aptamer recognition, and CRISPR-Cas12a-driven signal generation for facile and rapid point-of-care testing. Fe3O4@SiO2 nanoparticles capable of streamlining PC isolation via magnetic separation were employed to concentrate cancer-associated proteins from serum. A 6-plex aptamer panel, which selectively bound target proteins via forming protein-aptamer coronas (PACs), was used to initiate the in situ activation of CRISPR-Cas12a via complementary crRNAs for fluorescence signal generation. This entire workflow was miniaturized into a 3D-printed microfluidic chip and portable reader, achieving a fast sample-to-answer process. Clinical validation with 112 serum specimens demonstrated excellent diagnostic performance, achieving an area under the curve (AUC) of 0.95 and 88.24% accuracy. By integrating these modalities, the PACC platform overcomes serum matrix interference and biomarker scarcity, providing a scalable and low-cost avenue for detecting low-abundance protein biomarkers and advancing liquid biopsy toward precision oncology.},
}

Humans
*Aptamers, Nucleotide/chemistry
*Neoplasms/diagnosis/blood
*Protein Corona/chemistry
*Microfluidic Analytical Techniques
*Biomarkers, Tumor/blood
*Lab-On-A-Chip Devices
CRISPR-Cas Systems
Silicon Dioxide/chemistry

@article {pmid41565063,
year = {2026},
author = {Wu, Q and Du, F and Zhang, X and Lu, Z and Zheng, X and Li, A and Zhang, X and Zhang, R and Wang, Q},
title = {Development and evaluation of a one-pot CRISPR/Cas13-based assay for syphilis detection across multiple clinical sample types.},
journal = {International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases},
volume = {164},
number = {},
pages = {108414},
doi = {10.1016/j.ijid.2026.108414},
pmid = {41565063},
issn = {1878-3511},
mesh = {Humans ; Sensitivity and Specificity ; *Treponema pallidum/genetics/isolation & purification ; *Syphilis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; },
abstract = {OBJECTIVES: To develop and evaluate a rapid, one-pot molecular assay for the detection of Treponema pallidum subspecies pallidum (TPA), addressing the limitations of current diagnostic methods influenced by sample type and pathogen load.

METHODS: A one-pot assay integrating recombinase polymerase amplification (RPA) and Cas13a-based collateral cleavage activity was established for isothermal detection of TPA. The assay targeted the tpp47/tp0574 gene and was validated using 186 clinical specimens, including whole blood, lesion exudate, and cerebrospinal fluid (CSF) samples.

RESULTS: The one-pot RPA-Cas13a assay demonstrated high analytical sensitivity and specificity for TPA detection. Clinical sensitivities were 58.97% in whole blood, 84.21% in lesion exudate, and 57.14% in CSF, with 100% specificity across all sample types.

CONCLUSION: This one-pot isothermal assay enables rapid and accurate detection of T. pallidum directly from diverse clinical samples. Its high specificity and field-friendly format make it a promising complementary tool to conventional diagnostic approaches, particularly for point-of-care testing and screening in resource-limited or high-risk settings.},
}

Humans
Sensitivity and Specificity
*Treponema pallidum/genetics/isolation & purification
*Syphilis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
*CRISPR-Cas Systems

@article {pmid41491239,
year = {2026},
author = {Amelan, A and Collins, SC and Damseh, NS and Hamada, N and Salim, A and Dvir, E and Monderer-Rothkoff, G and Harel, T and Nagata, KI and Yalcin, B and Shifman, S},
title = {CRISPR knockout screens reveal genes and pathways essential for neuronal differentiation and implicate PEDS1 in neurodevelopment.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {592-603},
pmid = {41491239},
issn = {1546-1726},
support = {466/21//Israel Science Foundation (ISF)/ ; 1863/24//Israel Science Foundation (ISF)/ ; ANR-18-CE12-0009//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {Animals ; Mice ; *Cell Differentiation/genetics ; *Neurons/physiology ; *Neurogenesis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Neurodevelopmental Disorders/genetics ; Gene Knockout Techniques/methods ; Mice, Knockout ; CRISPR-Cas Systems ; Male ; },
abstract = {Neurodevelopmental disorders (NDDs) arise from disruptions in brain development, yet the underlying pathways remain incompletely understood. Here we demonstrate that genome-wide CRISPR knockout screens in mouse embryonic stem cells differentiating into neural lineages identify hundreds of essential genes, only a minority of which are currently implicated in NDDs. Dominant NDD genes were enriched for transcriptional regulators, whereas recessive NDD genes were predominantly involved in metabolic processes. Mouse models for eight genes (Eml1, Dusp26, Dynlrb2, Mta3, Peds1, Sgms1, Slitrk4 and Vamp3) revealed marked neuroanatomical abnormalities, including microcephaly in half of the cases. Focusing on PEDS1, a key enzyme in plasmalogen biosynthesis, we identified a bi-allelic variant in individuals with microcephaly, global developmental delay and congenital cataracts. In mice, Peds1 deficiency led to accelerated cell-cycle exit and impaired neuronal differentiation and migration. These pathways required for neural differentiation provide a genetic framework for discovering additional NDD genes.},
}

Animals
Mice
*Cell Differentiation/genetics
*Neurons/physiology
*Neurogenesis/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
*Neurodevelopmental Disorders/genetics
Gene Knockout Techniques/methods
Mice, Knockout
CRISPR-Cas Systems
Male

@article {pmid41413662,
year = {2026},
author = {Green, NFO and Sutton, GJ and Pérez-Burillo, J and Wang, J and Bagot, S and Danon, HG and Walsh, K and Gokool, A and Miles, SA and Yang, G and Herring, CA and Liang, Y and Pfundstein, G and Sytnyk, V and Alinejad-Rokny, H and Lister, R and Rosenbluh, J and Gagnon-Bartsch, JA and Voineagu, I},
title = {CRISPRi screening in cultured human astrocytes uncovers distal enhancers controlling genes dysregulated in Alzheimer's disease.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {703-716},
pmid = {41413662},
issn = {1546-1726},
support = {2020814//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Astrocytes/metabolism ; *Alzheimer Disease/genetics/metabolism ; *Enhancer Elements, Genetic/genetics ; Cells, Cultured ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis ; Machine Learning ; },
abstract = {Genetic variants associated with complex traits often lie in distal enhancers. While candidate enhancers have been mapped genome wide, their functional state and gene targets in specific cell types remain unclear. Here we present AstroREG, a resource of enhancer-gene interactions in human primary astrocytes, generated by combining CRISPR inhibition (CRISPRi), single-cell RNA-seq and machine learning. By functionally testing nearly 1,000 PsychENCODE enhancers, we identified more than 150 regulatory interactions, revealing enhancers that control key astrocyte functions and genes implicated in Alzheimer's disease. The CRISPRi screen also provided valuable ground-truth data from a primary cell type for training and benchmarking prediction models of enhancer activity. We thus developed EGrf, a random forest (RF) model trained on these data, and applied it genome wide to predict regulatory interactions with high specificity. Together, our data provide a comprehensive functional map of enhancer-mediated regulation in a key glial cell type, shedding light on brain function and disease.},
}

Humans
*Astrocytes/metabolism
*Alzheimer Disease/genetics/metabolism
*Enhancer Elements, Genetic/genetics
Cells, Cultured
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Single-Cell Analysis
Machine Learning

@article {pmid37762360,
year = {2023},
author = {Mak, MCE and Gurung, R and Foo, RSY},
title = {Applications of Genome Editing Technologies in CAD Research and Therapy with a Focus on Atherosclerosis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {18},
pages = {},
pmid = {37762360},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; Humans ; *Coronary Artery Disease/therapy/genetics ; Animals ; *Atherosclerosis/genetics/therapy ; CRISPR-Cas Systems ; Genetic Therapy/methods ; },
abstract = {Cardiovascular diseases, particularly coronary artery disease (CAD), remain the leading cause of death worldwide in recent years, with myocardial infarction (MI) being the most common form of CAD. Atherosclerosis has been highlighted as one of the drivers of CAD, and much research has been carried out to understand and treat this disease. However, there remains much to be better understood and developed in treating this disease. Genome editing technologies have been widely used to establish models of disease as well as to treat various genetic disorders at their root. In this review, we aim to highlight the various ways genome editing technologies can be applied to establish models of atherosclerosis, as well as their therapeutic roles in both atherosclerosis and the clinical implications of CAD.},
}

*Gene Editing/methods
Humans
*Coronary Artery Disease/therapy/genetics
Animals
*Atherosclerosis/genetics/therapy
CRISPR-Cas Systems
Genetic Therapy/methods

@article {pmid37762332,
year = {2023},
author = {Akhtar, J and Imran, M and Wang, G},
title = {CRISPR/Cas9-Mediated CtBP1 Gene Editing Enhances Chemosensitivity and Inhibits Metastatic Potential in Esophageal Squamous Cell Carcinoma Cells.},
journal = {International journal of molecular sciences},
volume = {24},
number = {18},
pages = {},
pmid = {37762332},
issn = {1422-0067},
support = {32070681//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Alcohol Oxidoreductases/genetics/metabolism ; *Esophageal Squamous Cell Carcinoma/genetics/pathology/drug therapy/metabolism ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/genetics ; *CRISPR-Cas Systems/genetics ; *Esophageal Neoplasms/genetics/pathology/drug therapy/metabolism ; Paclitaxel/pharmacology ; *DNA-Binding Proteins/genetics/metabolism ; Cell Proliferation/drug effects/genetics ; *Gene Editing/methods ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Survival/drug effects ; Neoplasm Metastasis ; },
abstract = {Innovative therapeutic strategies for esophageal squamous cell carcinoma (ESCC) are urgently required due to the limited effectiveness of standard chemotherapies. C-Terminal Binding Protein 1 (CtBP1) has been implicated in various cancers, including ESCC. However, the precise expression patterns and functional roles of CtBP1 in ESCC remain inadequately characterized. In this study, we aimed to investigate CtBP1 expression and its role in the resistance of ESCC to paclitaxel, an effective chemotherapeutic agent. Western blotting and immunofluorescence were applied to assess CtBP1 expression in the TE-1 and KYSE-50 cell lines. We observed the marked expression of CtBP1, which was associated with enhanced proliferation, invasion, and metastasis in these cell lines. Further, we successfully generated paclitaxel resistant ESCC cell lines and conducted cell viability assays. We employed the CRISPR/Cas9 genome editing system to disable the CtBP1 gene in ESCC cell lines. Through the analysis of the drug dose-response curve, we assessed the sensitivity of these cell lines in different treatment groups. Remarkably, CtBP1-disabled cell lines displayed not only improved sensitivity but also a remarkable inhibition of proliferation, invasion, and metastasis. This demonstrates that CtBP1 may promote ESCC cell malignancy and confer paclitaxel resistance. In summary, our study opens a promising avenue for targeted therapies, revealing the potential of CtBP1 inhibition to enhance the effectiveness of paclitaxel treatment for the personalized management of ESCC.},
}

Humans
*Alcohol Oxidoreductases/genetics/metabolism
*Esophageal Squamous Cell Carcinoma/genetics/pathology/drug therapy/metabolism
Cell Line, Tumor
*Drug Resistance, Neoplasm/genetics
*CRISPR-Cas Systems/genetics
*Esophageal Neoplasms/genetics/pathology/drug therapy/metabolism
Paclitaxel/pharmacology
*DNA-Binding Proteins/genetics/metabolism
Cell Proliferation/drug effects/genetics
*Gene Editing/methods
Gene Expression Regulation, Neoplastic/drug effects
Cell Survival/drug effects
Neoplasm Metastasis

@article {pmid37755318,
year = {2023},
author = {Ravichandran, K and Khargonkar, T and Samaddar, S and Banerjee, S},
title = {CRISPR Based Programmable RNA Editing in Primary Hippocampal Neurons.},
journal = {Current protocols},
volume = {3},
number = {9},
pages = {e886},
doi = {10.1002/cpz1.886},
pmid = {37755318},
issn = {2691-1299},
mesh = {*Hippocampus/cytology/metabolism ; *Neurons/metabolism/cytology ; *RNA Editing/genetics ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Cells, Cultured ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Investigating the RNA regulation landscape primarily relies on understanding how RNA-protein interactions are governed in various cell types, including neurons. Analysis of RNA-protein interactions in physiological environments warrants the development of new tools that rely on RNA manipulation. Recently, a CRISPR-based RNA-editing tool (dCas13b-ADAR2DD) was developed to mitigate disease-associated point mutations in cell lines. Here, we explored the targeted sequence editing potential of the tool (dCas13b-ADAR2DD system) by adapting it to manipulate RNA function to visualize RNA editing in primary hippocampal neurons. This two-component system includes a programmable guide RNA (gRNA) complementary to the target RNA and a catalytically dead version of the Cas13b enzyme fused to ADAR. The RNA editing protocol outlined in this article relies on gRNA-dependent targeting of the dCas13b-ADAR fusion protein to the mutant form of the Dendra2 transcript. Dendra2 is not required for intrinsic cellular functioning. It was ectopically expressed for fluorescent detection as a proof-of-principle demonstration of targeted RNA editing. We first abrogated the fluorescence of Dendra2 by introducing a nonsense mutation that precludes the formation of the functional protein. To visualize the efficacy of the RNA editing in neurons, we used the dCas13b-ADAR2DD system to edit specific nucleotides within the Dendra2 mRNA to restore the amino acid codes critical for Dendra2 fluorescence. This method lays the foundation for future studies on the dynamics of activity-induced RNA-protein interactions in neurons and can be extended to manipulate the endogenous RNome in diverse neuronal subtypes. Furthermore, this methodology will enable investigators to visualize the spatial and temporal resolution of RNA-protein interactions without altering the genomes via conventional methods. © 2023 Wiley Periodicals LLC. Support Protocol: Preparation of mouse primary hippocampal culture Basic Protocol: Targeted editing of RNA.},
}

*Hippocampus/cytology/metabolism
*Neurons/metabolism/cytology
*RNA Editing/genetics
Animals
Mice
*CRISPR-Cas Systems/genetics
Cells, Cultured
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid37741117,
year = {2023},
author = {Lee, J and Kim, DH and Lee, K},
title = {Research Note: Injection of adenoviral CRISPR/Cas9 system targeting melanophilin gene into different sites of embryos induced regional feather color changes in posthatch quail.},
journal = {Poultry science},
volume = {102},
number = {11},
pages = {103087},
pmid = {37741117},
issn = {1525-3171},
mesh = {Animals ; *CRISPR-Cas Systems ; *Feathers/physiology ; *Gene Editing/veterinary/methods ; *Coturnix/genetics/physiology ; Adenoviridae/genetics ; *Pigmentation/genetics ; *Avian Proteins/genetics/metabolism ; },
abstract = {Poultry species is an important animal model in both avian research and the poultry industry. To advance our understanding of genetic factors and benefit both fields, a gene of interest can be genetically edited, and consequential phenotypic changes can be investigated. Injection of adenovirus containing the CRISPR/Cas9 system into avian blastoderm induced genome editing in blastodermal cells randomly, including primordial germ cells, which results in generation of whole-body knockout in the offspring of the virus-injected quail. However, to observe phenotypic and functional changes in whole-body, homozygous knockout of genes using this genome editing technology requires at least 2 generations of breeding of chimeric, and heterozygotes birds. In the current study, we developed a strategy to investigate the gene function in 1-generation by inducing regional genome editing around the injection sites with CRISPR/Cas9 adenovirus. The adenoviral CRISPR/Cas9 vector targeting the melanophilin (Mlph) gene, regulating feather pigmentation, was injected into 2 different regions of embryos, the cervical flexure of quail embryos at HH stage 13 to 15 and the tip of the upper limb bud of embryos at HH stage 22 to 24, to induce genome editing in those regions. Indel mutations in the target loci of the Mlph gene were detected by extracting genomic DNA from the embryonic tissues, and consequential phenotypes, feather color changes, were analyzed at 1 mo after hatch. Injection of the adenovirus into the cervical flexure and the tip of the upper limb bud of embryos resulted in 8 to 21% efficiency of indel mutation in the embryonic cells of the injected regions. In the posthatch quail, gray feathers were shown on their upper back and primary wing feathers, corresponding to the injection sites at embryos. Successful validation of this strategy for inducing genome editing in parts of tissues within 1-generation will accelerate studies on genetic functions with advantages of less time and cost, facilitating avian research and providing foundations for future application for the poultry industry.},
}

Animals
*CRISPR-Cas Systems
*Feathers/physiology
*Gene Editing/veterinary/methods
*Coturnix/genetics/physiology
Adenoviridae/genetics
*Pigmentation/genetics
*Avian Proteins/genetics/metabolism

@article {pmid37718698,
year = {2023},
author = {Kumar, A and Stirling, PC},
title = {Turning up the heat on essential E. coli genes.},
journal = {Molecular systems biology},
volume = {19},
number = {10},
pages = {e11933},
pmid = {37718698},
issn = {1744-4292},
support = {RGPIN 2020-04360//Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; },
mesh = {*Escherichia coli/genetics ; *Genes, Essential ; CRISPR-Cas Systems ; Alleles ; Escherichia coli Proteins/genetics ; Hot Temperature ; Gene Expression Regulation, Bacterial ; Gene Editing/methods ; },
abstract = {Temperature-sensitive (TS) alleles create tunable thermoswitches to deplete essential cellular activities and are used to dissect gene function. In their recent study, Link and colleagues (Schramm et al 2023) use a CRISPR-based approach to systematically create TS alleles across essential genes in E. coli.},
}

*Escherichia coli/genetics
*Genes, Essential
CRISPR-Cas Systems
Alleles
Escherichia coli Proteins/genetics
Hot Temperature
Gene Expression Regulation, Bacterial
Gene Editing/methods

@article {pmid37702604,
year = {2023},
author = {Pescod, P and Bevivino, G and Anthousi, A and Shelton, R and Shepherd, J and Lombardo, F and Nolan, T},
title = {Measuring the Impact of Genetic Heterogeneity and Chromosomal Inversions on the Efficacy of CRISPR-Cas9 Gene Drives in Different Strains of Anopheles gambiae.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {419-429},
doi = {10.1089/crispr.2023.0029},
pmid = {37702604},
issn = {2573-1602},
mesh = {*Anopheles/genetics ; Animals ; *Chromosome Inversion/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; Gene Editing/methods ; *Genetic Heterogeneity ; Mosquito Vectors/genetics ; },
abstract = {The human malaria vector Anopheles gambiae is becoming increasingly resistant to insecticides, spurring the development of genetic control strategies. CRISPR-Cas9 gene drives can modify a population by creating double-stranded breaks at highly specific targets, triggering copying of the gene drive into the cut site ("homing"), ensuring its inheritance. The DNA repair mechanism responsible requires homology between the donor and recipient chromosomes, presenting challenges for the invasion of laboratory-developed gene drives into wild populations of target species An. gambiae species complex, which show high levels of genome variation. Two gene drives (vas2-5958 and zpg-7280) were introduced into three An. gambiae strains collected across Africa with 5.3-6.6% variation around the target sites, and the effect of this variation on homing was measured. Gene drive homing across different karyotypes of the 2La chromosomal inversion was also assessed. No decrease in gene drive homing was seen despite target site heterology, demonstrating the applicability of gene drives to wild populations.},
}

*Anopheles/genetics
Animals
*Chromosome Inversion/genetics
*CRISPR-Cas Systems/genetics
*Gene Drive Technology/methods
Gene Editing/methods
*Genetic Heterogeneity
Mosquito Vectors/genetics

@article {pmid37493514,
year = {2023},
author = {Wei, H and Li, J and Liu, Y and Cheng, W and Huang, H and Liang, X and Huang, W and Lin, L and Zheng, Y and Chen, W and Wang, C and Chen, W and Xu, G and Wei, W and Chen, L and Zeng, Y and Lu, Z and Li, S and Lin, Z and Wang, J and Lin, M},
title = {Rapid and Ultrasensitive Detection of Plasmodium spp. Parasites via the RPA-CRISPR/Cas12a Platform.},
journal = {ACS infectious diseases},
volume = {9},
number = {8},
pages = {1534-1545},
doi = {10.1021/acsinfecdis.3c00087},
pmid = {37493514},
issn = {2373-8227},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Plasmodium/genetics/isolation & purification ; Sensitivity and Specificity ; *Malaria/diagnosis/parasitology ; Recombinases/metabolism/genetics ; Plasmodium falciparum/genetics/isolation & purification ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Malaria, Falciparum/diagnosis ; },
abstract = {Microscopic examination of thick and thin blood smears stained with Giemsa dye is considered the primary diagnostic tool for the confirmation and management of suspected clinical malaria. However, detecting gametocytes is relatively insensitive, particularly in asymptomatic individuals with low-density Plasmodium infections. To complement existing diagnostic methods, a rapid and ultrasensitive point-of-care testing (POCT) platform for malaria detection is urgently needed and necessary. A platform based on recombinase polymerase amplification (RPA) followed by CRISPR/Cas12a (referred to as RPA-CRISPR/Cas12a) was developed and optimized for the determination of Plasmodium spp. parasites, particularly Plasmodium falciparum, using a fluorescence-based assay (FBDA), lateral flow test strips (LFTS), or naked eye observation (NEO). Then, the established platform was assessed with clinical malaria isolates. Under optimal conditions, the detection threshold was 1 copy/μL for the plasmid, and the limit of detection was 3.11-7.27 parasites/μL for dried blood spots. There was no cross-reactivity against blood-borne pathogens. For the accuracies of RPA-CRISPR/Cas12a, Plasmodium spp. and P. falciparum testing were 98.68 and 94.74%, respectively. The method was consistent with nested PCR results and superior to the qPCR results. RPA-CRISPR/Cas12a is a rapid, ultrasensitive, and reliable platform for malaria diagnosis. The platform requires no or minimal instrumentation for nucleic acid amplification reactions and can be read with the naked eye. Compared with similar diagnostic methods, this platform improves the reaction speed while reducing detection requirements. Therefore, this platform has the potential to become a true POCT for malaria parasites.},
}

Humans
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Plasmodium/genetics/isolation & purification
Sensitivity and Specificity
*Malaria/diagnosis/parasitology
Recombinases/metabolism/genetics
Plasmodium falciparum/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods
Point-of-Care Testing
Malaria, Falciparum/diagnosis

@article {pmid37482112,
year = {2023},
author = {Liu, L and Zhang, S and Zhi, F and Song, Y and Li, B and Gao, P and Zhang, Y and Ma, K and Xu, J and Jiang, B and Chu, Y and Li, Y and Qin, J},
title = {RNA helicase DExD/H-box 5 modulates intestinal microbiota in mice.},
journal = {Microbial pathogenesis},
volume = {182},
number = {},
pages = {106265},
doi = {10.1016/j.micpath.2023.106265},
pmid = {37482112},
issn = {1096-1208},
mesh = {Animals ; *Gastrointestinal Microbiome/genetics ; *DEAD-box RNA Helicases/genetics/metabolism ; Mice, Knockout ; Mice ; RNA, Ribosomal, 16S/genetics ; Ileum/microbiology/pathology ; Feces/microbiology ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Bacteria/classification/genetics/isolation & purification ; },
abstract = {The RNA helicase DExD/H-box (DDX) family of proteins plays a central role in host cellular RNA metabolism, including mRNA regulation, microRNA biogenesis, and ribosomal processing. DDX5, also known as p68, promotes viral replication and tumorigenesis. However, there have been no studies on the regulation of the intestinal microbiota by DDX family proteins. We constructed DDX5 knockout mice (Ddx5[+/-]) using CRISPR/CAS9 technology. Subsequently, DDX5 knockout mice were analyzed for PCR products, mRNA levels, protein expression, immunohistochemistry, and histopathological lesions. Fecal (n = 12) and ileum (n = 12) samples were collected from the Ddx5[+/-] and wild-type (Ddx5[+/+]) mice. The diversity, richness, and structural separation of the intestinal microbiota of the Ddx5[+/-] and Ddx5[+/+] mice were determined by 16S rRNA sequencing and analysis. Ddx5[+/-] mice were successfully established, and the ileum had normal morphology, a clear layer of tissue structures, and neatly arranged cupped cells. DDX5 knockout mice did not exhibit adverse effects on the ileal tissue. Microbial diversity and abundance were not significantly different, but the microbial structure of the intestinal microbiota was clustered separately between Ddx5[+/+] and Ddx5[+/-] mice. Furthermore, we found that the relative abundance of Akkermansia and Clostridium_sensu_stricto_1 in the Ddx5[+/-] mice was significantly lower than in the Ddx5[+/+] mice. These analyses indicated specific interactions between the intestinal microbiota and DDX5 protein. Our results indicate that DDX5 has a significant effect on the composition of the intestinal microbiota in mice, suggesting its potential as a promising novel target for the treatment of inflammation and tumorigenesis in the intestine.},
}

Animals
*Gastrointestinal Microbiome/genetics
*DEAD-box RNA Helicases/genetics/metabolism
Mice, Knockout
Mice
RNA, Ribosomal, 16S/genetics
Ileum/microbiology/pathology
Feces/microbiology
Mice, Inbred C57BL
CRISPR-Cas Systems
Bacteria/classification/genetics/isolation & purification

@article {pmid37477766,
year = {2023},
author = {Hu, J and Guan, X and Zhao, M and Xie, P and Guo, J and Tan, J},
title = {Genome-wide CRISPR-Cas9 Knockout Screening Reveals a TSPAN3-mediated Endo-lysosome Pathway Regulating the Degradation of α-Synuclein Oligomers.},
journal = {Molecular neurobiology},
volume = {60},
number = {11},
pages = {6731-6747},
pmid = {37477766},
issn = {1559-1182},
mesh = {Humans ; *Lysosomes/metabolism ; *alpha-Synuclein/metabolism ; HEK293 Cells ; *Tetraspanins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Proteolysis ; *Endosomes/metabolism ; *Gene Knockout Techniques ; *Protein Multimerization ; },
abstract = {Misfolding and aggregation of α-Synuclein (α-Syn), which are hallmark pathological features of neurodegenerative diseases such as Parkinson's disease (PD) and dementia with Lewy Bodies, continue to be significant areas of research. Among the diverse forms of α-Syn - monomer, oligomer, and fibril, the oligomer is considered the most toxic. However, the mechanisms governing α-Syn oligomerization are not yet fully understood. In this study, we utilized genome-wide CRISPR/Cas9 loss-of-function screening in human HEK293 cells to identify negative regulators of α-Syn oligomerization. We found that tetraspanin 3 (TSPAN3), a presumptive four-pass transmembrane protein, but not its homolog TSPAN7, significantly modulates α-Syn oligomer levels. TSPAN3 was observed to interact with α-Syn oligomers, regulate the amount of α-Syn oligomers on the cell membrane, and promote their degradation via the clathrin-AP2 mediated endo-lysosome pathway. Our findings highlight TSPAN3 as a potential regulator of α-Syn oligomers, presenting a promising target for future PD prevention and treatment strategies.},
}

Humans
*Lysosomes/metabolism
*alpha-Synuclein/metabolism
HEK293 Cells
*Tetraspanins/metabolism/genetics
*CRISPR-Cas Systems/genetics
*Proteolysis
*Endosomes/metabolism
*Gene Knockout Techniques
*Protein Multimerization

@article {pmid37451121,
year = {2023},
author = {Hu, Y and Qiao, Y and Li, XQ and Xiang, Z and Wan, Y and Wang, P and Yang, Z},
title = {Development of an inducible Cas9 nickase and PAM-free Cas12a platform for bacterial diagnostics.},
journal = {Talanta},
volume = {265},
number = {},
pages = {124931},
doi = {10.1016/j.talanta.2023.124931},
pmid = {37451121},
issn = {1873-3573},
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *Deoxyribonuclease I/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics ; CRISPR-Cas Systems ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/genetics/metabolism ; Humans ; },
abstract = {Rapid, efficient, specific and sensitive diagnostic techniques are critical for selecting appropriate treatments for drug-resistant bacterial infections. To address this challenge, we have developed a novel diagnostic method, called the Dual-Cas Tandem Diagnostic Platform (DTDP), which combines the use of Cas9 nickase (Cas9n) and Cas12a. DTDP works by utilizing the Cas9n-sgRNA complex to create a nick in the target strand's double-stranded DNA (dsDNA). This prompts DNA polymerase to displace the single-stranded DNA (ssDNA) and leads to cycles of DNA replication through nicking, displacement, and extension. The ssDNA is then detected by the Cas12a-crRNA complex (which is PAM-free), activating trans-cleavage and generating a fluorescent signal from the fluorescent reporter. DTDP exhibits a high sensitivity (1 CFU/mL or 100 ag/μL), high specificity (specifically to MRSA in nine pathogenic species), and excellent accuracy (100%). The dual RNA recognition process in our method improves diagnostic specificity by decreasing the limitations of Cas12a in detecting dsDNA protospacer adjacent motifs (PAMs) and leverages multiple advantages of multi-Cas enzymes in diagnostics. This novel approach to pathogenic microorganism detection has also great potential for clinical diagnosis.},
}

*CRISPR-Associated Proteins/metabolism/genetics
*Deoxyribonuclease I/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics
CRISPR-Cas Systems
DNA, Single-Stranded/genetics
*Bacterial Proteins/genetics/metabolism
Humans

@article {pmid37421327,
year = {2023},
author = {Yang, M and Wan, S and Chen, J and Chen, W and Wang, Y and Li, W and Wang, M and Guan, R},
title = {Mutation to a cytochrome P450 -like gene alters the leaf color by affecting the heme and chlorophyll biosynthesis pathways in Brassica napus.},
journal = {The Plant journal : for cell and molecular biology},
volume = {116},
number = {2},
pages = {432-445},
doi = {10.1111/tpj.16382},
pmid = {37421327},
issn = {1365-313X},
support = {32171974//National Natural Science Foundation of China/ ; 2022YFF1003500//the National Key R&D Program of China/ ; },
mesh = {*Chlorophyll/biosynthesis/metabolism ; *Brassica napus/genetics/metabolism ; *Plant Leaves/genetics/metabolism/physiology ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; *Heme/biosynthesis/metabolism ; *Plant Proteins/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; Photosynthesis ; Biosynthetic Pathways ; Gene Expression Regulation, Plant ; Gene Editing ; },
abstract = {The regulated biosynthesis of chlorophyll is important because of its effects on plant photosynthesis and dry biomass production. In this study, a map-based cloning approach was used to isolate the cytochrome P450 -like gene BnaC08g34840D (BnCDE1) from a chlorophyll-deficient mutant (cde1) of Brassica napus obtained by ethyl methanesulfonate (EMS) mutagenization. Sequence analyses revealed that BnaC08g34840D in the cde1 mutant (BnCDE1[I320T]) encodes a substitution at amino acid 320 (Ile320Thr) in the conserved region. The over-expression of BnCDE1[I320T] in ZS11 (i.e., gene-mapping parent with green leaves) recapitulated a yellow-green leaf phenotype. The CRISPR/Cas9 genome-editing system was used to design two single-guide RNAs (sgRNAs) targeting BnCDE1[I320T] in the cde1 mutant. The knockout of BnCDE1[I320T] in the cde1 mutant via a gene-editing method restored normal leaf coloration (i.e., green leaves). These results indicate that the substitution in BnaC08g34840D alters the leaf color. Physiological analyses showed that the over-expression of BnCDE1[I320T] leads to decreases in the number of chloroplasts per mesophyll cell and in the contents of the intermediates of the chlorophyll biosynthesis pathway in leaves, while it increases heme biosynthesis, thereby lowering the photosynthetic efficiency of the cde1 mutant. The Ile320Thr mutation in the highly conserved region of BnaC08g34840D inhibited chlorophyll biosynthesis and disrupted the balance between heme and chlorophyll biosynthesis. Our findings may further reveal how the proper balance between the chlorophyll and heme biosynthesis pathways is maintained.},
}

*Chlorophyll/biosynthesis/metabolism
*Brassica napus/genetics/metabolism
*Plant Leaves/genetics/metabolism/physiology
*Cytochrome P-450 Enzyme System/genetics/metabolism
*Heme/biosynthesis/metabolism
*Plant Proteins/genetics/metabolism
Mutation
CRISPR-Cas Systems
Photosynthesis
Biosynthetic Pathways
Gene Expression Regulation, Plant
Gene Editing

@article {pmid36960824,
year = {2023},
author = {Makrogkikas, S and Cheng, RK and Lu, H and Roy, S},
title = {A conserved function of Pkhd1l1, a mammalian hair cell stereociliary coat protein, in regulating hearing in zebrafish.},
journal = {Journal of neurogenetics},
volume = {37},
number = {3},
pages = {85-92},
doi = {10.1080/01677063.2023.2187792},
pmid = {36960824},
issn = {1563-5260},
mesh = {Animals ; Zebrafish ; *Zebrafish Proteins/genetics/metabolism/physiology ; *Hair Cells, Auditory/metabolism/physiology ; *Hearing/genetics/physiology ; *Stereocilia/metabolism ; Mice ; Hearing Loss/genetics ; Animals, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Pkhd1l1 is predicted to encode a very large type-I transmembrane protein, but its function has largely remained obscure. Recently, it was shown that Pkhdl1l1 is a component of the coat that decorates stereocilia of outer hair cells in the mouse ear. Consistent with this localization, conditional deletion of Pkhd1l1 specifically from hair cells, was associated with progressive hearing loss. In the zebrafish, there are two paralogous pkhd1l1 genes - pkhd1l1α and pkhd1l1β. Using CRISPR-Cas9 mediated gene editing, we generated loss-of-function alleles for both and show that the double mutants exhibit nonsense-mediated-decay (NMD) of the RNAs. With behavioural assays, we demonstrate that zebrafish pkhd1l1 genes also regulate hearing; however, in contrast to Pkhd1l1 mutant mice, which develop progressive hearing loss, the double mutant zebrafish exhibited statistically significant hearing loss even from the larval stage. Our data highlight a conserved function of Pkhd1l1 in hearing and based on these findings from animal models, we postulate that PKHD1L1 could be a candidate gene for sensorineural hearing loss (SNHL) in humans.},
}

Animals
Zebrafish
*Zebrafish Proteins/genetics/metabolism/physiology
*Hair Cells, Auditory/metabolism/physiology
*Hearing/genetics/physiology
*Stereocilia/metabolism
Mice
Hearing Loss/genetics
Animals, Genetically Modified
CRISPR-Cas Systems

@article {pmid36948605,
year = {2023},
author = {Deng, Y and Han, X and Chen, H and Zhao, C and Chen, Y and Zhou, J and de The, H and Zhu, J and Yuan, H},
title = {Ypel5 regulates liver development and function in zebrafish.},
journal = {Journal of molecular cell biology},
volume = {15},
number = {3},
pages = {},
pmid = {36948605},
issn = {1759-4685},
support = {31371479//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/genetics/metabolism/embryology ; *Zebrafish Proteins/metabolism/genetics ; *Liver/metabolism/embryology/growth & development ; Hepatocyte Nuclear Factor 4/metabolism/genetics ; Cell Proliferation/genetics ; Hepatocytes/metabolism ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Signal Transduction ; },
abstract = {YPEL5 is a member of the Yippee-like (YPEL) gene family that is evolutionarily conserved in eukaryotic species. To date, the physiological function of YPEL5 has not been assessed due to a paucity of genetic animal models. Here, using CRISPR/Cas9-mediated genome editing, we generated a stable ypel5-/- mutant zebrafish line. Disruption of ypel5 expression leads to liver enlargement associated with hepatic cell proliferation. Meanwhile, hepatic metabolism and function are dysregulated in ypel5-/- mutant zebrafish, as revealed by metabolomic and transcriptomic analyses. Mechanistically, Hnf4a is identified as a crucial downstream mediator that is positively regulated by Ypel5. Zebrafish hnf4a overexpression could largely rescue ypel5 deficiency-induced hepatic defects. Furthermore, PPARα signaling mediates the regulation of Hnf4a by Ypel5 through directly binding to the transcriptional enhancer of the Hnf4a gene. Herein, this work demonstrates an essential role of Ypel5 in hepatocyte proliferation and function and provides the first in vivo evidence for a physiological role of the ypel5 gene in vertebrates.},
}

Animals
*Zebrafish/genetics/metabolism/embryology
*Zebrafish Proteins/metabolism/genetics
*Liver/metabolism/embryology/growth & development
Hepatocyte Nuclear Factor 4/metabolism/genetics
Cell Proliferation/genetics
Hepatocytes/metabolism
Gene Expression Regulation, Developmental
CRISPR-Cas Systems
Signal Transduction

@article {pmid41779860,
year = {2026},
author = {Zhou, X and Pan, D and Zhou, J and Chen, W and Han, G and Zhang, R and Wang, C and Mao, Y and Du, Z and Zhang, F and Yue, H and Ma, J and Li, Z and Shen, RJ and Wang, B and Zhu, W and Peng, Y and Jin, K and Wu, DD and Wang, W and Zhou, B and Jin, ZB and Chen, L},
title = {Cis-regulatory evolution reveals sensory trade-offs as a genetic basis for temporal niche evolution in tapirs.},
journal = {Science advances},
volume = {12},
number = {10},
pages = {eadz4758},
pmid = {41779860},
issn = {2375-2548},
mesh = {Animals ; *Evolution, Molecular ; Mice ; *Regulatory Sequences, Nucleic Acid ; Biological Evolution ; CRISPR-Cas Systems ; Humans ; },
abstract = {Evolutionary shifts in diel activity patterns shape sensory remodeling across mammals, yet the genetic basis remains poorly understood. Tapirs represent a unique natural experiment, having reverted from a cathemeral ancestor to a nocturnal niche characterized by reduced vision but enhanced hearing and olfaction. Here, we investigate the genetic basis of this phenomenon by generating high-quality chromosome-level genomes for Tapirus terrestris and Tapirus indicus. Comparative analyses revealed extensive lineage-specific remodeling of genes and cis-regulatory elements linked to sensory pathways. Notably, functional validation via CRISPR-Cas9 editing of a tapir-specific conserved noncoding element (CNE74) upstream of the FLT1 gene in mice revealed coordinated sensory effects, including retinal degeneration and reduced visual acuity, yet enhanced auditory sensitivity. These findings suggest that regulatory element evolution may induce pleiotropic effects on competing sensory modalities, offering genetic insights into sensory evolution during temporal niche adaptation and potential relevance to human retinal vascular diseases.},
}

Animals
*Evolution, Molecular
Mice
*Regulatory Sequences, Nucleic Acid
Biological Evolution
CRISPR-Cas Systems
Humans

@article {pmid41779781,
year = {2026},
author = {Ju, X and Dong, L and Liu, T and Zhang, F and Sun, X and Schwoerer, MP and Ren, W and Gong, M and Ploss, A and Qin, W and Wu, X and Wang, L and Ding, Q},
title = {EIF4H and YBX1 are essential host factors for hepatitis E virus replication and pathogenesis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2529289123},
doi = {10.1073/pnas.2529289123},
pmid = {41779781},
issn = {1091-6490},
support = {2023YFC2306900//National Key Research and Development Plan of China/ ; 82341084//MOST | National Natural Science Foundation of China (NSFC)/ ; 82272302//MOST | National Natural Science Foundation of China (NSFC)/ ; 82522053//MOST | National Natural Science Foundation of China (NSFC)/ ; 20251080029//Tsinghua University Dushi Program/ ; Not applicable//SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine/ ; Not applicable//High Meadows Environmental Institute/ ; T32 GM007388/GM/NIGMS NIH HHS/United States ; },
mesh = {*Hepatitis E virus/physiology/pathogenicity/genetics ; *Virus Replication ; Humans ; *Hepatitis E/virology/metabolism/pathology/genetics ; Animals ; Rats ; *Y-Box-Binding Protein 1/metabolism/genetics ; *Eukaryotic Initiation Factors/metabolism/genetics ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Hepatocytes/virology ; RNA-Binding Proteins ; },
abstract = {Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Despite the availability of a vaccine in China, no direct-acting antivirals are approved, and host factors required for HEV replication remain poorly defined. Here, using a genome-wide CRISPR/Cas9 knockout screen in a replicon system, we identified Eukaryotic Translation Initiation Factor 4H (EIF4H) and Y-Box Binding Protein 1 (YBX1) as essential host factors for HEV replication and pathogenesis. Knockout of either factor markedly impaired replication of HEV genotypes 1, 3, and 4, as well as HEV infection and production in hepatocellular carcinoma cells and human induced pluripotent stem cell-derived hepatocyte-like cells, while leaving SARS-CoV-2, hepatitis B virus, hepatitis C virus, and Zika virus unaffected, underscoring their HEV-specific roles. Mechanistically, EIF4H interacts with ORF1 via its methyltransferase-Y-papain-like protease region, and EIF4H deficiency alters the composition of the ORF1-associated replication complex. By contrast, YBX1 is dispensable for ORF1 translation and RNA binding but is specifically required for ORF1 proteolytic processing, a prerequisite for assembling a functional replication machinery. EIF4H knockout rats and liver-specific YBX1 knockout rats were largely resistant to rat HEV-C1 infection, showing profound reductions in viral shedding, suppressed hepatic and intestinal viral loads, and protection from liver pathology. Together, our findings establish EIF4H and YBX1 as essential host factors for HEV infection and pathogenesis and reveal potential targets for antiviral intervention.},
}

*Hepatitis E virus/physiology/pathogenicity/genetics
*Virus Replication
Humans
*Hepatitis E/virology/metabolism/pathology/genetics
Animals
Rats
*Y-Box-Binding Protein 1/metabolism/genetics
*Eukaryotic Initiation Factors/metabolism/genetics
Host-Pathogen Interactions
CRISPR-Cas Systems
Hepatocytes/virology
RNA-Binding Proteins

@article {pmid41777069,
year = {2026},
author = {Zamperin, G and Palumbo, E and Castellan, M and Marciano, S and Fusaro, A and Monne, I},
title = {Metagenomic sequencing of zoonotic viruses: evaluation of a CRISPR-Cas-based rRNA depletion system.},
journal = {Veterinaria italiana},
volume = {62},
number = {2},
pages = {},
doi = {10.12834/VetIt.3908.38985.2},
pmid = {41777069},
issn = {1828-1427},
mesh = {*CRISPR-Cas Systems ; Animals ; *Metagenomics/methods ; *RNA, Ribosomal/genetics ; *Zoonoses/virology ; Genome, Viral ; },
abstract = {Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9-based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H-based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.},
}

*CRISPR-Cas Systems
Animals
*Metagenomics/methods
*RNA, Ribosomal/genetics
*Zoonoses/virology
Genome, Viral

@article {pmid41773018,
year = {2026},
author = {Yang, T and Tang, M and Xu, L and Jiang, L and Jiang, L and Zou, Y and Wang, J and Liu, Z and Chen, F and Ban, Y and Ren, W and Cheng, W},
title = {A tailored phosphorothioate coordinator enables CRISPR/Cas in-situ amplification.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773018},
issn = {1362-4962},
support = {U24A20751//National Natural Science Foundation of China/ ; 82372334//National Natural Science Foundation of China/ ; 82502827//National Natural Science Foundation of China/ ; CSTB2023NSCQ-LZX0022//Chongqing Education Commission/ ; CSTB2024NSCQ-QCXMX0006//New Chongqing Youth Innovative Talents Project/ ; //National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Phosphorothioate Oligonucleotides/chemistry/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics ; CRISPR-Associated Proteins/metabolism/genetics/chemistry ; RNA, Viral/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Uterine Cervical Neoplasms/virology/diagnosis ; Female ; HeLa Cells ; Endodeoxyribonucleases/metabolism/genetics ; },
abstract = {The CRISPR/Cas system is a powerful tool for molecular diagnostics, but its reliance on linear amplification constrains sensitivity, particularly for in situ imaging. Here, we discovered that phosphorothioate (PS)-modified activators can modulate Cas enzyme conformation via hydrophobic anchoring. By adjusting the PS modification sites, we achieved precise control over Cas activation and trans-cleavage resistance. Guided by this mechanism, we proposed a tailored design strategy featuring a "scattered" PS modification to engineer a linear "Coordinator" probe. This design effectively decouples Cas enzyme activation from substrate trans-cleavage resistance, enabling the construction of a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). SACA achieves exponential amplification without external enzymes, enhancing Cas12a and Cas13a sensitivity by 50 000-fold and 10 000-fold, respectively. Furthermore, the superior biostability and structural simplicity of these linear probes endow SACA with excellent compatibility, facilitating precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells. This study not only advances the understanding of Cas enzyme regulation by chemically modified nucleic acids but also establishes a new paradigm for precise and efficient molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
*Phosphorothioate Oligonucleotides/chemistry/genetics/metabolism
*Nucleic Acid Amplification Techniques/methods
Human papillomavirus 16/genetics
CRISPR-Associated Proteins/metabolism/genetics/chemistry
RNA, Viral/genetics/metabolism
RNA, Messenger/genetics/metabolism
Uterine Cervical Neoplasms/virology/diagnosis
Female
HeLa Cells
Endodeoxyribonucleases/metabolism/genetics

@article {pmid41773016,
year = {2026},
author = {Park, H and Yun, J and Lee, K and Kim, JH and Park, JH and Park, YJ and Park, JH and Lee, H and Kim, MG},
title = {Functional decoupling of crRNA enables customizable CRISPR diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773016},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; RS-2025-16063091//National Research Council of Science and Technology/ ; RS-2024-00411137//National Research Council of Science and Technology/ ; CRC22024-500//National Research Council of Science and Technology/ ; //National Research Foundation of Korea/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {One-pot CRISPR-based diagnostics have transformed nucleic acid testing, yet their design customizability remains constrained. Because target programming and cis-cleavage activity are simultaneously determined during CRISPR RNA (crRNA) design, optimizing cleavage activity to match isothermal amplification inevitably requires altering the programmed crRNA sequence. This requirement fundamentally constrains the range of compatible target sequences, imposing limitations on the flexible design of diagnostic assays. Here, we establish a customizable one-pot system by decoupling the dual functions inherent in crRNA design to enable their independent control. In this strategy, target programming remains defined by the crRNA sequence, whereas cis-cleavage activity is regulated by the reaction energy barrier. We selectively modulate this energy barrier through the introduction of a crRNA-complementary RNA oligonucleotide, achieving cleavage regulation without altering the crRNA sequence. Consequently, this approach ensures that cis-cleavage activity matches isothermal amplification conditions independent of the programmed target sequence, thereby realizing a customizable CRISPR diagnostic system. We validated the clinical applicability of this system using 120 patient-derived samples, achieving sensitivity and specificity comparable to quantitative polymerase chain reaction. Collectively, this work resolves a fundamental constraint of CRISPR diagnostics and establishes a customizable and clinically deployable platform for next-generation nucleic acid testing.},
}

*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Nucleic Acid Amplification Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41772759,
year = {2026},
author = {Donega, S and Gorospe, M and Harries, LW and Ferrucci, L},
title = {Loss of Splicing Homeostasis as a Hallmark of Aging.},
journal = {Molecular and cellular biology},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10985549.2026.2627235},
pmid = {41772759},
issn = {1098-5549},
abstract = {Alternative splicing is a fundamental mechanism that ensures accurate gene expression, supports cellular adaptability, and expands protein diversity beyond the limits of a fixed gene pool. With aging, splicing fidelity weakens, contributing to decline in RNA homeostasis and disrupting essential cellular functions, including mitochondrial oxidative phosphorylation, genome stability, and immune regulation, and in turn accelerating tissue and organ dysfunction. Evidence from senescent cells, aged tissues, and model organisms shows that altered levels of splicing factors and increased RNA polymerase II elongation rates impair co-transcriptional splicing and promote mis-spliced isoforms that reinforce senescence and drive pathology. Dysfunction of RNA-binding proteins further contributes to aberrant splicing, linking splicing defects to age-related diseases such as atherosclerosis, osteoarthritis, sarcopenia, and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Therapeutic strategies to correct splicing defects, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, ADAR-mediated editing, and RNA aptamers, can restore a homeostatic balance of mRNA isoforms. However, major challenges remain, including distinguishing adaptive physiological from pathological splicing 'noise' and achieving targeted delivery to tissues. Despite these obstacles, RNA splicing dysregulation represents a promising avenue to extend health span by reestablishing homeostatic RNA programs, and reinforces the idea that "transcriptomic instability" is a hallmark of aging.},
}

@article {pmid41771871,
year = {2026},
author = {Becerra, B and Wittibschlager, S and Patel, ZM and Kutschat, AP and Delano, J and Che, E and Tauber, A and Wu, T and Starrs, M and Horstmann, CS and Müller, S and Whittaker, MN and Sylvander, E and Lehner, M and Love, MI and Kleinstiver, BP and Jankowiak, M and Bauer, DE and Seruggia, D and Pinello, L},
title = {Nucleotide-resolution mapping of regulatory elements via allelic readout of tiled base editing.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-69918-8},
pmid = {41771871},
issn = {2041-1723},
abstract = {CRISPR tiling screens have enabled the characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing and enrichment analysis. This study introduces an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. As a proof of concept, we studied a putative CD19 enhancer, an immunotherapy target in leukemia, and identified alleles and single nucleotides crucial for CD19 regulation. Our visualization tools revealed transcription factor motifs corresponding to the top-ranked nucleotides. Validation experiments confirmed that mutations in MYB, PAX5, and EBF1 binding sites reduce CD19 expression. Critically, editing MYB and PAX5 motifs conferred resistance to CD19 CAR-T cell therapy, revealing how non-coding variants can drive immunotherapy escape. Taken together, this approach achieves nucleotide-resolution genotype-phenotype mapping at regulatory elements beyond conventional gRNA-based screens.},
}

@article {pmid41771837,
year = {2026},
author = {Schoger, E and Kim, R and Bleckwedel, F and Peralta, T and Priesmeier, L and Fischer, J and Stengel, L and Rocha, C and Santos, GL and Lutz, S and Boileau, E and Baumgarten, N and Schulz, MH and Dieterich, C and Müller, OJ and Cyganek, L and Cabrera-Orefice, A and Eberl, H and Maack, C and Streckfuss-Bömeke, K and Pavez-Giani, M and Doroudgar, S and Sossalla, ST and Zelarayán, LC},
title = {Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {41771837},
issn = {2059-3635},
mesh = {*Myocytes, Cardiac/metabolism/pathology ; *Kruppel-Like Transcription Factors/genetics/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; *Fibrosis/genetics/pathology ; *Cellular Reprogramming/genetics ; Humans ; Mice ; Fibroblasts/metabolism/pathology ; Gene Expression Regulation/genetics ; *Transcription Factors/genetics ; },
abstract = {Transcriptional activity perturbation holds promise for selectively modulating harmful transcriptional networks, but its therapeutic potential remains largely unexplored. We employed a network-based analysis of single-cell heart transcriptomes to identify transcription factor activities linked to pathological cardiomyocytes in vivo. This analysis revealed that transcriptional activity of Krüppel-like factor 15 (KLF15) exhibited the most significant change in pathological cardiomyocytes, characterized by less effective repression of disease-associated genes in stressed hearts, which correlated with reduced KLF15 expression. To restore KLF15 activity, we utilized CRISPR/nuclease-dead (d)Cas9-based transcriptional enhancement (CRISPRa) in cardiomyocytes, which effectively abolished fetal reprogramming by simultaneously suppressing pathological gene expression and restoring metabolic homeostasis under sustained stress conditions. Furthermore, we identified a novel cell-nonautonomous anti-fibrotic effect mediated by cardiomyocyte-fibroblast crosstalk, and revealed the contribution of KLF15-dependent Alpha-2-glycoprotein 1, zinc-binding (AZGP1) regulation in this process. We also elucidated the upstream mechanisms of KLF15 regulation, highlighting its role as a cell-specific downstream target of the broad TGF-β canonical signaling pathway, along with its downstream-dependent mechanisms in human cardiomyocytes. Finally, to enhance the therapeutic potential of this approach, we engineered and validated an adeno-associated viral (AAV) vector with a small CRISPRa system for endogenous regulation in human cardiomyocytes suitable for clinical applications. Overall, we elucidated a regulatory circuit involving TGF-β, KLF15, and AZGP1, which coordinates critical pathological responses through cellular crosstalk between cardiomyocytes and fibroblasts. Importantly, we demonstrated the efficacy of CRISPRa as an epigenetic intervention restoring a critical transcriptional function disrupted in non-genetic heart failure. This approach provides a promising blueprint for future adaptation targeting additional non-hereditary pathologies.},
}

*Myocytes, Cardiac/metabolism/pathology
*Kruppel-Like Transcription Factors/genetics/metabolism
Animals
CRISPR-Cas Systems/genetics
*Fibrosis/genetics/pathology
*Cellular Reprogramming/genetics
Humans
Mice
Fibroblasts/metabolism/pathology
Gene Expression Regulation/genetics
*Transcription Factors/genetics

@article {pmid41769381,
year = {2026},
author = {Mandal, S and Baloch, AR and Yuan, X and Chen, J and Saribas, AS and Zhu, Y and Zhang, D and Jaijyan, D and Xu, J and Hossain, R and Sisto, I and Wang, H and Yang, X and Li, Q and Hu, W},
title = {Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines.},
journal = {Materials today. Bio},
volume = {37},
number = {},
pages = {102939},
pmid = {41769381},
issn = {2590-0064},
abstract = {The persistence of HIV-1 latent reservoirs remains the principal barrier to a cure, as viral rebound occurs upon interruption of antiretroviral therapy. CRISPR/Cas genome editing offers a promising strategy to excise proviruses from host genome; however, the absence of a targeted and clinically viable delivery platform has hindered its translational application. Here, we report a chemistry-driven, CD4-targeted lipid nanoparticle (LNP) delivery platform employing a unique bipolar conjugation strategy to decorate dual CD4-targeted Designed Ankyrin Repeat Proteins (DARPins-55 and -57) on LNP (dual-DARPin-LNP). The N- and C-terminally modified DARPin-55/57 was thiolated stepwise, then bipolar maleimide-thiol coupling conjugated the thiolates to the maleimide-functionalized LNP surface. This coupling strategy ensured DARPin proper orientation on the LNP surface for efficient uptake by resting CD4 T cells. This dual-DARPin-LNP system was engineered for selective and efficient co-delivery of spCas9-GFP mRNA (Sp9m) and HIV-1-specific single-guide RNAs (sgRNAs) targeting LTR and Gag (LGsg) into HIV-1 latently infected CD4 T cells. In widely used HIV-1 latency models with defined proviral modifications (J-Lat 10.6 and 2D10 cell lines), dual-DARPin-LNP loaded with Sp9m/LGsg efficiently excised integrated HIV-1 proviral DNA, as confirmed by standard PCR genotyping, absolute digital PCR quantification, confocal microscopy, and flow cytometry. Importantly, proviral excision functionally blocked HIV-1 reactivation following stimulation with latency-reversing agents suberoylanilide hydroxamic acid (SAHA) and TNFα. Together, these findings establish a modular, non-viral, receptor-guided delivery platform for CD4 T cell targeting and provide proof-of-concept for precise HIV-1 DNA excision and reactivation blockade in established latency models. This new strategy represents a step toward next-generation curative interventions against persistent HIV-1 infection.},
}

@article {pmid41766888,
year = {2026},
author = {Li, X and Zhao, Y and Guo, X and Bai, Y and Wang, J},
title = {Characterization and diversity of defense systems in Providencia pathogen.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1755933},
pmid = {41766888},
issn = {1664-3224},
mesh = {*Providencia/genetics/immunology/virology ; Phylogeny ; Genome, Bacterial ; *Enterobacteriaceae Infections/microbiology/immunology ; CRISPR-Cas Systems ; Bacteriophages ; Humans ; },
abstract = {INTRODUCTION: Providencia species are emerging opportunistic pathogens associated with multidrug-resistant infections, yet their molecular defense mechanisms against phage or mobile genetic elements remain poorly characterized.

METHODS: We present a comprehensive pan-genomic analysis of antiviral defense systems across 73 complete genomes (or chromosomes) of Providencia stuartii (n = 31) and Providencia rettgeri (n = 42), using DefenseFinder and CRISPRCasFinder. We further expanded analysis of contig/scaffold assemblies to confirm conservation of core defense profiles across assembly types. BacMGEnet was employed to derive spacer-MGE interaction networks. Phylogenetic reconstruction and gene gain and loss modeling were performed to assess evolutionary patterns. To validate functionality, we experimentally tested the anti-phage activity of Gabija and Septu in heterologous E. coli assays, including point mutation analysis of conserved residues.

RESULTS: We reveal a diverse and complex defense repertoire dominated by restriction-modification systems and CRISPR-Cas Class 1 Type I-F, with significant contributions from toxin-antitoxin, GAPS2, PsyrTA, and Mokosh systems. Notably, defense genes are non-randomly distributed, often clustering into genomic islands suggestive of horizontal acquisition. Expanded analysis confirms conservation of core defense profiles across assembly types, supporting the utility of lower-quality data when complete genomes are scarce. Comparative analysis uncovers species-specific differences, with P. rettgeri harboring a higher abundance of non-CRISPR systems. BacMGEnet-derived spacer-MGE interaction networks further highlight species-specific dynamics, dense, hub-driven networks in P. stuartii versus sparser networks in P. rettgeri. Correlation analysis indicates potential associations between specific defense systems and virulence or antibiotic resistance genes. Phylogenetic reconstruction and gene gain and loss modeling further highlight dynamic evolutionary patterns. Both Gabija and Septu systems conferred robust, phage-specific protection; point mutations in conserved residues (GajA E465K and PtuB H53K) abolished defense.

DISCUSSION: Our findings unveil a multi-layered, modular immune architecture in Providencia, providing crucial insights into its genome plasticity, phage resistance, and adaptation in clinical environments. This work establishes a foundation for understanding the role of defense systems in the evolution and pathogenicity of the Providencia genus.},
}

*Providencia/genetics/immunology/virology
Phylogeny
Genome, Bacterial
*Enterobacteriaceae Infections/microbiology/immunology
CRISPR-Cas Systems
Bacteriophages
Humans

@article {pmid41764730,
year = {2026},
author = {D'Souza, LJ and Young, JN and Coffman, H and Petrow, EP and Bhattacharya, D},
title = {A genome-wide CRISPR screen reveals novel determinants of long-lived plasma cell secretory capacity.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {2},
pages = {},
doi = {10.1093/jimmun/vkaf354},
pmid = {41764730},
issn = {1550-6606},
support = {R01AI129945//National Institutes of Health (NIH)/ ; P30CA023074//Research, Innovation & Impact (RII) of the University of Arizona and National Cancer Institute/ ; S10 OD028466/GF/NIH HHS/United States ; },
mesh = {Animals ; *Plasma Cells/immunology/metabolism ; Mice ; Humans ; Myeloid Differentiation Factor 88/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Multiple Myeloma/immunology/genetics ; Mice, Inbred C57BL ; Mice, Knockout ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Plasma cell subsets vary in their lifespans and ability to sustain humoral immunity. We conducted a genome-wide CRISPR-Cas9 screen in myeloma cells for factors that promote surface expression of CD98, a marker of longevity in mouse plasma cells. A large fraction of genes found to promote CD98 expression in this screen are involved in secretory and other vesicles, including subunits of the V-type ATPase complex. Genetic ablation and chemical inhibition of V-type ATPases in myeloma cells and primary plasma cells, respectively, reduced antibody secretion. Mouse and human long-lived plasma cells had greater numbers of acidified vesicles than their short-lived counterparts, and this correlated with increased antibody secretory capacity. The screen also revealed a requirement for the signaling adapter MYD88 in CD98 expression. Plasma cell-specific deletion of Myd88 led to reduced survival and antibody secretion by antigen-specific cells in vivo and unresponsiveness to BAFF and APRIL ex vivo. These data reveal novel regulators that link plasma cell secretory capacity and lifespan.},
}

Animals
*Plasma Cells/immunology/metabolism
Mice
Humans
Myeloid Differentiation Factor 88/genetics/metabolism
CRISPR-Cas Systems/genetics
*Multiple Myeloma/immunology/genetics
Mice, Inbred C57BL
Mice, Knockout
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41628535,
year = {2026},
author = {Xie, K and Ren, H and Ban, D and Chen, L and Xin, X and Zhang, J and Tang, Q and Huang, L and Wei, J and Zhang, K and Liao, X},
title = {Silica-detoxified perovskite ECL: Cas13a-triggered signal-on sensing with CsPbBr3@SiO2@Au.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {170},
number = {},
pages = {109243},
doi = {10.1016/j.bioelechem.2026.109243},
pmid = {41628535},
issn = {1878-562X},
mesh = {*Silicon Dioxide/chemistry ; *Gold/chemistry ; *Biosensing Techniques/methods ; *Titanium/chemistry ; *MicroRNAs/analysis/blood ; *Oxides/chemistry ; *Calcium Compounds/chemistry ; Electrochemical Techniques/methods ; Limit of Detection ; Humans ; Luminescent Measurements/methods ; CRISPR-Cas Systems ; Nanocomposites/chemistry ; },
abstract = {Perovskite nanocrystals are attractive ECL emitters but suffer from poor water stability and potential toxicity. Here we report a signal-on electrochemiluminescent biosensor that integrates CsPbBr3@SiO2@Au nanocomposites with a CRISPR/Cas13a-Nb.BbvCI amplification cascade for ultrasensitive microRNA detection. The CsPbBr3 core provides bright emission, a conformal SiO2 shell enhances water compatibility and suppresses ion leakage, and surface Au nanoparticles offer abundant sites for thiolated ferrocene-hairpin (Fc-HP) immobilization. In the resting state, proximal Fc efficiently quenches the CsPbBr3 ECL. Target miRNA activates Cas13a to cleave a dumbbell probe and release an intermediate strand that hybridizes with Fc-HP; subsequent Nb.BbvCI nicking removes Fc from the electrode and is recycled, producing robust signal restoration. Morphology (TEM), composition (EDS/XPS), and stepwise electrochemistry (CV/EIS) verify a core-shell-Au architecture and a reliably assembled interface that follows the expected quench→restore behavior. Under optimized conditions (0.5 mg mL[-1] CsPbBr3@SiO2@Au, 2.0 μM Fc-HP, 40 min target incubation, 100 mM TPrA, 120 s pre-reaction), the assay affords a 1 aM-1.0 × 10[9] aM linear range with an estimated limit of detection (LOD) of 1.86 aM. The sensor shows high specificity against homologous sequences and achieves 95.22%-104.61% recoveries with RSD < 5% in spiked serum. Pilot measurements distinguish patient serum samples from healthy controls, underscoring clinical potential. This modular platform couples stable perovskite ECL emission with programmable CRISPR chemistry, offering a sensitive, selective, and water-compatible route for microRNA analysis and readily extensible nucleic-acid diagnostics.},
}

*Silicon Dioxide/chemistry
*Gold/chemistry
*Biosensing Techniques/methods
*Titanium/chemistry
*MicroRNAs/analysis/blood
*Oxides/chemistry
*Calcium Compounds/chemistry
Electrochemical Techniques/methods
Limit of Detection
Humans
Luminescent Measurements/methods
CRISPR-Cas Systems
Nanocomposites/chemistry

@article {pmid41534449,
year = {2026},
author = {Kirino, Y and Takeno, M},
title = {A critical look at animal and cellular models in autoinflammatory diseases.},
journal = {Current opinion in immunology},
volume = {99},
number = {},
pages = {102719},
doi = {10.1016/j.coi.2025.102719},
pmid = {41534449},
issn = {1879-0372},
mesh = {Animals ; Humans ; Disease Models, Animal ; *Autoimmune Diseases/immunology/genetics/etiology ; Gene Editing ; Induced Pluripotent Stem Cells ; *Inflammation/immunology ; CRISPR-Cas Systems ; *Hereditary Autoinflammatory Diseases ; },
abstract = {Autoinflammatory diseases (AIDs) comprise a diverse group of conditions arising from dysregulated immune control due to congenital or acquired genetic abnormalities in innate immune pathways, and patients typically require lifelong treatment. Owing to their rarity, access to patient samples is limited, making animal and cellular models indispensable for elucidating pathogenesis and advancing therapeutic development. Nevertheless, robust animal and cell-based models remain scarce. Recent advances in precision genome editing now enable lineage- and cell type-specific modeling of autoinflammation, steadily improving the fidelity with which disease phenotypes are recapitulated. In this review, we survey the current landscape of CRISPR-enabled knock-in/knock-out animal models, engineered cell lines, and patient-derived induced pluripotent stem cells for AIDs, and discuss how these platforms can be leveraged to dissect disease mechanisms and accelerate drug discovery.},
}

Animals
Humans
Disease Models, Animal
*Autoimmune Diseases/immunology/genetics/etiology
Gene Editing
Induced Pluripotent Stem Cells
*Inflammation/immunology
CRISPR-Cas Systems
*Hereditary Autoinflammatory Diseases

@article {pmid41506441,
year = {2026},
author = {Mokhles, F and Moosavi, MA and Gutierrez-Uzquiza, A and Velasco, G and Li, M and Cordani, M},
title = {Unraveling stress-adaptation pathways in cancer: Functional dissection through CRISPR-based genetic screens.},
journal = {Cancer letters},
volume = {644},
number = {},
pages = {218246},
doi = {10.1016/j.canlet.2026.218246},
pmid = {41506441},
issn = {1872-7980},
mesh = {Humans ; *Neoplasms/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; Animals ; Endoplasmic Reticulum Stress/genetics ; *Genetic Testing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Tumor Microenvironment ; Unfolded Protein Response/genetics ; *Stress, Physiological ; Adaptation, Physiological/genetics ; },
abstract = {Cancer cells face a hostile microenvironment characterized by hypoxia, nutrient deprivation, endoplasmic reticulum (ER) stress, and oxidative imbalance. To cope with these challenges, they activate an interconnected network of adaptive pathways including autophagy, the unfolded protein response, metabolic reprogramming, and the integrated stress response., which promote cell survival, therapy resistance, immune evasion, and metastasis. CRISPR-based functional genomics has emerged as a powerful strategy to systematically dissect these stress-adaptive networks, enabling the identification of key regulators and vulnerabilities across diverse contexts. In this review, we first summarize tumor progression in major stress conditions and then highlight how CRISPR screening strategies ranging from genome-wide loss-of-function studies to single-cell and combinatorial platforms, are unraveling critical stress regulators. We further discuss emerging tools, model systems, and translational perspectives, underscoring how the integration of CRISPR technologies with multi-omics, artificial intelligence, and advanced preclinical models is reshaping our understanding of cancer stress biology and guiding the development of novel therapeutic strategies. Finally, we addressed how these novel dissection technologies influence translational opportunities, specifically in the context of combining stress-pathway modulators with immunotherapy and targeted therapy drugs.},
}

Humans
*Neoplasms/genetics/pathology/metabolism
*CRISPR-Cas Systems
Animals
Endoplasmic Reticulum Stress/genetics
*Genetic Testing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
Signal Transduction
Tumor Microenvironment
Unfolded Protein Response/genetics
*Stress, Physiological
Adaptation, Physiological/genetics

@article {pmid41763755,
year = {2026},
author = {Ma, C and French, N and Wu, X and Gupta, SK and Gupta, TB},
title = {Molecular detection of Clostridium and Bacillus species in foods: recent advances and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {229},
number = {},
pages = {118370},
doi = {10.1016/j.foodres.2026.118370},
pmid = {41763755},
issn = {1873-7145},
mesh = {*Clostridium/isolation & purification/genetics ; *Bacillus/isolation & purification/genetics ; *Food Microbiology/methods ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence ; Humans ; Food Contamination/analysis ; },
abstract = {Spore-forming bacteria, especially Clostridium spp. and Bacillus spp., are ubiquitous in food systems, and their ingestion can cause serious diseases in humans and animals. Their persistence in diverse food matrices and resistance to conventional treatments make rapid and accurate detection essential for effective monitoring and control. Traditional culture-based and biochemical assays remain the standard for identifying these bacteria but are often time-consuming, labor-intensive and limited in sensitivity. In contrast, nucleic acid-based methods provide rapid, specific and sensitive alternatives by directly targeting genetic markers of pathogenic or spoilage strains. This review summarizes how nucleic acid methods, including PCR, FISH, LAMP, RPA, WGS, and the emerging CRISPR/Cas systems, have been applied specifically to detect Clostridium spp. and Bacillus spp. in food systems. Each method offers unique advantages and limitations. PCR-based methods enable accurate quantification but require thermal cycling. FISH-based methods are simple but require microscopy and have limited validation in food. WGS-based methods provide strain-level characterization but depend on informatics and specialized equipment. Isothermal techniques such as LAMP- and RPA-based methods allow rapid field detection but involve complex primer design or poor discrimination of closely related genes. CRISPR/Cas-based platforms further enhance simplicity, specificity, sensitivity for on-site detection, though the validation for spore-forming bacteria remains limited. Overall, this review provides an overview of gene targets, methodological adaptations, and analytical performance of nucleic acid-based assays for detecting Clostridium spp. and Bacillus spp., highlighting current progress and future opportunities for improving food safety monitoring.},
}

*Clostridium/isolation & purification/genetics
*Bacillus/isolation & purification/genetics
*Food Microbiology/methods
Nucleic Acid Amplification Techniques/methods
Polymerase Chain Reaction
CRISPR-Cas Systems
In Situ Hybridization, Fluorescence
Humans
Food Contamination/analysis

@article {pmid41762821,
year = {2026},
author = {Golla, DA and Sun, C and Haugh, L and Straub, N and Gao, X},
title = {Advances in multiplex precision genome editing in eukaryotic and prokaryotic systems.},
journal = {Current opinion in biotechnology},
volume = {99},
number = {},
pages = {103470},
doi = {10.1016/j.copbio.2026.103470},
pmid = {41762821},
issn = {1879-0429},
abstract = {Multiplex genome editing (MGE) enables coordinated modification of multiple genomic loci and is foundational for engineering complex biological traits. Traditional CRISPR-Cas nuclease-based strategies rely on DNA double-strand breaks (DSBs), which limit precision and pose scaling challenges for incorporating simultaneous edits across different loci. Recent advances in genome editing technologies that operate without generating DSBs have expanded the accuracy and feasibility of multiplexed genomic manipulation. This review focuses on emerging strategies for precise MGE, including base editing, prime editing, and related genome rewriting platforms. We highlight key engineering principles that impact the success of scalable multiplexing, including the choice of editing platform, edit size, and guide RNA architecture, and discuss applications across mammalian, plant, fungal, and bacterial systems. Together, these technologies establish MGE as a versatile framework for precise multigene control in biotechnology and agriculture.},
}

@article {pmid41761908,
year = {2026},
author = {Perry, TN and Mais, CN and Sanchez-Londono, M and Steinchen, W and Plitzko, PA and Randau, L and Pausch, P and Innis, CA and Bange, G},
title = {Structural basis of Cas8-independent Cas3 recruitment in Type I-F2 CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41761908},
issn = {1362-4962},
support = {//Deutsche Forschungsgemeinschaft/ ; 260989694//DFG/ ; 324652314//DFG/ ; 405858511//DFG/ ; 3869//DFG/ ; //Inserm/ ; 5342-2023//Research Council of Lithuania/ ; //Marburg University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; DNA/chemistry/metabolism/genetics ; Models, Molecular ; DNA Helicases/chemistry/metabolism/genetics ; Protein Domains ; Protein Binding ; *Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity in prokaryotes by targeting and degrading invasive genetic elements. Among them, the Type I-F2 system represents the most compact Type I CRISPR-Cas variant, distinguished by the complete absence of both large (Cas8) and small (Cas11) subunits. In other Type I systems, Cas8 is essential for protospacer adjacent motif (PAM) recognition and for triggering Cas3 recruitment, while Cas11 stabilizes the Cascade backbone and guides the nontarget DNA strand during R-loop formation. To elucidate how I-F2 executes interference in their absence, we determined the cryo-electron microscopy structure of the I-F2 Cascade bound to target DNA and Cas3. Our structure reveals that Cas5 alone mediates PAM sensing, while Cas7 subunits directly recruit Cas3, which adopts a helicase-loaded conformation compatible with DNA engagement. We show how the helicase and C-terminal domains of Cas3 capture the displaced nontarget strand to initiate directional unwinding and degradation. These findings uncover key mechanistic adaptations that enable efficient interference without canonical large and small subunits and emphasize the mechanistic diversity among closely related Type I systems, including I-E, I-F1, and I-F2. These insights provide a structural basis for engineering the hypercompact I-F2 system for genome editing and biotechnological applications.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
Cryoelectron Microscopy
DNA/chemistry/metabolism/genetics
Models, Molecular
DNA Helicases/chemistry/metabolism/genetics
Protein Domains
Protein Binding
*Bacterial Proteins/chemistry/metabolism/genetics

@article {pmid41759621,
year = {2026},
author = {Yan, X and Chen, M and Yang, S and Guo, Y and Dai, Y and Chen, Y and Zhong, H and Ma, T and Zha, D and He, Y and Li, B and Jia, X and Guo, L and Hu, J and Wei, Y and Chen, X},
title = {Mitochondrial genome editing tools: prospects in animal breeding.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2026.02.018},
pmid = {41759621},
issn = {1673-8527},
abstract = {Mitochondria are vital organelles responsible for driving cellular energy metabolism and regulating key biological processes. Their circular mitochondrial DNA (mtDNA) encodes 13 subunits of the respiratory chain proteins but is susceptible to mutations due to high levels of reactive oxygen species and limited repair mechanisms. Mutant phenotypes manifest only when heteroplasmy surpasses a critical threshold. Understanding the consequences of mtDNA mutations has long been hampered by the lack of precise editing tools. Recently, CRISPR-free, protein-only mitochondrial base editors have enabled C·G-to-T·A and A·T-to-G·C transitions. These breakthroughs facilitate the creation of relevant disease models and offer unique opportunities for animal breeding, as specific mtDNA variants are known to influence economically important traits in livestock, including production, reproduction, and stress tolerance. This review summarizes recent advances in mitochondrial genome editing technologies, including CRISPR/Cas-based systems, restriction endonucleases, double-stranded DNA deaminase toxin A (DddA)-based cytosine and adenine base editors, and DddA-free base editors, along with their delivery strategies and optimization avenues. Furthermore, we outline the associations between mtDNA polymorphisms, copy number variation, and economic traits in livestock and poultry. Finally, we discuss the potential applications of mitochondrial genome editing in animal breeding and highlight the critical safety and ethical considerations that require careful attention.},
}

@article {pmid41759529,
year = {2026},
author = {Cao, Z and Yu, S and Peng, J and Barrett, DR and Liu, Y and Sussman, JH and Chen, C and Thadi, A and Liu, L and Alikarami, F and Xu, J and Carroll, MP and Tan, K and Bernt, KM and Shi, J},
title = {CRISPR-based functional genomics for dissecting therapeutic dependency in primary acute myeloid leukemia samples.},
journal = {Molecular cell},
volume = {86},
number = {5},
pages = {968-985.e7},
doi = {10.1016/j.molcel.2026.02.003},
pmid = {41759529},
issn = {1097-4164},
support = {R01 CA262260/CA/NCI NIH HHS/United States ; U01 CA243072/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/pathology/drug therapy/therapy ; *CRISPR-Cas Systems ; Animals ; *Genomics/methods ; Mice ; Single-Cell Analysis ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Gene Editing ; Gene Expression Regulation, Leukemic ; Mice, Inbred NOD ; },
abstract = {Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functional interrogation of heterogeneous primary tumors offers a powerful opportunity to evaluate therapeutic targets and uncover cancer dependencies or resistance mechanisms. Here, we developed an optimized CRISPR-based platform for functional genomics in patient-derived xenograft and primary acute myeloid leukemia (AML) samples harboring diverse pathogenic mutations. Integrated in vitro and in vivo CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) dropout screens validated known AML-biased targets and identified cis-regulatory elements essential for leukemic growth. Coupling pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq) further resolved the perturbation-induced alterations in regulatory networks, cell cycle states, and cellular hierarchies in primary AML samples. Together, these studies establish a general and robust framework for leveraging CRISPR-based functional genomics to directly dissect cancer dependencies and cellular heterogeneity in primary AML patient samples.},
}

Humans
*Leukemia, Myeloid, Acute/genetics/pathology/drug therapy/therapy
*CRISPR-Cas Systems
Animals
*Genomics/methods
Mice
Single-Cell Analysis
Mutation
*Clustered Regularly Interspaced Short Palindromic Repeats
Cell Line, Tumor
Xenograft Model Antitumor Assays
Gene Editing
Gene Expression Regulation, Leukemic
Mice, Inbred NOD

@article {pmid41742419,
year = {2026},
author = {Zhu, M and Yuan, J and Meng, Q and Yu, J and Xu, X and Xu, M and Ren, X and Hu, Y and Wei, G and Jia, Z and Yuan, G and Zang, L and Liu, S and Yang, Y and Zheng, Y and Wang, J and Cong, T and Xie, W and Lan, X and Cong, L and Ma, T and Ding, S and Guo, W and Zhang, X and Li, Y},
title = {Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity.},
journal = {Cell stem cell},
volume = {33},
number = {3},
pages = {470-486.e14},
doi = {10.1016/j.stem.2026.01.015},
pmid = {41742419},
issn = {1875-9777},
mesh = {*Gene Editing/methods ; *Chromatin/metabolism/genetics ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Neural Stem Cells/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; CCCTC-Binding Factor/metabolism ; },
abstract = {Although CRISPR-Cas9 holds therapeutic promise, broader application demands an understanding of complications in vast non-coding regions. We found that CRISPR-Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach and identified editing-induced chromatin accessibility changes, with their scale varying by cell type. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and, ultimately, loss of stemness identity. To minimize chromatin perturbations and preserve cell identity, we refined gene-editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for the safer and broader application of genome-editing technologies.},
}

*Gene Editing/methods
*Chromatin/metabolism/genetics
Animals
Mice
CRISPR-Cas Systems/genetics
Cell Differentiation/genetics
*Neural Stem Cells/metabolism/cytology
Mouse Embryonic Stem Cells/metabolism/cytology
CCCTC-Binding Factor/metabolism

@article {pmid41679300,
year = {2026},
author = {Wang, Y and Hu, W and Xia, R and Yang, X and Gu, Y and Ning, Z and Yang, T and Yu, C and Zhang, L and Li, D and Jin, Y and Li, J and Zhang, F and Xu, Y and Xu, C and Wang, Z and Jing, N and Chen, L and Wang, G},
title = {CLIM-TIME identifies metastatic microenvironment modulators for T cell therapy response.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1555-1572.e23},
doi = {10.1016/j.cell.2025.12.042},
pmid = {41679300},
issn = {1097-4172},
mesh = {*Tumor Microenvironment/immunology/genetics ; Animals ; *T-Lymphocytes/immunology ; Mice ; Humans ; Immunotherapy/methods ; Laser Capture Microdissection/methods ; Mice, Inbred C57BL ; Lung Neoplasms/secondary/immunology/therapy ; Cell Line, Tumor ; Extracellular Matrix/metabolism ; CRISPR-Cas Systems ; Neoplasm Metastasis ; Neoplasms/immunology/therapy/pathology ; },
abstract = {The tumor microenvironment (TME) poses a major barrier to effective immunotherapy, yet high-throughput perturbation-mapping approaches to dissect TME spatial complexity and its contextual immune modulators remain lacking. Here, we introduce CRISPR-laser-captured microdissection (LCM) integration mapping of the tumor-immune microenvironment (CLIM-TIME), a scalable platform that integrates CRISPR screening with LCM of metastatic tumors for transcriptomic, deconvolution, and immunofluorescence analyses. CLIM-TIME enables spatially resolved mapping of how tumor suppressor gene (TSG) loss reshapes the TME and modulates immune responses. We identified seven distinct TME subtypes, revealing that DNA repair and Polycomb repressive complex (PRC) TSG loss is linked to immune-infiltrated TMEs sensitive to T cell therapy. In contrast, knockouts of TSGs in the Hippo pathway promoted immune evasion and therapy resistance by fostering myeloid-enriched but T cell-excluded TMEs with elevated extracellular matrix (ECM). Targeting the ECM-crosslinking enzyme LOXL2 effectively remodeled the metastatic TME, enhancing T cell infiltration and improving therapeutic efficacy in lung metastases across multiple cancers.},
}

*Tumor Microenvironment/immunology/genetics
Animals
*T-Lymphocytes/immunology
Mice
Humans
Immunotherapy/methods
Laser Capture Microdissection/methods
Mice, Inbred C57BL
Lung Neoplasms/secondary/immunology/therapy
Cell Line, Tumor
Extracellular Matrix/metabolism
CRISPR-Cas Systems
Neoplasm Metastasis
Neoplasms/immunology/therapy/pathology

@article {pmid41636073,
year = {2026},
author = {Zhu, X and Gu, G and Shen, Y and Abdurazik, M and Sun, G},
title = {CRISPR/Cas13a-induced self-priming cyclic amplification enables liquid biopsy of exosomal circular RNA in non-small cell lung cancer.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1413-1419},
doi = {10.1039/d5an01345c},
pmid = {41636073},
issn = {1364-5528},
mesh = {*RNA, Circular/genetics/blood ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; Humans ; *Lung Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Exosomes/genetics/chemistry ; Liquid Biopsy/methods ; *Nucleic Acid Amplification Techniques/methods ; DNA Primers/genetics ; },
abstract = {The precise and reliable identification of circular RNA (circRNA) is essential for both biological studies and clinical diagnostics of non-small cell lung cancer (NSCLC), especially the exosomal circRNA. In this study, we utilize a CRISPR/Cas13a system to specifically recognize the unique back-splice junction of target circRNA and develop a novel detection platform termed CRISPR/Cas13a-induced self-priming cyclic amplification. This method enables highly sensitive and specific circRNA detection. A pair of stem-loop DNA primers was carefully designed, each incorporating complementary single-stranded DNA sequences and five ribouridine (rU) residues at the 3' end serving as an overhang. When Cas13a binds to the target circRNA, its trans-cleavage activity is activated, leading to the cleavage of the rU residues. This cleavage permits the 3' ends of the stem-loop primers to extend along one another, generating multiple double stem-loop DNA structures that initiate successive cycles of self-priming chain elongation. By leveraging the sustained trans-cleavage activity of Cas13a and the high amplification efficiency of the self-priming cyclic reaction, the assay achieves sensitive detection of circRNA at concentrations as low as 564 aM within 90 min. In addition, the proposed method has been successfully applied for the analysis of exosomal hsa_circ_0003026 expression level in normal samples and NSCLC samples and demonstrated the potential of exosomal hsa_circ_0003026 in regulating the pathological progression. Owing to the high specificity of Cas13a, the proposed method can be directly applied to detect circRNA in complex biological samples without prior isolation of corresponding linear RNAs.},
}

*RNA, Circular/genetics/blood
*Carcinoma, Non-Small-Cell Lung/genetics/diagnosis
Humans
*Lung Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
*Exosomes/genetics/chemistry
Liquid Biopsy/methods
*Nucleic Acid Amplification Techniques/methods
DNA Primers/genetics

@article {pmid41622198,
year = {2026},
author = {Speth, ZJ and Pokhrel, V and Featherston, KM and Rehard, DG and Reid, WR and Franz, AWE},
title = {Monoallelic knockout of r2d2 affects the antiviral RNAi response to Mayaro virus and the reproductive potential in Aedes aegypti.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {},
pmid = {41622198},
issn = {1756-3305},
support = {R01 AI134661/AI/NIAID NIH HHS/United States ; R56 AI167980/AI/NIAID NIH HHS/United States ; R01-AI134661 (awarded to A.W.E.F), R56-AI167980 (awarded to A.W.E.F), and R56-AI180215 (awarded to Dr. Stefan Rothenburg, UC Davis)//National Institutes of Health - National Institute for Allergy and Infectious Diseases (NIH-NIAID)/ ; },
mesh = {Animals ; *Aedes/virology/genetics/physiology/immunology ; *RNA Interference ; Female ; *Alphavirus/physiology/immunology ; Mosquito Vectors/virology/genetics ; Gene Knockout Techniques ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Reproduction ; Fertility ; Alleles ; Argonaute Proteins/genetics ; RNA, Small Interfering/genetics ; },
abstract = {BACKGROUND: Aedes aegypti is an important vector for several human-pathogenic arboviruses. RNAi is the principal antiviral immune pathway in mosquitoes. Key steps of antiviral RNAi are processing of long dsRNAs into siRNA duplexes by dicer-2; loading of the siRNA duplexes onto Argonaute-2 with the help of R2D2; RISC formation via incorporation of Argonaute-2, which contains an siRNA; RISC-mediated targeting and degradation of homologous viral RNAs. Here, we generated an r2d2 knockout mosquito line to reveal how RNAi impairment during RISC loading complex (RLC) formation would affect arbovirus infection of Ae. aegypti.

METHODS: CRISPR/Cas9 gene editing has been used to knock out r2d2 in Ae. aegypti. Crossing experiments were conducted to reveal the effects of loss of r2d2 function on fecundity and fertility. Mayaro virus (Togaviridae: MAYV) infection and RNAi pathway gene expression levels were monitored using time-course RT-qPCR assays. Small RNA profiling was conducted to determine small RNA abundance in ΔR2D2[(+/-)] mosquitoes.

RESULTS: We show that in Ae. aegypti, the r2d2 allele is linked to the sex determination locus on chromosome 1. It was not possible to generate homozygous ΔR2D2[(-/-)] mosquitoes, indicating that complete loss of r2d2 function is lethal to Ae. aegypti. Our observations suggest that r2d2 function is not limited to RNAi but also affects mosquito fecundity/fertility, likely through follicle development. Monoallelic disruption of r2d2 increased the replication of MAYV, and r2d2 expression was also increased in infected mosquitoes. MAYV infection of ΔR2D2[(+/-)] mosquitoes was associated with an increase in abundance of putative vpiRNAs. However, impairment of r2d2 did not affect the function of dicer-2, as there was no difference in the 21 nt siRNA profiles between the ΔR2D2[(+/-)] mosquitoes and the non-transgenic control.

CONCLUSIONS: The RNAi pathway gene, r2d2, is an essential gene, and it is not possible to generate mosquitoes with biallelic (complete) loss of r2d2 function. Monoallelic impairment of r2d2 compromises the siRNA pathway downstream of dicer-2 function, at the point of RLC formation. In MAYV-infected mosquitoes, this defect in siRNA pathway function is compensated for by an increased piRNA pathway activity, which moderates increases in viral replication over a 10-day period.},
}

Animals
*Aedes/virology/genetics/physiology/immunology
*RNA Interference
Female
*Alphavirus/physiology/immunology
Mosquito Vectors/virology/genetics
Gene Knockout Techniques
*Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Reproduction
Fertility
Alleles
Argonaute Proteins/genetics
RNA, Small Interfering/genetics

@article {pmid41610849,
year = {2026},
author = {Samelson, AJ and Ariqat, N and McKetney, J and Rohanitazangi, G and Parra Bravo, C and Bose, RS and Travaglini, KJ and Lam, VL and Goodness, D and Ta, T and Dixon, G and Marzette, E and Jin, J and Tian, R and Tse, E and Abskharon, R and Pan, HS and Carroll, EC and Lawrence, RE and Gestwicki, JE and Rexach, JE and Eisenberg, DS and Kanaan, NM and Southworth, DR and Gross, JD and Gan, L and Swaney, DL and Kampmann, M},
title = {CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1517-1534.e19},
doi = {10.1016/j.cell.2025.12.038},
pmid = {41610849},
issn = {1097-4172},
support = {U54 NS123746/NS/NINDS NIH HHS/United States ; R01 AG075802/AG/NIA NIH HHS/United States ; K99 AG080116/AG/NIA NIH HHS/United States ; R01 AG085357/AG/NIA NIH HHS/United States ; U19 AG060909/AG/NIA NIH HHS/United States ; R01 AG070895/AG/NIA NIH HHS/United States ; R00 AG080116/AG/NIA NIH HHS/United States ; R01 AG082141/AG/NIA NIH HHS/United States ; U54 NS100717/NS/NINDS NIH HHS/United States ; F32 AG063487/AG/NIA NIH HHS/United States ; R01 AG062359/AG/NIA NIH HHS/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; R01 AG060477/AG/NIA NIH HHS/United States ; U24 AG072458/AG/NIA NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *tau Proteins/metabolism/genetics ; *Neurons/metabolism/cytology ; *Proteostasis ; Tauopathies/metabolism/pathology/genetics ; Ubiquitin-Protein Ligases/metabolism ; *CRISPR-Cas Systems/genetics ; Ubiquitination ; Mitochondria/metabolism ; Proteasome Endopeptidase Complex/metabolism ; },
abstract = {Aggregation of the protein tau defines tauopathies, the most common age-related neurodegenerative diseases, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to tau aggregation, dysfunction, and death. However, molecular mechanisms underlying cell-type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi screen in induced pluripotent stem cell (iPSC)-derived neurons. The screen uncovered both known and unexpected pathways, including UFMylation and GPI anchor biosynthesis, which control tau oligomer levels. We discovered that the E3 ubiquitin ligase CRL5[SOCS4] controls tau levels in human neurons, ubiquitinates tau, and is correlated with resilience to tauopathies in human disease. Disruption of mitochondrial function promotes proteasomal misprocessing of tau, generating disease-relevant tau proteolytic fragments and changing tau aggregation in vitro. These results systematically reveal principles of tau proteostasis in human neurons and suggest potential therapeutic targets for tauopathies.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*tau Proteins/metabolism/genetics
*Neurons/metabolism/cytology
*Proteostasis
Tauopathies/metabolism/pathology/genetics
Ubiquitin-Protein Ligases/metabolism
*CRISPR-Cas Systems/genetics
Ubiquitination
Mitochondria/metabolism
Proteasome Endopeptidase Complex/metabolism

@article {pmid41431195,
year = {2026},
author = {Spezzani, E and Capelli, L and Di Lena, D and Chamorro-Garcia, A and Ippodrino, R and Porchetta, A and Bertucci, A},
title = {MARPLE: A Proximity-Triggered CRISPR-Cas13 Platform for Ultrasensitive Antibody Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {13},
pages = {e17799},
pmid = {41431195},
issn = {2198-3844},
support = {//National Recovery and Resilience Plan/ ; MUR 2023-2027//'Departments of Excellence' program of the Italian Ministry for University and Research/ ; 31108//Fondazione AIRC per la ricerca sul cancro ETS/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Antibodies/analysis ; Immunoassay/methods ; },
abstract = {Monitoring clinically relevant antibodies-as biomarkers of disease or therapeutic response-is essential for informed clinical decision-making. Traditional immunoassays like ELISA offer reliable quantification but often involve multistep workflows and limited point-of-care utility. New approaches coupling antibody recognition with signal amplification are therefore highly desirable. The CRISPR-Cas13 system, known for its potent collateral cleavage activity, has emerged as a powerful diagnostic tool for nucleic acid detection. However, its application to protein biomarkers such as antibodies remains underdeveloped. Here, we introduce MARPLE (Modular Antibody Recognition via Proximity-triggered Linker Exchange), a modular CRISPR-Cas13-based platform for ultrasensitive antibody detection. MARPLE harnesses antibody-induced proximity to trigger a strand displacement reaction that releases a sequestered RNA target, activating Cas13-mediated collateral cleavage of fluorescent RNA reporters. This cascade enables detection of antibodies at femtomolar concentrations. We demonstrate MARPLE's versatility across diverse targets-including anti-digoxigenin, anti-cholesterol, anti-HA, trastuzumab, and anti-MUC1-highlighting applications in infectious disease monitoring, cancer diagnostics, and therapeutic drug tracking. The assay is isothermal, one-pot, and retains robust performance in complex matrices such as human serum. These features establish MARPLE as a promising tool for immunodiagnostics, extending CRISPR-based sensing beyond nucleic acids to protein biomarker detection.},
}

*CRISPR-Cas Systems/genetics
Humans
*Antibodies/analysis
Immunoassay/methods

@article {pmid40898426,
year = {2026},
author = {Sahin, U},
title = {Cas9 beyond CRISPR - SUMOylation, effector-like potential and pathogenic adaptation.},
journal = {The FEBS journal},
volume = {293},
number = {5},
pages = {1285-1296},
doi = {10.1111/febs.70256},
pmid = {40898426},
issn = {1742-4658},
support = {223Z048//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; 121C230//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; YIN IG3336//European Molecular Biology Organization/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Sumoylation/genetics ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; Gene Editing ; Protein Processing, Post-Translational ; *Bacteria/pathogenicity/genetics ; Host-Pathogen Interactions/genetics ; Animals ; },
abstract = {The CRISPR/Cas9 system has revolutionized molecular biology and gene editing, yet key aspects of its regulation, especially within eukaryotic environments, remain enigmatic. In this Viewpoint article, I will speculate on and explore the provocative hypothesis that Cas9 may possess previously unrecognized effector-like functions when expressed in host cells, potentially shaped by host-mediated post-translational modifications (PTMs). Of particular interest is SUMOylation at lysine 848, a key residue for DNA binding within the catalytic site, raising the possibility that this modification is not incidental, but functionally significant and precisely regulated. SUMOylation, a eukaryotic PTM, is increasingly recognized as a mechanism that also targets bacterial and viral effector proteins and virulence factors during infection, exerting context-dependent effects that may either enhance or hinder pathogen replication. Could Cas9, beyond its canonical role in bacterial CRISPR immunity, act as a host-modulating effector during infection, akin to known bacterial nucleomodulins such as transcription activator-like (TAL) effectors? If so, this would imply that certain pathogenic bacteria may have evolved Cas9 variants capable of exploiting host PTM machinery and targeting the host genome-an adaptation with potential implications for microbial virulence, host-pathogen interactions, and co-evolutionary dynamics. This perspective underscores the importance of systematically mapping Cas9 PTMs and examining their evolutionary conservation, functional significance, and pharmacological tunability, not only for basic biological insight and to deepen our understanding of microbial strategies, but also to refine the precision and safety of Cas9-based therapeutic platforms.},
}

*CRISPR-Cas Systems/genetics
*Sumoylation/genetics
Humans
*CRISPR-Associated Protein 9/genetics/metabolism
Gene Editing
Protein Processing, Post-Translational
*Bacteria/pathogenicity/genetics
Host-Pathogen Interactions/genetics
Animals

@article {pmid41759295,
year = {2026},
author = {Molina, C and Knight, AL and Lisi, GP},
title = {Comparative thermodynamic and kinetic properties governing the nucleic acid interactions of CRISPR-Cas9 and Cas12a.},
journal = {Physical biology},
volume = {},
number = {},
pages = {},
doi = {10.1088/1478-3975/ae4b7f},
pmid = {41759295},
issn = {1478-3975},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeat-associated proteins (CRISPR-Cas) biochemistry has been leveraged in genome editing applications in biochemical research and therapeutics. CRISPR-Cas9 and CRISPR-Cas12a are the two most widely used RNA-guided endonucleases and while Cas9 and Cas12a have a shared function, both have unique biophysical properties that alter their specificity and efficiency. The thermodynamic and kinetic properties governing their molecular interactions, recognition and binding of target DNA, and R-loop formation can differ. In some cases, these critical biophysical metrics have not been resolved. Distinctions between Cas9 and Cas12a enzymes are also prevalent in RNA:DNA hybrid binding affinities, DNA localization relative to the preferred PAM site and DNA cleavage mechanism. In this review, we examine the biophysical properties of both endonucleases, focused on the nucleic acid interactions that confer specificity and function. Complementing this biophysical overview, we discuss case studies in disparate model organisms that compare the genome editing and fidelity of Cas9 and Cas12a.},
}

@article {pmid41758946,
year = {2026},
author = {Saito, R and Umemura, Y and Makino, S and Fukaya, T},
title = {Decoding the molecular logic of rapidly evolving ZAD zinc finger proteins in Drosophila.},
journal = {Science advances},
volume = {12},
number = {9},
pages = {eady7568},
pmid = {41758946},
issn = {2375-2548},
mesh = {Animals ; *Drosophila Proteins/metabolism/genetics/chemistry ; *Zinc Fingers ; *Transcription Factors/metabolism/genetics ; *Evolution, Molecular ; Protein Binding ; *Drosophila/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism/embryology ; DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; CCCTC-Binding Factor ; Microtubule-Associated Proteins ; Nuclear Proteins ; },
abstract = {The zinc finger-associated domain (ZAD)-containing C2H2 zinc finger proteins (ZAD-ZnFs) represent the most abundant class of transcription factors that emerged during insect evolution, yet their molecular diversity and biological functions remain largely unclear. Here, we established a systematic CRISPR-based protein-tagging approach that enables direct, unambiguous comparison of nuclear localization and genome-wide binding profiles of endogenous ZAD-ZnFs in developing Drosophila embryos. Evidence is provided that a subset of ZAD-ZnFs forms nuclear condensates through the stacking of the N-terminal ZAD dimerization surface. Disruption of condensation activity leads to misregulation of genome-wide binding profiles and lethality, underscoring its functional and physiological significance in development. Integrative chromatin immunoprecipitation sequencing and Micro-C analyses reveal that many ZAD-ZnFs colocalize with core insulator proteins such as CCCTC-binding factor and Centrosomal protein 190 kD to control the formation of topological boundaries. We suggest that the diverse molecular functions of ZAD-ZnFs have evolutionarily arisen from their ancestral role as insulator-binding proteins.},
}

Animals
*Drosophila Proteins/metabolism/genetics/chemistry
*Zinc Fingers
*Transcription Factors/metabolism/genetics
*Evolution, Molecular
Protein Binding
*Drosophila/genetics/metabolism
*Drosophila melanogaster/genetics/metabolism/embryology
DNA-Binding Proteins/metabolism/genetics
CRISPR-Cas Systems
CCCTC-Binding Factor
Microtubule-Associated Proteins
Nuclear Proteins

@article {pmid41758452,
year = {2026},
author = {Bharti S, AK and Mukherjee, AG and Gopalakrishnan, AV and Gajendran, B and Vashishth, R and Prince, SE},
title = {From bench to bedside: stem cell therapy as a transformative approach against HIV.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {41758452},
issn = {1573-4978},
mesh = {Humans ; *HIV Infections/therapy/virology/immunology ; *Hematopoietic Stem Cell Transplantation/methods ; Gene Editing/methods ; HIV-1 ; *Stem Cell Transplantation/methods ; Induced Pluripotent Stem Cells ; Translational Research, Biomedical ; Receptors, CCR5/genetics ; CRISPR-Cas Systems ; Virus Latency ; },
abstract = {Human immunodeficiency virus (HIV) remains a persistent global health burden, as combination antiretroviral therapy (ART) achieves sustained viral suppression but fails to eliminate long-lived latent reservoirs. Stem cell-based therapeutic strategies have emerged as transformative approaches with the potential to induce durable remission and, ultimately, a functional cure. Clinical proof-of-concept has been established through allogeneic hematopoietic stem cell transplantation (HSCT) using CCR5Δ32/Δ32 donor cells, demonstrating that durable resistance to viral entry can result in prolonged HIV remission. Building on these landmark observations, recent advances in autologous gene-edited hematopoietic stem and progenitor cells and induced pluripotent stem cell (iPSC)-derived immune effectors have accelerated the development of scalable, patient-specific interventions. The convergence of stem cell biology with precision genome-editing platforms, including CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), has enabled targeted disruption of viral entry pathways and host dependency factors, while offering new strategies to address viral latency and immune reconstitution. Despite significant challenges related to treatment-associated toxicity, manufacturing complexity, long-term safety, and ethical considerations, rapid progress in cellular engineering and translational immunology continues to advance the field toward curative outcomes. This review critically synthesizes recent progress in stem cell-based HIV therapeutics, elucidates the underlying mechanistic frameworks, evaluates emerging clinical and preclinical evidence, and outlines future directions required to achieve a durable functional cure.},
}

Humans
*HIV Infections/therapy/virology/immunology
*Hematopoietic Stem Cell Transplantation/methods
Gene Editing/methods
HIV-1
*Stem Cell Transplantation/methods
Induced Pluripotent Stem Cells
Translational Research, Biomedical
Receptors, CCR5/genetics
CRISPR-Cas Systems
Virus Latency

@article {pmid41758321,
year = {2026},
author = {Pathak, A and Singh, J and Swati, and Dwibedi, V},
title = {Deciphering microbial biofilm: mechanism, infection, and advanced approaches for control.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {41758321},
issn = {1874-9356},
abstract = {Microbial biofilms are densely organised microbial communities that adhere to biotic and abiotic surfaces, encased within an extracellular polymeric substance (EPS). Microorganisms within these biofilm structures gain enhanced protection, versatility, and resistance to external stresses, antibiotics, and host immune systems. The biofilm formation follows a series of steps, including initial microbial adherence, microcolony establishment, EPS production, regulation by quorum sensing (QS), and dispersal. This flexibility enables biofilm survival in multiple environments, such as medical devices and natural systems, posing serious challenges in healthcare, agricultural, and industrial sectors. The review focuses on the mechanisms involved in biofilm formation and discusses the role of EPS in promoting biofilm stability and resistance to antimicrobials. It addresses biofilm-associated infections in medical environments, such as chronic wounds, cystic fibrosis, urinary tract infections (UTIs), and complications with implanted medical devices. The capacity of biofilm-forming microorganisms to evade immune responses and persist through extended antibiotic use highlights the urgent demand for novel therapeutic approaches. The discussion includes emerging strategies for biofilm control, including anti-biofilm agents, QS inhibitors, enzymatic treatments, and innovative combination therapies combining antibiotics with biofilm-disrupting agents. Emerging technologies, like antimicrobial peptides (AMPs), CRISPR-Cas systems, nanotechnology, and bioelectric therapies, present innovative biofilm disruption and removal approaches. This paper discusses the effectiveness of natural products, plant-derived compounds, and bacteriophage therapies for mitigating biofilm-associated infections linked to biofilms. The review examines the dynamic challenges posed by biofilms, particularly their role in chronic and device-related infections, which contribute to significant healthcare complications. The study highlights the significance of adopting new approaches to overcome biofilm-induced antimicrobial resistance (AMR) and improve therapeutic outcomes. Furthermore, this paper discusses the promising potential of emerging technologies, such as nanomaterials, QS interference, and biofilm-specific antimicrobial agents, in enhancing biofilm control and prevention measures across clinical, industrial, and environmental domains.},
}

@article {pmid41757824,
year = {2026},
author = {Chen, J and Davison, CW and Ellis, J and Blevins, B and Presley, W and Myers, MT and Kong, D and Hou, Z and Mian, SI and Prasov, L and Zhang, Y},
title = {CRISPR Base Editing Correction of TGFBI Mutations in Autosomal Dominant Corneal Dystrophies.},
journal = {Investigative ophthalmology & visual science},
volume = {67},
number = {2},
pages = {60},
pmid = {41757824},
issn = {1552-5783},
mesh = {Humans ; *Corneal Dystrophies, Hereditary/genetics/therapy/metabolism ; *Gene Editing/methods ; *Mutation ; *Transforming Growth Factor beta/genetics/metabolism ; *Genetic Therapy/methods ; Epithelium, Corneal/metabolism/pathology ; *Extracellular Matrix Proteins/genetics ; *CRISPR-Cas Systems ; Dependovirus/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {PURPOSE: Lattice and granular corneal dystrophy comprise two common TGFBI-associated autosomal dominant corneal disorders. Existing therapies are only temporizing and carry significant morbidity. Here, we develop a novel therapeutic approach using an adenine base editor (ABE) to correct common TGFBI mutations.

METHOD: We generated two human corneal epithelial (HCE) cell models harboring a copy of the most common disease-causing TGBFI mutations, R124C or R555W. These lines were electroporated with an ABE8e-NG-encoding mRNA and guide RNAs targeting the mutations. The resulting A•T-to-G•C editing efficiencies and off-target (OT) effects were assessed by amplicon sequencing. GFP-expressing adeno-associated viruses (AAVs) with different capsid types were transduced into HCE cells and healthy human corneal donor tissues, and GFP fluorescence was evaluated.

RESULTS: Using all-RNA delivery for ABE8e-NG, we achieved 91% and 62% correction of the pathogenic adenines in HCE TGFBIR124C/WT and TGFBIR555W/WT cells, without editing the wild-type allele. Indel formation was negligible (<0.2%), bystander adenine editing was minimal (<0.7%), and editing at top computationally predicted OT sites was modest (<1.2% at all but 1 of the 20 OT sites analyzed), suggesting minimal safety concerns. Correction of TGFBIR124C/WT in HCEs rescued the aberrant lysosomal localization of TGFBI. We further identified AAV1 as the most effective serotype for gene delivery into both human corneal donor tissue and HCE cells.

CONCLUSIONS: Our study demonstrates the feasibility and safety of CRISPR adenine base editing as a new therapeutic strategy for correcting common TGFBI mutations in corneal dystrophies, paving the way for further preclinical testing.},
}

Humans
*Corneal Dystrophies, Hereditary/genetics/therapy/metabolism
*Gene Editing/methods
*Mutation
*Transforming Growth Factor beta/genetics/metabolism
*Genetic Therapy/methods
Epithelium, Corneal/metabolism/pathology
*Extracellular Matrix Proteins/genetics
*CRISPR-Cas Systems
Dependovirus/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41757451,
year = {2026},
author = {Molina-Márquez, A and Kelterborn, S and Hegemann, P and Pérez-Rodríguez, M and Vigara, J and León, R},
title = {Characterization of Phytoene Desaturase Knockout Carotenoid-Deficient Microalgal Mutants Generated by Cas9-Ribonucleoprotein Complexes.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70811},
pmid = {41757451},
issn = {1399-3054},
support = {2019-110438RB-C22//Agencia Estatal de Investigación-MCIN/AEI/10.13039/501100011033/ ; PID2022-140995OB-C21//Agencia Estatal de Investigación-MCIN/AEI/10.13039/501100011033/ ; 426566805//German Research Foundation (DFG)/ ; //Hertie Foundation/ ; },
mesh = {*Carotenoids/metabolism ; *Oxidoreductases/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; Gene Knockout Techniques ; *Microalgae/genetics/metabolism ; *Chlamydomonas reinhardtii/genetics/metabolism ; Mutation/genetics ; Chloroplasts/metabolism/ultrastructure ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Phytoene desaturase (PDS; EC 1.3.5.5) is a key enzyme of the carotenoid biosynthetic pathway, catalyzing the desaturation of phytoene, precursor of all carotenoids. In this study, several PDS-knockout (PDS-KO) transformants of the chlorophyte microalga Chlamydomonas reinhardtii were generated using a reverse genetics strategy. Two single guide RNAs (sgRNA) were designed to target the first exon of the PDS gene, and pre-assembled Cas9 ribonucleoprotein (RNPs) complexes were delivered into microalgal nuclei by electroporation. Multiple white PDS-KO transformants were successfully obtained by this approach, and three independent transformant lines were subsequently characterized. By integrating ultrastructural, pigment and transcriptomic analyses of dark-grown cells of several PDS-KO carotenoid-deficient mutants in comparison with the parental strain, it was demonstrated that carotenoids are indispensable components of multiple cellular architectures. Chromatographic analysis confirmed that the only carotenoid accumulated in these transformants was phytoene, which lacks the critical structural and photoprotective functions of its colored derivatives. Transmission Electron Microscopy (TEM) observations revealed profound ultrastructure alterations, including poorly developed chloroplasts and effects on other cellular structures that were either absent or severely disorganized. Consistently, clustering differentially expressed genes into functional groups revealed downregulation of pathways associated with photosynthesis, chlorophyll and carotenoid biosynthesis, ribosome biogenesis, and vesicle and membrane trafficking in the PDS-KO lines. Conversely, upregulation of regulatory and retrotransposon-inducing genes was observed. These findings underscore the central metabolic role of colored carotenoids in plant cells, highlighting their essential contribution to cellular homeostasis and photosynthetic competence.},
}

*Carotenoids/metabolism
*Oxidoreductases/genetics/metabolism
*Ribonucleoproteins/metabolism/genetics
Gene Knockout Techniques
*Microalgae/genetics/metabolism
*Chlamydomonas reinhardtii/genetics/metabolism
Mutation/genetics
Chloroplasts/metabolism/ultrastructure
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid41757335,
year = {2026},
author = {Shafiq, T and Khan, N and Kausar, T and Ahmed, W and Zhang, Z and Liang, Y and Duan, L},
title = {Red Blood Cell-Derived Extracellular Vesicles for Gene and RNA Therapeutics: Biological, Engineering, and Translational Challenges.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {579975},
pmid = {41757335},
issn = {1178-2013},
mesh = {Humans ; *Extracellular Vesicles/chemistry/metabolism ; *Genetic Therapy/methods ; *Erythrocytes/cytology/chemistry/metabolism ; CRISPR-Cas Systems ; Animals ; RNA, Messenger/genetics/administration & dosage/therapeutic use ; *RNA/therapeutic use ; Gene Transfer Techniques ; },
abstract = {Gene therapy has great prospects of DNA/RNA manipulations and protein modulations. Its use in clinic is, however, stifled by risks of immunogenicity, low target specificity, and adverse effects. The red blood cell (RBC-EVs) extracellular vesicles can serve as a solution to this issue since they are biocompatible, long-term stable, and with low immunogenicity. RBC-EVs permit the accurate delivery of therapeutic cargo to space and time, thus minimizing systemic toxicity. This review presents the most recent developments on the expansion of the use of RBC-EVs to encapsulate the components of mRNA and CRISPR-Cas. Through the addition of the means to address these deficiencies, including stimulus-sensitive release mechanisms (eg, pH- or light-activated systems) and tissue-selective targeting approaches, RBC-EVs can be applied to enable the precise application in genetic diseases, inflammatory diseases, and cancer. Such innovations have the potential to overcome the clinical need and enable the biological complexity of mRNA- and CRISPR-Cas-based agents to provide a powerful delivery platform. Moreover, the review also demonstrates the unprecedented benefits of red blood cell EVs, which include immune evasion, scalability, and universal loading capacity, which can establish them as the next-generation delivery vehicles. Red blood cell EVs have the potential to increase the efficacy of precision medicine by increasing its feasibility. Lastly, we note the potential and translational issues in the provision of red blood cell EV-based mRNA and CRISPR-Cas therapeutic delivery of gene therapy.},
}

Humans
*Extracellular Vesicles/chemistry/metabolism
*Genetic Therapy/methods
*Erythrocytes/cytology/chemistry/metabolism
CRISPR-Cas Systems
Animals
RNA, Messenger/genetics/administration & dosage/therapeutic use
*RNA/therapeutic use
Gene Transfer Techniques

@article {pmid41755886,
year = {2026},
author = {Kaniganti, S and Saini, H and Chaitanya, AK and Hegde, N and Shah, P and Magar, ND and Rijal, R and Kaushik, JJ and Nanda, D and Sachan, S and Kumar, A and Bhoite, R and Jamedar, HR},
title = {CRISPR/Cas Genome Editing and Its Applications in Cereal Crop Improvement.},
journal = {Plant-environment interactions (Hoboken, N.J.)},
volume = {7},
number = {2},
pages = {e70133},
pmid = {41755886},
issn = {2575-6265},
abstract = {CRISPR/Cas-based genome editing has emerged as a transformative tool for precise genetic improvement of cereal crops. Recent advances in CRISPR technologies, including Cas9, Cas12, Cas13, base editing, and prime editing, have enabled targeted modification of genes and regulatory elements controlling yield, stress tolerance, and grain nutritional quality in major cereals such as rice, wheat, maize, and barley. This review summarizes current progress in CRISPR-mediated genome editing systems, delivery strategies, and representative applications in cereal crop improvement. Emphasis is placed on how genome editing reprograms enzymatic activities and biological pathways underlying complex agronomic traits rather than acting through single-gene effects. The review also discusses challenges related to trait complexity, regulatory considerations, and prospects for translating genome-edited cereal crops from laboratory research to field-level application. Collectively, this review highlights the potential of CRISPR/Cas genome editing as a powerful approach for developing high-yielding, resilient, and nutritionally improved cereal crops.},
}

@article {pmid41755634,
year = {2026},
author = {Hu, Z and Liu, Y and Han, Y and Li, M and Deng, K and Lu, X and Huang, Y and Liang, C and Wang, Y and Fu, Y and Xu, A},
title = {CRISPR/Cas9 screening with destabilized bicistronic fluorescent protein reporter revealed PABPN1 as a hub of regulators for alternative polyadenylation.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755634},
issn = {1362-4962},
support = {2022YFA1103900//National Key Research and Development Program of China/ ; 32470586//National Natural Science Foundation of China/ ; 91942301//National Natural Science Foundation of China/ ; 81430099//National Natural Science Foundation of China/ ; 31930084//National Natural Science Foundation of China/ ; 32500472//National Natural Science Foundation of China/ ; 2023B1212060028//Guangdong Science and Technology Department/ ; 2022YFA1103900//National Key Research and Development Program of China/ ; },
mesh = {*Polyadenylation/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Genes, Reporter ; *Poly(A)-Binding Protein I/genetics/metabolism ; HEK293 Cells ; DEAD-box RNA Helicases/metabolism/genetics ; *Luminescent Proteins/genetics/metabolism ; Cell Proliferation/genetics ; Polypyrimidine Tract-Binding Protein/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {Alternative polyadenylation (APA) is intricately intertwined with diverse biological processes. Efficient approaches for screening the regulatory factors of specific APA events are essential to elucidate their regulation mechanisms. Here, we first engineered a destabilized bicistronic fluorescent protein reporter (dBFPR) to enhance the sensitivity of APA detection. Then, we developed a robust high-throughput screening platform for APA regulators by integrating CRISPR/Cas9, dBFPR, and fluorescence-activated cell sorting. With this method, we successfully screened the library of RNA binding proteins and found that PTBP1, ELAVL1, and DDX3X play significant roles in regulating APA and promoting cell proliferation through interaction with PABPN1, suggesting that PABPN1 is an important hub for APA regulation.},
}

*Polyadenylation/genetics
Humans
*CRISPR-Cas Systems/genetics
Genes, Reporter
*Poly(A)-Binding Protein I/genetics/metabolism
HEK293 Cells
DEAD-box RNA Helicases/metabolism/genetics
*Luminescent Proteins/genetics/metabolism
Cell Proliferation/genetics
Polypyrimidine Tract-Binding Protein/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics

@article {pmid41755633,
year = {2026},
author = {Aguilar-González, A and Martos-Jamai, I and Ramos-Hernández, I and Molina-Estévez, FJ and Villao, NV and Puig-Serra, P and Rodríguez-Perales, S and Torres, R and Labun, K and Sánchez-Martín, RM and Díaz-Mochón, JJ and Martín, F},
title = {A novel Dual-guide CRISPR-Cas13 strategy improves specificity for single-nucleotide variant detection.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755633},
issn = {1362-4962},
support = {MCIN)/AEI/10.13039/501100011033//Spanish Ministry of Science and Innovation/ ; PID2022-141065OB-I00//European Union Next Generation/ ; CV20-77741//Consejería de Economía y Conocimiento/Project/ ; PI21/00298//Instituto de Salud Carlos III/ ; PI24/00888//Instituto de Salud Carlos III/ ; RD21/0017/0004//Instituto de Salud Carlos III/ ; RD24/0014/0005//Instituto de Salud Carlos III/ ; PI-0236-2024//Consejería de Salud y Familias/ ; PIP-0004-2025//Consejería de Salud y Familias/ ; GeneHumdi-CA21113//European Cooperation in Science and Technology/ ; FPU22/03455//Spanish Ministry of Science, Innovation and Universities/ ; RHJ-0053-2025//Consejería de Salud y Familias, Junta de Andalucía/ ; //European Social Fund/ ; //Universidad de Granada/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *SARS-CoV-2/genetics/isolation & purification ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *COVID-19/diagnosis/virology/genetics ; RNA, Viral/genetics ; *Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins p21(ras)/genetics ; Sensitivity and Specificity ; Leishmania/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {The emergence of CRISPR-Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13's heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first Dual-guide RNA system for Cas13 that improves mismatch discrimination and enhances target specificity. This system employs two distinct RNAs-dcrRNA and dtracrRNA-which cooperatively recognize the target and reduce off-target activity. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, distinguished KRAS G12D and G12C mutations, and differentiated mucocutaneous from cutaneous Leishmania sequences in analytical assays, with clinical validation confirming accurate detection of positive and negative samples. These results highlight the Dual-guide Cas13 platform's potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a substantial advance over conventional Cas13 systems, offering improved specificity while maintaining clinically relevant sensitivity, and provides a generalizable tool for next-generation molecular diagnostics and precision RNA targeting and regulation.},
}

*CRISPR-Cas Systems/genetics
Humans
*SARS-CoV-2/genetics/isolation & purification
*RNA, Guide, CRISPR-Cas Systems/genetics
*COVID-19/diagnosis/virology/genetics
RNA, Viral/genetics
*Polymorphism, Single Nucleotide
Proto-Oncogene Proteins p21(ras)/genetics
Sensitivity and Specificity
Leishmania/genetics
CRISPR-Associated Proteins/genetics

@article {pmid41740934,
year = {2026},
author = {Sun, W and Zhu, S and Dong, Q and Tang, S and Liu, Q and Sha, Y and Chen, T and Wang, R and Chen, Y and Ying, H},
title = {Engineering Escherichia coli cell Factories for continuous 5'-cytidine monophosphate production via biofilm-anchored dual-enzyme cascade catalysis.},
journal = {Bioresource technology},
volume = {447},
number = {},
pages = {134267},
doi = {10.1016/j.biortech.2026.134267},
pmid = {41740934},
issn = {1873-2976},
mesh = {*Biofilms/growth & development ; *Escherichia coli/metabolism/genetics/enzymology ; *Cytidine Monophosphate/biosynthesis ; Biocatalysis ; CRISPR-Cas Systems/genetics ; Uridine Kinase/metabolism ; },
abstract = {5'-cytidine monophosphate (5'-CMP) serves as a crucial intermediate for diverse nucleotide derivatives and finds extensive applications in the food and pharmaceutical industries. However, existing enzymatic production processes suffer from low catalytic efficiency and poor economic feasibility. In this study, we developed a continuous 5'-CMP production system based on a cell-enzyme co-immobilized biocatalytic platform. First, the pgaABCD gene cluster was integrated into ClearColi BL21(DE3) using CRISPR-Cas9, enhancing its biofilm-forming capacity by 168.93% and enabling robust cell immobilization on the carrier. Second, a dual-anchoring strategy utilizing ice-nucleation protein (INP) and autotransporter (AIDA-I) enabled surface display of uridine kinase (UDK) and acetate kinase (AckA) on the cell surface. This approach successfully addressed the instability and recovery issues of free enzymes by using biofilm engineering to co-immobilize cells and enzymes. The modified strain achieved a 5'-CMP productivity of 1.77 mmol/L/h, 5.98-fold higher than free intracellular enzyme catalysis, and was reused for ten consecutive cycles under the tested conditions while maintaining a cytidine conversion rate above 73.79%, and a 5'-CMP yield above 59.26%. This work demonstrates the first successful realization of continuous 5'-CMP biosynthesis and establishes an efficient route for its industrial production.},
}

*Biofilms/growth & development
*Escherichia coli/metabolism/genetics/enzymology
*Cytidine Monophosphate/biosynthesis
Biocatalysis
CRISPR-Cas Systems/genetics
Uridine Kinase/metabolism

@article {pmid41719905,
year = {2026},
author = {Yu, F and Yue, D and Wang, F and Fu, B and Qin, G and Wei, S and Zang, W and Zhang, Q and Cui, L and Wang, T},
title = {Structure-initiated CHA variant coordinating SDA for cascade amplification in CRISPR/Cas12a-based miRNA analysis.},
journal = {Talanta},
volume = {304},
number = {},
pages = {129558},
doi = {10.1016/j.talanta.2026.129558},
pmid = {41719905},
issn = {1873-3573},
mesh = {*MicroRNAs/genetics/analysis/blood ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) are well-established biomarkers for tumor diagnosis and monitoring. Herein, we report a novel biosensing platform by engineering a structure-initiated variant of catalytic hairpin assembly (VCHA) that coordinates with strand displacement amplification (SDA) to drive cascade amplification for CRISPR/Cas12a-based detection. This system employs three hairpin probes which, upon recognizing the target miRNA, self-assemble into a key 5'-end dangling three-way conjugate (5'-DTC) structure. This structure serves as the exclusive trigger, simultaneously propagating the VCHA cycle and priming the SDA process through the coordinated action of polymerase and nicking enzyme. Consequently, VCHA and SDA operate synergistically within a unified circuit, generating abundant single-stranded activator DNA (acDNA) products. These acDNA molecules then activate the trans-cleavage activity of CRISPR/Cas12a, yielding a significantly amplified fluorescence readout. The VCHA-SDA/Cas12a platform demonstrated excellent performance for miRNA-155 detection, achieving a broad dynamic range from 1 pmol/L to 10 nmol/L with an ultra-low detection limit of 0.166 pmol/L. Furthermore, the platform successfully quantified miRNA levels in clinical plasma specimens and various cell lines, confirming its considerable potential as a robust tool for molecular diagnostics and clinical translation.},
}

*MicroRNAs/genetics/analysis/blood
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41713574,
year = {2026},
author = {Hu, T and Hou, Z and Zhang, Y and Jing, P and Dai, X and Wang, H},
title = {Development of a one-pot integrated rapid detection method for white spot syndrome virus based on RAA-CRISPR/Cas12a technology.},
journal = {Journal of invertebrate pathology},
volume = {216},
number = {},
pages = {108569},
doi = {10.1016/j.jip.2026.108569},
pmid = {41713574},
issn = {1096-0805},
mesh = {*White spot syndrome virus 1/isolation & purification ; Animals ; *Penaeidae/virology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Aquaculture ; Sensitivity and Specificity ; },
abstract = {Pathogenic microorganisms, particularly white spot syndrome virus (WSSV), pose a major threat to global shrimp aquaculture, causing mass mortalities and substantial economic losses. To address the urgent need for rapid, accurate, and field-deployable detection methods, this study developed an innovative one-pot RAA-CRISPR/Cas12a assay. The platform integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technology using sucrose-mediated density gradient phase separation in a closed-tube format: sucrose acts as a density modifier to form distinct layers, spatially isolating RAA amplification reagents from CRISPR/Cas12a detection components to avoid cross-interference and enable sequential reactions without manual intervention. Under isothermal conditions at 37℃ for 60 min, the optimized assay achieves a limit of detection as low as 1 copy/μL, validated by both fluorescence and lateral flow dipstick (LFD) readouts. High specificity was confirmed by the absence of cross-reactivity with four other prevalent shrimp pathogens: infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND). Clinical validation with 30 field samples showed concordant results with the chinese national detection standard (GB/T 28630.2-2012). This novel nucleic acid detection platform combines highly sensitive, excellent specificity, and user-friendly visual interpretation, making it highly suitable for point-of-care testing and large-scale disease surveillance in shrimp aquaculture.},
}

*White spot syndrome virus 1/isolation & purification
Animals
*Penaeidae/virology
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Aquaculture
Sensitivity and Specificity

@article {pmid41690121,
year = {2026},
author = {Yin, B and Wu, X and Zhou, H and Meng, Y and Liu, J and Ding, L and Zhu, C and Tai, Z and Zhu, Q and Chen, Z},
title = {Topical ionic liquid-mediated GLUT1 gene editing ameliorates psoriasis and prevents recurrence.},
journal = {Biomaterials},
volume = {330},
number = {},
pages = {124058},
doi = {10.1016/j.biomaterials.2026.124058},
pmid = {41690121},
issn = {1878-5905},
mesh = {Animals ; *Gene Editing/methods ; *Psoriasis/therapy/genetics/pathology ; *Glucose Transporter Type 1/genetics ; Mice ; *Ionic Liquids/chemistry/administration & dosage ; Humans ; CRISPR-Cas Systems/genetics ; Recurrence ; Mice, Inbred C57BL ; Keratinocytes/metabolism ; Disease Models, Animal ; Administration, Topical ; },
abstract = {Psoriasis is a chronic inflammatory skin disorder characterized by immune dysregulation and a high relapse rate. Current therapies seldom achieve lasting remission. Aberrant overexpression of glucose transporter 1 (GLUT1) in keratinocytes enhances glycolysis, fueling inflammation and immune imbalance, thus positioning GLUT1 as a promising therapeutic target. In this work, a composite ionic liquid-mediated transdermal platform was established for the delivery of CRISPR-Cas9 ribonucleoprotein (CIL-RNP), aiming to achieve efficient GLUT1 gene editing in keratinocytes. The CIL-RNP achieved 76.6% editing efficiency, downregulated pyruvate kinase M (PKM) expression, and reduced inflammatory cytokine secretion. In a psoriasis mouse model, topical administration of CIL-RNP decreased lesion severity by 50% PASI (Psoriasis Area and Severity Index) score, alleviating epidermal hyperplasia and immune infiltration. Furthermore, the treatment inhibited M1 macrophage polarization, reduced reactive oxygen species generation, rebalanced Th17/regulatory T cells (Tregs) responses, and diminished the accumulation of tissue-resident memory T cells (TRMs), thereby lowering the risk of relapse. This study establishes ionic liquid-based CRISPR-RNP transdermal editing of GLUT1 as a novel and effective strategy for restoring immune homeostasis in psoriasis, with potential for long-term remission and broader applications in cutaneous immunopathological conditions.},
}

Animals
*Gene Editing/methods
*Psoriasis/therapy/genetics/pathology
*Glucose Transporter Type 1/genetics
Mice
*Ionic Liquids/chemistry/administration & dosage
Humans
CRISPR-Cas Systems/genetics
Recurrence
Mice, Inbred C57BL
Keratinocytes/metabolism
Disease Models, Animal
Administration, Topical

@article {pmid41685943,
year = {2026},
author = {Wang, C and Li, D and Yu, R and Xue, J and Xie, W and Zhang, Q and Gui, X and Wang, L and Guo, S and Xie, Y and Jiang, Y and Liu, G and Wu, J},
title = {A CRISPR/Cas9-regulated dual-ring topological allosteric probe for detection of the EGFR L858R resistance mutation in CTCs.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {9},
pages = {1815-1825},
doi = {10.1039/d5ay02124c},
pmid = {41685943},
issn = {1759-9679},
mesh = {Humans ; ErbB Receptors/genetics ; *Neoplastic Cells, Circulating/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Lung Neoplasms/genetics/blood ; *Carcinoma, Non-Small-Cell Lung/genetics/blood ; Polymorphism, Single Nucleotide ; *Drug Resistance, Neoplasm/genetics ; Mutation ; Fluorescent Dyes/chemistry ; Cell Line, Tumor ; },
abstract = {A single-nucleotide polymorphism (SNP) is a point mutation occurring at a defined genomic locus, and its precise and rapid detection in circulating tumor cells (CTCs) is essential for early diagnosis and therapeutic monitoring of non-small cell lung cancer (NSCLC). In this study, a CRISPR/Cas9-regulated dual-ring topological allosteric probe was developed for ultrasensitive and specific detection of the EGFR L858R mutation. The recognition ring selectively hybridizes with the target sequence and is cleaved by the Cas9-sgRNA complex, triggering the release of the reporter ring. The released reporter ring then serves as a template for rolling circle amplification (RCA), generating products that hybridize with dual-labeled fluorescent probes to yield measurable signals. This assay clearly distinguished L858R from the wild-type sequence and detected mutation frequencies as low as 1.0% with high specificity against other common EGFR variants. Its robustness was further validated using clinical blood samples, enabling sensitive detection of low-abundance L858R mutations. These results demonstrate that the integration of programmable target recognition, efficient signal amplification, and fluorescence readout provides a promising platform for SNP analysis in liquid biopsy, supporting precision diagnosis and treatment monitoring in NSCLC.},
}

Humans
ErbB Receptors/genetics
*Neoplastic Cells, Circulating/pathology/metabolism
*CRISPR-Cas Systems/genetics
*Lung Neoplasms/genetics/blood
*Carcinoma, Non-Small-Cell Lung/genetics/blood
Polymorphism, Single Nucleotide
*Drug Resistance, Neoplasm/genetics
Mutation
Fluorescent Dyes/chemistry
Cell Line, Tumor

@article {pmid41666850,
year = {2026},
author = {Zhao, R and Chen, J and Li, Y and Jin, M and Liu, K and Liu, Y and Gao, L and Yang, G and Yuan, X and Chu, X and Wang, JG},
title = {CRISPR/Cas9-mediated knockout of the 22 kDa α-prolamin genes orchestrates the regulation of functional amino acid content in foxtail millet.},
journal = {Journal of plant physiology},
volume = {318},
number = {},
pages = {154723},
doi = {10.1016/j.jplph.2026.154723},
pmid = {41666850},
issn = {1618-1328},
mesh = {*CRISPR-Cas Systems/genetics ; *Setaria Plant/genetics/metabolism ; *Amino Acids/metabolism ; Gene Knockout Techniques ; *Prolamins/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Genes, Plant ; },
abstract = {Improving the nutritional quality of cereal crops remains a primary objective in modern breeding programs. The composition and content of prolamins directly affect the overall nutritional value. This study elucidates the role of two 22 kDa α-prolamin genes (Seita.9G301400 and Seita.9G406400) in foxtail millet using CRISPR/Cas9-mediated knockout. While simultaneous disruption of both genes in double mutants reduced prolamin content, amino acids, and soluble sugars, single-gene mutants exhibited the opposite effect. These single mutants displayed increased grain size alongside significantly enhanced levels of essential amino acids and sugars. Starch pasting properties were also improved in single mutants but compromised in double mutants. Our findings demonstrate that individual knockout of the two prolamin genes enhances nutritional and sensory quality, providing a potential strategy for developing improved foxtail millet varieties.},
}

*CRISPR-Cas Systems/genetics
*Setaria Plant/genetics/metabolism
*Amino Acids/metabolism
Gene Knockout Techniques
*Prolamins/genetics/metabolism
*Plant Proteins/genetics/metabolism
Genes, Plant

@article {pmid41653514,
year = {2026},
author = {Li, M and He, L and Wang, Z and Wang, L and Pan, Q and Sun, P and Li, D and Zhang, C},
title = {Amplified ferroptosis and immunomodulation triggered by NIR-II photothermal-controllable CRISPR/Cas9 nanoplatform to treat osteosarcoma and prevent postsurgical implant-associated infections.},
journal = {Biomaterials},
volume = {330},
number = {},
pages = {124043},
doi = {10.1016/j.biomaterials.2026.124043},
pmid = {41653514},
issn = {1878-5905},
mesh = {*Ferroptosis/drug effects ; Humans ; Animals ; *CRISPR-Cas Systems ; *Osteosarcoma/therapy/immunology ; *Immunomodulation ; Mice ; Cell Line, Tumor ; Infrared Rays ; HSP70 Heat-Shock Proteins/genetics ; NF-E2-Related Factor 2/genetics/metabolism ; },
abstract = {Ferroptosis has been proven as a promising therapeutic approach with immunomodulatory effect; however, intracellular antioxidant system maintains redox balance and diminishes its efficacy. Nuclear factor erythroid 2-related factor 2 (Nrf2) is identified as a central transcription factor for regulating oxidative homeostasis. Herein, we have developed a thermal-controllable genome-editing nanoplatform BF/pHCN. Specifically, a CRISPR/Cas9 plasmid with an upstream HSP70 promoter sequence (HSP70-Cas9-sgNrf2, named pHCN) was constructed. Subsequent Fe(II) and pHCN were co-loaded into organic small-molecule BTP with near infrared II (NIR-II) absorption and coated with DSPE-mPEG2000. The generated BF/pHCN (BTP@Fe/pHCN) could be internalized within osteosarcoma cells. Subsequent NIR-II laser-triggered hyperthermia at 42 °C activated the HSP70 promoter and facilitated the precise inhibition targeting Nrf2 genomic sequences while promoting Fe(II) release, ultimately disrupting oxidative stress states. Moreover, the amplified ferroptosis fully triggered immunogenic cell death, thus reprogramming macrophages, promoting maturation of dendritic cells, and activating cellular antitumoral immunity. Additionally, BF/pHCN exhibited direct bactericidal activity against planktonic bacteria, and effectively eliminated intracellular bacteria through iron metabolic disorders strategy targeting macrophages, thereby initiating adaptive antimicrobial immunity. Overall, our NIR-II thermal-controllable genome-editing nanoplatform BF/pHCN exhibits remarkable antitumoral properties alongside robust antiinfection and immunomodulation, providing feasible strategies toward effective management of osteosarcoma, and preventing postsurgical implant-associated infections.},
}

*Ferroptosis/drug effects
Humans
Animals
*CRISPR-Cas Systems
*Osteosarcoma/therapy/immunology
*Immunomodulation
Mice
Cell Line, Tumor
Infrared Rays
HSP70 Heat-Shock Proteins/genetics
NF-E2-Related Factor 2/genetics/metabolism

@article {pmid41649281,
year = {2026},
author = {Zhu, Z-J and Cui, M-L and Liu, Y and Yao, X-Q and Lu, M-J and Wang, M-C and Liu, J-H and Li, J-F and Li, E-Z},
title = {CRISPR/Cas14a combined with RPA for visual detection of Marek's disease virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {3},
pages = {e0262525},
pmid = {41649281},
issn = {2165-0497},
support = {22A310017//Key Scientific Research Projects of Universities in Henan/ ; },
mesh = {Animals ; *Marek Disease/diagnosis/virology ; *CRISPR-Cas Systems ; *Herpesvirus 2, Gallid/isolation & purification/genetics ; *Poultry Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Chickens/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; Recombinases/metabolism/genetics ; *Mardivirus/isolation & purification/genetics ; },
abstract = {Marek's disease, a highly contagious avian immunosuppressive disorder caused by the α-herpesvirus MDV-1, poses a significant threat to poultry health. The development of rapid visual detection methods capable of distinguishing epidemic MDV-1 strains from vaccine strains is crucial for early disease warning, vaccine efficacy evaluation, and precise disease control. We developed a novel isothermal detection system that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology for the visual identification of epidemic MDV-1 strains. This method operates at a constant temperature of 37°C and allows for either real-time analysis or endpoint visual readout without the need for complex instrumentation. Our results showed no cross-reactivity with Newcastle disease virus, infectious bursal disease virus, MDV-1 vaccine strains, or herpesvirus of turkeys. Plasmid DNA standards were used to determine the sensitivity of the assay, and the detection limit was 24.6 copies/μL. Clinical evaluation using 24 field samples confirmed that the method successfully identified all Marek's disease virus-positive cases, demonstrating its diagnostic reliability. In conclusion, we have developed a rapid, highly specific nucleic acid detection platform for MDV-1 that enables visual readout without complex instrumentation by combining the sensitivity of RPA with the specificity of CRISPR/Cas14a technology, offering promising potential for field-based diagnostics and disease surveillance.IMPORTANCEMarek's disease virus (MDV-1) is a highly contagious and economically important avian pathogen. Existing diagnostic methods are unable to reliably distinguish between epidemic and vaccine strains in field settings, which hampers effective surveillance and evaluation of vaccination programs. To address this challenge, we developed a portable isothermal detection assay that combines recombinase polymerase amplification with CRISPR/Cas14a technology. This approach enables highly sensitive (24.6 copies/μL) and specific visual detection of epidemic MDV-1 strains without cross-reactivity with vaccine strains or related viruses. The assay demonstrated 100% agreement with reference methods when evaluated using clinical samples. As a cost-effective method that avoids the need for complex detection instruments, it offers a practical solution for rapid on-site diagnosis, facilitating enhanced outbreak control and improved poultry health management globally.},
}

Animals
*Marek Disease/diagnosis/virology
*CRISPR-Cas Systems
*Herpesvirus 2, Gallid/isolation & purification/genetics
*Poultry Diseases/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
Chickens/virology
Sensitivity and Specificity
*Molecular Diagnostic Techniques/methods
Recombinases/metabolism/genetics
*Mardivirus/isolation & purification/genetics

@article {pmid41638449,
year = {2026},
author = {Qu, Y and Li, Y and Shao, T and Kuang, J and Qi, Y and Yang, J and Liu, Y and Wang, J and Fu, X and Liu, J and Zhang, X and Peng, T and Yuan, Q and Zhu, L},
title = {Optimizing prime editing: Advances in efficiency enhancement.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108815},
doi = {10.1016/j.biotechadv.2026.108815},
pmid = {41638449},
issn = {1873-1899},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Protein Engineering/methods ; },
abstract = {Prime editing (PE) enables the precise installation of targeted insertions, deletions, and all possible base-to-base conversions without introducing double-strand breaks or donor DNA templates. However, its efficiency remains highly variable across genomic contexts. To address this, multi-faceted optimization strategies have been developed: protein engineering has yielded editor variants with enhanced reverse transcriptase activity and stability; structural refinements to pegRNA design improve its functional integrity and resistance to degradation; regulation of the PE-Flap-mismatch repair (MMR) process favors the retention of desired edits; and the development of protospacer adjacent motif (PAM)-relaxed Cas variants dramatically expands targetable sites. This review systematically consolidates these advances, illustrating how the integration of structural, mechanistic and targeting enhancements is overcoming fundamental bottlenecks. Together, these developments establish PE as a versatile and efficient system for precision genome engineering, paving the way for its reliable application in diverse biological settings.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
Protein Engineering/methods

@article {pmid41506374,
year = {2026},
author = {Marschhofer, M and Chen, S and Molbay, M and Winkeljann, B and Villano, E and Giancaspro, C and Kourou, A and Berninghausen, O and Rieder, S and Ungewickell, C and Beckmann, R and Popper, B and Torres, AM and Vidal, A and Merkel, OM and Carneiro, SP},
title = {Optimized lipid nanoparticles for pulmonary delivery of CRISPR/Cas9 targeting KRAS G12S in lung cancer.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114607},
doi = {10.1016/j.jconrel.2026.114607},
pmid = {41506374},
issn = {1873-4995},
mesh = {Animals ; *Lung Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles/administration & dosage/chemistry ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Lipids/chemistry/administration & dosage ; Gene Editing ; A549 Cells ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy ; Mice ; *CRISPR-Associated Protein 9/genetics ; Mice, Nude ; RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics ; Female ; RNA, Messenger/administration & dosage/genetics ; Apoptosis ; Cell Line, Tumor ; Liposomes ; },
abstract = {KRAS G12S mutations in non-small cell lung cancer (NSCLC) remain refractory to current targeted therapies, with few clinical options and frequent resistance. While CRISPR/Cas9 enables mutation-specific gene disruption, its pulmonary application is limited by systemic clearance, hepatic tropism, and airway mucus barriers. Here, we present lipid nanoparticles (LNPs) specifically engineered for pulmonary delivery of Cas9 mRNA and KRAS G12S-targeting sgRNA, optimized through mRNA surrogate screening and orthogonal mixture design to guide lipid composition and Cas9:sgRNA weight-to-weight ratios. Two lead LNP formulations, A6 3:1 and A8 1:1, exhibited robust critical quality attributes, including particle sizes below 120 nm, low polydispersity, near-neutral zeta potential, and over 80 % encapsulation efficiency. Cryo-TEM revealed distinct morphologies correlated with enhanced transfection. In vitro, A8 1:1 achieved up to 90 % on-target gene editing in A549 cells and a 3.6-fold increase in apoptosis, while A6 3:1 induced a 3.7-fold apoptotic response. Both formulations efficiently traversed airway mucus in air-liquid interface cultures and preserved over 80 % cell viability across doses. In vivo, repeated pulmonary administration was well tolerated, with no signs of systemic toxicity or cytokine elevation in healthy or tumor-bearing mice. In an orthotopic A549-luc lung tumor model, intratracheal delivery of A6 3:1 and A8 1:1 modestly suppressed tumor growth, with histological evidence of tumor cell apoptosis for A8 1:1. Quantification confirmed a statistically significant increase of apoptosis in the A8 1:1 group, consistent with effective KRAS disruption in vivo. Overall, lead LNPs, particularly A8 1:1, enabled efficient and localized RNA-based gene editing that induced downstream apoptotic signaling, demonstrating a preliminary, yet promising, proof-of-concept for CRISPR/Cas9 therapy in NSCLC.},
}

Animals
*Lung Neoplasms/genetics/therapy
*CRISPR-Cas Systems
Humans
*Nanoparticles/administration & dosage/chemistry
*Proto-Oncogene Proteins p21(ras)/genetics
*Lipids/chemistry/administration & dosage
Gene Editing
A549 Cells
*Carcinoma, Non-Small-Cell Lung/genetics/therapy
Mice
*CRISPR-Associated Protein 9/genetics
Mice, Nude
RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics
Female
RNA, Messenger/administration & dosage/genetics
Apoptosis
Cell Line, Tumor
Liposomes

@article {pmid41489552,
year = {2026},
author = {Fan, R and Tong, Y and Luo, S and He, Y and Yang, C and Li, W and Liu, J and Pan, J and Zhu, Y and Zhang, X and Zhu, J and Zhu, Y and Guo, Y and Li, L and Situ, B and Yan, X and Ma, W and Chang, L and Zhang, Y},
title = {Integrated Electroporated-Lysis Electrochemical Platform Enables Sensitive and Rapid EV Protein and miRNA Profiling Based on Multiplex-Responsive CRISPR/Cas12a.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {13},
pages = {e13331},
doi = {10.1002/smll.202513331},
pmid = {41489552},
issn = {1613-6829},
support = {2024YFC2419100//National Key R&D Program of China/ ; 82272424//National Natural Science Foundation of China/ ; 82572682//National Natural Science Foundation of China/ ; 82372343//National Natural Science Foundation of China/ ; 82402753//National Natural Science Foundation of China/ ; 22304006//National Natural Science Foundation of China/ ; 62471021//National Natural Science Foundation of China/ ; 2023A1515010909//Guang Dong Basic and Applied Basic Research Foundation of China/ ; JCYJ20230807142204008//Shenzhen Science and Technology Innovation Committee/ ; 2024J004//Southern Medical University/ ; 2024M750606//China Postdoctoral Science Foundation/ ; 2025T180607//China Postdoctoral Science Foundation/ ; T2425021//National Science Fund for Distinguished Young Scholars/ ; },
mesh = {*MicroRNAs/metabolism/genetics ; Humans ; *Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Electroporation/methods ; },
abstract = {Proteins and miRNAs in extracellular vesicles (EVs) have emerged as crucial biomarkers for tumor diagnosis. While CRISPR/Cas12a-based platforms have shown great promise in nucleic acid and protein detection, their susceptibility to off-target activation and structural instability remains a significant limitation. Here, we have developed an electroporation-lysis electrochemical platform integrated with DNA cube-cage-locked CRISPR/Cas12a (DC-Cas12a), termed EL-DC-Cas12a. This platform utilizes an electric field to rapidly lyse EVs, releasing their internal proteins and miRNAs. These released molecules then activate the DC-Cas12a system, thereby triggering the displacement of two distinct crRNA/Cas12a complexes that correspond to EV proteins and miRNAs, respectively. These complexes then specifically recognize and cleave electrochemical probes, generating quantifiable electrochemical signals that enable synchronous and accurate analysis of the two biomarkers. The integrated workflow for EV lysis and detection can be completed within 40 min, greatly simplifying the overall operation. The detection limits (LOD) of this platform for EV PD-L1 protein and miR-1246 were 5.44 × 10[4] particles/mL and 3.59 × 10[3] particles/mL, respectively. Moreover, by applying machine learning algorithms to analyze the EV-associated proteins and miRNAs profiling, the platform demonstrated a diagnostic accuracy of 98.3% in distinguishing healthy donors from early-stage GC patients, and 99% in differentiating early-stage from advanced-stage GC patients in a clinical gastric cancer cohort. Therefore, the proposed platform offers a promising strategy for multiplexed detection of EV biomarkers and precise discrimination of GC.},
}

*MicroRNAs/metabolism/genetics
Humans
*Extracellular Vesicles/metabolism
*CRISPR-Cas Systems/genetics
*Electrochemical Techniques/methods
*Electroporation/methods

@article {pmid41477961,
year = {2026},
author = {Gong, P and Tao, D and Chen, Q and Yang, Y and Xie, S and Chen, X and Shi, S and Wang, Y and Wang, L and Qian, Y and Ye, S},
title = {A rapid, visual, ultrasensitive and highly specific method for detecting adeno-associated virus 2020 based on the LAMP-CRISPR/Cas12a system.},
journal = {Poultry science},
volume = {105},
number = {2},
pages = {106344},
pmid = {41477961},
issn = {1525-3171},
mesh = {Animals ; *Poultry Diseases/diagnosis/virology ; *Parvoviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *CRISPR-Cas Systems ; *Dependovirus/isolation & purification/genetics ; *Ducks ; *Molecular Diagnostic Techniques/veterinary/methods ; Sensitivity and Specificity ; Phylogeny ; },
abstract = {Avian parvovirus infection would lead to growth retardation, weight loss, physical deformities and increased mortality in poultry, causing substantial economic losses to the poultry industry. Therefore, the development of a rapid, visual, ultrasensitive and highly specific method is essential for timely diagnosis and effective control of the avian parvovirus infection. In this study, we developed a detection platform based on loop-mediated isothermal amplification (LAMP) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system. Firstly, we have identified a novel avian parvovirus strain from diseased Muscovy ducks. Through genome sequencing, sequence assembly and phylogenetic tree analysis, we have identified this novel avian parvovirus as an adeno-associated virus (AAA) belonging to the family Parvoviridae, subfamily Parvovirinae and genus Dependoparvovirus. So, the novel virus strain was named AAV-2020. Next, specific sgRNAs and LAMP primers targeting the 3 capsid proteins (Cap) genes of AAV-2020 were designed and optimized. Moreover, the CRISPR/Cas12a-based system demonstrated a limit of detection as low as 2 copies/μL for AAV-2020. Importantly, the system could effectively distinguish AAV-2020 from 3 closely related AAV strains with high sequence similarity, indicating excellent specificity. In summary, we developed a novel, rapid, visual, ultrasensitive and highly specific detection system for AAV-2020, offering a reliable tool for early diagnosis and on-site detection of avian parvovirus infections, which would aid in the prevention and control of avian parvovirus infection in poultry industry.},
}

Animals
*Poultry Diseases/diagnosis/virology
*Parvoviridae Infections/veterinary/diagnosis/virology
*Nucleic Acid Amplification Techniques/veterinary/methods
*CRISPR-Cas Systems
*Dependovirus/isolation & purification/genetics
*Ducks
*Molecular Diagnostic Techniques/veterinary/methods
Sensitivity and Specificity
Phylogeny

@article {pmid41423250,
year = {2026},
author = {Sharma, A and Pathangey, L and Chirackal, SS and Shim, KG and Fonseca, R and Swaminathan, S},
title = {Ferritin H Knockout Induces Differential Immunomodulatory Drug Responses in Multiple Myeloma Cell Lines.},
journal = {European journal of haematology},
volume = {116},
number = {4},
pages = {391-401},
doi = {10.1111/ejh.70084},
pmid = {41423250},
issn = {1600-0609},
support = {//Paula and Rodger Riney Foundation/ ; },
mesh = {Humans ; *Multiple Myeloma/genetics/metabolism/drug therapy/pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Gene Knockout Techniques ; *Ferritins/genetics ; Reactive Oxygen Species/metabolism ; Iron/metabolism ; CRISPR-Cas Systems ; *Immunomodulating Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Oxidative Stress ; Oxidoreductases ; },
abstract = {BACKGROUND: Immunomodulatory agents (IMiDs) are a cornerstone in the successful management of multiple myeloma (MM). However, acquired IMiD resistance leading to disease relapses remains a major barrier. Hydrogen peroxide generation and oxidative stress are key mediators that determine IMiD's effectiveness in MM. Iron plays a key role in the generation of oxidative stress; therefore, cellular iron levels are tightly governed. FTH1 is the major iron storage protein that tightly regulates cellular iron availability. Hence, the present study is targeted to investigate the role of FTH1 in MM and IMiD resistance.

METHODS: IMiD-sensitive and IMiD-resistant MM cells were analyzed for expression of iron-metabolism genes. CRISPR-cas9-mediated knockout of FTH1 was performed and the after-effects were assessed through multiple experiments.

RESULTS: Initial analysis showed a positive correlation between FTH1 expression and IMiD resistance in MM cells. FTH1-KO reduced IMiD sensitivity in the KMS11 cell line but had no effect on the RPMI8226 cell line. RNA-seq data showed downregulation of ER-stress and calcium signaling genes after FTH1-KO. Further, KMS11-FTH1KO cells exhibited lower intracellular ROS, labile-iron, and mitochondrial superoxide levels along with increased CD63, suggesting activation of L-ferritin secretory pathways.

CONCLUSION: Data reveals a link between FTH1, labile iron, ROS, and IMiD resistance in MM cells.},
}

Humans
*Multiple Myeloma/genetics/metabolism/drug therapy/pathology
Cell Line, Tumor
Drug Resistance, Neoplasm/genetics
Gene Knockout Techniques
*Ferritins/genetics
Reactive Oxygen Species/metabolism
Iron/metabolism
CRISPR-Cas Systems
*Immunomodulating Agents/pharmacology
Gene Expression Regulation, Neoplastic/drug effects
Oxidative Stress
Oxidoreductases

@article {pmid41376159,
year = {2026},
author = {Ha, AS and Kalter, N and Rosenberg, M and Acevedo, LA and Liang, B and Liu, W and Paruthiyil, S and Sinha, M and Vu, A and Nguyen, V and Qi, Z and Krishnappa, N and Shu, J and Yu, J and Catanzaro, J and Bluestone, JA and Tang, Q and Urnov, FD and Marson, A and Hendel, A and Herold, KC and Shy, BR and Esensten, JH},
title = {Gene-corrected regulatory T cell therapy for IL2RA deficiency.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1367-1381},
doi = {10.1016/j.ymthe.2025.12.004},
pmid = {41376159},
issn = {1525-0024},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology/metabolism/transplantation ; *Interleukin-2 Receptor alpha Subunit/genetics/deficiency ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Gene Editing ; Male ; Female ; Interleukin-2 ; Mutation ; },
abstract = {Bi-allelic germline deficiency of IL2RA causes a rare autoimmune disease with impaired regulatory T cell (Treg) function and interleukin-2 (IL-2) signaling. Definitive treatment is currently limited to allogeneic hematopoietic stem cell transplantation, which carries significant morbidity and mortality risks. We previously identified a family with three siblings affected by compound heterozygous mutations in their IL2RA gene, resulting in dysfunctional Tregs. Here, we introduce a novel therapeutic approach involving ex vivo generation of gene-corrected autologous regulatory T cells (gcTregs). One of the two disease-causing mutations in patient-derived Tregs was corrected with CRISPR-Cas9-mediated homology-directed repair, restoring IL2RA expression. The resulting gcTregs demonstrated robust suppressive activity in vitro. Clinical-scale manufacturing from a patient with IL2RA deficiency showed efficient gene correction, restored IL2RA expression, and functional equivalence to healthy donor Tregs. This work establishes a Good Manufacturing Practice-compatible manufacturing process for personalized gcTreg therapies, potentially providing a safer treatment option for patients with IL2RA deficiency as well as a framework for treating other inborn errors of immunity.},
}

Humans
*T-Lymphocytes, Regulatory/immunology/metabolism/transplantation
*Interleukin-2 Receptor alpha Subunit/genetics/deficiency
*Genetic Therapy/methods
CRISPR-Cas Systems
Gene Editing
Male
Female
Interleukin-2
Mutation

@article {pmid41376155,
year = {2026},
author = {Jin, X and Wu, X and Song, J and Luo, M and Ye, Q and Ren, C and Song, L and Li, M and Hu, M and An, Y and Su, J and Fu, J and Xu, Q and Luo, M and Liu, F and Liu, M and Li, Q and Yao, S and Chen, L and Yang, Y},
title = {Comparative evaluation of liver-directed knockin strategies with viral and nonviral vectors in mouse inherited disease models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1775-1793},
doi = {10.1016/j.ymthe.2025.12.012},
pmid = {41376155},
issn = {1525-0024},
mesh = {Animals ; *Genetic Vectors/genetics/administration & dosage ; Mice ; Disease Models, Animal ; Dependovirus/genetics ; *Liver/metabolism ; *Gene Knock-In Techniques/methods ; Genetic Therapy/methods ; Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Recombinational DNA Repair ; *Hemophilia B/genetics/therapy ; Transgenes ; },
abstract = {CRISPR-Cas9-mediated gene knockin has emerged as a promising strategy for early-onset genetic disease intervention. However, the therapeutic efficacy and editing outcomes of different knockin strategies remain incompletely understood. Here, we systematically evaluated three major liver-directed knockin strategies, namely homology-directed repair (HDR), homology-independent targeted integration (HITI), and homology-mediated end joining (HMEJ), using neonatal mouse models of mucopolysaccharidosis type I and hemophilia B. Although all three approaches effectively rescued disease phenotypes, we observed distinct editing outcomes. Notably, the HMEJ approach, delivered via a combined adeno-associated virus-lipid nanoparticle (AAV-LNP) system, exhibited superior integration efficiency (5.8%-5.9%) and fidelity (97%-98%) compared with HDR and HITI. In contrast, whole-genome sequencing indicated that HITI induced a higher risk of random AAV donor integration than HDR or HMEJ. Furthermore, long-read sequencing analyses revealed that the frequencies of inverted terminal repeat (ITR)-mediated transgene integration differed between the 5' and 3' genomic junctions among the three strategies. Specifically, in HDR- and HMEJ-treated mice, ITR-mediated integration events were 7.7- to 19.7-fold more common at the 3' junctions than at the 5' junctions. These findings highlight the comprehensive advantages of the AAV-LNP-mediated HMEJ approach for liver-directed knockin therapy and suggest its strong potential for clinical translation.},
}

Animals
*Genetic Vectors/genetics/administration & dosage
Mice
Disease Models, Animal
Dependovirus/genetics
*Liver/metabolism
*Gene Knock-In Techniques/methods
Genetic Therapy/methods
Gene Editing/methods
Humans
CRISPR-Cas Systems
Recombinational DNA Repair
*Hemophilia B/genetics/therapy
Transgenes

@article {pmid41370232,
year = {2026},
author = {Alok, A and Raman, V and D'Agostino, L and Kshetry, AO and Rai, KM and Wang, C and Gunapati, S and Stupar, RM and Patil, GB and Zhang, F},
title = {Developmental regulators enable rapid and efficient soybean transformation and CRISPR-mediated genome editing.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
doi = {10.1093/plphys/kiaf640},
pmid = {41370232},
issn = {1532-2548},
support = {IOS-2040218//National Science Foundation/ ; IOS-2206920//National Science Foundation/ ; #2021-67013-34565//USDA NIFA/ ; //Texas Governor's University Research/ ; },
mesh = {*Glycine max/genetics/growth & development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Plant Growth Regulators/metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {Soybean (Glycine max) transformation remains challenging and has not kept pace with rapid advances in genetic engineering technologies due to low efficiency, lengthy timelines, and genotype dependency. Here, we developed a streamlined transformation method by leveraging developmental regulators (DRs) to promote de novo shoot regeneration directly from growing soybean plants. By evaluating multiple DR combinations, our results showed that co-expression of WUSCHEL2 (WUS2) and the gene encoding isopentenyltransferase (IPT) achieved higher transformation efficiencies (14.6% to 22.3%) in Williams 82 and Bert varieties than individual DRs without requiring exogenous hormones or selection agents. Moreover, this method produced heritable transgenic events within 9 to 11 weeks and successfully delivered CRISPR-Cas9 components, generating heritable mutations with 20% efficiency. The temporal transcriptomic and gene regulatory network analyses revealed that WUS2/IPT synergistically modulates stress responses and activates developmental pathways, orchestrating a transition from initial stress adaptation to regenerative programming. Our findings demonstrate that this DR-enabled approach significantly enhances soybean transformation frequency, reduces tissue culture requirements, and offers a promising genome-editing platform for soybean improvement.},
}

*Glycine max/genetics/growth & development
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
*Transformation, Genetic
*Plant Growth Regulators/metabolism
Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism

@article {pmid41332134,
year = {2026},
author = {Inuzuka, T and Mouzannar, K and Zhang, M and Umarova, R and Park, SB and Uchida, T and Ma, CD and Liang, TJ},
title = {A CRISPR-based genome-wide loss-of-function screen defines a role of host metabolism in regulating hepatitis B virus infection.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1616-1632},
pmid = {41332134},
issn = {1525-0024},
support = {Z01 DK054500/ImNIH/Intramural NIH HHS/United States ; Z01 DK054504/ImNIH/Intramural NIH HHS/United States ; Z99 DK999999/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Hepatitis B virus/genetics/physiology ; *Hepatitis B/metabolism/virology/genetics ; *CRISPR-Cas Systems ; Hep G2 Cells ; Virus Replication/genetics ; Hepatocytes/metabolism/virology ; *Host-Pathogen Interactions/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Hepatitis B virus (HBV) co-opts and interacts with an extensive array of host factors for productive infection. Herein, we develop an HBV reporter virus expressing red fluorescent protein (HBV-RFP) that is suitable for a CRISPR-based genome-wide screen for HBV host dependency factors. HepG2[NTCP/Cas9] cells were transduced with a pooled lentiviral library of single-guide RNA (sgRNA) targeting 19,114 human genes, edited and infected with HBV-RFP. RFP-low cells were sorted using fluorescence-activated cell sorting. The sorted cells were expanded and underwent two additional rounds of infection and sorting to enrich for sgRNA-targeted proviral host factors. By next-generation sequencing and bioinformatic analyses, we identified 63 genes as candidate host proviral factors, including known HBV proviral factors: RXRA, POLL, LDLR, and NTCP. Among the novel candidate genes, knockout of 12 genes significantly decreased HBV replication markers. Validation using siRNA knockdown in primary human hepatocytes confirmed several factors including the monoacylglycerol acyltransferase 2 (MOGAT2) gene as a bona fide HBV proviral factor. Further analysis with MGAT2 inhibitors demonstrated that inhibition of MOGAT2 activity impairs HBV transcription and replication. Our study demonstrates the value of the HBV reporter system in identifying previously unrecognized host metabolic factors important for HBV infection, offering a potential avenue for therapeutic development.},
}

Humans
*Hepatitis B virus/genetics/physiology
*Hepatitis B/metabolism/virology/genetics
*CRISPR-Cas Systems
Hep G2 Cells
Virus Replication/genetics
Hepatocytes/metabolism/virology
*Host-Pathogen Interactions/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41267399,
year = {2026},
author = {Dua, PH and Simon, BMJ and Marley, CBE and Feliciano, CM and Watry, HL and Cowan, QT and Steury, D and Abraham, A and Gilbertson, EN and Ramey, GD and Capra, JA and Conklin, BR and Judge, LM},
title = {Haplotype editing with CRISPR-Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1633-1651},
doi = {10.1016/j.ymthe.2025.11.026},
pmid = {41267399},
issn = {1525-0024},
support = {R01 NS119678/NS/NINDS NIH HHS/United States ; U01 ES032673/ES/NIEHS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Mutation, Missense ; *Haplotypes ; *Charcot-Marie-Tooth Disease/genetics/therapy ; Motor Neurons/metabolism ; Polymorphism, Single Nucleotide ; Alleles ; Genetic Therapy/methods ; Phenotype ; },
abstract = {Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplosufficient. We previously edited a dominant NEFL missense mutation causing Charcot-Marie-Tooth type 2E (CMT2E) with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause CMT2E. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach in two iPSC lines with different missense mutations and demonstrated phenotypic rescue in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent by-product of excision editing, failed to reliably disrupt mutant allele expression. We deployed novel molecular assays to optimize our approach and achieve therapeutic levels of editing in immature iPSC-motor neurons. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Induced Pluripotent Stem Cells/metabolism/cytology
*Mutation, Missense
*Haplotypes
*Charcot-Marie-Tooth Disease/genetics/therapy
Motor Neurons/metabolism
Polymorphism, Single Nucleotide
Alleles
Genetic Therapy/methods
Phenotype

@article {pmid41247434,
year = {2026},
author = {Liu, F and Ge, D and Lian, G and Han, Z and Chai, J},
title = {Unraveling plant immunity: from pathogen perception to resistance engineering.},
journal = {Science China. Life sciences},
volume = {69},
number = {3},
pages = {779-793},
pmid = {41247434},
issn = {1869-1889},
mesh = {*Plant Immunity/genetics ; *Disease Resistance/genetics/immunology ; *Plant Diseases/immunology/microbiology ; Receptors, Pattern Recognition/metabolism/immunology ; NLR Proteins/metabolism/genetics ; Signal Transduction ; *Plants/immunology/genetics ; Calcium Signaling ; Plant Proteins/genetics/metabolism ; Host-Pathogen Interactions/immunology ; CRISPR-Cas Systems ; },
abstract = {Unlike animals, which rely on circulatory systems and mobile immune cells, each plant cell must autonomously detect and respond to pathogenic threats. Plant immunity operates through two major layers: pattern-triggered immunity (PTI), initiated by cell-surface pattern recognition receptors (PRRs), and effector-triggered immunity (ETI), primarily mediated by intracellular nucleotide-binding leucine-rich repeat (NLR) receptors. Recent advances have substantially enhanced our understanding of PTI and ETI signaling. Notably, some NLRs following activation by specific recognition of pathogen effectors form higher-order oligomeric complexes termed resistosomes that act as Ca[2+]-permeable channels to trigger immune signaling. Increasing evidence points to extensive crosstalk and mutual potentiation between PTI and ETI, with Ca[2+] functioning as a pivotal second messenger in both pathways. Elucidating the molecular basis of these pathways, combined with emerging tools like CRISPR/Cas9, offers new strategies for engineering durable disease resistance in crops. This review highlights current insights into PTI and ETI, with an emphasis on the central role of Ca[2+] signaling and key challenges in engineering NLR receptors.},
}

*Plant Immunity/genetics
*Disease Resistance/genetics/immunology
*Plant Diseases/immunology/microbiology
Receptors, Pattern Recognition/metabolism/immunology
NLR Proteins/metabolism/genetics
Signal Transduction
*Plants/immunology/genetics
Calcium Signaling
Plant Proteins/genetics/metabolism
Host-Pathogen Interactions/immunology
CRISPR-Cas Systems

@article {pmid41170937,
year = {2026},
author = {Soliman, H and Akram, N and Saleh, M},
title = {CRISPR-Enhanced RAA-SHERLOCK Assay for Point-of-Care Detection of Cyprinid Herpesvirus-3: Development, Validation and Clinical Application.},
journal = {Journal of fish diseases},
volume = {49},
number = {4},
pages = {e70079},
pmid = {41170937},
issn = {1365-2761},
support = {//Veterinary Medicine University/ ; //Academy of Scientific Research and Technology/ ; },
mesh = {*Fish Diseases/diagnosis/virology ; Animals ; *Herpesviridae/isolation & purification/genetics ; *Herpesviridae Infections/veterinary/diagnosis/virology ; *Carps ; CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Point-of-Care Systems ; Recombinases/metabolism ; },
abstract = {Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus-3 (CyHV-3), poses a significant threat to global aquaculture due to its high mortality rates and economic impact. Current diagnostic methods, such as PCR, are limited by equipment dependency and procedural complexity, hindering point-of-care (POC) applications. To address this, we developed an integrated assay combining recombinase-aided amplification (RAA) with CRISPR-Cas13a-mediated SHERLOCK technology and lateral flow detection (LFD) for rapid and visual detection of CyHV-3 in clinical samples. The KHV-SHERLOCK assay targets a conserved region of the CyHV-3 thymidine kinase (TK) gene, demonstrating exceptional specificity with no cross-reactivity to related pathogens or host DNA. Sensitivity evaluations revealed a detection limit of 100 ag/μL for CyHV-3 plasmid DNA, tenfold more sensitive than the conventional PCR (1 fg/μL) assay, even in the presence of 100 ng of carp genomic DNA as background interference. Clinical validation using 50 archived samples showed 100% concordance with reference PCR results, confirming diagnostic reliability. The assay's isothermal RAA step (37°C, 40 min) and CRISPR-Cas13a detection (37°C, 1 h) enable equipment-free operation, while LFD provides unambiguous visual results within minutes. This platform merges high sensitivity with POC practicality, offering a transformative tool for field-based KHVD surveillance.},
}

*Fish Diseases/diagnosis/virology
Animals
*Herpesviridae/isolation & purification/genetics
*Herpesviridae Infections/veterinary/diagnosis/virology
*Carps
CRISPR-Cas Systems
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/veterinary/methods
*Point-of-Care Systems
Recombinases/metabolism

@article {pmid41078118,
year = {2026},
author = {Wang, Y and Yang, J and Hou, H and Song, L and Cheng, X and Liu, YX},
title = {Advancing Plant Microbiome Research Through Host DNA Depletion Techniques.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1189-1203},
pmid = {41078118},
issn = {1467-7652},
support = {32470055//National Natural Science Foundation of China/ ; U23A20148//National Natural Science Foundation of China/ ; CAAS-BRC-CB-2025-01//Basic Research Center for Crop Biosafety Sciences/ ; CAAS-ZDRW202308//Agricultural Science and Technology Innovation Program/ ; },
mesh = {*Microbiota/genetics ; *Plants/microbiology/genetics ; *Metagenomics/methods ; *DNA, Plant/genetics/isolation & purification ; CRISPR-Cas Systems ; },
abstract = {Plants provide ecological habitats for diverse microorganisms, making accurate metagenomic sequencing essential for understanding the complex interactions that support plant growth, development and disease resistance. However, host DNA contamination poses a major challenge in plant microbiome studies, obscuring microbial genetic signatures and complicating the accurate analysis of microbial genomes. This review provides a comprehensive overview of current host DNA depletion strategies, including physical separation (e.g., filtration, gradient centrifugation), selective lysis and enzymatic treatments targeting plant cell walls. Advanced techniques such as targeted sequence capture with magnetic beads, methylation-based enrichment and nanopore selective sequencing offer additional options for host DNA removal. Despite these advances, current methods still face challenges in efficiency, specificity and applicability, emphasising the need for tailored strategies and the exploration of novel approaches for microbial enrichment. Innovations like CRISPR-Cas9 and chromatin immunoprecipitation-based host DNA depletion methods are proposed to provide novel directions for addressing current limitations. The development and refinement of host depletion techniques tailored to plant systems are crucial for enabling high-resolution, cost-effective metagenomic studies. These efforts promise to deepen our understanding of microbial diversity and functionality, ultimately accelerating microbiome-based innovations in crop improvement, sustainable agriculture and ecosystem resilience.},
}

*Microbiota/genetics
*Plants/microbiology/genetics
*Metagenomics/methods
*DNA, Plant/genetics/isolation & purification
CRISPR-Cas Systems

@article {pmid40744834,
year = {2026},
author = {Qin, Z and Deng, Z and Li, C and Wang, D and Ji, X and Chen, Z},
title = {AI sheds new light on genome editing.},
journal = {Trends in biotechnology},
volume = {44},
number = {3},
pages = {696-709},
doi = {10.1016/j.tibtech.2025.07.011},
pmid = {40744834},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; *Artificial Intelligence ; CRISPR-Cas Systems/genetics ; Humans ; Biotechnology ; },
abstract = {Artificial intelligence (AI) has revolutionized life sciences, driving transformative advances in engineering clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)-based genome editors for therapeutic and agricultural applications. Recent breakthroughs demonstrate how deep learning accelerates the discovery, engineering, and design of next-generation genome editing tools. In this review, we explore how AI-driven approaches are supercharging genome editing in three aspects: (i) structure-based methods for discovering novel genome editors neglected by conventional methods, (ii) engineering genome editors with enhanced properties, and (iii) the de novo design of entirely new genome editors endowed with bespoke functions. Finally, we discuss the current challenges and envision the future potential of data-driven AI to unlock new possibilities in genome editing, catalyzing innovations across biology and biotechnology.},
}

*Gene Editing/methods
*Artificial Intelligence
CRISPR-Cas Systems/genetics
Humans
Biotechnology

@article {pmid41754561,
year = {2026},
author = {Wupori, K and Garnett, L and Bello, A and Strong, JE},
title = {CRISPR-Based Detection of Viral Hemorrhagic Fevers at the Point of Care.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754561},
issn = {1999-4915},
support = {8//Genomics Research and Development Initiative/ ; CSSP-2022-CP-2546//Canadian Safety and Security Program/ ; },
mesh = {Humans ; *Hemorrhagic Fevers, Viral/diagnosis/virology ; *Point-of-Care Systems ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Point-of-Care Testing ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Viral hemorrhagic fevers (VHFs) are highly lethal diseases that often present non-specific, influenza-like symptoms in their early stages, making clinical recognition and differentiation from other febrile illnesses difficult. This overlap underscores the critical need for diagnostic tests that are both sensitive and specific. Point-of-care (POC) diagnostic tests are an invaluable tool for detecting and controlling the spread of pathogens that threaten public health, such as VHFs, as these require fast, accurate diagnostics to ensure biosafety and appropriate mobilization of resources during outbreaks. Current molecular and serological diagnostic tests, while efficient and effective, lack the characteristics required of a POC test (POCT) to quickly and easily respond to a VHF outbreak while maintaining a low cost. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tests have gained popularity as POCTs due to their inherent attractive qualities, including high sensitivity and specificity, adaptability, low cost, quick turnaround time, and ease of use. However, studies on the development of CRISPR-based POC diagnostic tests for VHFs are limited. This review summarizes the current CRISPR-based POCTs for VHFs, including Ebola virus (EBOV), Lassa virus (LASV), Dengue virus (DENV), and Crimean-Congo hemorrhagic fever virus (CCHF). The isothermal pre-amplification methods commonly paired with CRISPR-based tests, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are also discussed.},
}

Humans
*Hemorrhagic Fevers, Viral/diagnosis/virology
*Point-of-Care Systems
*Molecular Diagnostic Techniques/methods
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Point-of-Care Testing
Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity

@article {pmid41754535,
year = {2026},
author = {Li, H and Wang, R and Li, J and Duan, W and Liang, Y and Sun, Q and Zhou, J and Zhang, Y},
title = {SHFL Post-Transcriptionally Restricts Coxsackievirus A16 In Vitro and In Vivo.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754535},
issn = {1999-4915},
support = {GJJKJ-2024-ZY//National Disease Control and Prevention Administration Public Health Talent Training Support Project/ ; ZDGWNLJS25-36//National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID)/ ; 2021YFC2302003//National Key Research and Development Program of China/ ; },
mesh = {Animals ; Mice ; Virus Replication ; *Coxsackievirus Infections/virology ; *Enterovirus A, Human/genetics/physiology ; Humans ; *Enterovirus/genetics/physiology ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Hand, Foot and Mouth Disease/virology ; Cell Line, Tumor ; Viral Load ; Interferon-beta/pharmacology ; },
abstract = {Coxsackievirus A16 (CVA16), a major etiological agent of hand, foot, and mouth disease, is increasingly contributing to neurological complications, with no vaccines or virus-specific antivirals currently available. To identify CVA16-restricting host factors, we investigated the role of the interferon-stimulated gene shiftless (SHFL), previously implicated in the control of other RNA viruses. Using CRISPR-Cas 9, we generated SHFL knockout rhabdomyosarcoma cells and assessed viral replication, cytopathic effects, and replication stage dynamics. We evaluated disease progression and tissue injury in neonatal mice infected with a mouse-adapted CVA16 strain. SHFL expression was strongly induced during CVA16 infection and was inducible by exogenous interferon-β treatment, and its loss markedly increased infectious virus production, accelerated early replication, and exerted severe cytopathic effects. In vivo, SHFL deficiency led to rapid weight loss, pronounced neurological signs, increased viral burden across multiple tissues, and uniform mortality, together with high viral loads and extensive pathological damage in the central nervous system, lungs, and skeletal muscle. Transcriptomic analyses revealed SHFL-dependent modulation of adhesion- and mitogen-activated protein kinase-related pathways. Overall, our results suggest SHFL as a key determinant of host resistance to CVA16, acting mainly at the post-transcriptional stage to limit viral spread and tissue injury, and highlight SHFL-linked pathways as promising host-directed antiviral targets.},
}

Animals
Mice
Virus Replication
*Coxsackievirus Infections/virology
*Enterovirus A, Human/genetics/physiology
Humans
*Enterovirus/genetics/physiology
Host-Pathogen Interactions
CRISPR-Cas Systems
Hand, Foot and Mouth Disease/virology
Cell Line, Tumor
Viral Load
Interferon-beta/pharmacology

@article {pmid41753780,
year = {2026},
author = {Palanisamy, V and Bosilevac, JM and Barkhouse, DA and Velez, SE and Dass, SC},
title = {Unraveling the Coevolutionary Dynamics of Phage and Bacterial Protein Warfare Occurring in the Drains of Beef-Processing Plants.},
journal = {Microorganisms},
volume = {14},
number = {2},
pages = {},
pmid = {41753780},
issn = {2076-2607},
support = {2020-67017-30776//USDA-NIFA/ ; },
abstract = {Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic analysis. Metagenomic data collected from 75 drain samples from beef-processing plants were analyzed to investigate phage-bacterial interactions. First, assembled contigs were screened to identify viral sequences, which were then taxonomically annotated to determine the viral composition, including phages. Functional annotation of these viral sequences provided information about the viral genes and their roles in bacterial interactions specifically associated with attack and counterattack of bacteria. In parallel, bacterial contigs were examined to identify genes associated with antiphage defense systems, providing insights into the strategies adapted by bacteria to resist phage infection. Taxonomic annotation of viral sequences from the bulk metagenomic data revealed the presence of phages targeting Pseudomonas, Klebsiella, and Enterococcus. The higher abundance of Pseudomonas phages aligns with our previous study, where Pseudomonas was identified as the dominant bacterial genus, suggesting potential copersistence of phages and their hosts. Functional annotation of phage contigs revealed infective and lysis-related genes, highlighting their potential role in bacterial attack. Conversely, bacterial contigs encoded antiphage defense systems, including CRISPR-Cas, restriction-modification, and other defense-related genes. The study also uncovered the presence of anti-CRISPR proteins in phages, suggesting a counterattack on the bacterial defense. These findings provide evidence for phage attack, bacterial defense, and phage counterattack and may showcase the ongoing coevolutionary arms race between phages and bacteria. While this evidence looks promising, these results remain preliminary and further studies are needed to validate these findings. Still, this study provides a foundational understanding of bacteria-phage coexistence in beef-processing plant drains and paves the way for further explorations of these intricate interactions and their possible applications in controlling pathogenic microorganisms within biofilms.},
}

@article {pmid41752145,
year = {2026},
author = {Fayed, S and Amer, S and Badawy, M and Bou Malhab, L and Omran, N and Khoder, G and Ghemrawi, R and Haider, M and Hamoudi, R and Harati, R},
title = {The Role of CRISPR and Its Therapeutic Applications in Glioblastoma.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41752145},
issn = {1422-0067},
support = {210111350//University of Sharjah/ ; 2201110368//University of Sharjah/ ; 23010902146//University of Sharjah/ ; VRI-20-10//ASPIRE Precision Medicine Research Institute Abu Dhabi/ ; },
mesh = {Humans ; *Glioblastoma/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Brain Neoplasms/therapy/genetics ; Animals ; *Genetic Therapy/methods ; },
abstract = {Glioblastoma multiforme (GBM) remains the most aggressive and treatment-refractory form of primary brain tumor in adults, characterized by rapid proliferation, intratumoral heterogeneity and resistance to current therapies. Despite therapeutic advancements in surgical resection, radiotherapy and chemotherapy, clinical outcomes remain poor, underscoring the need for innovative molecular strategies. This review examines the therapeutic potential of CRISPR/Cas9 genome-editing technologies in GBM, highlighting their ability to model, dissect and potentially correct the genetic alterations that drive GBM tumorigenesis. Key molecular targets, such as EGFR, PTEN, TP53, NF1 and PIK3CA, are discussed within the context of GBM's mutational and signaling landscape. We further outline emerging CRISPR applications in preclinical models, the current status of CRISPR-based clinical trials and the major barriers hindering translation, including off-target effects, immunogenicity and the challenge of delivering gene-editing systems across the blood-brain barrier. Particular emphasis is placed on delivery technologies, viral and non-viral vectors, including lipid nanoparticles, polymeric systems, inorganic nanocarriers and DNA nanostructures, which are rapidly evolving to improve precision, safety and CNS penetrance. Collectively, this review highlights CRISPR/Cas9 as a powerful tool whose integration with molecular neuro-oncology and precision medicine may ultimately shift GBM treatment toward more personalized and durable therapeutic interventions.},
}

Humans
*Glioblastoma/therapy/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Brain Neoplasms/therapy/genetics
Animals
*Genetic Therapy/methods

@article {pmid41751979,
year = {2026},
author = {Eskildsen, J and Dong, M and Hanak, T and Madsen, CK and Holme, I and Plaszkó, T and Vestergård, M and Nicolaisen, M and Thordal-Christensen, H and Brinch-Pedersen, H},
title = {Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751979},
issn = {1422-0067},
support = {NNF19OC0056580//Novo Nordisk Foundation/ ; BarleyMicroBreed, 101060057//EU Horizon research and innovation/ ; },
mesh = {*CRISPR-Cas Systems ; *Hordeum/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Ascomycota/pathogenicity ; *Microbiota/genetics ; Alleles ; Plant Roots/microbiology/genetics ; *Plant Proteins/genetics ; Mutation ; },
abstract = {Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein's extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5-5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein's extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa.},
}

*CRISPR-Cas Systems
*Hordeum/genetics/microbiology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics
*Ascomycota/pathogenicity
*Microbiota/genetics
Alleles
Plant Roots/microbiology/genetics
*Plant Proteins/genetics
Mutation

@article {pmid41751840,
year = {2026},
author = {Begum, SN and Hasan, SK},
title = {Prime Editing Driven Functional Genomics: Bridging Genotype to Phenotype in the Post-Genomic Era.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751840},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genomics/methods ; Phenotype ; Genotype ; Animals ; },
abstract = {The post-genomic era, defined by large-scale sequencing initiatives, has generated an unprecedented catalogue of human genetic variation. Yet, the vast majority of genetic variants remain classified as variants of uncertain significance or are located within poorly characterized non-coding regions, thereby hindering the effective translation of genomic data into meaningful biological understanding and clinical application. Bridging this genotype-to-phenotype gap requires precise, high-throughput functional genomics. Early CRISPR-Cas9 knockout and CRISPR interference/activation (CRISPRi/a) screens mapped gene-level functions but could not assess single nucleotide variants (SNVs). Bridging this genotype-to-phenotype gap demands precise, high-throughput functional genomics. Multiplexed assays of variant effect (MAVEs), like saturation genome editing, systematically test all possible mutations using CRISPR-Cas9 and donor libraries. Base editors allow targeted single-base changes without double-strand breaks but are limited in scope, while prime editing can introduce any small substitution, insertion, or deletion without double-strand breaks (DSBs) or donor templates. This review traces the evolution of functional screens from gene-level knockouts to saturation genomic editing (SGE), and highlights how prime editing is driving a new paradigm for the systematic functional characterization of thousands of variants across disease-relevant genes. We also detail the architecture, mechanism, and progressive optimization of PE systems and their delivery methods. Collectively, prime editing stands as a transformative platform poised to accelerate precision functional genomics and advance the diagnosis and treatment of genetic diseases.},
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems
*Genomics/methods
Phenotype
Genotype
Animals

@article {pmid41751799,
year = {2026},
author = {Cai, X and Liang, X and Zou, P and Xiao, R and Wang, Y},
title = {CRISPRi Screening Identifies Essential E. coli Virulence Factors for Placental Barrier Breach in a Maternal-Fetal Infection Model.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751799},
issn = {1422-0067},
support = {82574169//National Natural Science Foundation of China/ ; 7232009, 7244289//Natural Science Foundation of Beijing Municipality/ ; 20240484724//Cross-cooperation project of Beijing Science and Technology New Star Program/ ; Subject leaders-03-02//High Level Public Health Technical Personnel Construction Project/ ; //Research Foundation of Capital Institute of Pediatrics/ ; //Beijing Chaoyang District Postdoctoral Research Foundation 2024/ ; },
mesh = {Female ; Pregnancy ; Animals ; *Virulence Factors/genetics/metabolism ; *Escherichia coli Infections/microbiology/genetics ; *Escherichia coli/pathogenicity/genetics ; Rats ; *Placenta/microbiology/metabolism ; Humans ; Disease Models, Animal ; Escherichia coli Proteins/genetics/metabolism ; Host-Pathogen Interactions ; Trophoblasts/microbiology/metabolism ; CRISPR-Cas Systems ; *Pregnancy Complications, Infectious/microbiology ; Neonatal Sepsis/microbiology ; },
abstract = {Early-onset neonatal sepsis caused by Escherichia coli (E. coli) threatens neonates' lives due to the pathogen's high virulence and multidrug resistance. The mechanisms that enable its placental barrier breach are poorly understood. Using a clinically isolated ST95 ExPEC strain from a neonatal sepsis case, along with a pregnant rat model and an in vitro placental barrier model, we performed CRISPR interference screening. This screen targeted 264 virulence factor genes and identified virulence factors for motility, iron acquisition, hemolysin secretion, and adherence/invasion as critical. We demonstrated that hlyB is essential for uterine infection, and we elucidated a mechanism for ibeA that facilitates syncytial trophoblast cell layer penetration by interacting with the host receptor(s) PSF/VIM to enhance bacterial internalization. Host cells countered ibeA+ E. coli infection via a novel host defense pathway involving upregulation of ASPHD1. This study systematically mapped the virulence factors required for E. coli placental translocation and delineated key host-pathogen interactions.},
}

Female
Pregnancy
Animals
*Virulence Factors/genetics/metabolism
*Escherichia coli Infections/microbiology/genetics
*Escherichia coli/pathogenicity/genetics
Rats
*Placenta/microbiology/metabolism
Humans
Disease Models, Animal
Escherichia coli Proteins/genetics/metabolism
Host-Pathogen Interactions
Trophoblasts/microbiology/metabolism
CRISPR-Cas Systems
*Pregnancy Complications, Infectious/microbiology
Neonatal Sepsis/microbiology

@article {pmid41751614,
year = {2026},
author = {Sun, Q and Guo, Y and Wang, L and Jia, L and Wei, P and Ma, S},
title = {CRISPR-Mediated Silkworm: The Oncoming Agricultural Revolutions and a Rising Model Organism.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751614},
issn = {2073-4425},
support = {32570591//National Natural Science Foundation of China/ ; },
mesh = {*Bombyx/genetics/growth & development ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Agriculture/methods ; Silk/genetics ; Genome, Insect ; Genomics/methods ; },
abstract = {The silkworm (Bombyx mori) is essential to sericulture and is also becoming a key model organism in genomics and agriculture. For decades, genetic studies of the silkworm were limited by inefficient and inflexible genome tools. CRISPR genome editing allows precise and scalable alterations to genes regulating development, physiology, and industrial traits. This review summarizes silkworm genome-editing breakthroughs, highlighting CRISPR's evolution from simple gene knockouts to large-scale genome-wide screening. We highlight how these advancements contribute to disease resistance, higher yields, and the development of new silk-based materials, as well as how they influence the development and growth rate of the sericulture. The creation of high-quality reference genomes, pangenomes, and genome-wide screening systems has made the silkworm a major model for integrating multiple biological datasets and approaches, such as genomic, transcriptomic, and proteomic. By considering the unique biological characteristics of the silkworm, this provides new insights for research on silk biology, piRNA synthetic biology, and hormonal signaling regulation. Finally, we examine new areas at the intersection of CRISPR, pangenomics, and artificial intelligence (AI) and suggest future paths for molecular breeding, pest control, and synthetic biology. Moreover, AI-assisted prediction of CRISPR outcomes is utilized to inform the design of targeted trait modifications, representing an approach to enhancing biomanufacturing efficiency and eco-friendly silk production. Together, these advances have made the silkworm a flexible genetic platform and an important part of sustainable agriculture and biomanufacturing.},
}

*Bombyx/genetics/growth & development
Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Agriculture/methods
Silk/genetics
Genome, Insect
Genomics/methods

@article {pmid41751560,
year = {2026},
author = {Lee, S and Park, S and Bang, H and Kim, SU and Park, YH and Wee, G and Chae, U and Kim, E},
title = {VPS35 Deficiency Markedly Reduces the Proliferation of HEK293 Cells.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751560},
issn = {2073-4425},
mesh = {Humans ; *Cell Proliferation/genetics ; *Vesicular Transport Proteins/genetics/deficiency/metabolism ; HEK293 Cells ; Apoptosis/genetics ; CRISPR-Cas Systems ; Mitochondrial Dynamics/genetics ; Mitochondria/metabolism/genetics ; Gene Knockout Techniques ; },
abstract = {Background/Objectives: The retromer protein complex is involved in various physiological processes, especially endosomal trafficking, and its dysregulation has been linked to Alzheimer's disease and Parkinson's disease, as well as VPS35 knockout (KO), causing early embryonic lethality. We aimed to investigate the cellular consequences of VPS35 deficiency. Methods: To investigate the effects of VPS35 loss, we used CRISPR/Cas9 to generate VPS35 KO human embryonic kidney 293 (HEK293) cells. We analyzed changes in retromer component expression, cell proliferation, apoptosis, and mitochondrial dynamics using Western blotting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy. Results: VPS35 KO led to a significant reduction in cell proliferation and decreased expression of VPS29 and VPS26, both essential for retromer complex assembly. Consequently, retromer formation was impaired. Compared to control cells, KO cells exhibited elevated levels of cleaved caspase-3, poly(ADP-ribose) polymerase, cytochrome C, and p21, while the expression of Ki-67, CDK4, and cyclin D was reduced. Additionally, VPS35 deletion also promoted mitochondrial fragmentation, associated with increased expression of mitochondrial fission-related proteins. Finally, the rescue experiment using the human VPS35 gene confirmed that the recovery of VPS35 not only led to the recovery of the essential elements constituting the retromer but also the recovery of molecules related to the cell cycle, restoring cell death to a normal level. Conclusions: These findings suggest that VPS35 plays a critical role in cell growth and survival by modulating apoptosis, mitochondrial dynamics, and cell cycle progression.},
}

Humans
*Cell Proliferation/genetics
*Vesicular Transport Proteins/genetics/deficiency/metabolism
HEK293 Cells
Apoptosis/genetics
CRISPR-Cas Systems
Mitochondrial Dynamics/genetics
Mitochondria/metabolism/genetics
Gene Knockout Techniques

@article {pmid41751536,
year = {2026},
author = {Hawkins, V and Rudiger, SR and McLaughlan, CJ and Kelly, JM and Lehnert, K and Jacobsen, JC and Handley, RR and Henare, K and Verma, PJ and Snell, RG},
title = {Foundations of an Ovine Model of Fragile X Syndrome.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751536},
issn = {2073-4425},
support = {3914//Curekids/ ; 20/259//Health Research Council of New Zealand/ ; },
mesh = {Animals ; *Fragile X Syndrome/genetics/pathology ; *Disease Models, Animal ; *Fragile X Messenger Ribonucleoprotein 1/genetics ; Sheep/genetics ; Male ; Female ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Humans ; },
abstract = {BACKGROUND: Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder characterised by intellectual disability, developmental delays, anxiety, and social and behavioural challenges. Currently, no effective treatments exist to address the root cause of FXS. Mouse models are the most widely used for studying molecular pathogenesis and conducting preclinical treatment testing. However, therapeutic interventions that show promise in rodent models have yet to succeed in clinical trials. After evaluating the current models, we have developed an ovine model to address this clinical translation gap. We expect this model to more accurately reflect the human condition in brain size, structure, and neurodevelopmental trajectory. We aim to establish this model as a valuable preclinical platform for testing therapies for FXS.

METHODS: To generate the sheep model, we used CRISPR-Cas9 dual-guide editing to knock out the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene in ovine embryos.

RESULTS: Two founder animals were created, one ram (male) and one ewe (female), both of which carried FMR1 gene knockouts. The ewe carries inactivating mutations on both alleles, with the edits in both animals resulting in no detectable Fragile X Messenger Ribonucleoprotein (FMRP) as expected. Both founders have undergone molecular characterisation and basic health checks, with the female founder showing increased joint flexibility, a characteristic of FXS. The ram has been used for breeding, with the successful transmission of the edited allele to his offspring. Importantly, specific lamb cohorts for postnatal treatment testing can be produced efficiently utilising accelerated breeding methods and preimplantation selection.},
}

Animals
*Fragile X Syndrome/genetics/pathology
*Disease Models, Animal
*Fragile X Messenger Ribonucleoprotein 1/genetics
Sheep/genetics
Male
Female
CRISPR-Cas Systems
Gene Editing
Gene Knockout Techniques
Humans

@article {pmid41750315,
year = {2026},
author = {Schulze, A and Kainz, K and Bauer, MA and Carmona-Gutierrez, D},
title = {Editing Candida: Origins and Advances of CRISPR Tools.},
journal = {Biomolecules},
volume = {16},
number = {2},
pages = {},
pmid = {41750315},
issn = {2218-273X},
support = {10.55776/P37278//FWF Austrian Science Fund/ ; not applicable//University of Graz/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Candida/genetics/pathogenicity ; Humans ; Virulence/genetics ; Genome, Fungal ; Candidiasis/microbiology/genetics ; },
abstract = {Pathogens causing candidiasis encompass a diverse group of ascomycetous yeasts that have become essential models for studying fungal adaptability, pathogenicity, and host-pathogen interactions. Although many candidiasis-promoting species exist as commensals within host microbiota, several have acquired virulence traits that enable opportunistic infections, positioning them as a leading cause of invasive fungal disease in humans. Deciphering the molecular and genetic determinants that underpin the biology of organisms responsible for candidiasis has long been a central objective in medical and molecular mycology. However, research progress has been constrained by intrinsic biological challenges, including noncanonical codon usage and the absence of a complete sexual cycle in diploid species, which have complicated traditional genetic manipulation. CRISPR-Cas9 genome editing has overcome many of these limitations, providing a precise, efficient, and versatile framework for targeted genomic modification. This system has facilitated functional genomic studies ranging from single-gene deletions to high-throughput mutagenesis, yielding new insights into the mechanisms governing virulence, antifungal resistance, and stress adaptation. Since its initial application in Candida albicans, CRISPR-Cas9 technology has been refined and adapted for other clinically and industrially relevant species, including Nakaseomyces glabratus (formerly referred to as Candida glabrata), Candida parapsilosis, and Candida auris. The present work provides an overview of the evolution of genetic approaches employed in research directed against candidiasis-associated species, with a particular focus on the development and optimization of CRISPR-based systems. It highlights how recent advancements have improved the genetic tractability of these pathogens and outlines emerging opportunities for both fundamental and applied studies in fungal biology.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Candida/genetics/pathogenicity
Humans
Virulence/genetics
Genome, Fungal
Candidiasis/microbiology/genetics

@article {pmid41748831,
year = {2026},
author = {Moreno, DS and Carvalho, JP and Murray, E and Colombo, NSR and Lamas, A and Cobas, AC and Hill, C and Azeredo, J and Domingues, L},
title = {Evaluation of the delivery of an anti-Listeria endolysin via CRISPR-Cas9 engineered probiotic Saccharomyces boulardii.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41748831},
issn = {1432-0614},
mesh = {*Listeria monocytogenes/drug effects ; *Probiotics ; *CRISPR-Cas Systems ; *Endopeptidases/genetics/pharmacology/metabolism ; *Saccharomyces boulardii/genetics/metabolism ; Humans ; Bacteriophages/enzymology ; *Anti-Bacterial Agents/pharmacology/metabolism ; Saccharomyces cerevisiae/genetics ; },
abstract = {Listeriosis is a foodborne infection caused by Listeria monocytogenes that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, Listeria is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of L. monocytogenes, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. Saccharomyces boulardii is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered Saccharomyces cerevisiae through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against L. monocytogenes, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast S. boulardii and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce L. monocytogenes levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for Listeria. Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic S. boulardii. KEY POINTS: CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared, both allowing the expression and activity of endolysin Ply511 against L. monocytogenes. Endolysin Ply511 retained its activity against L. monocytogenes in simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI). Using fecal samples from human donors, the anti-Listeria effect was reduced potentially due to the lower metabolic activity of S. boulardii and the higher competition with the intestinal microbiome.},
}

*Listeria monocytogenes/drug effects
*Probiotics
*CRISPR-Cas Systems
*Endopeptidases/genetics/pharmacology/metabolism
*Saccharomyces boulardii/genetics/metabolism
Humans
Bacteriophages/enzymology
*Anti-Bacterial Agents/pharmacology/metabolism
Saccharomyces cerevisiae/genetics

@article {pmid41719925,
year = {2026},
author = {Park, YJ and Song, DY and Jeon, HB and Kim, DM},
title = {Nucleic acid detection via protein readout through Cas-controlled gating of cell-free protein synthesis.},
journal = {Biosensors & bioelectronics},
volume = {301},
number = {},
pages = {118514},
doi = {10.1016/j.bios.2026.118514},
pmid = {41719925},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Cell-Free System ; *Protein Biosynthesis/genetics ; *Escherichia coli O157/genetics ; Bacillus anthracis/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Ribosomal, 16S/genetics/analysis ; *Nucleic Acids/analysis/genetics ; Bacterial Proteins/genetics ; },
abstract = {We present a modular platform that converts CRISPR target recognition into programmable protein outputs for nucleic acid detection. This system couples Cas-mediated collateral cleavage with cell-free protein synthesis. In the presence of a target, Cas-mediated collateral cleavage releases an extension-competent trigger DNA that gates reporter expression. Although collateral cleavage is inherently indiscriminate, we achieve deterministic fragment generation by employing chemically programmed precursors bearing backbone modifications-exemplified here by phosphorothioate linkages-that constrain cleavage to predefined sites. Using Bacillus anthracis and E. coli O157:H7 as a model, the developed CRIVER assay enables concurrent readouts of 16S rRNA together with the species-specific capB or ecf1 loci by integrating Cas13a-mediated RNA recognition and Cas12a-mediated DNA recognition into a dual-channel workflow. Taken together, proposed method establishes addressable signaling at the protein layer, supports protein-based outputs in a modular workflow, and provides a general route to sensitive, dual-channel nucleic acid detection.},
}

*Biosensing Techniques/methods
Cell-Free System
*Protein Biosynthesis/genetics
*Escherichia coli O157/genetics
Bacillus anthracis/genetics
*CRISPR-Cas Systems/genetics
*RNA, Ribosomal, 16S/genetics/analysis
*Nucleic Acids/analysis/genetics
Bacterial Proteins/genetics

@article {pmid41700332,
year = {2026},
author = {Huang, Y and Zhao, Z and Li, J and Wang, X and Qu, H and Zheng, Y},
title = {An aptamer-CRISPR/Cas12a biosensor for rapid and sensitive detection of florfenicol.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {9},
pages = {1910-1916},
doi = {10.1039/d5ay01903f},
pmid = {41700332},
issn = {1759-9679},
mesh = {*Biosensing Techniques/methods ; *Thiamphenicol/analogs & derivatives/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/analysis ; Limit of Detection ; Animals ; Food Contamination/analysis ; },
abstract = {Florfenicol (FF), a broad-spectrum antibacterial agent widely used in livestock and poultry farming, has raised significant food safety concerns due to the accumulation of its residues in animal-derived products (e.g., eggs), posing potential threats to human health. Herein, we developed a novel aptamer-CRISPR/Cas12a biosensor for the rapid and sensitive detection of FF. The biosensor employs streptavidin-modified magnetic beads (SA-MBs) as a solid carrier to achieve efficient enrichment of FF-specific aptamers (APT), while integrating the dual advantages of the APT's high-specificity target recognition and the CRISPR/Cas12a system's powerful signal amplification capability. The detection mechanism is based on a competitive displacement: APT pre-hybridizes with its complementary strand (APT-c) to form stable duplexes. The presence of FF triggers the release of APT-c from APT, and the liberated APT-c then activates the trans-cleavage activity of the CRISPR/Cas12a system. This process converts the small-molecule FF into a CRISPR/Cas12a-detectable nucleic acid signal and enables quantitative FF detection. Under optimized conditions, the biosensor demonstrated a linear detection range of 10 nM to 100 µM for FF (R[2] = 0.9907) and a limit of detection (LOD) of 1.41 nM. The accuracy and practicality were confirmed through spiked recovery experiments in egg samples, yielding recoveries between 97.1% and 100.8%. Furthermore, the modular design of this platform allows its easy adaptation for detecting other antibiotics simply by replacing the specific APT and its corresponding APT-c, highlighting its considerable potential for broad applications in food safety monitoring.},
}

*Biosensing Techniques/methods
*Thiamphenicol/analogs & derivatives/analysis
*Aptamers, Nucleotide/chemistry/genetics
*CRISPR-Cas Systems
*Anti-Bacterial Agents/analysis
Limit of Detection
Animals
Food Contamination/analysis

@article {pmid41693124,
year = {2026},
author = {Xin, W and Tang, Z and Wang, S and Song, ZL and Luo, X},
title = {Scaffold-Proximal DNA Extensions Enhance Cas12a Trans-cleavage for Direct and Broad-Scope Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {8},
pages = {6149-6162},
doi = {10.1021/acs.analchem.5c07125},
pmid = {41693124},
issn = {1520-6882},
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; *MicroRNAs/analysis/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; Polymorphism, Single Nucleotide ; *RNA, Messenger/analysis ; *Bacterial Proteins/metabolism ; },
abstract = {The CRISPR/Cas12a system has revolutionized nucleic acid diagnostics, but its direct application for RNA detection remains constrained by the requisite reverse transcription step, insufficient sensitivity, and poor compatibility across diverse RNA targets such as microRNAs (miRNAs), long mRNAs, and single nucleotide polymorphisms (SNPs). Here, we introduce a rationally engineered DNA activator architecture that markedly enhances Cas12a trans-cleavage activity and expands its analytical utility. A key mechanistic finding is the strong positional dependence of activator extensions: appending an overhang specifically at the scaffold-proximal end of the DNA activator (termed Proximal-Extended Activator, PEA) potently boosts Cas12a activation through ribonucleoprotein (RNP) stabilization, whereas distal extensions are inhibitory due to steric hindrance of essential interdomain motions. This optimized PEA system facilitates direct, amplification-free RNA detection, achieving exceptional sensitivity with detection limits of 1.3 fM for miRNA and 93 fM for mRNA, all without reverse transcription. Furthermore, a Split-PEA format confers exceptional discriminatory power for SNPs, enabling robust identification of the EGFR T790 M mutation at a 0.1% allelic frequency. This work establishes a facile and versatile platform where simple sequence modification enables highly sensitive and specific analysis of a broad range of nucleic acid targets, effectively overcoming a significant hurdle in CRISPR-based diagnostics.},
}

*CRISPR-Associated Proteins/metabolism/chemistry
CRISPR-Cas Systems
*DNA/chemistry/metabolism/genetics
*MicroRNAs/analysis/genetics
Humans
*Endodeoxyribonucleases/metabolism/genetics
Polymorphism, Single Nucleotide
*RNA, Messenger/analysis
*Bacterial Proteins/metabolism