@article {pmid40506596,
year = {2025},
author = {Sajjad, MW and Muzamil, F and Naqvi, RZ and Amin, I},
title = {QBEmax redefines the precise base editing in crop plants.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {127},
pmid = {40506596},
issn = {1438-7948},
mesh = {*Crops, Agricultural/genetics ; *CRISPR-Cas Systems ; Genome, Plant ; Cytidine Deaminase/genetics/chemistry/metabolism ; *Gene Editing ; },
abstract = {Hu et al.‘s new study, published in Nature Biotechnology, introduces QBEmax; a tiny, conformationally sound editing tool with a cytidine deaminase buried within a looping permuted Cas9 (cpCas9). Supported by molecular dynamics models and AlphaFold3 structural predictions, this unique internal fusion creates a structurally protected complex that improves editing accuracy and lowers typical artifacts such as indels and impure base conversions (Hu et al. Nature Biotechnology:1-7, 2025). High precision editing (up to 99.8% purity), lower indels, and lower off target impacts well suit imminent plant transformation events. Its tiny, stable architecture and wider editing window at PAM sites increase its ability for precise and adaptable trait change in complex plant genomes.},
}

*Crops, Agricultural/genetics
*CRISPR-Cas Systems
Genome, Plant
Cytidine Deaminase/genetics/chemistry/metabolism
*Gene Editing

@article {pmid40999513,
year = {2025},
author = {Lee, S and Yu, Y and Kim, DH and Bock, M and Kim, Y and An, SB and Choi, H and Shin, HE and Hwang, DY and Han, I},
title = {Enhanced disc regeneration through CRISPR/Cas9-mediated SOX9 and TGFβ1 coexpression in tonsil-derived mesenchymal stromal cells.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {501},
pmid = {40999513},
issn = {1757-6512},
support = {RS-2024-00347403//National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT: Ministry of Science and ICT)/ ; RS-2023-KH141187//Korean Cell-Based Artificial Blood Project funded by the Korean government (The Minis-try of Science and ICT, The Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety)/ ; },
mesh = {*SOX9 Transcription Factor/genetics/metabolism ; *Transforming Growth Factor beta1/genetics/metabolism ; *Mesenchymal Stem Cells/metabolism/cytology ; Animals ; Rats ; Humans ; *Intervertebral Disc Degeneration/therapy/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Regeneration ; Rats, Sprague-Dawley ; *Palatine Tonsil/cytology/metabolism ; *Intervertebral Disc/physiology/metabolism ; Male ; Cell Differentiation ; Chondrogenesis ; },
abstract = {BACKGROUND: Intervertebral disc (IVD) degeneration, a primary cause of chronic low back pain, currently lacks treatments that target its underlying pathological mechanisms. Tonsil-derived mesenchymal stromal cells (ToMSCs) have shown promise for IVD regeneration; however, their therapeutic potential is limited by the harsh microenvironment of degenerated discs. This study investigated whether ToMSCs engineered to co‐overexpress SOX9 and TGFβ1 using a tetracycline‐off (Tet‐off) regulatory system could enhance extracellular matrix (ECM) restoration and reduce inflammation in degenerative IVDs. METHODS: We used CRISPR/Cas9 technology to generate ToMSCs that express SOX9, TGFβ1, or both factors under Tet-off regulation. Gene expression was confirmed by Western blot and qRT-PCR analyses. In vitro studies assessed chondrogenic differentiation capacity, while in vivo assessments were performed using a rat tail needle puncture model of IVD degeneration. After administering the CRISPR-engineered ToMSCs, we monitored mechanical allodynia with the von Frey test over six weeks. Therapeutic outcomes were evaluated through T2‐weighted MRI and histological analysis. RESULTS: In vitro experiments showed that ToMSCs co-expressing SOX9 and TGFβ1 exhibited superior chondrogenic differentiation compared to cells expressing a single factor. In vivo studies demonstrated that dual-factor expressing ToMSCs significantly improved disc hydration (as confirmed by MRI), enhanced ECM synthesis—particularly aggrecan and type II collagen—and reduced inflammation compared to single-factor treatments. These improvements were accompanied by reduced mechanical allodynia, indicating functional recovery. CONCLUSION: Our study demonstrates that ToMSCs engineered to co-express SOX9 and TGFβ1 effectively promote IVD regeneration by enhancing ECM production and reducing inflammation. This dual-factor approach represents a promising therapeutic strategy for treating degenerative disc disease and warrants further investigation for clinical application.},
}

*SOX9 Transcription Factor/genetics/metabolism
*Transforming Growth Factor beta1/genetics/metabolism
*Mesenchymal Stem Cells/metabolism/cytology
Animals
Rats
Humans
*Intervertebral Disc Degeneration/therapy/genetics/pathology/metabolism
*CRISPR-Cas Systems/genetics
*Regeneration
Rats, Sprague-Dawley
*Palatine Tonsil/cytology/metabolism
*Intervertebral Disc/physiology/metabolism
Male
Cell Differentiation
Chondrogenesis

@article {pmid41003970,
year = {2026},
author = {Kanafi, MM and Moazzami, R},
title = {Leveraging CRISPR/Cas9 To Overcome Hypoxic Barriers in Regenerative Dentistry.},
journal = {Stem cell reviews and reports},
volume = {22},
number = {1},
pages = {82-86},
pmid = {41003970},
issn = {2629-3277},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Dental Pulp/cytology/metabolism ; *Stem Cells/metabolism/cytology ; Animals ; *Dentistry/methods ; Cell Hypoxia ; *Regeneration ; *Regenerative Medicine/methods ; Oxidative Stress ; Gene Editing/methods ; },
abstract = {Dental pulp stem cells (DPSCs) have gained increasing attention as a valuable cell source for regenerative dentistry owing to their accessibility, high proliferative potential, and capacity for multilineage differentiation. Despite these advantages, their therapeutic efficacy is substantially compromised by pathological hypoxia, a common feature of injured or poorly vascularized oral tissues. Hypoxic stress not only impairs DPSC survival but also diminishes their regenerative capacity, creating a major barrier to effective clinical translation. Addressing this limitation is therefore essential to harness the full therapeutic potential of DPSCs. Recent advances in genome-editing technologies, particularly the CRISPR/Cas9 system, have created novel opportunities to enhance the resilience of DPSCs against hypoxic stress. By enabling precise genetic modifications, CRISPR offers a powerful platform to reprogram cellular pathways associated with oxygen deprivation, oxidative stress, and apoptosis. Current preclinical investigations have focused on key targets such as HIF1α, PHD2, NRF2, BAX, and VEGF, exploring their modulation through CRISPR-mediated activation, inhibition, or knockout strategies. Upregulation of HIF1α and VEGF has demonstrated the ability to enhance angiogenesis and promote cell survival in oxygen-deficient microenvironments. Similarly, activation of NRF2 improves antioxidant defense mechanisms and mitigates oxidative damage, while suppression of pro-apoptotic genes such as BAX increases overall viability. Collectively, these strategies represent a multifaceted approach to strengthening DPSC performance in adverse conditions. The integration of CRISPR/Cas9 into regenerative dentistry represents a paradigm shift in addressing hypoxia-induced barriers to stem cell therapy. While early findings are promising, several critical challenges remain, including the potential for off-target effects, the need for stable and long-term genetic modifications, and concerns regarding biosafety and ethical considerations. Robust preclinical validation and carefully designed translational studies will be required before CRISPR-engineered DPSCs can be considered for clinical application. In summary, CRISPR/Cas9-based modulation of hypoxia-responsive pathways offers a transformative strategy to enhance the therapeutic efficacy of DPSCs. By improving survival, stress tolerance, and angiogenic potential under hypoxic conditions, this approach may significantly expand the clinical applicability of stem cell–based interventions in dentistry. Continued research is essential to ensure the safety, reliability, and long-term benefits of this promising therapeutic avenue.},
}

Humans
*CRISPR-Cas Systems/genetics
*Dental Pulp/cytology/metabolism
*Stem Cells/metabolism/cytology
Animals
*Dentistry/methods
Cell Hypoxia
*Regeneration
*Regenerative Medicine/methods
Oxidative Stress
Gene Editing/methods

@article {pmid41171582,
year = {2026},
author = {Feger, M and Tsapara, A and Hülße, S and Rausch, S and Barholz, M and Stournaras, C and Föller, M},
title = {Chorein Regulates Key Osteoblast Genes in UMR-106 Cells.},
journal = {Cell biochemistry and biophysics},
volume = {84},
number = {1},
pages = {1245-1252},
pmid = {41171582},
issn = {1559-0283},
mesh = {*Osteoblasts/metabolism/cytology ; Humans ; *Vesicular Transport Proteins/genetics/metabolism/antagonists & inhibitors ; Cell Line ; Fibroblast Growth Factor-23 ; Fibroblast Growth Factors/metabolism/genetics ; PHEX Phosphate Regulating Neutral Endopeptidase/metabolism/genetics ; Cell Differentiation ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; Animals ; *Gene Expression Regulation ; },
abstract = {Chorein is an endoplasmic reticulum protein expressed in many cell types. Loss-of-function mutations of the gene encoding chorein (VPS13A) are the cause of chorea-acanthocytosis, a rare and severe neurodegenerative disease with chorea-like movements, loss of mental function, progressive muscle weakness and misshaped erythrocytes (acanthocytes). Chorein regulates diverse cellular functions including the cytoskeleton, apoptosis, Ca2+ entry, or autophagy. Since its role in bone is enigmatic, we aimed to explore the function of chorein in osteoblasts. To this end, we generated UMR-106 osteoblast-like cells with stable chorein knockdown (KD) using a CRISPR/Cas9-based approach and compared them to cells undergoing CRISPR/Cas9 with a non-targeting sequence (NT). Gene expression was assessed by qPCR and protein by Western blotting and ELISA. Gene and protein expression of chorein and fibroblast growth factor 23 (FGF23), an osteoblast-derived hormonal regulator of phosphate metabolism, were decreased in KD compared to NT cells. Moreover, FGF23 regulator Phex was down- and Galnt3 was up-regulated in KD compared to NT cells. The expression of further genes regulating osteoblast and osteoclast differentiation was affected by chorein knockdown. Taken together, chorein is expressed in UMR-106 osteoblasts and modulates the expression of various genes affecting osteoblast and osteoclast differentiation and function.},
}

*Osteoblasts/metabolism/cytology
Humans
*Vesicular Transport Proteins/genetics/metabolism/antagonists & inhibitors
Cell Line
Fibroblast Growth Factor-23
Fibroblast Growth Factors/metabolism/genetics
PHEX Phosphate Regulating Neutral Endopeptidase/metabolism/genetics
Cell Differentiation
CRISPR-Cas Systems
Gene Knockdown Techniques
Animals
*Gene Expression Regulation

@article {pmid41250247,
year = {2025},
author = {Qian, Y and Hui, F and Niu, W and Wang, D and Hao, Y and Meng, Q and Ren, S and Kong, D and Gong, H and Wu, J and Chen, K and Alariqi, M and Gao, J and Li, Z and Jin, S},
title = {Double-stranded DNA deaminase DddA[E1347A] can increase the efficiency and targeting range of cytidine base editors.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {391},
pmid = {41250247},
issn = {1474-760X},
support = {2024M761134//The China Postdoctoral Fund/ ; KY2020YC0002//The China Tobacco Hunan Industrial Co., Ltd. Research Project/ ; 2023YFF1000204//The National Key R&D Program of China/ ; 32272128//The National Science Foundation of China/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; },
mesh = {*Cytidine/metabolism/genetics ; *Gene Editing/methods ; *Cytidine Deaminase/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; *Cytosine Deaminase/genetics/metabolism ; DNA ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {BACKGROUND: Cytidine base editors (CBEs) consist of a single-strand specific cytidine deaminase fused to Cas9 nickase, enabling efficient C-to-T conversion across diverse organisms. Enhancing editing range and efficiency of these tools is essential for expanding their applications. RESULTS: In this study, we report that fusing a double-stranded DNA-specific cytosine deaminase DddAE1347A to CBEs significantly improves editing activity and broadens the editing window in cell lines, embryos, tobacco, and cotton. Compared to BE4max, the optimized DddAE1347A-BE4max exhibits up to a 93- fold increase in editing efficiency, achieving up to 52% efficiency at C14 and C15 in cell lines. Further investigation reveals that DddAE1347A is compatible with various Cas9 variants (SpCas9, SpaCas9, and Nme2Cas9) and deaminase variants (rA1, A3G, and A3A). Additionally, we demonstrate that cytosine deaminases with single-stranded DNA activity fail to enhance the CBE system. In contrast, various DddA variants can improve CBE editing activity at PAM-proximal cytosine positions, highlighting the modularity of fusion between DddAs and CBEs. CONCLUSIONS: These findings suggest that the double-stranded DNA-specific cytosine deaminase protein can act as an engineered fusion module in the CBE system, altering the performance (window/efficiency) of CBEs.},
}

*Cytidine/metabolism/genetics
*Gene Editing/methods
*Cytidine Deaminase/genetics/metabolism
Humans
CRISPR-Cas Systems
*Cytosine Deaminase/genetics/metabolism
DNA
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid41381576,
year = {2025},
author = {Palavesam, A and Karthik Raj, BN and Madan, N and Sri, SSL and Babitha, R and Tirumurugaan, KG},
title = {CRISPR-Cas12a-based rapid detection of Babesia gibsoni and Ehrlichia canis in dogs using fluorometer platform.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {402},
pmid = {41381576},
issn = {2045-2322},
mesh = {Animals ; Dogs ; *Ehrlichia canis/genetics/isolation & purification ; *Babesia/genetics/isolation & purification ; *Dog Diseases/diagnosis/microbiology/parasitology ; *Ehrlichiosis/diagnosis/veterinary/microbiology ; *Babesiosis/diagnosis/parasitology ; *CRISPR-Cas Systems/genetics ; *Fluorometry/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; },
abstract = {Canine babesiosis and canine monocytic ehrlichiosis are important tick-borne diseases caused by Babesia gibsoni (B. gibsoni) and Ehrlichia canis (E. canis) in dogs. Early diagnosis is important for effective clinical management, as these infections can result in other complications if untreated. This study aimed to develop a CRISPR-Cas12a-based detection assay for B. gibsoni and E. canis in a fluorometer platform. Custom designed guide RNAs (gRNAs) were synthesised with the spacer sequence targeting the B. gibsoni18S rRNA and E. canis p43 genes, located 20 bp downstream of the PAM site (5’-TTTV- 3’). Following PCR amplification of the short fragments encompassing the above target regions, the specific gRNA binding the LbaCas12a-gRNA complex initiated the collateral cleavage of FAM- labelled AT rich ssDNA probe for detection using a fluorometer and Biotin-digoxigenin (DIG) labelled GT-rich ssDNA probe for visual detection in LFA.The limit of detection (LOD) using the fluorometer based detection platform was 6 × 108and 6 × 109 for B. gibsoni and E. canis respectively. The LOD was comparable to that of real-time PCR, but more sensitive than point-of-care methods such as LFA, indicating its potential applicability in veterinary clinical settings.},
}

Animals
Dogs
*Ehrlichia canis/genetics/isolation & purification
*Babesia/genetics/isolation & purification
*Dog Diseases/diagnosis/microbiology/parasitology
*Ehrlichiosis/diagnosis/veterinary/microbiology
*Babesiosis/diagnosis/parasitology
*CRISPR-Cas Systems/genetics
*Fluorometry/methods
Rapid Diagnostic Tests
Sensitivity and Specificity

@article {pmid41437324,
year = {2025},
author = {Jun, Y and Han, J and Kim, Y and Choi, S and Kim, SL and Oh, JH and Suh, EJ and Kang, SH and Park, HR and Jeong, HC and Park, SR and Mo, Y},
title = {Phenotypic and transcriptomic characterization of OsSWEET14-edited rice (cv. Samkwang) with enhanced bacterial blight resistance.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1771},
pmid = {41437324},
issn = {1471-2229},
support = {RS-2022-RD010034//Rural Development Administration/ ; RS-2024-00322166//Rural Development Administration/ ; },
mesh = {*Oryza/genetics/microbiology/immunology ; *Xanthomonas/physiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; Phenotype ; *Transcriptome ; Plants, Genetically Modified ; *Plant Proteins/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; },
abstract = {BACKGROUND: Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) poses a serious threat to rice production. The pathogen promotes infection by targeting effector binding elements in the promoter regions of rice susceptibility genes such as SWEET (Sugars Will Eventually be Exported Transporters) genes. Previous studies have shown that natural or induced variations in SWEET genes can effectively enhance BB resistance in rice. However, the effects of variations in SWEET genes on disease resistance and agronomic performance vary depending on the Xoo strains and the genetic background of rice cultivars, highlighting the need for precise evaluations in breeding applications. RESULTS: In this study, we developed a CRISPR/Cas9-edited OsSWEET14 knockout line (SK-s14) in the background of the elite Korean rice cultivar Samkwang. The SK-s14 line exhibited enhanced resistance against the Xoo strain KACC10859. Transcriptomic analysis revealed that defense-related genes, including pathogenesis-related genes and salicylic acid signaling genes, were downregulated in SK-s14 relative to Samkwang under Xoo inoculation. These results suggest that the enhanced resistance may result from pathogen starvation due to restricted sugar efflux to apoplast, rather than from classical defense activation. Field evaluations showed that SK-s14 plants headed 1–7 days earlier, had longer panicles (+ 8.0%), and exhibited modest reductions in grain weight (-3.0%) and grain fertility (-4.4%) compared to Samkwang. CONCLUSIONS: Our results show that knocking out OsSWEET14 in the Samkwang background enhances BB resistance against the KACC10859 strain, likely through a pathogen starvation mechanism. Despite a modest grain yield reduction (-7.2%) observed under field conditions, SK-s14 represents a promising breeding material for disease-prone rice growing regions.},
}

*Oryza/genetics/microbiology/immunology
*Xanthomonas/physiology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics/immunology
Phenotype
*Transcriptome
Plants, Genetically Modified
*Plant Proteins/genetics/metabolism
Gene Editing
CRISPR-Cas Systems
Gene Expression Profiling
Gene Expression Regulation, Plant

@article {pmid41513714,
year = {2026},
author = {Antunes, M and Moura, F and Sebastian, IR and Alves, P and Gomes-Alves, P and Escandell, JM},
title = {Streamlined rAAV HeLaS3 producer cell line generation via GS selection.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {4658},
pmid = {41513714},
issn = {2045-2322},
support = {UID/04462: iNOVA4Health//Fundação para a Ciência e a Tecnologia/ ; 10.55776/W1224//Austrian Science Fund/ ; },
mesh = {*Dependovirus/genetics ; *Glutamate-Ammonia Ligase/genetics/metabolism ; *Genetic Vectors/genetics ; Humans ; Animals ; CRISPR-Cas Systems ; HeLa Cells ; Cricetulus ; CHO Cells ; Gene Knockout Techniques ; },
abstract = {The high cost and complexity of manufacturing recombinant adeno-associated virus vectors continue to limit the broader application of gene therapies, which offer life-changing potential for individuals affected by genetic diseases. Although stable producer cell lines represent a scalable and cost-effective alternative to transient transfection methods, their development is often delayed by inefficient selection strategies and extended timelines. In this study, we present a novel application of the glutamine synthetase-based selection system - commonly used in CHO cells - to a HeLaS3-based rAAV production platform. By generating glutamine synthetase-knockout HeLaS3 cells via CRISPR-Cas9 and applying glutamine deprivation under serum-free conditions, we significantly streamlined the PCL generation process, reducing the timeline to approximately two months while maintaining rAAV productivity (>1x1011 vg/mL) and product quality (~70% full capsids). This work establishes a robust and scalable workflow for rAAV manufacturing, with the potential to enhance accessibility and reduce viral vector production costs for applications in gene therapy.},
}

*Dependovirus/genetics
*Glutamate-Ammonia Ligase/genetics/metabolism
*Genetic Vectors/genetics
Humans
Animals
CRISPR-Cas Systems
HeLa Cells
Cricetulus
CHO Cells
Gene Knockout Techniques

@article {pmid41746183,
year = {2026},
author = {Liu, S and Ding, Z and Lu, X and Liu, Z and Ma, W and Xu, H and Zhang, H and Dai, X and Shen, M and Huang, Y and Gao, M and Bao, J and Chen, M},
title = {Deoxyribonucleic Acid Activator-Triggered Entropy-Driven Catalysis-Modulated CRISPR/Cas12a-Based Portable Biosensor for Simultaneous Detection of Multiple Pathogenic Bacteria.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4311-4324},
doi = {10.1021/acssensors.5c03012},
pmid = {41746183},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems/genetics ; Pseudomonas aeruginosa/isolation & purification ; Bacterial Proteins/genetics ; beta-Lactamases/genetics/analysis/metabolism ; *Methicillin-Resistant Staphylococcus aureus/isolation & purification ; Catalysis ; *Klebsiella pneumoniae/isolation & purification ; Lab-On-A-Chip Devices ; *DNA/chemistry ; Rapid Diagnostic Tests ; Limit of Detection ; },
abstract = {Rapid and sensitive detection of antibiotic-resistant bacteria (ARB) remains a critical challenge in clinical and public health settings. This study describes the successful construction of a portable DNA activator-triggered entropy-driven catalysis-modulated CRISPR/Cas12a-based sensor (PSDA) for the ultrasensitive and rapid detection of multiple ARB. This PSDA platform utilizes a CRISPR/Cas12a-mediated signal transduction strategy, in which a target-specific DNA activator initiates an entropy-driven dynamic DNA network for signal amplification. To further enhance detection performance, a 3D-printed microfluidic chip device with a smartphone-based readout system has been integrated into the sensor, using green-emitting Zn2GeO4:Mn persistent luminescent nanoparticles as a novel molecular beacon for fluorescence enhancement. This platform enables the simultaneous detection of methicillin-resistant Staphylococcus aureus, carbapenem-resistant Pseudomonas aeruginosa, and Klebsiella pneumoniae carbapenemase 2 (KPC-2)-expressing Klebsiella pneumoniae (KPC-2 KP) with a broad dynamic range (1-10[7] CFU/mL), an ultralow detection limit (1 CFU/mL), and rapid analysis (∼45 min). The assay results are also highly consistent with those of conventional plate counting methods (95.48-115.15%). Overall, this study presents a cost-effective, rapid-response biosensing platform for the simultaneous detection of multiple ARB, with direct applications in clinical diagnostics, food safety monitoring, and environmental surveillance.},
}

*Biosensing Techniques/methods/instrumentation
*CRISPR-Cas Systems/genetics
Pseudomonas aeruginosa/isolation & purification
Bacterial Proteins/genetics
beta-Lactamases/genetics/analysis/metabolism
*Methicillin-Resistant Staphylococcus aureus/isolation & purification
Catalysis
*Klebsiella pneumoniae/isolation & purification
Lab-On-A-Chip Devices
*DNA/chemistry
Rapid Diagnostic Tests
Limit of Detection

@article {pmid42136104,
year = {2026},
author = {Purman, C and Lu, C and Modi, A and Vijaykumar, V and Flister, MJ and den Hollander, AI and Kadri, S and Stender, JD},
title = {Optimization of Genome-Wide CRISPR Screens Using Dual-Guide RNA Infection with Cas9 Electroporation (DICE).},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {141-150},
doi = {10.1177/25731599261448141},
pmid = {42136104},
issn = {2573-1602},
mesh = {Humans ; *Electroporation/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lentivirus/genetics ; *Gene Editing/methods ; HEK293 Cells ; THP-1 Cells ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Single-guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE) enables CRISPR screening in primary cell types that require transient Cas9 expression, yet is limited by scalability and robustness. Here, we introduce dual guide RNA infection with Cas9 electroporation (DICE), which expresses two guides from the same lentiviral construct that target the same gene. In genome-wide screens, DICE outperformed SLICE in defining essential genes and modulators of PD-L1 expression in Interferon-gamma-activated THP1 cells. Collectively, these data demonstrate that DICE can be utilized for reduced-scale CRISPR screens in cell types with transient Cas9 protein expression without sacrificing screening quality.},
}

Humans
*Electroporation/methods
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Lentivirus/genetics
*Gene Editing/methods
HEK293 Cells
THP-1 Cells
*CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid42160542,
year = {2026},
author = {Liu, S and Hu, Y and Zheng, M and Zhang, F and Yu, Z and Sun, Q and Tang, D and Weng, Z and Ye, Z},
title = {A CRISPR/Cas12a-MXene Nanozyme Platform for Universal Detection of Trace DNA.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4345-4356},
doi = {10.1021/acssensors.5c03702},
pmid = {42160542},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; Colorimetry/methods ; *Biosensing Techniques/methods ; Proto-Oncogene Mas ; Methicillin-Resistant Staphylococcus aureus/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; Limit of Detection ; Bacterial Proteins/genetics ; *DNA, Bacterial/analysis/genetics ; *Endodeoxyribonucleases/genetics/chemistry ; *DNA/analysis ; Nitrites ; Transition Elements ; },
abstract = {The instrument-free detection of specific DNA sequences is critical for point-of-care diagnostics yet remains challenging. To address this, we developed a universal colorimetric biosensing platform integrating the CRISPR/Cas12a system with MXene nanozymes. Target recognition by the Cas12a/crRNA complex triggers trans-cleavage of single-stranded DNA inhibitors, restoring the peroxidase-like activity of MXenes to produce a visible signal. The platform achieved a detection limit of 132 copies/μL for the methicillin-resistant Staphylococcus aureus mecA gene. By incorporating an exponential circular DNA (CirDNA) amplification strategy and rationally designed crRNAs for single-base discrimination, sensitivity was further enhanced, enabling detection of the B-raf proto-oncogene V600E mutation down to 109 aM with 0.5% variant allele frequency. The platform also showed excellent reproducibility and high recovery rates in simulated clinical samples. This work provides a low-cost, label/instrument-free, and highly sensitive universal approach through the synergistic combination of CRISPR/Cas12a programmability, MXene nanozyme activity, and CirDNA amplification.},
}

*CRISPR-Cas Systems/genetics
Colorimetry/methods
*Biosensing Techniques/methods
Proto-Oncogene Mas
Methicillin-Resistant Staphylococcus aureus/genetics
Humans
*CRISPR-Associated Proteins/genetics/chemistry
Limit of Detection
Bacterial Proteins/genetics
*DNA, Bacterial/analysis/genetics
*Endodeoxyribonucleases/genetics/chemistry
*DNA/analysis
Nitrites
Transition Elements

@article {pmid42163774,
year = {2026},
author = {Urbaitis, T and Trinkuniene, L and Lenkaite, I and Petrauskyte, M and Krasauskas, R and Stitilyte, M and Sabaliauskas, M and Sasnauskas, G and Tamulaitiene, G and Young, JK and Siksnys, V and Gasiunas, G},
title = {A Potent CRISPR-Cas12l Double-Strand Break Gene Editor.},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {126-140},
doi = {10.1177/25731599261448428},
pmid = {42163774},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA Breaks, Double-Stranded ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Endonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Recently, a new family of CRISPR-Cas12 endonucleases from an unexplored phylum of bacteria, Armatimonadota, was discovered. Named Cas12l, they are compact (800-900 aa), recognize a 5' C-rich protospacer adjacent motif, and present an N-terminal domain that stretches from the beginning to the end of the ribonucleoprotein-bound DNA target site, effectively locking it in place. Here, structure-guided rational design supplemented with AI-based large protein language model predictions was used to improve rates of DNA target cleavage of a family member, Asp2Cas12l. Compared to the wild-type, engineered variants exhibited an approximately 10-fold increase in double-strand break (DSB) editing efficiency in human cells with less target-to-target variation. Moreover, frequencies of editing were comparable to those of SpCas9 at overlapping target sites, and their DSBs efficiently corrected by homology-directed repair (39-56% of editing outcomes). Altogether, this study extends our understanding of CRISPR-Cas12 protein engineering and offers a potent new alternative for DSB-mediated genome editing in human cells.},
}

*CRISPR-Cas Systems/genetics
Humans
*DNA Breaks, Double-Stranded
*Gene Editing/methods
*CRISPR-Associated Proteins/genetics/metabolism
Endonucleases/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42213084,
year = {2026},
author = {Wang, XY and Wang, JX and Li, QN and Pang, XZ and Yang, QF and Zhu, LN and Kong, DM},
title = {A Review of Activator Strand Engineering Strategies for Smart CRISPR/Cas12a Diagnostics.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4224-4244},
doi = {10.1021/acssensors.6c00469},
pmid = {42213084},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; DNA/genetics ; Biosensing Techniques/methods ; *Genetic Engineering/methods ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system has emerged as a transformative tool in molecular diagnostics and biosensing, leveraging its high-efficiency DNA-targeting and unique trans-cleavage activity. However, its practical deployment is hindered by persistent challenges such as elevated background signals, constrained target versatility, and insufficient controllability. The activator strand (AS), serving as the molecular trigger for Cas12a activation, presents a promising engineering target to systematically enhance system performance. This review comprehensively summarizes recent advances in AS-driven regulation of the CRISPR/Cas12a system, focusing on four core engineering strategies: terminal modification engineering, split activator design, PAM (protospacer adjacent motif) engineering and regulation, and topological conformation engineering. By redesigning AS architecture, introducing allosteric control, and refining spatial assembly, these approaches significantly improve detection sensitivity, specificity, and versatility. AS engineering has effectively mitigated background interference, expanded target scope to include non-nucleic acid analytes, and enabled precise conditional activation of Cas12a. We further discuss current challenges and future directions, aiming to guide the development of next-generation CRISPR diagnostic systems with enhanced robustness, programmability, and adaptability for real-world applications.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
DNA/genetics
Biosensing Techniques/methods
*Genetic Engineering/methods
*Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins

@article {pmid42215315,
year = {2026},
author = {Zhang, J and Liang, S and Sun, Y and Zhan, C and Xue, G and Zhou, X},
title = {Glycine/PVP-Enabled One-Pot CRISPR/Cas13a Detection of Vibrio parahaemolyticus by Tetrahedron-Mediated Electrochemistry.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4980-4990},
doi = {10.1021/acssensors.6c00902},
pmid = {42215315},
issn = {2379-3694},
mesh = {*Vibrio parahaemolyticus/isolation & purification/genetics ; *Electrochemical Techniques/methods ; *Glycine/chemistry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Povidone/chemistry ; Limit of Detection ; },
abstract = {Vibrio parahaemolyticus is a major foodborne pathogen widely distributed in aquatic environments and seafood supply chains, necessitating rapid and ultrasensitive detection strategies adaptable to diverse testing scenarios. Here, we present a glycine/ PVP- and tetrahedron-integrated one-pot CRISPR sensing platform (termed GPT-CRISPR) for robust and ultrasensitive nucleic acid detection. The platform introduces chemical regulation into a multienzyme one-pot RAA-CRISPR/Cas13a network, where glycine may help reduce nonspecific Cas13a background activity, possibly through weak competitive interactions, while polyvinylpyrrolidone (PVP) may enhance reaction compatibility through macromolecular crowding and spatial shielding. This coordinated microenvironment enables stable amplification and CRISPR activation within a single closed vessel while minimizing background interference. Upon target recognition, activated Cas13a cleaves uracil-containing, surface-immobilized DNA tetrahedra, translating molecular recognition into amplified electrochemical signals. This transduction strategy enables quantitative detection with a linear dynamic range of 1.5 to 3 × 10[3] copies μL[-1] and a limit of detection of 0.38 copies μL[-1]. The same chemically regulated one-pot CRISPR framework remains compatible with fluorescence and lateral flow readouts. The assay operates under isothermal conditions and delivers results within 30 min without complex sample preparation. Validation across real-world samples demonstrates robustness in complex matrices. Collectively, GPT-CRISPR integrates chemical stabilization of a one-pot CRISPR framework with electrochemical transduction, defining a robust sensing architecture with adaptable readout capability.},
}

*Vibrio parahaemolyticus/isolation & purification/genetics
*Electrochemical Techniques/methods
*Glycine/chemistry
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Povidone/chemistry
Limit of Detection

@article {pmid42263201,
year = {2026},
author = {Sun, Y and Wang, R and Sun, H and Chen, J and Liang, C and Zhang, Z and Feng, Y and Chen, L and Wang, X},
title = {Modular CRISPR-Cas12a-Activated Gold Nanoparticle Assay for Rapid Visual Detection of Hepatocellular Carcinoma-Related miRNAs.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4991-5002},
doi = {10.1021/acssensors.6c00916},
pmid = {42263201},
issn = {2379-3694},
mesh = {*MicroRNAs/genetics/analysis ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Carcinoma, Hepatocellular/genetics/diagnosis ; Humans ; *Liver Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; Colorimetry/methods ; *CRISPR-Associated Proteins/metabolism ; Rapid Diagnostic Tests ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; },
abstract = {MicroRNAs (miRNAs) are emerging biomarkers for early hepatocellular carcinoma (HCC) detection, but most CRISPR-Cas12a assays rely on reverse transcription and preamplification and often lack simple visual readouts. Here, we develop a reverse transcription-free, cleavage-guided strategy that converts miRNA recognition into visual outputs. In the presence of the target miRNA, a scaffold RNA forms an active crRNA that triggers Cas12a trans-cleavage using a preformed dsDNA activator, enabling two readout formats. In a homogeneous non-crosslinking colorimetric assay (mC-NCA), Cas12a cleavage regulates π-π-stacking-mediated gold nanoparticle (AuNP) aggregation to produce a rapid visual colorimetric response. In a heterogeneous versatile lateral flow assay (mC-vLFA), the cleavage of partially hybridized DNA-magnetic bead probes generates a target-dependent test line while maintaining a built-in control line. Using miRNA-21 and miRNA-122 as targets, mC-NCA shows calculated limits of detection (LODs) of 1.62 and 1.64 fM with a linear range of 50 fM-500 pM, whereas mC-vLFA shows calculated LODs of 1.59 and 1.40 fM with a linear range of 10 fM-10 nM. In a preliminary clinical evaluation, both formats show good agreement with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and clinical assessment while enabling faster detection with minimal instrumentation. Overall, this dual-readout platform couples RT-free CRISPR-Cas12a miRNA recognition with cleavage-directed visual signal transduction, enabling rapid and low-instrument-dependence CRISPR diagnostics for miRNAs.},
}

*MicroRNAs/genetics/analysis
*Gold/chemistry
*Metal Nanoparticles/chemistry
*Carcinoma, Hepatocellular/genetics/diagnosis
Humans
*Liver Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
Colorimetry/methods
*CRISPR-Associated Proteins/metabolism
Rapid Diagnostic Tests
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins

@article {pmid42272131,
year = {2026},
author = {Peng, CL and Kamau, WS and Freeman, J and Hill, ZJ and Esvelt, KM},
title = {Increasing the Effective Gene Drive Homing Rate by Targeting the Haploinsufficient Spermatogenesis Gene Klhl10.},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {167-176},
doi = {10.1177/25731599261452112},
pmid = {42272131},
issn = {2573-1602},
mesh = {Animals ; *Spermatogenesis/genetics ; Male ; Mice ; *Haploinsufficiency/genetics ; CRISPR-Cas Systems ; Frameshift Mutation ; Female ; DNA End-Joining Repair/genetics ; Spermatozoa/metabolism ; *Gene Targeting/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-based gene drives represent a powerful new technology for limiting disease transmission and controlling invasive populations. These systems rely on homology-directed repair (HDR) to "drive" a genetic element through a population. However, mammals tend to favor non-homologous end joining (NHEJ), which generates mutations that halt further drive propagation. Here, we describe the experimental characterization of a putative target locus for a gene drive system targeting the haploinsufficient spermatogenesis gene Klhl10 in the laboratory mouse. Using a newly designed "coding sequence cassette," we introduce downstream guide RNAs within the gene, ensuring that sperm undergoing NHEJ are selectively removed from the population. As a proof of principle, we demonstrate that targeting Klhl10 with constitutively expressed LbCas12a results in strong selection against frameshift-containing sperm, validating the core purification mechanism required for this drive strategy. Unexpectedly, we also observed that female offspring lacked most frameshift mutations, suggesting a previously unrecognized role for Klhl10 in oogenesis or early embryonic development.},
}

Animals
*Spermatogenesis/genetics
Male
Mice
*Haploinsufficiency/genetics
CRISPR-Cas Systems
Frameshift Mutation
Female
DNA End-Joining Repair/genetics
Spermatozoa/metabolism
*Gene Targeting/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42290195,
year = {2026},
author = {Michalski, MN and Diegel, CR and Zhong, ZA and Marshall, ME and Foxa Wiartalla, GE and Stevens, PD and Suino-Powell, K and Blazer, LL and Adams, JJ and Melcher, K and Sidhu, SS and Angers, S and Williams, BO},
title = {Clarifying Frizzled 2 function in development through genetically validated mouse models.},
journal = {Disease models & mechanisms},
volume = {19},
number = {7},
pages = {},
doi = {10.1242/dmm.052410},
pmid = {42290195},
issn = {1754-8411},
support = {K08DE031039/DE/NIDCR NIH HHS/United States ; K08DE031039//National Institute of Health/ ; /DE/NIDCR NIH HHS/United States ; //Van Andel Research Institute/ ; },
mesh = {Animals ; *Frizzled Receptors/metabolism/genetics/deficiency ; Phenotype ; Gene Deletion ; Mice, Knockout ; Mice ; Reproducibility of Results ; Integrases/metabolism ; *Embryonic Development/genetics ; CRISPR-Cas Systems/genetics ; Extremities/embryology ; Base Sequence ; Embryo, Mammalian/metabolism ; Models, Animal ; },
abstract = {Wnt receptors of the Frizzled (Fzd) family are widely considered to exhibit substantial functional redundancy, complicating efforts to therapeutically target individual receptors. Fzd2 was believed to be functionally redundant with Fzd1 and Fzd7, based on previously published global knockout mouse studies. By contrast, homozygosity for a Fzd2 global knockout mouse allele developed by the International Mouse Phenotype Consortium (IMPC) has been reported to cause embryonic lethality, suggesting that Fzd2 is critical for early embryonic development. If global deletion of Fzd2 leads to early lethality, conditional deletion models are necessary to identify tissue-specific phenotypes. We found that a previously published Fzd2 conditional deletion model does not eliminate Fzd2. We have generated a new conditional model to address the contradictory previous studies and allow tissue-specific studies of Fzd2. We successfully inserted two loxP sites around the Fzd2 gene and confirmed that subsequent Cre-mediated recombination creates a Fzd2 null allele. Global deletion of Fzd2 in this model does not cause embryonic lethality while limb-specific deletion causes limb shortening. This work supports the hypothesis that Fzd2 regulates limb development and emphasizes the importance of thoroughly validating newly generated mouse models.},
}

Animals
*Frizzled Receptors/metabolism/genetics/deficiency
Phenotype
Gene Deletion
Mice, Knockout
Mice
Reproducibility of Results
Integrases/metabolism
*Embryonic Development/genetics
CRISPR-Cas Systems/genetics
Extremities/embryology
Base Sequence
Embryo, Mammalian/metabolism
Models, Animal

@article {pmid42299032,
year = {2026},
author = {Porenta, D and Benne, N and Sijts, A and Broere, F and Mastrobattista, E},
title = {Induction of SpCas9-Directed Immune Responses Using Lipid Nanoparticles and Identification of SpCas9-Derived T Cell Epitopes in C57BL/6 Mice.},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {151-166},
doi = {10.1177/25731599261456887},
pmid = {42299032},
issn = {2573-1602},
mesh = {Animals ; Mice, Inbred C57BL ; Mice ; *Epitopes, T-Lymphocyte/immunology/genetics ; *Nanoparticles/chemistry/administration & dosage ; Lipids/chemistry ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/immunology/genetics/metabolism ; Gene Editing/methods ; Female ; Interferon-gamma ; Liposomes ; },
abstract = {A major goal of clinically oriented CRISPR-Cas9-based applications is safe and effective in vivo gene editing (knockout or correction) with precise targeting. Substantial efforts have been devoted to the preclinical development of novel drug delivery platforms that enable efficient, targeted delivery. However, the immune responses induced by CRISPR-Cas9 treatment are often overlooked. Preexisting immunity to clinically relevant Cas9 proteins has already been established as a consequence of natural exposure to Cas9-bearing bacteria, which may implicate the safety and efficacy of CRISPR-Cas9-based therapies. Naturally, CRISPR-Cas9 therapies should be nonimmunogenic to avoid amplifying existing Cas9-specific immunity, especially cytotoxic T cell responses. Nonviral delivery systems, such as lipid nanoparticles (LNPs), are widely regarded as less immunogenic than more traditionally used viral vectors, even though LNPs are suitable as a vaccination platforms. In this study, we investigate the induction of SpCas9-directed immunity in C57BL/6 mice upon repeated dosing of LNPs encapsulating Cas9-coding mRNA in two different settings: (1) a vaccination-resembling setting using intramuscularly administered adjuvanted LNPs, and (2) a therapy-resembling setting using intravenously injected, liver-targeting LNPs. In both settings, Cas9-specific T cell responses were detected by evaluating increased total IFN-γ levels upon ex vivo restimulation of isolated splenocytes. However, undetectable Cas9-reactive antibodies induced in the therapeutic setting emphasize the discrepancy between humoral and cellular responses. To improve future monitoring of Cas9-specific T cell responses, we report six Cas9-derived epitopes recognized by CD8[+] T cells, as well as a CD4[+] T cell polypeptide carrying one of the CD8[+] T cell epitopes that induced strong IFN-γ production ex vivo. This work is intended to facilitate the preclinical monitoring of Cas9-specific T cell responses in C57BL/6 mice and support the development of safe CRISPR-Cas9-based therapies.},
}

Animals
Mice, Inbred C57BL
Mice
*Epitopes, T-Lymphocyte/immunology/genetics
*Nanoparticles/chemistry/administration & dosage
Lipids/chemistry
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/immunology/genetics/metabolism
Gene Editing/methods
Female
Interferon-gamma
Liposomes

@article {pmid42328786,
year = {2026},
author = {Xu, C and Yang, Q and Niu, X and Ke, A},
title = {Structure basis for single-strand nucleic acid targeting by IscB and variants.},
journal = {Nucleic acids research},
volume = {54},
number = {12},
pages = {},
pmid = {42328786},
issn = {1362-4962},
support = {R35GM118174/NH/NIH HHS/United States ; S10OD023603/NH/NIH HHS/United States ; //Brookhaven National Laboratory─Laboratory for BioMolecular Structure/ ; KP1607011//DOE Office of Biological and Environmental Research/ ; //Yale CryoEM Resource/ ; },
mesh = {*DNA, Single-Stranded/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; Gene Editing/methods ; Models, Molecular ; Nucleic Acid Conformation ; Humans ; },
abstract = {Transposon-encoded IscB was defined as the evolutionary ancestor of CRISPR-Cas9. This compact RNA-guided endonuclease has since been engineered for genome-editing applications. We previously repurposed IscB and related Cas9s as efficient RNA editors by removing their double-stranded DNA (dsDNA) recognition module, the target-adjacent motif (TAM)/protospacer adjacent motif-interacting domain. Here, we report four cryo-electron microscopy structures of IscB, with or without TAM-interaction domain (TID), in complex with single-stranded nucleic acid (ssNA) targets. Structures reveal that, regardless of TID presence, IscB engages ssNA using the same mechanism. IscB initially facilitates formation of a 10-nt seed duplex with ssNA; further base-pairing is blocked by an alternatively positioned HNH nuclease that acts as a roadblock. In this intermediate state, neither HNH nor RuvC is competent for target cleavage. Only upon full duplex formation is the HNH roadblock dislodged by the duplex extension between guide RNA and ssNA. HNH and RuvC nuclease active sites become exposed as the result. A similar set of conformational rearrangements likely governs IscB activity during dsDNA target interrogation. Guided by the structural and mechanistic insights, we introduced mutations to either improve ssNA binding or ease HNH dislodging. Both approaches improved the RNA-targeting efficiency of IscB in vitro and in human cells.},
}

*DNA, Single-Stranded/chemistry/metabolism/genetics
Cryoelectron Microscopy
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
Gene Editing/methods
Models, Molecular
Nucleic Acid Conformation
Humans

@article {pmid42329468,
year = {2026},
author = {Ahmad, S and Aftab, MN and Ghalib, M and Aslam, MS},
title = {Overcoming cellular secretion bottlenecks: advanced secretion engineering and molecular tailoring for next-generation microbial α-amylases with enhanced industrial performance.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {7},
pages = {},
pmid = {42329468},
issn = {1573-0972},
mesh = {Bacillus subtilis/genetics/enzymology/metabolism ; *alpha-Amylases/metabolism/genetics/chemistry ; *Metabolic Engineering/methods ; Aspergillus niger/genetics/enzymology/metabolism ; Escherichia coli/genetics/metabolism ; Recombinant Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Industrial Microbiology ; Pichia/genetics/enzymology ; Carbohydrate Binding Modules ; Protein Engineering ; Synthetic Biology ; Saccharomycetales ; },
abstract = {α-amylases are indispensable industrial biocatalysts, yet their recombinant production faces significant biochemical and cellular bottlenecks. Recent scientific advancement shifts the paradigm from traditional cloning toward a design-parameter framework, where host selection predominantly Bacillus subtilis, Pichia pastoris, and Aspergillus niger is dictated by secretion capacity, folding landscapes, and metabolic compatibility. While Escherichia coli remains a common host, its lack of efficient extracellular secretion often leads to inclusion body formation and metabolic stress. Advanced strategies are being employed to overcome these limits, including signal peptide optimization, chaperone co-expression, and the fusion of carbohydrate-binding modules (CBMs) to enhance raw-starch degradation. Furthermore, how rational design, aided by artificial intelligence and molecular dynamics simulations, enables the engineering of hyper-thermostable and alkaline-tolerant variants capable of withstanding extreme industrial processing conditions. The advent of CRISPR-Cas technology has further revolutionized the field, allowing for precise genome editing and metabolic rewiring to achieve record-breaking enzyme titers, such as 102,893 U/mL in engineered B. subtilis. By balancing transcriptional levels with enhanced secretion pathways and stress-mitigation systems, modern synthetic biology provides the tools to tailor α-amylases for specific needs, ranging from biofuel production to high-purity malto-oligosaccharide synthesis. Therefore, this comprehensive analysis underscores that reconciling host biology with enzyme biochemistry is essential for meeting global industrial demands.},
}

Bacillus subtilis/genetics/enzymology/metabolism
*alpha-Amylases/metabolism/genetics/chemistry
*Metabolic Engineering/methods
Aspergillus niger/genetics/enzymology/metabolism
Escherichia coli/genetics/metabolism
Recombinant Proteins/metabolism/genetics
CRISPR-Cas Systems
Industrial Microbiology
Pichia/genetics/enzymology
Carbohydrate Binding Modules
Protein Engineering
Synthetic Biology
Saccharomycetales

@article {pmid42330666,
year = {2026},
author = {Ling, H and Su, N and Huang, L and Wang, M and Ren, L},
title = {Cleavage-responsive DNA/AgNCs enable accelerated Cas12a trans-cleavage for rapid multigene methylation diagnosis.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118929},
doi = {10.1016/j.bios.2026.118929},
pmid = {42330666},
issn = {1873-4235},
abstract = {DNA methylation, particularly 5-methylcytosine (5 mC), is a key epigenetic modification involved in the regulation of gene expression and genomic stability, and has emerged as a promising biomarker for early cancer screening and molecular stratification. CRISPR-Cas12a systems have been increasingly exploited to convert methylation-associated sequence information into detectable signals owing to their programmability and collateral cleavage-mediated signal amplification. However, many CRISPR-based assays remain constrained by the high background and relatively slow response kinetics of conventional fluorophore-quencher reporters. To overcome these limitations, we developed a cleavage-responsive DNA-templated silver nanocluster (DNA/AgNC) reporter that translates Cas12a trans-cleavage activity into a green-to-red ratiometric fluorescence shift. In this design, the AgNC-templating DNA scaffold itself serves as an enzymatically cleavable signal transducer, rather than relying on a terminal fluorophore-quencher pair. Compared with a representative F-ssDNA-Q reporter, the DNA/AgNC reporter exhibited stronger apparent association with Cas12a and an approximately two-fold improvement in apparent catalytic efficiency. When incorporated into an MSRE-RPA-Cas12a workflow, the platform achieved a limit of detection of 74.5 aM, while completing the Cas12a reporting step within 30 min. Coupling this assay with a miniaturized optoelectronic device further enabled spatially resolved profiling of five genomic loci, with relative errors of approximately 5%. Overall, this strategy establishes a ratiometric reporter format for CRISPR-based DNA methylation profiling and offers potential for point-of-care epigenetic biosensing.},
}

@article {pmid42333809,
year = {2026},
author = {Hou, S and Bi, W and Liu, W and Nie, H and Jiang, J and Zeng, L},
title = {Beyond DNA editing: how Cas13 redefined programmable RNA manipulation and what still limits its therapeutic promise.},
journal = {Nucleic acids research},
volume = {54},
number = {12},
pages = {},
pmid = {42333809},
issn = {1362-4962},
support = {2024ZD0541500//Noncommunicable Chronic Diseases-National Science and Technology Major Project/ ; 82222038//National Natural Science Foundation of China/ ; 01-SWKJYCJJ06//Outstanding Young Talents of National Defense Biotechnology/ ; 2023XRC01//High-Level Talent Program Cultivation Project of Army Medical University/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA Editing/genetics ; *RNA/genetics/metabolism ; *Gene Editing/methods ; Animals ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The Type VI Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas13 system, evolved from prokaryotic immunity, has become a versatile, programmable RNA-targeting platform with broad biotechnological potential. Guided by CRISPR RNA (crRNA), Cas13 cleaves single-stranded RNA via higher eukaryotic and prokaryotic nucleotide-binding domains, exerting specific (cis) and nonspecific (trans) collateral cleavage, which enables ultrasensitive nucleic acid detection while introducing cytotoxicity risks in eukaryotic cells. Diversification of Cas13 subtypes, including compact variants, enhances targetability and delivery compatibility, and inhibitory strategies (anti-CRISPR proteins, crRNA mimicry/degradation) enable activity modulation for improved safety. Building on mechanistic foundations, Cas13 is repurposed for targeted RNA knockdown, nucleic acid diagnostics, live-cell RNA imaging with catalytically inactive variants, programmable RNA base editing through deaminase fusions, splicing regulation, epitranscriptomic editing of multiple RNA chemical marks, interaction mapping of RNA-protein and RNA-RNA networks, and translational control, with preliminary clinical translation in antiviral therapies, pathogenic transcript correction, and cancer therapy. Furthermore, Cas13-integrated diagnostics and functional genomics are accelerating biomarker discovery and personalized treatment. Nevertheless, successful clinical translation hinges on overcoming critical bottlenecks, including tissue-specific delivery, mitigation of collateral cytotoxicity, and management of host immunogenicity. This review synthesizes Cas13 classification, structure-function principles, regulatory inhibitors, application modalities, and translational challenges to inform next-generation engineering and responsible deployment of RNA-targeted technologies.},
}

Humans
*CRISPR-Cas Systems/genetics
*RNA Editing/genetics
*RNA/genetics/metabolism
*Gene Editing/methods
Animals
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid42334452,
year = {2026},
author = {Lin, Y and Liou, B and Fannin, V and Adler, S and Mayhew, CN and Hammonds, JE and Hu, YC and Tchieu, J and Zhang, W and Zhao, X and Beres, RL and Setchell, KDR and Kaynak, A and Qi, X and Feldman, RA and Sun, Y},
title = {Patient-specific midbrain organoids with CRISPR correction recapitulate neuronopathic Gaucher disease phenotypes and enable evaluation of novel therapies.},
journal = {eLife},
volume = {15},
number = {},
pages = {},
pmid = {42334452},
issn = {2050-084X},
support = {R21HD1027881//National Institute of Health/ ; R21OD033660//National Institute of Health/ ; R01NS138309//National Institute of Health/ ; R01NS103931//National Institute of Health/ ; Center of Pediatric Genomics Award//Cincinnati Children's Hospital Medical Center/ ; Research Innovation Pilot Funding Program Award//Cincinnati Children's Hospital Medical Center/ ; 2UL1TR001425-05A1, CHMC-CTSA 00003827/NH/NIH HHS/United States ; },
mesh = {*Gaucher Disease/genetics/therapy/pathology ; Humans ; *Organoids/pathology/metabolism ; *Mesencephalon/pathology ; *Glucosylceramidase/genetics/metabolism ; Phenotype ; *CRISPR-Cas Systems ; Mutation ; Induced Pluripotent Stem Cells ; Dopaminergic Neurons ; },
abstract = {Neuronopathic Gaucher disease (nGD) is a lysosomal storage disorder caused by GBA1 mutations, leading to defective acid β-glucosidase (GCase) and accumulation of glycosphingolipid substrates, causing inflammation and neurodegeneration. Patients with nGD manifest severe neurological symptoms, but current animal models fail to fully recapitulate the human condition, posing a major barrier to the development of effective therapies targeting the brain. To bridge this gap, we have developed midbrain-like organoids (MLOs) from human induced pluripotent stem cells of nGD patients with GBA1[L444P/P415R] and GBA1[L444P/RecNcil] mutations to model nGD brain pathogenesis. These nGD MLOs exhibited GCase deficiency, resulting in diminished enzymatic function, accumulation of lipid substrates, widespread transcriptomic changes, and impaired dopaminergic neuron differentiation, mirroring nGD pathology. GBA1 mutation correction mediated by CRISPR/Cas9 restored GCase activity, normalized lipid substrate levels, and rescued dopaminergic neuron function, confirming the causal role of GBA1 mutations during early brain development. Using this novel platform, we further evaluated therapeutic strategies, including SapC-DOPS nanovesicles delivering GCase, AAV9-GBA1 gene therapy, and substrate reduction therapy with GZ452, a glucosylceramide synthase inhibitor currently under clinical investigation. These treatments either restored GCase activity, reduced lipid substrate accumulation, improved autophagic and lysosomal abnormalities, or ameliorated dysregulated genes involved in neural development. These patient-specific, 3D neural models offer a transformative, physiologically relevant platform for unraveling disease mechanisms and accelerating the discovery of therapies for patients with nGD.},
}

*Gaucher Disease/genetics/therapy/pathology
Humans
*Organoids/pathology/metabolism
*Mesencephalon/pathology
*Glucosylceramidase/genetics/metabolism
Phenotype
*CRISPR-Cas Systems
Mutation
Induced Pluripotent Stem Cells
Dopaminergic Neurons

@article {pmid42334517,
year = {2026},
author = {Zhang, XL and Luo, JN and Cao, Y and Zhao, JX and Zhang, Y and Xu, SF and Zhuang, LN and He, J},
title = {Generation of transgenic pigs with targeted insertion of a wildtype copy of human PKD2 gene.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42334517},
issn = {1573-4978},
support = {2021YFA0805902//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Animals, Genetically Modified/genetics ; Swine/genetics ; *TRPP Cation Channels/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; *Polycystic Kidney, Autosomal Dominant/genetics/metabolism ; Female ; Disease Models, Animal ; Kidney/metabolism/pathology ; },
abstract = {BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD), primarily driven by PKD1 or PKD2 mutations, is a prevalent hereditary nephropathy for which large-animal models capturing human renal anatomy and disease tempo remain urgently needed. This study aimed to establish a site-specific human PKD2 (hPKD2) transgenic porcine model as a versatile platform capable of supporting two temporally distinct applications: longitudinal evaluation of whether sustained PKD2 overexpression is sufficient to induce renal pathology, and-should overexpression prove phenotypically silent-future crossbreeding-based functional rescue of PKD2-knockout lines.

METHODS AND RESULTS: A CRISPR/Cas9-mediated, homology-recombination-independent strategy was employed to target full-length hPKD2 cDNA into the porcine pH11 safe harbor locus. Somatic cell nuclear transfer yielded five F0 transgenic founders, and natural mating of two founders with wild-type sows produced four F1 transgenic offspring, confirming stable germline transmission. Quantitative real-time PCR and whole-genome sequencing validated single-copy, site-specific transgene integration at the designated locus. Robust hPKD2 mRNA and FLAG-tagged polycystin-2 (PC-2) expression were detected in renal and other tissues across both generations. During the initial 12-month monitoring period, serum blood urea nitrogen and creatinine levels remained within normal ranges and no gross histological abnormalities were evident. However, given that these biomarkers are insensitive to early-stage renal impairment, extended observation with more comprehensive phenotyping is required before definitive conclusions regarding renal function can be drawn.

CONCLUSIONS: A transgenic porcine model with stable, single-copy hPKD2 integration at the pH11 safe harbor locus was successfully generated and shown to permit germline transmission. This platform provides a foundation for long-term investigation of PKD2 overexpression pathophysiology and, alternatively, for functional complementation of PKD2-deficient models, thereby advancing both mechanistic and translational ADPKD research.},
}

Animals
*Animals, Genetically Modified/genetics
Swine/genetics
*TRPP Cation Channels/genetics/metabolism
Humans
CRISPR-Cas Systems
*Polycystic Kidney, Autosomal Dominant/genetics/metabolism
Female
Disease Models, Animal
Kidney/metabolism/pathology

@article {pmid42337238,
year = {2026},
author = {Shi, LD and Kolody, BC and Wang, S and Valentin-Alvarado, LE and Lei, S and Sachdeva, R and Banfield, JF},
title = {Jumbo circular extrachromosomal elements of methane-oxidizing archaea with variably extensive metabolic and defense gene repertoires.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-74423-z},
pmid = {42337238},
issn = {2041-1723},
support = {DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; INV-037174/GATES/Gates Foundation/United States ; },
abstract = {Archaeal extrachromosomal elements (ECEs) are arguably the least well understood of all genetic elements, and few have >200 kbp (jumbo) genomes. Here, we report circular, jumbo ECEs with genomes of up to 535 kbp in length that associate with anaerobic methane-oxidizing Methanoperedens archaea. Notably, a 409-kbp genome related to jumbo ECEs is integrated into a subset of the ~4.2 Mbp Methanoperedens chromosomes at the tRNA-Asp genes. This represents the largest integrative element in Archaea and supports the jumbo ECE-host association. Multiple genome alignments and phylogenetic analyses suggest that the large ECE sizes developed by extensive DNA acquisition from Methanoperedens. The newly identified ECEs encode, and in some cases express, metabolic genes such as tetrahydromethanopterin S-methyltransferase exclusively involved in methane metabolism, and genes for nitrogen and sulfur compound transformations. Also encoded are defense systems, some of which are absent in hosts, such as hybrid Type I/Type III-A CRISPR-Cas systems. In contrast to viruses and plasmids, they have host-like replication machinery and occur at stable copy ratios of 1.44 ± 0.24:1 to the host. Overall, our results reveal a spectrum of jumbo ECEs of Methanoperedens, ranging from plasmid-like to minichromosome-like.},
}

@article {pmid42339381,
year = {2026},
author = {Bashir, T and Rashid, R and Malik, AR and Sundouri, AS and Bhat, KM and Mir, MA and Rehman, MU and Masood, M and Tehleelah, },
title = {Reframing fruit biofortification for sustainable micronutrient security: from crop innovation to nutritional impact.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1853898},
pmid = {42339381},
issn = {1664-462X},
abstract = {Micronutrient malnutrition, commonly referred to as "hidden hunger, " remains a persistent global health challenge, particularly in regions with limited dietary diversity. Although agricultural intensification has substantially improved caloric availability, it has not ensured adequate micronutrient density in food systems, highlighting the urgent need for nutrition-sensitive crop improvement strategies. Biofortification has emerged as a sustainable and cost-effective approach to enhance the micronutrient content of food crops through agronomic, genetic, and biotechnological interventions. While biofortification research has predominantly focused on staple cereals and legumes, horticultural fruit crops have received comparatively limited attention despite their widespread consumption, high consumer acceptance, and natural richness in bioactive compounds. This review advances beyond existing overviews by providing a critical and comparative evaluation of agronomic, conventional breeding, and biotechnological approaches for fruit crop biofortification, with particular emphasis on their effectiveness, scalability, limitations, and translational potential. Current advances aimed at enhancing iron, zinc, iodine, selenium, and provitamin A concentrations are comprehensively synthesized. Special attention is given to the physiological and molecular mechanisms regulating micronutrient uptake, transport, accumulation, and storage in fruit tissues. In addition, advanced biotechnological tools, including CRISPR/Cas-mediated genome editing, are critically assessed in relation to biosafety, regulatory considerations, and practical applicability. Evidence from major fruit crops, including apple, banana, mango, pomegranate, strawberry, and papaya, demonstrates that integrated biofortification strategies can improve micronutrient density while maintaining fruit yield and quality. Importantly, this review addresses a major knowledge gap by linking crop-level nutrient enhancement with micronutrient bioavailability and human nutritional outcomes, emphasizing the influence of food matrix interactions and nutrient absorption efficiency. Key constraints, including genotype × environment interactions, postharvest nutrient instability, climate-driven variability, and limited clinical validation, are also discussed. Finally, a systems-level framework integrating plant science, human nutrition, postharvest biology, and policy perspectives is proposed to support the large-scale adoption of nutrition-sensitive fruit biofortification. Collectively, fruit crop biofortification represents a promising strategy for improving global micronutrient security and advancing sustainable food systems.},
}

@article {pmid42340480,
year = {2026},
author = {Guo, Z and Yang, Z and Liu, Z and Zheng, G and Zou, S},
title = {Fgf8, a gene knockout that leads to intermuscular bones-reduced of crucian carp (Carassius auratus), acts as a potentially regulatory factor in osteogenic development.},
journal = {Fish physiology and biochemistry},
volume = {52},
number = {4},
pages = {},
pmid = {42340480},
issn = {1573-5168},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Carps/genetics/growth & development ; *Fibroblast Growth Factor 8/genetics/metabolism ; *Osteogenesis/genetics/physiology ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; },
abstract = {Although fibroblast growth factor 8 (FGF8) is a critical regulator of skeletal morphogenesis in vertebrates, its specific role in the formation and development of intermuscular bones (IBs) in teleost fish remains insufficiently characterized. In this study, we generated double mutants (fgf8a[+/-] + fgf8b[+/-]) in diploid Chongming crucian carp (Carassius auratus) using CRISPR/Cas9-mediated gene editing. Compared to wild-type fish, the double mutants exhibited significantly reduced IB number (p < 0.01), demonstrating a synergistic role of fgf8a and fgf8b in IB formation. Notably, reduced number of IBs did not compromise overall growth, muscle architecture, or reproductive performance. Integrated transcriptomic and metabolomic analyses revealed that the reduced IB phenotype was linked to modifications in relevant signaling pathways and a concomitant upregulation of metabolites beneficial for muscle quality and health. Our findings highlight the crucial role of fgf8 in regulating IB formation and development in crucian carp, providing insights into the genetic mechanisms underlying this process in teleosts.},
}

Animals
*Carps/genetics/growth & development
*Fibroblast Growth Factor 8/genetics/metabolism
*Osteogenesis/genetics/physiology
Gene Knockout Techniques
CRISPR-Cas Systems
Gene Expression Regulation, Developmental

@article {pmid42342668,
year = {2026},
author = {Mitsuda, Y and Sugaya, M and Ishikawa, J and Nagahata, N and Okazaki, S and Hiraizumi, M and Kato, K and Gootenberg, JS and Abudayyeh, OO and Osawa, T and Yamashita, K and Nishimasu, H},
title = {Structural mechanism of SAM-AMP and SAM-AMP2 synthesis by the type III-D2 CRISPR effector complex.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42342668},
issn = {2041-1723},
support = {25H00436//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; *S-Adenosylmethionine/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Escherichia coli/metabolism/genetics/virology ; Adenosine Triphosphate/metabolism ; Escherichia coli Proteins/metabolism/genetics/chemistry ; },
abstract = {The type III-D2 CRISPR-Cas system comprises multiple Cas subunits and a CRISPR RNA, and is likely an evolutionary intermediate between the well-studied type III-A and III-E systems. Here we show that the type III-D2 complex synthesizes two distinct second messengers, SAM-AMP and SAM-AMP2, from S-adenosylmethionine (SAM) and ATP in response to target RNA recognition. We determined cryo-electron microscopy structures of the type III-D2 effector complex in different functional states, providing mechanistic insights into target RNA cleavage and second messenger synthesis. The structures reveal how SAM and ATP are recognized by the Cas10 subunit within the effector complex. Furthermore, our biological data suggest that both SAM-AMP and SAM-AMP2 act on the CorA ancillary effector, inducing growth arrest of infected bacterial cells and thereby conferring immunity. Thus, our study establishes the type III-D2 system as a unique anti-phage defense mechanism that employs both SAM-AMP and SAM-AMP2 as second messengers, expanding the repertoire of second messenger strategies in bacterial defense systems and highlighting the remarkable functional diversity of CRISPR-Cas systems.},
}

*CRISPR-Cas Systems/genetics
Cryoelectron Microscopy
*S-Adenosylmethionine/metabolism/chemistry
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
Escherichia coli/metabolism/genetics/virology
Adenosine Triphosphate/metabolism
Escherichia coli Proteins/metabolism/genetics/chemistry

@article {pmid42342872,
year = {2026},
author = {Van Der Straeten, D and Bulut, M and Cao, D and Aharoni, A and Bouis, H and Granell, A and Gruissem, W and Lindberg Møller, B and Martin, C and Puchta, H and Sreenivasulu, N and Tissier, A and Tripathi, L and Van Montagu, M and Fernie, AR},
title = {Genetic technologies to enhance crop nutritional value under climate change.},
journal = {Nature},
volume = {654},
number = {8120},
pages = {877-891},
pmid = {42342872},
issn = {1476-4687},
mesh = {Humans ; *Biofortification/methods ; *Climate Change ; CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/metabolism/chemistry ; *Gene Editing/methods ; Metabolic Engineering ; Micronutrients/deficiency/analysis ; Nutritive Value ; },
abstract = {At present, more than 700 million people live with caloric hunger, and more than two billion suffer from micronutrient deficiencies, known as 'hidden hunger'. From an agricultural viewpoint, three major objectives need to be worked towards simultaneously to achieve zero hunger (the United Nations Sustainable Development Goal 2): (1) enhanced yield; (2) higher vitamin and mineral density to sustain recommended daily intake (multi-biofortification); and (3) enhanced climate-change resilience. Although the Green Revolution increased global calorie production, it exacerbated hidden hunger by prioritizing high yield over nutritional quality. Stress from global climate change has been shown to reduce the densities of several micronutrients. CRISPR-Cas, which allows genome editing with extremely high precision, has emerged as a groundbreaking breeding technology that has already been adopted by many countries. Here we examine how CRISPR-Cas-based approaches could be used to achieve biofortification targets by enhancing micronutrient densities to the levels necessary to alleviate dietary vitamin and mineral deficiencies. Given the limited time frame available to achieve zero hunger, we argue that CRISPR-Cas technologies should be combined with metabolic engineering based on transformation and other technologies. We also consider untapped resources beyond metabolic pathways and current CRISPR-Cas methodologies to address one of the most important societal issues of the twenty-first century.},
}

Humans
*Biofortification/methods
*Climate Change
CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics/metabolism/chemistry
*Gene Editing/methods
Metabolic Engineering
Micronutrients/deficiency/analysis
Nutritive Value

@article {pmid42342925,
year = {2026},
author = {Gong, Q and Liu, H and Nie, Q and Tong, J and Li, W and Pak, M and Mei, Y and He, K and Wang, D and Wang, NN},
title = {GmENDO-like 1, a structurally divergent homolog of S1/P1-type endonuclease, modulates the vegetative-to-reproductive phase transition in soybean.},
journal = {Plant cell reports},
volume = {45},
number = {7},
pages = {},
pmid = {42342925},
issn = {1432-203X},
support = {24JCQNJC01940//Natural Science Foundation of Tianjin/ ; 2023ZD040710207//Biological Breeding-National Science and Technology Major Project/ ; 32070317//National Natural Science Foundation of China, Grant/ ; 32270359//National Natural Science Foundation of China/ ; },
mesh = {*Glycine max/genetics/enzymology/physiology/growth & development ; *Plant Proteins/genetics/metabolism/chemistry ; Amino Acid Sequence ; Gene Expression Regulation, Plant ; *Endonucleases/genetics/metabolism/chemistry ; Plant Leaves/genetics/physiology/growth & development ; Gene Knockout Techniques ; Plants, Genetically Modified ; Flowers/genetics/physiology ; Plant Senescence/genetics ; Reproduction/genetics ; Phylogeny ; CRISPR-Cas Systems ; Phenotype ; Mutation ; },
abstract = {GmENDO-like 1, a structurally divergent S1/P1 endonuclease homolog, modulates soybean vegetative-to-reproductive transition; its knockout delays flowering and accelerates leaf senescence. Soybean is a monocarpic crop in which the transition from vegetative to reproductive growth triggers the onset of senescence in vegetative tissues. In this study, we identified a soybean homolog of the S1/P1-type endonuclease gene, GmENDO-like 1, which was significantly upregulated during leaf senescence. Sequence analysis revealed that GmENDO-like 1 has substantial deletions at both the N- and C-termini, resulting in the loss of key residues essential for the active center of canonical S1/P1-type nucleases. Specifically, among the nine conserved amino acids responsible for metal ion coordination, one tryptophan (Trp), two histidines (His), and one aspartic acid (Asp) residue are absent. Although these deletions result in a significant rearrangement of the trinuclear metal-binding center, GmENDO-like 1 remarkably retains intrinsic catalytic activity. Using CRISPR/Cas9-mediated genome editing, we generated two knockout mutant lines of the GmENDO-like 1 gene. Phenotypic analysis revealed a markedly delayed flowering time and an obviously precocious leaf senescence in the GmENDO-like 1 mutant plants. These observations were further corroborated by the altered expression levels of flowering- and senescence-associated marker genes. Both the 100-seed weight and per-plant yield of the mutants were significantly decreased. Collectively, these findings establish GmENDO-like 1 as a functional, evolutionarily divergent nuclease and an essential modulator regulating the vegetative-to-reproductive phase transition in soybean.},
}

*Glycine max/genetics/enzymology/physiology/growth & development
*Plant Proteins/genetics/metabolism/chemistry
Amino Acid Sequence
Gene Expression Regulation, Plant
*Endonucleases/genetics/metabolism/chemistry
Plant Leaves/genetics/physiology/growth & development
Gene Knockout Techniques
Plants, Genetically Modified
Flowers/genetics/physiology
Plant Senescence/genetics
Reproduction/genetics
Phylogeny
CRISPR-Cas Systems
Phenotype
Mutation

@article {pmid42343762,
year = {2026},
author = {Han, N and Xiao, Y and Zhang, J and Guo, L and Li, Q and Yang, P and Li, X and Lin, X and Xu, L and Zhang, H},
title = {[Research progress of the CRISPR-Cas system in the detection and elimination of antibiotic resistance genes].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {5},
pages = {2051-2068},
doi = {10.13345/j.cjb.250847},
pmid = {42343762},
issn = {1872-2075},
support = {2023-YJ-zd1//the Key Research and Development Program of Health and Healthcare of Fuzhou First General Hospital/ ; 2024-YJ-ZK04//the Fujian Province Clinical Key Specialty Construction Research Project/ ; 2024J011257//the General Program of Fujian Provincial Natural Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; *Gene Editing/methods ; },
abstract = {The abuse of antibiotics has led to increasingly severe antibiotic resistance, which poses a serious threat to global public health security. Therefore, the rapid detection and effective elimination of antibiotic resistance genes are crucial for controlling the spread of drug-resistant bacteria. Conventional methods for detecting antibiotic resistance genes, such as PCR, quantitative real-time PCR, and isothermal amplification, have shortcomings including time-consuming procedures, labor intensiveness, high costs, and poor specificity. Therefore, there is an urgent need to find new solutions to combat bacterial resistance, making the development of novel and efficient technologies for detecting and eliminating antibiotic resistance genes. In recent years, gene editing technology has become a research hotspot. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas proteins) consisted of the CRISPR-Cas system. As one of the adaptive immune systems in prokaryotes, it possesses unique sequence targeting ability and high sensitivity, which enable it to accurately recognize and cleave target nucleic acid sequences. Thus, it shows great potential in the detection and elimination of antibiotic resistance genes. This review summarizes the classification, structures, and action mechanisms of CRISPR-Cas systems, elaborates on the application of different CRISPR-Cas systems in the detection and elimination of antibiotic resistance genes, and finally discusses the challenges faced by this technology and its future development directions, aiming to provide new ideas for addressing antibiotic resistance in pathogenic bacteria.},
}

*CRISPR-Cas Systems/genetics
*Drug Resistance, Bacterial/genetics
*Bacteria/genetics/drug effects
Anti-Bacterial Agents/pharmacology
*Gene Editing/methods

@article {pmid42343773,
year = {2026},
author = {Chen, Y and Jin, W and Ma, X and Feng, R and Yang, Y},
title = {[YTHDF1 enhances the expression of exogenous proteins in the CHO-K1-GS[‒/‒] cell line].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {5},
pages = {2219-2232},
doi = {10.13345/j.cjb.250823},
pmid = {42343773},
issn = {1872-2075},
support = {2025-2-177//the Lanzhou Science and Technology Program/ ; 26YFFA026//the Gansu Science and Technology Program/ ; 31920250053//the Fundamental Research Funds for the Central Universities/ ; 2025CXZX-242//the "Innovation Star" Project for Postgraduate Students in Gansu Province/ ; 25FNNA002//the Gansu Province Research-Production Integration Sci-Tech Project Empowerment Initiative/ ; },
mesh = {Animals ; CHO Cells ; Cricetulus ; *Glutamate-Ammonia Ligase/genetics ; *RNA-Binding Proteins/genetics/metabolism ; Cricetinae ; Recombinant Proteins/genetics/biosynthesis ; Humans ; *RNA Splicing Factors/genetics ; CRISPR-Cas Systems ; },
abstract = {YTH domain family protein 1 (YTHDF1) is an RNA-binding protein and belongs to the reader modified by N[6]-methyladenosine (m[6]A). It recognizes and binds to m[6]A modifications on RNA through specific domains, thereby performing corresponding biological functions. This study aims to explore the effects of YTHDF1 on the expression levels of exogenous proteins in the Chinese hamster ovary (CHO) cell line and evaluate its application potential in the optimization of the expression system of CHO cells. Firstly, the glutamine synthetase (GS) gene of CHO-K1 cells was knocked out by CRISPR/Cas9 to obtain the CHO-K1-GS[‒/‒] cell line. The enhancement effects of YTHDF1 overexpression on the expression of exogenous proteins in CHO-K1-GS[‒/‒] cells were further analyzed. Western blotting, qPCR, and fluorescence microscopy observations showed that the overexpression of YTHDF1 significantly up-regulated the expression levels of human serum albumin, single-chain antibody, and green fluorescent protein in cells. Moreover, this process did not have a significantly negative impact on the long-term proliferation and survival rate of the cells. In addition, the experiment with the translation inhibitor cycloheximide (CHX) confirmed that YTHDF1 mainly enhanced the protein expression by promoting translation in cells. This study demonstrates that YTHDF1 can enhance the cell's ability to synthesize exogenous proteins by promoting the translation process of mRNA. This strategy provides a new theoretical basis and technical direction for constructing efficient cell lines for producing biopharmaceuticals.},
}

Animals
CHO Cells
Cricetulus
*Glutamate-Ammonia Ligase/genetics
*RNA-Binding Proteins/genetics/metabolism
Cricetinae
Recombinant Proteins/genetics/biosynthesis
Humans
*RNA Splicing Factors/genetics
CRISPR-Cas Systems

@article {pmid42343789,
year = {2026},
author = {Han, C and Li, Y and Zhang, H},
title = {[Advances in genome editing technologies in Komagataella phaffii and their applications in biomanufacturing].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {6},
pages = {2414-2438},
doi = {10.13345/j.cjb.250908},
pmid = {42343789},
issn = {1872-2075},
support = {1012050205238420//the Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Saccharomycetales/genetics/metabolism ; Recombinant Proteins/genetics/biosynthesis ; Metabolic Engineering/methods ; },
abstract = {Komagataella phaffii is widely recognized as a premier host for the production of recombinant proteins and value-added metabolites, owing to its low background secretion of endogenous proteins, strong capacity for heterologous protein secretion, and robust growth and metabolic performance under industrially relevant fermentation conditions. In recent years, rapid progress in genome editing technologies and synthetic biology toolkits has markedly improved the precision and efficiency of gene function interrogation, metabolic pathway reconstruction, and dynamic regulation in K. phaffii, thereby continuously strengthening its performance as a microbial cell factory. Consequently, beyond its established roles in producing recombinant proteins, industrial enzymes, and vaccine antigens, K. phaffii has also demonstrated substantial potential for the biosynthesis of natural products, biopharmaceutical molecules, and emerging biomaterials. This review systematically summarizes the evolution of genome engineering technologies in K. phaffii, spanning the transition from conventional recombination-based methods to next-generation precision editing tools. We highlight recent advances, optimization strategies, and engineering practices of CRISPR/Cas and related systems in this host. Moreover, in light of emerging research trends, we discuss key challenges and opportunities associated with improving editing efficiency, enabling rapid assembly of complex metabolic pathways, and accelerating industrial translation, thereby providing a reference for the rational engineering of Komagataella phaffii and its broader applications in synthetic biology and biomanufacturing.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Saccharomycetales/genetics/metabolism
Recombinant Proteins/genetics/biosynthesis
Metabolic Engineering/methods

@article {pmid42343816,
year = {2026},
author = {Wang, B and DU, C and Gege, R and Zhao, Q and Wang, H and Li, B and Wang, J and Mei, J and Zhang, S and Bao, F},
title = {[CRISPR/Cas9-mediated knockout of the Soc gene in T4 bacteriophage and mutant construction].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {6},
pages = {2839-2848},
doi = {10.13345/j.cjb.250870},
pmid = {42343816},
issn = {1872-2075},
support = {32260893//the National Natural Science Foundation of China/ ; 2022MS03019//the Natural Science Foundation of Inner Mongolia Autonomous Region/ ; NJYT23094//the Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region/ ; 2023YFXZ0002//the Science and Technology Project of Inner Mongolia Autonomous Region/ ; BR250101//the Basic Scientific Research Fund of Directly Affiliated Universities in Inner Mongolia Autonomous Region/ ; },
mesh = {*Bacteriophage T4/genetics ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; Escherichia coli/genetics/metabolism ; Mutation ; },
abstract = {To address the antigen display limitations of current vaccine carriers, we engineered the T4 bacteriophage into a high-capacity platform. The T4 bacteriophage has advantages such as structural stability, high loading capacity, and easy production. However, the presence of high-copy endogenous Soc protein on its capsid surface severely restricts the effective display of exogenous proteins. To overcome the aforementioned spatial limitations, we employed the CRISPR/Cas9 system to precisely knockout the Soc gene of T4 bacteriophage. We successfully achieved the knockout of the Soc gene by co-transferring the three plasmid systems-pCas, pTargetF-sgRNA, and pMD19-T-Soc-arm-into Escherichiacoli TG1, inducing the expression of Cas9 with L-arabinose, and then infecting the engineered bacteria with the wild-type T4 bacteriophage (named T4 WT). The PCR, SDS-PAGE, and sequencing results confirmed a Soc gene-deficient T4 bacteriophage mutant strain (named T4ΔSoc) was successfully constructed. This mutant strain had comparable growth, thermal stability, and pH stability to the wild type, and maintained complete infectivity. After continuous passage for five generations, it remained stable in terms of genome, protein composition, and phage plaque phenotype, with no occurrence of revertant mutations. In conclusion, T4ΔSoc is stable both genetically and phenotypically, effectively breaking through the limitation of the restricted display space of T4 WT. This study provides a reliable vector tool and technical foundation for constructing an efficient and stable antigen display and vaccine delivery platform and has good application potential in vaccine research and the construction of targeted delivery systems.},
}

*Bacteriophage T4/genetics
*CRISPR-Cas Systems
Gene Knockout Techniques
Escherichia coli/genetics/metabolism
Mutation

@article {pmid41454999,
year = {2025},
author = {Vadakkan, K and Raphael, R and Korattil, TT and Mapranathukaran, VO and Ramadas, V and Kizhakkepeedika, RD},
title = {A Critical Review of Quorum-Sensing Inhibition Strategies in Klebsiella Pneumoniae.},
journal = {Current microbiology},
volume = {83},
number = {2},
pages = {103},
pmid = {41454999},
issn = {1432-0991},
abstract = {Klebsiella pneumoniae is an antibiotic-resistant microbe that causes pneumonia, urinary tract infections, bloodstream infections, and liver abscesses. Its pathogenicity is fueled by the polysaccharide capsule, lipopolysaccharides, and quorum-sensing-regulated biofilm formation, which enhance immune evasion and antibiotic resistance. The rise of multidrug-resistant and hypervirulent pathogens poses a significant challenge to treatment. Targeting quorum sensing with quorum quenching is a promising method for disrupting bacterial interaction and reducing virulence without increasing resistance. This review examines the pathogenesis of K. pneumoniae, the significance of quorum sensing, and emerging therapeutics, including quorum-sensing inhibitors and advanced techniques such as CRISPR-Cas and computational drug development, while emphasizing the need for further investigation.},
}

@article {pmid41762232,
year = {2026},
author = {Deepika, and Sharma, S and Kumar, P and Rathore, V and Chauhan, A and Kumar, A and Dogra, RK and Handa, A},
title = {Insights into physiological, biochemical and molecular mechanisms of abiotic stress tolerance in Persian walnut (Juglans regia L.).},
journal = {Protoplasma},
volume = {263},
number = {4},
pages = {1099-1116},
pmid = {41762232},
issn = {1615-6102},
abstract = {Walnut (Juglans regia) is an economically and nutritionally valuable tree species that often encounters diverse abiotic stresses such as drought, salinity, cold, heat and heavy metal toxicity. These stresses adversely affect its growth, productivity and survival by altering physiological functions, disturbing cellular homeostasis and triggering oxidative damage. In response, J. regia deploy a multifaceted adaptive system comprising morphological changes, biochemical adjustments and intricate molecular signaling pathways. The review aims to analyze current knowledge on the physiological, morphological changes observed under stress with biochemical defense mechanism. These include the crucial antioxidant defense system (increase in Superoxide Dismutase (SOD), Peroxidase (POD) and Catalase (CAT) activity), the accumulation of protective soluble solutes and amino acids and the biosynthesis of secondary metabolites through the Methylerythritol Phosphate (MEP) pathway involved in mitigating oxidative stress caused by Reactive Oxygen Species (ROS). Crucially, we synthesize the understanding of molecular regulation that underpins stress adaptation. This encompasses stress-responsive gene expression including Jr (Juglans regia) VHAG1 (V-ATPase H+ transporting ATPase subunit G1), JrDREB (Dehydration-Responsive Element Binding protein), JrRD29 (Responsive to Dehydration 29), transcriptional regulation by myeloblastosis (MYB), Dehydration-responsive element-binding proteins (DREB) and WRKY-TF families and their interaction with hormonal (abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene) which play a pivotal role in signal transduction and stress tolerance. These mechanisms employed by walnut under abiotic stresses, highlighting key genetic and hormonal pathways that can be targeted for the development of stress-resilient cultivars and ensuring sustainable production under changing climatic conditions. Recent advancements in genomics, transcriptomics and proteomics got attention that provides new insights into the regulatory networks and candidate genes conferring stress resilience. Furthermore, it explores biotechnological approaches for improving stress tolerance, highlighting the prospects of latest high-throughput techniques, including molecular breeding, genetic engineering, Next-Generation Sequencing (NGS), microRNA (miRNA)-based regulation and Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas) gene editing. This integrated review connects multi-level stress response mechanisms, highlighting potential molecular markers and biotechnological interventions for accelerating walnut improvement and ensuring sustainable production under changing climatic conditions.},
}

@article {pmid41820648,
year = {2026},
author = {Hanif, N and Naveed, M and Salah Ud Din, M and Aziz, T and Shami, A and Al-Joufi, FA},
title = {Genome-wide characterization and comparative analysis of Bacillus paranthracis MBBL1 reveals genomic plasticity features.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {41820648},
issn = {1438-7948},
support = {PNURSP2026R31//Princess Nourah Bint Abdulrahman University/ ; },
abstract = {Bacillus paranthracis is a member of the Bacillus cereus group with close phylogenetic proximity to pathogenic species, yet its genomic diversity and pathogenic potential remain poorly understood. In this study, we present a comprehensive whole-genome analysis of B. paranthracis strain MBBL1, an environmental isolate, to explore its genomic architecture, evolutionary relationships, and potential virulence traits. Comparative genomic and pangenome analyses revealed that MBBL1 clusters closely with other B. paranthracis strains while maintaining measurable genomic similarity to B. cereus and B. anthracis. Notably, the genome lacks plasmids and known serotype markers, distinguishing it from many pathogenic members of the group. Despite this, MBBL1 harbors multiple chromosomally encoded virulence-associated genes, mobile genetic elements, and genomic islands, indicating considerable genomic plasticity. The presence of an active Type I CRISPR-Cas system may contribute to genome stability and the absence of plasmids. Pathogenicity prediction suggests a low but detectable potential for human pathogenicity. Collectively, these findings highlight the importance of environmental Bacillus strains as reservoirs of hidden virulence traits and provide a valuable genomic framework for future functional and risk assessment studies.},
}

@article {pmid41964738,
year = {2026},
author = {Karwa, P and Kharul, A and Parekar, V and Labhade, S and Sakle, N and Bhole, R and Kapare, H},
title = {Next-Generation Therapies for Genetic Diseases: The Synergy of CRISPR and Gene Therapy.},
journal = {Biochemical genetics},
volume = {},
number = {},
pages = {},
pmid = {41964738},
issn = {1573-4927},
abstract = {The introduction of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has changed the game of gene therapy and promises surgical precision and efficacy in the process of addressing genetic disorders in humans. Gene therapy that commonly assumes insertion of functional DNA with the help of viral vectors has developed or rather undergone adaptation with the introduction of CRISPR-Cas mechanisms that make it possible to edit the genome, correct or regulate it and silence. It is a critical study that reviews mechanistic disparities among traditional and CRISPR bases types of gene therapy, based on benefits, shortcomings, and states of improvement till date, as far as clinical development is concerned. It talks about their historical evolution, molecular principles, delivery modes, and therapeutic promise of the CRISPR systems such as Cas9, Cas12, Cas13, base editing, and prime editing. It is of significant value to mention the application of CRISPR as a treatment in monogenic illnesses such as 2-thalassemia and Duchenne muscular dystrophy as well as the future use of CRISPR in complex and multifactorial diseases. Also, safety concerns, ethical issues, and delivery translational problems are discussed by the review and they are paramount to successful clinical translation of CRISPR-based therapeutics. The discussion highlights the revolutionary possibility of CRISPR in precision medicine and predetermines its expansion in the future healthcare genomic practice.},
}

@article {pmid42105238,
year = {2026},
author = {Wang, DM and Tiruppathi, C},
title = {Protocol to generate endothelial cell-specific knockout mouse models using Cas9/Cdh5-Cre mice coupled with sgRNA.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104551},
pmid = {42105238},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Endothelial Cells/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mice, Knockout ; Integrases/genetics/metabolism ; Cadherin 5 ; *Cadherins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Antigens, CD/genetics/metabolism ; *Gene Knockout Techniques/methods ; },
abstract = {The vascular endothelium is a critical regulator of vascular homeostasis and tissue fluid balance, and mouse models are essential for studying these processes in vivo. Here, we present a protocol to generate adult endothelial cell (EC)-specific gene knockout (KO) mouse models. We describe steps for Cas9-active and Cdh5-Cre-positive (Cas9/Cdh5-Cre) mouse line generation, single guide RNA (sgRNA) design for vector construction, plasmid DNA generation, and liposome preparation. We then detail procedures for liposome/plasmid complex injection, lung harvest, homogenization, protein quantification, and verification with western blotting. For complete details on the use and execution of this protocol, please refer to Wang et al.[1].},
}

Animals
Mice
*Endothelial Cells/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics
Mice, Knockout
Integrases/genetics/metabolism
Cadherin 5
*Cadherins/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Antigens, CD/genetics/metabolism
*Gene Knockout Techniques/methods

@article {pmid42163310,
year = {2026},
author = {Jiang, Y and Caban, KM and Peitzsch, M and Herrmann, C and Mayr, D and Stöckl, JB and Fröhlich, T and Mayerhofer, A and Müller-Taubenberger, A and Welter, H},
title = {Knockout of filamin A in KGN granulosa tumor cells impairs proliferation, cell cycle progression, migration, and cytoskeletal organization under mechanical stress.},
journal = {Biological research},
volume = {59},
number = {1},
pages = {},
pmid = {42163310},
issn = {0717-6287},
mesh = {*Filamins/genetics/metabolism ; Humans ; Female ; *Cell Movement/physiology ; *Cell Proliferation/physiology ; *Granulosa Cell Tumor/pathology/metabolism/genetics ; *Cytoskeleton/physiology ; Stress, Mechanical ; *Cell Cycle/physiology ; Cell Line, Tumor ; Immunohistochemistry ; *Ovarian Neoplasms/pathology ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Cell Adhesion ; },
abstract = {BACKGROUND: Filamin A (FLNA) is an actin-binding protein that regulates mechanosensitivity and functions as an intracellular signaling scaffold in various cell types. It has also been implicated in tumor growth. We recently reported FLNA expression in human ovarian granulosa cells and in KGN cells, a granulosa cell tumor (GCT) line.

RESULTS: Immunohistochemistry analysis of 51 GCT samples revealed heterogeneous FLNA expression, with approximately 20% showing weak, 18% strong, and the majority moderate expression. We therefore conducted functional studies in KGN cells using CRISPR/Cas9 gene editing. A proteomic approach revealed marked changes in protein abundance upon FLNA depletion: proteins with increased abundance were predominantly related to adhesion, cytoskeletal organization, regulation of cell shape, and lipid metabolic process, whereas those with decreased abundance were associated with DNA replication, cell division, and cell cycle regulation. FLNA-knockout cells showed enlarged cell sizes, reduced proliferation, and slightly affected steroidogenesis. Disruption of FLNA further reduced migration velocity, altered actin cytoskeletal alignment under flow, and modified expression of genes involved in cytoskeletal architecture, adhesion, and mechanosensing under shear stress.

CONCLUSIONS: Our results identify crucial roles of FLNA in shaping the cellular architecture, motility, and proliferation of KGN cells. Consequently, alterations in FLNA expression may influence intracellular signaling, and responsiveness to mechanical cues in both physiological and pathological contexts.},
}

*Filamins/genetics/metabolism
Humans
Female
*Cell Movement/physiology
*Cell Proliferation/physiology
*Granulosa Cell Tumor/pathology/metabolism/genetics
*Cytoskeleton/physiology
Stress, Mechanical
*Cell Cycle/physiology
Cell Line, Tumor
Immunohistochemistry
*Ovarian Neoplasms/pathology
CRISPR-Cas Systems
Gene Knockout Techniques
Cell Adhesion

@article {pmid42172127,
year = {2026},
author = {Hong, JP and Nussenzweig, MC},
title = {Protocol for an in vivo CRISPR screen for germinal center B cells in mice using ecotropic retrovirus.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104586},
pmid = {42172127},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Germinal Center/cytology/immunology ; *B-Lymphocytes/metabolism/cytology ; *Retroviridae/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The lack of an in vitro model that recapitulates germinal center (GC) B cell biology necessitates the use of animal models for genetic studies. Here, we present a protocol for an in vivo CRISPR screen for GC B cells in mice using an ecotropic retrovirus. We describe steps for constructing a single guide (sgRNA) library and performing a genetic screen in a mouse model of protein immunization, including procedures for sequencing and data analysis.},
}

Animals
Mice
*Germinal Center/cytology/immunology
*B-Lymphocytes/metabolism/cytology
*Retroviridae/genetics
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42213768,
year = {2026},
author = {Hassan, AZ and Zhang, X and Ward, HN and Billmann, M and Bajjali, S and Brown, KR and Bhojoo, U and Chan, K and Lin, K and Costanzo, M and Andrews, B and Boone, C and Moffat, J and Myers, CL},
title = {Orobas: A computational approach for scoring and analysis of quantitative chemical-genetic interactions from CRISPR-Cas9 screens.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104594},
pmid = {42213768},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Computational Biology/methods ; *Software ; },
abstract = {We present Orobas, a computational approach for transforming raw read count data from CRISPR-Cas9 chemical-genetic screens into quantitative interaction scores. We describe steps for computing differential interaction scores with statistical tests that account for multiple CRISPR guides per gene. We then outline approaches for post-processing differential log2-fold-change scores across multiple screens, incorporating normalization to reduce technical artifacts and correct batch effects.},
}

*CRISPR-Cas Systems/genetics
*Computational Biology/methods
*Software

@article {pmid42213769,
year = {2026},
author = {Li, Z and Roberts, A and Nimse, U and Vu, L and Fei, J},
title = {Protocol for generating endogenous degron tags in essential transcription factors in human iPSCs via CRISPR-Cas9.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104602},
pmid = {42213769},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/genetics/metabolism ; Degrons ; *Gene Editing/methods ; Gene Knock-In Techniques/methods ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Precise genome engineering in human pluripotent stem cells remains inefficient, limiting endogenous fluorescent tagging needed to study phase-separated membraneless nuclear compartments. Here, we present a protocol for generating precise knockin alleles of essential genes in human induced pluripotent stem cells (iPSCs) via CRISPR-Cas9 editing. We describe steps for combining transient p53 inhibition, optimized transfection conditions, and fluorescence-activated cell sorting. This protocol enables recovery of viable edited clones that would otherwise be lost due to editing-induced stress or essential gene perturbation. For complete details on the use and execution of this protocol, please refer to Li et al.[1].},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Transcription Factors/genetics/metabolism
Degrons
*Gene Editing/methods
Gene Knock-In Techniques/methods
Tumor Suppressor Protein p53/genetics

@article {pmid42226724,
year = {2026},
author = {Jong, U and Lim, D and Lee, SH},
title = {Prime editing updates: technological evolution, methodological expansion, and delivery strategies for in vivo applications.},
journal = {BMB reports},
volume = {59},
number = {6},
pages = {313-320},
pmid = {42226724},
issn = {1976-670X},
mesh = {*Gene Editing/methods/trends ; CRISPR-Cas Systems/genetics ; Humans ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Prime editing is a next-generation genome editing technology that enables precise base substitutions, insertions, and deletions at target genomic loci without inducing double-strand breaks or requiring exogenous donor DNA. This system operates through a fusion protein composed of Cas9 nickase and reverse transcriptase together with prime editing guide RNA; it has emerged as a precise genome editing platform that overcomes the limitations of conventional double-strand break-inducing CRISPR-Cas9 systems. Since first reported in 2019, diverse methodological improvements from PE1 to PE7 have been achieved, leading to rapid advances in editing efficiency, expansion of the editable target range, correction of large genomic regions, and development of in vivo delivery technologies. In this review, we comprehensively discuss the fundamental working mechanism of prime editing, its methodological evolution, recent expansion strategies, and delivery platforms for therapeutic applications, and provide perspectives for future development. [BMB Reports 2026; 59(6): 313-320].},
}

*Gene Editing/methods/trends
CRISPR-Cas Systems/genetics
Humans
Animals
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid42264452,
year = {2026},
author = {Li, Y and Du, H and Chen, J and Xiao, J and Ma, Y and Zhang, Y and Wang, Q},
title = {The development of RPA-CRISPR/Cas12a assay for the detection of Pseudomonas plecoglossicida.},
journal = {FEMS microbiology letters},
volume = {373},
number = {},
pages = {},
doi = {10.1093/femsle/fnag067},
pmid = {42264452},
issn = {1574-6968},
support = {T2023328//Shanghai Agricultural Science and Technology Innovation Project/ ; //MOF/ ; },
mesh = {*Pseudomonas/genetics/isolation & purification ; Animals ; *Fish Diseases/microbiology/diagnosis ; *CRISPR-Cas Systems ; *Pseudomonas Infections/veterinary/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Perciformes/microbiology ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; Bacterial Proteins/genetics ; DNA, Bacterial/genetics ; Recombinases/metabolism ; },
abstract = {Pseudomonas plecoglossicida is the etiological agent of visceral white spot disease, which induces significant mortality in economically important fish such as the large yellow croaker. In this study, we integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection to establish a fluorescence-based assay for rapid identification of P. plecoglossicida. The complete single-tube, two-stage RPA-CRISPR/Cas12a workflow can be performed within ~45 min. Using purified genomic DNA, the assay achieved an analytical detection limit of 1.65 copies μl-1 and showed no cross-reactivity with several other common fish pathogens. Its applicability was further evaluated using crude DNA extracts from spleen, liver, and kidney tissues of experimentally infected large yellow croakers. Overall, with its rapid turnaround, minimal equipment requirement, and high sensitivity, the RPA-CRISPR/Cas12a assay represents a promising diagnostic tool for rapid detection of P. plecoglossicida, thereby helping to control the spread of infection.},
}

*Pseudomonas/genetics/isolation & purification
Animals
*Fish Diseases/microbiology/diagnosis
*CRISPR-Cas Systems
*Pseudomonas Infections/veterinary/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Perciformes/microbiology
Rapid Diagnostic Tests
Sensitivity and Specificity
Bacterial Proteins/genetics
DNA, Bacterial/genetics
Recombinases/metabolism

@article {pmid42265371,
year = {2026},
author = {Su, F and Dong, Y and Guo, R and Xie, H and Zhang, Y and Liu, J and Cao, X and Xie, H and Zhou, M and Sun, X and Wang, M and Zhang, J and Zhu, JK},
title = {Efficient prime editors for heritable multiplex precision genome editing in soybean.},
journal = {Nature plants},
volume = {12},
number = {6},
pages = {1252-1263},
pmid = {42265371},
issn = {2055-0278},
support = {32188102//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Glycine max/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; CRISPR-Cas Systems ; Genetic Engineering/methods ; RNA Editing ; },
abstract = {Prime editing (PE) is limited by low efficiency in dicot plants. Here we develop an optimized PE system for soybean, GmPEplus, by deleting the RNase H domain, introducing a V223A substitution within the reverse transcriptase domain, inserting a viral nucleocapsid protein between Cas9 and reverse transcriptase, and co-expressing a dominant-negative engineered allele of the endogenous GmMLH1. GmPEplus achieves editing efficiencies of up to 81.3% in stable transgenic lines. Subsequently, we show that nicking the non-edited strand using an additional sgRNA via the tRNA processing system enhances editing efficiency, and that optimizing its expression with an independent AtU6 cassette boosts efficiency by 13.1-fold. Importantly, a Csy4-mediated multiplex PE system (CMMPE) is established for simultaneous editing of 2-12 genes in soybean hairy roots and up to 3 genes in stable transgenic lines. GmPEplus and CMMPE offer powerful, versatile tools for precise, multiplex and heritable genome editing in soybean breeding.},
}

*Glycine max/genetics
Plants, Genetically Modified/genetics
*Genome, Plant
CRISPR-Cas Systems
Genetic Engineering/methods
RNA Editing

@article {pmid42267397,
year = {2026},
author = {Xie, X and Meng, X and Han, B and Zhang, X and Qiu, M and Zhang, J and Zhao, F and Jiang, Y and Zhang, X},
title = {A Near-Infrared Light-Driven Photoelectrochemical Biosensor Based on CRISPR/Cas12a for Highly Sensitive Detection of Pseudomonas fluorescens in Dairy Products.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {24},
pages = {19094-19104},
doi = {10.1021/acs.jafc.6c01081},
pmid = {42267397},
issn = {1520-5118},
mesh = {*Pseudomonas fluorescens/genetics/isolation & purification/radiation effects ; *Biosensing Techniques/methods/instrumentation ; *Electrochemical Techniques/methods/instrumentation ; *Dairy Products/microbiology/analysis ; CRISPR-Cas Systems ; Infrared Rays ; Food Contamination/analysis ; Gold/chemistry ; Limit of Detection ; },
abstract = {Pseudomonas fluorescens (P. fluorescens) is a psychrotrophic bacterium causing dairy spoilage. Plate count and PCR methods, as conventional detection standards, are either time-consuming or dependent on expensive instruments, restricting widespread application. Herein, a near-infrared (NIR)-driven photoelectrochemical (PEC) biosensor based on CRISPR/Cas12a was constructed by using SH-ssDNA/AuNPs/AgBiS2 as the working electrode. AgBiS2 possesses broad-spectrum absorption and an appropriate bandgap for efficient NIR utilization. In the presence of P. fluorescens, the CRISPR/Cas12a system was activated by the LAMP amplification products of the target DNA to perform the trans-cleavage activity toward the SH-ssDNA modified on the AuNPs/AgBiS2 electrode surface. This cleavage released SH-ssDNA from the electrode, reducing steric hindrance and accelerating interfacial electron transfer, thereby enhancing the PEC signal. The constructed biosensor achieved sensitive detection of P. fluorescens with a limit of detection (LOD) of 1 CFU/mL in the range of 10[1]-10[8] CFU/mL, offering a reliable strategy for detecting the psychrotrophic bacteria in dairy products.},
}

*Pseudomonas fluorescens/genetics/isolation & purification/radiation effects
*Biosensing Techniques/methods/instrumentation
*Electrochemical Techniques/methods/instrumentation
*Dairy Products/microbiology/analysis
CRISPR-Cas Systems
Infrared Rays
Food Contamination/analysis
Gold/chemistry
Limit of Detection

@article {pmid42274413,
year = {2026},
author = {Liu, L and Wu, M and Sun, X and Lu, X and Yuan, C and Li, CY},
title = {Hierarchical Amplification-Interlinked CRISPR-Cas14a Luminescent Biosensor Coupled with Portable Photonic Crystal Biochip-Boosted Time-Delayed Signaling for the Diagnosis of Pediatric Mycoplasma pneumoniae Pneumonia.},
journal = {Analytical chemistry},
volume = {98},
number = {24},
pages = {18288-18298},
doi = {10.1021/acs.analchem.6c03088},
pmid = {42274413},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Pneumonia, Mycoplasma/diagnosis ; *Mycoplasma pneumoniae/isolation & purification/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Luminescent Measurements ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Luminescence ; Rapid Diagnostic Tests ; },
abstract = {Mycoplasma pneumoniae pneumonia (MPP) is a respiratory infection that readily propagates within pediatric populations, and thus, the development of a robust assay approach for its timely diagnosis is of clinical importance. In this contribution, we build a hierarchical amplification-interlinked CRISPR-Cas14a luminescent biosensor coupled with portable photonic crystal biochip-boosted time-delayed signaling to fulfill this need. To first perform highly sensitive detection, an initial DNA walker module and a later rolling circle amplification module are integrated to construct a hierarchical amplification, which is then utilized to interlink CRISPR-Cas14a systems. To further improve applicability in complicated biosamples, a time-delayed signaling actualized by afterglow luminescence is introduced to circumvent background autoluminescence from biological media. After employing a photonic crystal self-assembled from polystyrene nanospheres to manufacture a portable biochip, the naturally attenuated afterglow luminescence is dramatically boosted. Leveraging these strategies, our biosensing platform achieves a limit of detection as low as 2.27 fM for the M. pneumoniae sequence while maintaining good specificity. Moreover, the luminescent biosensor permits precise analysis of targets in throat swab samples from a pediatric cohort (n = 250) comprising severe and mild MPP patients as well as healthy controls, based on which a convolutional neural network-implemented deep learning model is finally established for accurate automated disease stratification, thereby holding great potential as an efficient and promising diagnostic tool.},
}

*Biosensing Techniques/methods
*Pneumonia, Mycoplasma/diagnosis
*Mycoplasma pneumoniae/isolation & purification/genetics
Humans
*CRISPR-Cas Systems/genetics
Luminescent Measurements
Nucleic Acid Amplification Techniques
Limit of Detection
Luminescence
Rapid Diagnostic Tests

@article {pmid42299927,
year = {2026},
author = {Xia, Q and Guo, R and Xiao, J and Zeng, L and Huang, Y and Li, J and Chen, X and Huang, T and Xiao, B and Miao, C and Liu, W and Liang, QL and Lau, CH and Zhu, H},
title = {A rapid, multiplex, one-pot CRISPR/Dx system for visual detection of influenza A, influenza B, and respiratory syncytial viruses.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {24},
pages = {5052-5060},
doi = {10.1039/d6ay00425c},
pmid = {42299927},
issn = {1759-9679},
mesh = {*Influenza B virus/isolation & purification/genetics ; *Influenza A virus/isolation & purification/genetics ; Humans ; *CRISPR-Cas Systems ; *Respiratory Syncytial Viruses/isolation & purification/genetics ; Rapid Diagnostic Tests ; Respiratory Syncytial Virus Infections/diagnosis/virology ; },
abstract = {The detection of Influenza A Virus (IAV), Influenza B Virus (IBV), and Respiratory Syncytial Virus (RSV) presents significant diagnostic challenges due to the high similarity of clinical symptoms with other respiratory infections, leading to the need for multiplexed, rapid testing. Herein, we have established a one-pot, multiplex CRISPR/Dx detection system based on reverse transcription-recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a. It completes the detection within 30 minutes at a constant temperature of 40 °C, with the ability to detect 10 copies per µL of IAV, 10 copies per µL of IBV, and 8 copies per µL of RSV. No cross-reactivity between these respiratory viruses was observed. When equipped with our customized miniature device, it allows visual fluorescence readout without specialized equipment. Compared with conventional RT-qPCR and two-tube RT-RPA-CRISPR/Cas12a approaches, this one-pot detection system offers a simplified workflow and shorter detection time and enables visual detection, making it especially suitable for point-of-care testing and field deployment. In essence, our CRISPR/Dx system provides a novel and practical molecular diagnostic strategy for rapid and multiplex detection of respiratory pathogens to improve patient management, rational antiviral use, and epidemic control.},
}

*Influenza B virus/isolation & purification/genetics
*Influenza A virus/isolation & purification/genetics
Humans
*CRISPR-Cas Systems
*Respiratory Syncytial Viruses/isolation & purification/genetics
Rapid Diagnostic Tests
Respiratory Syncytial Virus Infections/diagnosis/virology

@article {pmid42325637,
year = {2026},
author = {Adegoke, SC and Karim, MA and Jr, MC and Yao Yawlui, IS and LaJeunesse, D},
title = {Advancements in Technologies Targeting Horizontal Gene Transfer(?)Routes to Control Drug Resistance Evolution.},
journal = {ACS bio & med chem Au},
volume = {6},
number = {3},
pages = {210-236},
pmid = {42325637},
issn = {2694-2437},
abstract = {The global rise of multidrug-resistant (MDR) bacteria poses a major public health crisis, threatening the effectiveness of modern medicine. Traditional antibiotic development struggles to keep pace with bacterial evolution, largely due to the rapid dissemination of antibiotic resistance genes via horizontal gene transfer (HGT). HGT mechanisms both canonical and noncanonical enable bacteria to acquire resistance traits defining species and even special challenges. In this review, we cover the current understanding of HGT in spreading antibiotic resistance and explore possible strategies to control HGT and slow the spread of antimicrobial resistance. Recent advances highlight the potential of synthetic competence inhibitors, advanced oxidation processes (AOPs), CRISPR-Cas technologies, gene drives, and antiplasmids to disrupt horizontal gene flow and mitigate resistance evolution. Despite promising laboratory results, challenges remain in translating these approaches into clinical and environmental applications. Blocking HGT could complement antimicrobial stewardship programs and traditional antibiotic therapies by curbing the emergence of new resistant strains at their genetic roots. By targeting the foundational mechanisms of resistance acquisition, these strategies offer a proactive pathway to extend the efficacy of existing antibiotics and prevent a "postantibiotic" era. Ongoing research into bacterial pathogenesis, genome defense systems, and innovative gene-editing technologies will be critical to developing effective, scalable solutions for managing MDR infections worldwide.},
}

@article {pmid42326420,
year = {2026},
author = {Alzahrani, AJ},
title = {Bacteriophage therapy against multidrug resistant bacterial infections demonstrates clinical advances and engineering innovations between 2020-2026.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1865548},
pmid = {42326420},
issn = {1664-302X},
abstract = {PURPOSE: The global crisis of antimicrobial resistance has reached critical levels, with multi-drug resistant (MDR) bacterial pathogens threatening to render conventional antibiotics ineffective. This mini review synthesizes recent evidence from 2020 to 2026 on bacteriophage therapy against MDR bacteria, examining clinical applications, engineering advances, mechanistic insights, and emerging technologies.

METHODS: A comprehensive literature search was conducted across Embase, Scopus, and Cochrane Library databases, supplemented by PubMed, Google Scholar, and ArXiv searches, focusing on phage therapy for MDR bacterial infections published between 2020 and 2026.

RESULTS: Recent developments include expanded clinical experience through specialized phage centers, sophisticated genetic engineering techniques including CRISPR-based systems, successful compassionate-use programs, and innovative combination therapies with antibiotics. Clinical reports demonstrate safety and preliminary efficacy signals in selected refractory infections, though randomized controlled data remain limited. Engineering advances have produced phage-delivered CRISPR antimicrobials, hybrid delivery platforms, and synthetic phage particles that expand therapeutic capabilities.

CONCLUSION: While challenges remain in regulatory standardization, scalable manufacturing, and resistance management, the field has demonstrated significant progress toward clinical translation. The convergence of synthetic biology, personalized medicine approaches, and growing clinical evidence positions phage therapy as a viable complementary strategy in the fight against MDR bacterial infections.},
}

@article {pmid42328080,
year = {2026},
author = {Singh, AP and Haider, S and Sawarkar, A and Karki, S and Atta, K and Al-Zharani, M and Barasarathi, J and Rebouh, NY and Ahmed, ZFR},
title = {Programmable nanocarriers for precision plant engineering: converging nanotechnology, CRISPR, and next-generation breeding.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1845328},
pmid = {42328080},
issn = {1664-462X},
abstract = {The convergence of nanotechnology and genome editing in plant sciences is redefining modern precision breeding through efficient, transgene free, tissue culture independent pathways for genetic improvement in crops. Conventional breeding and transgenic tools are limited to genotype dependency, inefficient gene delivery, unpredictable transgene insertions, thereby restricting their application in elite germplasm. Nanoparticles-mediated gene delivery systems have revolutionized the genetic transformation in plants through targeted and transgene free delivery of CRISPR/Cas ribonucleoproteins (RNPs), DNA, and RNA into plant cells, while minimizing genome interference. Nanocarriers are the engineered delivery systems wherein the material component is a nanoparticle. DNA-free delivery refers to the absence of exogenous DNA during editing, whereas transgene free plants are those that do not retain integrated foreign DNA after regeneration. Firstly, this review summarizes current progress in designing nanocarriers, including lipid, polymeric, mesoporous silica nanoparticles, carbon-based nanoparticles, layered double hydroxides, and DNA-based nanoparticles; harnessing the function of their physicochemical traits in modulating plant cellular uptake, cargo stability, controlled delivery, and tissue specific targeting in plants. Secondly, the broad-spectrum roles of nano particles in genome editing, crop protection via RNA interference, organelle-targeted modifications are discussed, stressing transgene free approaches to mitigate somaclonal variation and regulatory concerns to foster public acceptance. The integration of nano-mediated delivery with speed breeding, meristem transformation, multiplexed editing in elite germplasm is proposed as an approach for prompt trait stacking and validation. Thirdly, the collaborative roles of experts in the field of nanotechnology, plant breeding, plant physiology, and agronomy are mentioned for mitigating multifaceted climatic effects and glitches. Moreover, current challenges including nanotoxicity, scalability and field translation, regulatory concerns, and public perception are also discussed. While nanocarrier mediated delivery shows strong potential for improving plant genome engineering, current evidence is largely confined to controlled experimental systems, and significant challenges remain before routine integration into breeding pipelines becomes feasible.},
}

@article {pmid42328450,
year = {2026},
author = {Corno, C and Costantino, M and Pettinari, P and Mirra, L and Stucchi, S and Arrighetti, N and Perta, N and Di Muccio, G and Robin, M and Beretta, GL and Corna, E and Carenini, N and Cleris, L and Colombo, D and Luison, E and Ciniselli, CM and Lecchi, M and Verderio, P and Figini, M and Linder, S and Tosi, D and La Teana, A and D'Arcy, P and Perego, P},
title = {USP18 Impacts Cisplatin Resistance in Ovarian Cancer Cells by Modulating DNA Repair.},
journal = {International journal of biological sciences},
volume = {22},
number = {11},
pages = {5780-5798},
pmid = {42328450},
issn = {1449-2288},
mesh = {Female ; *Cisplatin/therapeutic use/pharmacology ; Humans ; *Ovarian Neoplasms/metabolism/drug therapy/genetics ; *DNA Repair/genetics/drug effects ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; Animals ; *Ubiquitin Thiolesterase/metabolism/genetics ; Mice ; CRISPR-Cas Systems ; Antineoplastic Agents/therapeutic use/pharmacology ; DNA Damage ; },
abstract = {Deubiquitinases (DUBs) are proteases with emerging roles in cancer, yet their contribution to drug resistance in ovarian cancer remains underexplored. Ovarian cancer patients often fail to benefit from platinum-based therapy, highlighting the need to identify novel factors driving drug resistance. Thus, we performed a CRISPR/Cas9 screen targeting the DUB family to identify genes essential for cisplatin-resistant ovarian carcinoma cell survival. CRISPR/Cas9 DUB knockout screens, preclinical pharmacology approaches, RNA sequencing, proteomic analyses, computational tools, surface plasma resonance were applied. We identified USP18 as a survival factor in cisplatin-resistant ovarian cancer cells. USP18 expression was elevated at the mRNA and protein levels across five cisplatin-resistant variants. Knockdown and CRISPR/Cas9 editing of USP18 sensitized cells to cisplatin, coinciding with impaired repair of cisplatin-induced DNA damage. Enhanced sensitivity to cisplatin was evident from studies in mice. RNA-seq of USP18 RNA interfered and edited cells revealed the modulation of pathways including DNA repair. A peptide-based USP18 inhibitor suppressed growth of cisplatin-resistant cells, supporting USP18 role in sustaining their growth. We identified USP18 as a novel mediator of cisplatin resistance in ovarian cancer, acting through DNA repair modulation. Targeting USP18 may offer a therapeutic strategy to improve outcomes in platinum-resistant ovarian cancer.},
}

Female
*Cisplatin/therapeutic use/pharmacology
Humans
*Ovarian Neoplasms/metabolism/drug therapy/genetics
*DNA Repair/genetics/drug effects
*Drug Resistance, Neoplasm/genetics
Cell Line, Tumor
Animals
*Ubiquitin Thiolesterase/metabolism/genetics
Mice
CRISPR-Cas Systems
Antineoplastic Agents/therapeutic use/pharmacology
DNA Damage

@article {pmid42328522,
year = {2026},
author = {Wang, W and Zheng, S and Xiang, G and Feng, T and Peng, Y and Wu, Y and Du, X and Zhu, P and Ru, Y and Zhang, J and Wu, S and Han, H and Huang, T and Cao, G and Zheng, H and Zhao, Y and Wang, H and Zhang, R},
title = {Robust production of heavy-chain-only antibodies in mice by CRISPR/Cas mediated in situ modification of IgH locus.},
journal = {National science review},
volume = {13},
number = {11},
pages = {nwag270},
pmid = {42328522},
issn = {2053-714X},
}

@article {pmid42328613,
year = {2025},
author = {Selim, HMRM and Gomaa, FAM and Alshahrani, MY and Aboshanab, KM},
title = {Role of CRISPR-Cas system as a new approach in fighting the antimicrobial resistance of bacterial and viral pathogens.},
journal = {Infectious diseases & immunity},
volume = {5},
number = {2},
pages = {127-137},
pmid = {42328613},
issn = {2693-8839},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Caspase (Cas) system acts as a natural defense of bacteria against invasion by mobile genetic elements, such as plasmids, transposons, and bacteriophages. The review discusses the different classes and types of CRISPR-Cas systems in terms of principles of their action, limitations, and future perspectives. Also, the role of the CRISPR-Cas system as a new arsenal in fighting multidrug-resistant pathogens and clinically relevant pathogenic viruses and up-to-date clinical trials have been discussed and highlighted. Moreover, the utilization mode, regulation, and the link of CRISPR-Cas to quorum sensing for targeting cell chromosome or antimicrobial-resistant gene(s) of some clinically relevant pathogens, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa have been discussed. The information provided in this review can act as a platform for researchers for the future use of the CRISPR-Cas system as a smart antimicrobial agent for combating infections caused by life-threatening pathogens, particularly those with limited therapeutic options.},
}

@article {pmid41061927,
year = {2026},
author = {Sun, H and Zou, J and Tu, S and Luo, D and Xiao, R and Du, Y and Xiong, C and Xie, S and Liu, H and Jin, M and Chen, H and Zhou, H},
title = {Genome-wide CRISPR screen identifies STK11 as a critical regulator of sialic acid clusters important for influenza A virus attachment.},
journal = {Journal of advanced research},
volume = {85},
number = {},
pages = {1207-1222},
doi = {10.1016/j.jare.2025.09.059},
pmid = {41061927},
issn = {2090-1224},
mesh = {Animals ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Virus Replication ; Swine ; *Influenza A virus/physiology ; CRISPR-Cas Systems ; *N-Acetylneuraminic Acid/metabolism ; *Virus Attachment ; AMP-Activated Protein Kinase Kinases ; Dogs ; Mice ; *Orthomyxoviridae Infections/virology/genetics ; Host-Pathogen Interactions ; Humans ; },
abstract = {INTRODUCTION: Swine influenza virus (SIV) is a highly contagious respiratory pathogen in pigs that causes substantial economic losses in the pig industry. Importantly, pigs act as "mixing vessels" for diverse influenza A viruses (IAVs), facilitating the emergence of novel pandemic strains through reassortment, which represents a continuous global public health threat. IAV replication relies heavily on host cellular machinery, underscoring the importance of elucidating virus-host protein interactions for the development of targeted antiviral therapeutics.

OBJECTIVES: This study aims to identify host genes required for SIV replication via a genome-wide CRISPR screen and elucidate the mechanism by which STK11 modulates viral replication.

METHODS: A pig genome-scale CRISPR knockout (PigGeCKO) screen was performed in newborn pig trachea (NPTr) cells to identify host genes required for SIV replication. Candidate genes were further validated by generating knockout cell lines using CRISPR/Cas9-mediated gene editing, followed by assessing their impact on IAV replication. The specific lifecycle stage regulated by STK11 and its mechanistic role in viral attachment were determined via Western blotting, confocal microscopy, transmission electron microscopy, and stimulated emission depletion (STED) imaging. In vivo validation of STK11 knockdown effects on IAV replication was conducted in BALB/c mice treated with STK11-targeting siRNA, with outcomes evaluated by survival analysis, body weight monitoring, lung viral titers quantification, immunofluorescence, and histopathology.

RESULTS: STK11 promotes replication of different IAV subtypes in vitro, and STK11 knockdown significantly suppresses SIV replication in vivo. Mechanistically, STK11 depletion impairs viral attachment by altering the organization of sialic acid clusters, mediated through reduced intracellular actin stress fibers via inhibition of RhoA signaling pathway.

CONCLUSION: We identify STK11 as a novel regulator of IAV attachment and elucidate its mechanistic role in facilitating viral entry. These findings highlight the potential of STK11 to serve as an ideal antiviral target against IAV infection.},
}

Animals
*Protein Serine-Threonine Kinases/genetics/metabolism
Virus Replication
Swine
*Influenza A virus/physiology
CRISPR-Cas Systems
*N-Acetylneuraminic Acid/metabolism
*Virus Attachment
AMP-Activated Protein Kinase Kinases
Dogs
Mice
*Orthomyxoviridae Infections/virology/genetics
Host-Pathogen Interactions
Humans

@article {pmid41692276,
year = {2026},
author = {Piseddu, I and Endres, R and Lanzl, F and Hammann, L and Bérouti, M and Thaler, M and Fahr, L and Fischer, H and Varlamova, V and Gärtig, J and Nixdorf, D and Layritz, P and Marx, C and Hörth, C and Witte, C and Bulut, A and Illig, D and Senz, AM and Holdt, L and Regel, I and Gottschlich, A and Subklewe, M and Mayerle, J and Anz, D and Kobold, S and Linder, A and Hornung, V},
title = {STING Ablation in T Cells Is Required for the Efficacy of STING Agonists in CAR-T Cell Immunotherapy of Pancreatic Cancer.},
journal = {Gastroenterology},
volume = {171},
number = {1},
pages = {50-65},
doi = {10.1053/j.gastro.2026.01.031},
pmid = {41692276},
issn = {1528-0012},
mesh = {Animals ; *Pancreatic Neoplasms/therapy/immunology/pathology/genetics/metabolism ; STING Protein ; *Membrane Proteins/agonists/genetics/metabolism ; *Immunotherapy, Adoptive/methods ; Humans ; *Receptors, Chimeric Antigen/immunology/metabolism/genetics ; Cell Line, Tumor ; cGAS-STING Signaling Pathway ; Mice ; Xenograft Model Antitumor Assays ; T-Cell Exhaustion ; Tumor Microenvironment/immunology ; Interferon-gamma/metabolism ; *T-Lymphocytes/immunology/metabolism/transplantation/drug effects ; Tumor Necrosis Factor-alpha/metabolism ; Cell Proliferation ; CRISPR-Cas Systems ; Signal Transduction ; Female ; Immunologic Memory ; },
abstract = {BACKGROUND & AIMS: Chimeric antigen receptor (CAR) T cells have shown great potential in hematological cancers, but lack efficacy in solid tumors, highlighting the need for novel strategies. Stimulator of interferon genes (STING) activation was shown to inflame the tumor microenvironment, but combination of STING agonists and CAR-T cells might be limited by detrimental outcomes of T cell-intrinsic STING activation. In this study, we evaluated the potential of combining STING agonists and CAR-T cells in the context of pancreatic cancer.

METHODS: We assessed the synergy of CRISPR-Cas9-edited CAR-T cells and the STING agonist diABZI within a T cell exhaustion model in vitro and both xenograft and syngeneic mouse models in vivo.

RESULTS: Combination of STING-ablated CAR-T cells and diABZI resulted in enhanced cancer cell killing, increased CAR-T cell proliferation, reduced exhaustion, and expansion of an effector-memory phenotype in vitro. Mechanistically, superior CAR-T cell functionality required genetic ablation of STING in CAR-T cells and was dependent on cancer cell-intrinsic STING signaling on STING-agonistic treatment. Moreover, we identified a synergistic feedback loop comprising the T cell-secreted cytokines interferon-γ and tumor necrosis factor, which prime STING signaling within cancer cells, thereby potentiating the outcomes of cancer cell-intrinsic STING activation in inducing ameliorated CAR-T cell states. Ultimately, we could demonstrate that combination of STING deficient CAR-T cells and diABZI was able to provide enhanced tumor control in both xenograft and syngeneic mouse models. This was accompanied by increased intratumoral CAR-T cell numbers and reprogramming of the tumor microenvironment in vivo.

CONCLUSIONS: Our findings suggest that STING deficient CAR-T cells stand to benefit from STING agonists to improve CAR-T cell therapy for immune-deprived cancers such as pancreatic cancer.},
}

Animals
*Pancreatic Neoplasms/therapy/immunology/pathology/genetics/metabolism
STING Protein
*Membrane Proteins/agonists/genetics/metabolism
*Immunotherapy, Adoptive/methods
Humans
*Receptors, Chimeric Antigen/immunology/metabolism/genetics
Cell Line, Tumor
cGAS-STING Signaling Pathway
Mice
Xenograft Model Antitumor Assays
T-Cell Exhaustion
Tumor Microenvironment/immunology
Interferon-gamma/metabolism
*T-Lymphocytes/immunology/metabolism/transplantation/drug effects
Tumor Necrosis Factor-alpha/metabolism
Cell Proliferation
CRISPR-Cas Systems
Signal Transduction
Female
Immunologic Memory

@article {pmid41886459,
year = {2026},
author = {Yang, M and Chen, G and Xiao, J and Zhang, X and Hu, Z and Zhou, B},
title = {Protocol for enhancing Cas9 efficiency and fidelity through structure-guided phosphate-locking loop engineering.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104452},
pmid = {41886459},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Phosphates/chemistry/metabolism ; DNA/genetics/chemistry/metabolism ; *Protein Engineering/methods ; },
abstract = {The phosphate-locking loop (PLL), stabilizing Cas9-DNA interactions, is a key target for optimizing efficiency and specificity. Here, we present a protocol for enhancing Cas9 efficiency and fidelity through structure-guided PLL engineering. We describe steps for identifying PLL engineering targets through sequence alignment and structural analysis, constructing variants via inverse PCR, evaluating efficiency using amplicon sequencing, and assessing specificity through Genome-wide Unbiased Identification of DSBs Evaluated by sequencing (GUIDE-seq (GUIDE-seq). This protocol provides a generalizable framework for Cas9 engineering across orthologs. For complete details on the use and execution of this protocol, please refer to Yang et al.[1].},
}

*CRISPR-Cas Systems/genetics
*Phosphates/chemistry/metabolism
DNA/genetics/chemistry/metabolism
*Protein Engineering/methods

@article {pmid41903144,
year = {2026},
author = {Zhou, Z and Zhu, S and Pan, D and Lee, HY and Liu, N},
title = {Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104468},
pmid = {41903144},
issn = {2666-1667},
mesh = {Humans ; K562 Cells ; CRISPR-Cas Systems/genetics ; *DNA Transposable Elements/genetics ; Genome, Human/genetics ; },
abstract = {The composite transposon SINE-VNTR-Alu (SVA) is hominid-specific and composed of five parts, but the regulatory mechanism of SVA transcription is still unclear. Here, we present a protocol to identify SVA regulators using genome-wide screening in human K562 cells. We describe steps for constructing an SVA-GFP reporter and performing genome-wide CRISPR-Cas9 screening in human K562 cells to identify genes that control SVA transcription. This protocol provides a representative procedure of genome-wide screening for transposon regulators. For complete details on the use and execution of this protocol, please refer to Zhou et al.[1].},
}

Humans
K562 Cells
CRISPR-Cas Systems/genetics
*DNA Transposable Elements/genetics
Genome, Human/genetics

@article {pmid41957359,
year = {2026},
author = {Wang, Y and Zhao, Y and Hu, J and Wang, Z and Pattarayan, D and Li, S and Zhang, Y and Wang, X and Wang, Y and Xie, W and Zhang, M and Yang, D},
title = {CRISPR activation screens identify oncogenic lncRNAs that are susceptible to CDK4/6 inhibitor treatment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41957359},
issn = {2041-1723},
support = {R01CA255196//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01CA222274//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA255196/CA/NCI NIH HHS/United States ; R01CA272866//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA282704/CA/NCI NIH HHS/United States ; R01CA282704//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Cyclin-Dependent Kinase 4/antagonists & inhibitors/metabolism/genetics ; *Cyclin-Dependent Kinase 6/antagonists & inhibitors/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; *Breast Neoplasms/genetics/drug therapy/pathology/metabolism ; Piperazines/pharmacology/therapeutic use ; Cell Line, Tumor ; Pyridines/pharmacology/therapeutic use ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Cell Proliferation/drug effects/genetics ; Animals ; Estrogen Receptor alpha/metabolism/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The roles of long non-coding RNAs (lncRNAs) in tumorigenesis and therapeutic response remain largely unknown. Here we perform genome-wide and focused CRISPR activation screens to identify lncRNAs regulating palbociclib response in breast cancer cells. A synchronized two-stage proliferation screen not only characterizes tumor growth-regulating lncRNAs, but also reveals a strong negative correlation between lncRNA-mediated regulation of tumor proliferation and CDK4/6 inhibitor sensitivity. By integrating CRISPRa screen results with drug response data from 815 cancer cell lines, we identify and functionally validate that TENM3-AS1, LINC01117, and ENSG00000226706 can increase breast cancer sensitivity to CDK4/6i while promoting tumor proliferation. In breast cancer patients, all three lncRNA signatures are associated with CDK4/6 inhibitor response. Mechanistically, we have shown that lncRNA TENM3-AS1 is a potential ERα-interacting lncRNA, and its regulation of CDK4/6 inhibitor sensitivity is dependent on ERα expression. Our integrated strategy characterizes oncogenic lncRNAs as potential therapeutic biomarkers for CDK4/6 inhibitor treatment in cancer.},
}

Humans
*Cyclin-Dependent Kinase 4/antagonists & inhibitors/metabolism/genetics
*Cyclin-Dependent Kinase 6/antagonists & inhibitors/metabolism
*RNA, Long Noncoding/genetics/metabolism
*Breast Neoplasms/genetics/drug therapy/pathology/metabolism
Piperazines/pharmacology/therapeutic use
Cell Line, Tumor
Pyridines/pharmacology/therapeutic use
Female
Gene Expression Regulation, Neoplastic/drug effects
*Protein Kinase Inhibitors/pharmacology/therapeutic use
Cell Proliferation/drug effects/genetics
Animals
Estrogen Receptor alpha/metabolism/genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41986330,
year = {2026},
author = {Nguyen, QV and Lan, YJ and Chang, JC and Shih, HA and Faustine, J and Chen, CC and Ho, SY and Cheng, CW and Chao, TL and Lin, S},
title = {Genome-wide CRISPR screens in primary human natural killer cells identify countermeasures against immunosuppressive environment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41986330},
issn = {2041-1723},
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *CRISPR-Cas Systems ; Dinoprostone/metabolism ; Tumor Microenvironment/immunology/genetics ; Interleukin-2/metabolism ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Serine-Threonine Kinases/genetics ; Signal Transduction ; },
abstract = {Natural killer (NK) cells are promising effectors for cancer immunotherapy, but their efficacy is limited by immunosuppressive tumor microenvironments. To uncover strategies for enhancing NK cell function, we establish a CRISPR loss-of-function screening platform for primary human NK cells by combining BaEVRless-pseudotyped lentiviral transduction of sgRNA libraries with Cas9 protein electroporation. This platform enables genome-scale interrogation of gene function in non-transformed NK cells. Kinome-focused and genome-wide screens identify key regulators of NK cell proliferation, cytotoxicity, and resistance to prostaglandin E2 (PGE2)-mediated suppression. STK17B deletion enhances NK cell expansion, while loss of CCDC53 boosts degranulation and cytotoxicity. We also uncover the CRL5 complex-including RNF7, UBE2F, and CISH-as critical inhibitors of IL-2 signaling and effector function under PGE2 stress. These findings establish a scalable platform for CRISPR-based functional genomics in primary NK cells and reveal engineering targets to enhance NK cell persistence and efficacy in tumor microenvironments.},
}

Humans
*Killer Cells, Natural/immunology/metabolism
*CRISPR-Cas Systems
Dinoprostone/metabolism
Tumor Microenvironment/immunology/genetics
Interleukin-2/metabolism
Cell Proliferation
Clustered Regularly Interspaced Short Palindromic Repeats
Protein Serine-Threonine Kinases/genetics
Signal Transduction

@article {pmid42012982,
year = {2026},
author = {Djamshidi, M and Tanida, R and Heshmatzad, K and Krowicki, H and Hill, A and Yang, Y and Riabowol, K},
title = {Protocol for enhancing CRISPR-Cas9 genome editing using histone deacetylase inhibition and engineered virus-like particle delivery.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104493},
pmid = {42012982},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Histone Deacetylase Inhibitors/pharmacology ; *Gene Editing/methods ; Humans ; *Virion/genetics ; },
abstract = {We present a 10-fold faster, accurate, and more efficient (FAME)-CRISPR-Cas9 gene editing workflow utilizing histone deacetylase inhibitor (HDACi)-mediated chromatin relaxation and engineered virus-like particle (eVLP) delivery of Cas9. We describe steps for optimizing HDACi concentration, euchromatinization timing, and Cas9 delivery/expression to improve CRISPR-Cas9 editing efficiency and efficacy. This protocol can eliminate the need for single-cell cloning and reduce experimental timelines up to 10-fold while minimizing HDACi-mediated toxicity. For complete details on the use and execution of this protocol, please refer to Djamshidi et al.[1].},
}

*CRISPR-Cas Systems/genetics
*Histone Deacetylase Inhibitors/pharmacology
*Gene Editing/methods
Humans
*Virion/genetics

@article {pmid42025580,
year = {2026},
author = {Shukla, SK and Singh, A and Yadav, R and Kumar, A},
title = {Advances in molecular diagnostic strategies during the SARS-CoV-2 pandemic.},
journal = {Expert review of molecular diagnostics},
volume = {26},
number = {4},
pages = {293-308},
doi = {10.1080/14737159.2026.2665263},
pmid = {42025580},
issn = {1744-8352},
mesh = {Humans ; *COVID-19/diagnosis/epidemiology/virology ; *SARS-CoV-2/genetics/isolation & purification ; *Molecular Diagnostic Techniques/methods ; Pandemics ; COVID-19 Testing/methods ; COVID-19 Nucleic Acid Testing/methods ; Rapid Diagnostic Tests ; Pandemic Preparedness ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: The SARS-CoV-2 pandemic provided critical insights into pandemic preparedness. The community spread can be slowed down or contained through effective, rapid, and robust diagnosis of infected individuals.

AREA COVERED: During the pandemic, substantial advances were made in developing rapid and cost-effective diagnostic approaches. Self-collected gargle samples offer clear advantages over conventional NSP/OPS methods by reducing reliance on trained personnel and personal protective equipment. Colorimetric assays further improve accessibility, enabling rapid, instrument-free, and visually interpretable detection at low cost. CRISPR-based diagnostics present a promising alternative to RT-PCR, facilitating scalable mass screening with reduced technical dependence. Concurrently, optimization of RT-PCR workflows-particularly through minimization of pre-PCR steps-can enhance speed and affordability. The integration of digital technologies and artificial intelligence further leverages diagnostic capabilities. Despite this, improved regulatory frameworks and resilient supply chains are critical for ensuring scalable, equitable access, and effective pandemic preparedness.

EXPERT OPINION: Global efforts were made to develop sensitive, rapid, cost-effective, and noninvasive technologies to identify the pandemic virus; however, variations in sensitivity/specificity and limited sample size validation hampered their utility in routine diagnostics. The COVID-19 pandemic has ended, but global efforts are still needed to combat the early infection of subsequent waves or similar disease waves.},
}

Humans
*COVID-19/diagnosis/epidemiology/virology
*SARS-CoV-2/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods
Pandemics
COVID-19 Testing/methods
COVID-19 Nucleic Acid Testing/methods
Rapid Diagnostic Tests
Pandemic Preparedness
CRISPR-Cas Systems

@article {pmid42033729,
year = {2026},
author = {He, Z and Chen, S and Huang, A and Jiang, H},
title = {Protocol for the generation of DDT signaling reporter cell line for CRISPR screening.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104523},
pmid = {42033729},
issn = {2666-1667},
mesh = {*Signal Transduction/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Cells respond to perturbations through signaling pathways that often induce characteristic transcriptional changes. Here, we present a protocol for generating a double death trap (DDT) reporter that converts pathway activity into a binary survival-death outcome. The DDT reporter employs puromycin resistance and FKBP12(F36V)-ΔCaspase9 constructs driven by pathway-specific response elements. We describe the steps for DDT reporter plasmid construction, cell line generation, and genome-wide CRISPR screening in DDT cells. We further detail procedures for next-generation sequencing (NGS) sample preparation, sequencing, and downstream analysis. For complete details on the use and execution of this protocol, please refer to He et al.[1].},
}

*Signal Transduction/genetics
Humans
*CRISPR-Cas Systems/genetics
*Genes, Reporter/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Line
High-Throughput Nucleotide Sequencing/methods

@article {pmid42033731,
year = {2026},
author = {Sarmah, H and Iannello, G and Wantroba, R and Wu, C and Idiarte, J and Munoz, A and Déry, O and Rubio de la Torre, E and Lebayle, E and Seminara, S and Sirabella, D and Huynh, S and Issa, R and Kissner, M and Corneo, B},
title = {Protocol for clonal isolation of gene-edited hiPSCs using droplet and microfluidic sorting.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104518},
pmid = {42033731},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Microfluidics/methods ; Electroporation ; *Cell Separation/methods ; },
abstract = {Genetically engineered human induced pluripotent stem cells (hiPSCs) are vital for disease modeling and drug discovery, yet generating clonal lines efficiently post-editing remains challenging. Here, we present a protocol to generate clonal hiPSC lines after gene editing using either electrostatic droplet- or microfluidics-based sorting platforms. We describe steps for culturing hiPSCs, CRISPR-RNP electroporation, single-cell sorting, and expansion of gene-edited clones. Using this protocol, we generated over 100 clonal lines across seven knock-in/knock-out experiments, demonstrating broad utility and reproducibility. For additional details on the use and execution of this protocol, please refer to Patel et al.[1].},
}

Humans
*Induced Pluripotent Stem Cells/cytology/metabolism
*Gene Editing/methods
CRISPR-Cas Systems/genetics
*Microfluidics/methods
Electroporation
*Cell Separation/methods

@article {pmid42044055,
year = {2026},
author = {Li, X and Gao, X and Dong, J and Gu, T and Li, Q and Wang, L and Deng, F and Hou, J and Hou, C and Huo, D},
title = {Aptamer Nanoswitch-Mediated Lock-Expose Mechanism Enables Highly Sensitive In Vitro Detection and Precise In Situ Membrane Imaging of HER2 Protein.},
journal = {ACS sensors},
volume = {11},
number = {5},
pages = {4014-4023},
doi = {10.1021/acssensors.6c00371},
pmid = {42044055},
issn = {2379-3694},
mesh = {Humans ; *Erb-b2 Receptor Tyrosine Kinases/metabolism/analysis/genetics ; *Aptamers, Nucleotide/chemistry/metabolism/genetics ; Cell Line, Tumor ; Limit of Detection ; *Biosensing Techniques/methods ; Neoplastic Cells, Circulating ; Fluorescent Dyes/chemistry ; CRISPR-Cas Systems ; Breast Neoplasms ; },
abstract = {Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer correlates with high aggressiveness, an increased recurrence rate, and poor survival, holding significant diagnostic value. In this work, a HER2-specific aptamer (Apt) was engineered into a Apt nanoswitch (hApt) and revealed the interaction of HER2-aptamers through molecular docking and quantified the binding energy and dynamic behavior through molecular dynamics simulations. A lock-expose mechanism was designed by combining the target-induced conformational switch of hApt with template-prefabricated rolling circle amplification (rRCA), forming a T-hApt-rRCA sandwich structure. Coupled with CRISPR/Cas12a and fluorescent probes, this sensor enabled highly sensitive detection, with a linear range of 10 fg/mL to 10 ng/mL and a limit of detection of 1.42 fg/mL. Using HUVEC, A549, MCF-7, and SK-BR-3 cell lines to model HER2 heterogeneity in circulating tumor cells (CTCs) enabled in situ imaging, differentiation, and quantitative detection of membrane HER2 expression, thereby providing direct visualization of expression levels and highlighting the translational promise of this approach. The accurate detection and clear differentiation between the eight healthy samples and the twenty-eight breast cancer patient samples further underscore the practical applicability of this sensing strategy.},
}

Humans
*Erb-b2 Receptor Tyrosine Kinases/metabolism/analysis/genetics
*Aptamers, Nucleotide/chemistry/metabolism/genetics
Cell Line, Tumor
Limit of Detection
*Biosensing Techniques/methods
Neoplastic Cells, Circulating
Fluorescent Dyes/chemistry
CRISPR-Cas Systems
Breast Neoplasms

@article {pmid42066814,
year = {2026},
author = {Nie, H and Zheng, Z and Fan, W and Yan, M and Shao, W and Meng, Y and An, D and Zhao, S and Yuan, L and Yang, J and Wang, H},
title = {Homozygous IbGBSS1 knockouts in hexaploid sweet potato enable amylose-free starch without a yield trade-off.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {369},
number = {},
pages = {113172},
doi = {10.1016/j.plantsci.2026.113172},
pmid = {42066814},
issn = {1873-2259},
mesh = {*Ipomoea batatas/genetics/metabolism/growth & development ; *Starch Synthase/genetics/metabolism ; *Amylose/metabolism ; *Starch/metabolism ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Homozygote ; Polyploidy ; CRISPR-Cas Systems ; },
abstract = {Sweet potato (Ipomoea batatas) is a major crop and an important industrial starch source; however, its hexaploid genome has hindered the generation of complete gene knockouts. Because the amylose-to-amylopectin ratio determines starch functionality, the production of amylose-free (waxy) starch is of considerable interest for food, pharmaceutical, and industrial applications. It was hypothesized that the complete knockout of all six alleles of IbGBSS1, which encodes granule-bound starch synthase I, would abolish amylose biosynthesis without compromising plant growth or yield. CRISPR/Cas9 mutagenesis combined with the Hi-TOM high-throughput mutation detection platform was used to generate homozygous Ibgbss1 mutants with confirmed edits across all alleles. These mutants contained < 1% amylose and exhibited normal growth and unchanged yield under both greenhouse and field conditions. Physicochemical analyses showed that amylose-free starch displayed larger granules, an altered amylopectin chain-length distribution (reduced DP 6-12 and enriched > DP 36), and numerous surface pores. Differential scanning calorimetry indicated increased gelatinization onset and peak temperatures, along with higher gelatinization enthalpy. Transcriptome analysis revealed broad reprogramming of starch and sucrose metabolism, accompanied by increased accumulation of glucose, fructose, and sucrose in storage roots. These results demonstrate that IbGBSS1 is essential for amylose biosynthesis and establish a strategy for generating complete multi-allelic knockouts in hexaploid sweet potato. Amylose-free germplasm was obtained without a yield penalty, providing potential for food and industrial applications.},
}

*Ipomoea batatas/genetics/metabolism/growth & development
*Starch Synthase/genetics/metabolism
*Amylose/metabolism
*Starch/metabolism
Gene Knockout Techniques
*Plant Proteins/genetics/metabolism
Homozygote
Polyploidy
CRISPR-Cas Systems

@article {pmid42080968,
year = {2026},
author = {Yang, Y and Arro, J and Zou, C and Oravec, M and Reisch, B and Zhong, GY},
title = {First brassinosteroid-based dwarf mutant discovered and characterized in grapevine.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {5},
pages = {},
pmid = {42080968},
issn = {1432-2242},
mesh = {*Vitis/genetics/growth & development ; *Brassinosteroids/biosynthesis ; Phenotype ; Chromosome Mapping ; *Cytochrome P-450 Enzyme System/genetics ; Mutation ; *Plant Proteins/genetics/metabolism ; Genes, Plant ; CRISPR-Cas Systems ; Genetic Association Studies ; },
abstract = {In this study, we investigated the genetic control of dwarfism in naturally occurring dwarf mutant lines of grapevines. Through trait-segregation and marker-trait association analyses, we identified a major locus on Chromosome 14 tightly associated with the dwarf trait. Subsequently, we conducted a bulked RNA-seq analysis, fine mapped the dwarf trait and identified VviBR6OX1, a cytochrome P450 enzyme involved in brassinosteroid synthesis, as a candidate gene for the observed dwarfism. RNA-seq sequence analyses revealed two in-frame deletions in the gene: a 12-bp deletion in exon 1 and a 9-bp deletion in exon 4. A survey of the two indels in Vitis germplasm suggested that the 9-bp deletion is most likely the cause of dwarfism in the mutant. We recreated similar dwarf grapevines by knocking out VviBR6OX1 using CRISPR/Cas9 gene editing and confirmed VviBR6OX1's role in controlling vine architecture. Additionally, we observed several vines with an extreme compact dwarf phenotype and determined that the compact dwarfing phenotype was a result of simultaneous editing of a second BR6OX gene, VviBR6OX2. The discovery of BR-related dwarfism in grapevine provides an important genetic avenue for developing desirable vine architecture for various breeding purposes.},
}

*Vitis/genetics/growth & development
*Brassinosteroids/biosynthesis
Phenotype
Chromosome Mapping
*Cytochrome P-450 Enzyme System/genetics
Mutation
*Plant Proteins/genetics/metabolism
Genes, Plant
CRISPR-Cas Systems
Genetic Association Studies

@article {pmid42084357,
year = {2026},
author = {Stone, S and Elsharkawy, A and Patterson, LD and Natekar, JP and Jiang, H and Viktoria Hyddmark, E and Camarillo, J and Zhao, G and Kumar, M},
title = {A Novel Humanized Lethal Mouse Model of SARS-CoV-2-Associated Disease.},
journal = {Journal of medical virology},
volume = {98},
number = {5},
pages = {e70959},
doi = {10.1002/jmv.70959},
pmid = {42084357},
issn = {1096-9071},
mesh = {Animals ; *Disease Models, Animal ; Humans ; *COVID-19/pathology/virology ; Mice ; *SARS-CoV-2/pathogenicity ; *Angiotensin-Converting Enzyme 2/genetics/metabolism ; Mice, Inbred C57BL ; Lung/pathology/virology ; Mice, Transgenic ; Female ; Gene Knock-In Techniques ; Brain/pathology/virology ; Viral Load ; CRISPR-Cas Systems ; Cytokines ; },
abstract = {Mice are valuable small animal models for studying SARS-CoV-2 pathogenesis. Ancestral SARS-CoV-2 strains do not efficiently utilize murine Ace2, rendering wild-type mice resistant to infection. Although human ACE2 transgenic models such as K18-hACE2 have provided critical insights, they express multiple copies of both murine and human ACE2, and random transgene insertion can result in non-physiological receptor expression. To overcome these limitations, we employed a human ACE2 knock-in (hACE2-KI) model in which the murine Ace2 coding sequence is replaced with human ACE2 using CRISPR/Cas9 technology, generating an mAce2-null background. This design allows human ACE2 expression under endogenous regulatory control while eliminating murine Ace2 expression, thereby providing a more physiologically relevant platform to investigate SARS-CoV-2 pathogenesis and evaluate therapeutic and preventive strategies. In this study, SARS-CoV-2-associated disease was evaluated and compared among hACE2-KI, K18-hACE2 and C57BL/6J mice. Mice were intranasally inoculated with 10[5] plaque-forming units of SARS-CoV-2 lineages B.1 or B.1.351. Both hACE2-KI and K18-hACE2 mice developed severe disease after SARS-CoV-2 infection. Following infection with B.1, both K18-hACE2 mice and hACE2-KI mice exhibited significant weight loss and mortality, with high viral loads detected in the lungs and brain. hACE2-KI mice infected with SARS-CoV-2 B.1.351 also showed significant weight loss and viral loads, resulting in high mortality. The pathology and inflammatory response within the lungs and brain of infected hACE2-KI mice revealed robust expression of viral nucleocapsid protein, histopathological changes, and upregulated cytokine and chemokine responses. Together, these findings demonstrate that the hACE2-KI knock-in mouse model supports robust SARS-CoV-2 replication and mimics severe COVID-19 disease.},
}

Animals
*Disease Models, Animal
Humans
*COVID-19/pathology/virology
Mice
*SARS-CoV-2/pathogenicity
*Angiotensin-Converting Enzyme 2/genetics/metabolism
Mice, Inbred C57BL
Lung/pathology/virology
Mice, Transgenic
Female
Gene Knock-In Techniques
Brain/pathology/virology
Viral Load
CRISPR-Cas Systems
Cytokines

@article {pmid42096138,
year = {2026},
author = {Zhang, Q and Li, Y and Li, Y and Han, L and Yan, J and Zhan, M and Liu, T and Ke, P and Wang, Q and Huang, X},
title = {Anti-CRISPR Protein Regulates CRISPR/Cas12a Fusogenic-Nanovesicle-Based Platform for Extracellular Vesicle-Encapsulated Non-Nucleic Acid Target In-Vesicle Detection.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {65},
number = {26},
pages = {e6795217},
doi = {10.1002/anie.6795217},
pmid = {42096138},
issn = {1521-3773},
support = {2024ZD0533400//National Key Research and Development Program of China/ ; 2024ZD0533401//National Key Research and Development Program of China/ ; 0720240230//Guangdong Special Support Program Health and health talents List Project of Provincial Health Commission/ ; 2024A1515030043//Natural Science Foundation of Guangdong Province/ ; 2026A1515010500//Natural Science Foundation of Guangdong Province/ ; SZ2022QN09//"Young Talents Program" of Guangdong Academy of Traditional Chinese Medicine/ ; SKLKY2024B0011//State Key Laboratory of Traditional Chinese Medicine Syndrome Projects/ ; 2023A03J0755//Guangzhou Science and Technology Plan Projects/ ; 2023B110008//Guangdong Provincial Clinical Research Guangdong Provincial Clinical Research Center for Laboratory Medicine/ ; 2025JY-A1004//Guangdong Provincial Clinical Research Guangdong Provincial Clinical Research Center for Laboratory Medicine/ ; //Science and Technology Innovation Center of Guangzhou University of Chinese Medicine/ ; },
mesh = {*Extracellular Vesicles/chemistry/metabolism ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; Aptamers, Nucleotide/chemistry/metabolism ; *Endodeoxyribonucleases/metabolism ; },
abstract = {The detection of non-nucleic acid targets encapsulated in extracellular vesicles (EVs) faces two major challenges: (1) difficulties in efficient isolation and the risk of content degradation, and (2) the low abundance of target molecules encapsulated in EVs always leads to failed signal transduction and inadequate output signal intensity. To overcome these limitations, we propose a high-efficiency in-vesicle analysis strategy that integrates targeting probe delivery and regulation by protein signal amplification. By applying aptamer-mediated membrane fusion and "locked-activated" CRISPR-Cas12a-AcrVA1 (LACA) for protein signal regulation, we fabricated a yly12-aptamer-functionalized self-assembled nanovesicle which encapsulate LACA-system (yly12-lipo@Cas12a nanovesicle) as an in-vesicle bioanalytical platform. Leveraging the high specificity of the aptamer and the regulatory function of AcrVA1 in selectively modulating Cas12a activity, the platform enables highly specifiec and sensitive detection, offering advantages of simple operation and versatility across platforms within only 2.5 h. Clinical analysis demonstrated effective differentiation between patients and healthy controls, yielding high diagnostic performance with an AUC of 0.965. The proposed platform shows great potential for EV-carrying protein biomarker analysis and has broad prospects for the disease's diagnosis in clinical settings.},
}

*Extracellular Vesicles/chemistry/metabolism
*CRISPR-Cas Systems
Humans
*CRISPR-Associated Proteins/metabolism
*Bacterial Proteins/metabolism
Aptamers, Nucleotide/chemistry/metabolism
*Endodeoxyribonucleases/metabolism

@article {pmid42017197,
year = {2026},
author = {Wafer, R and Tandon, P and Minchin, J},
title = {A quantitative in vivo CRISPR-imaging platform identifies regulators of hyperplastic and hypertrophic adipose morphology in zebrafish.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42017197},
issn = {2050-084X},
support = {BB/X009467/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; REA PhD Studentship/BHF_/British Heart Foundation/United Kingdom ; },
mesh = {Animals ; *Zebrafish/genetics ; *Adipose Tissue/metabolism/pathology ; Hyperplasia/genetics ; Humans ; Hypertrophy/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Zebrafish Proteins/genetics/metabolism ; Adipocytes ; CRISPR-Cas Systems ; },
abstract = {Adipose tissues exhibit a remarkable capacity to expand, regress, and remodel in response to energy status. The cellular mechanisms underlying adipose remodelling are central to metabolic health. Hypertrophic remodelling - characterised by the enlargement of existing adipocytes - is associated with insulin resistance, type 2 diabetes, and cardiovascular disease. In contrast, hyperplastic remodelling - in which new adipocytes are generated - is linked to improved metabolic outcomes. Despite its clinical importance, the regulation of hypertrophic and hyperplastic adipose morphology remains poorly understood. Here, we integrate human transcriptomic data with a quantitative CRISPR-imaging platform in zebrafish to identify regulators of adipose morphology. We developed an image-based phenotyping pipeline that captures lipid droplet size, number, and spatial patterning, and applied generalised additive modelling to quantify hyperplastic versus hypertrophic morphology signatures. Using this platform, we conducted an F0 CRISPR screen targeting 25 candidate genes and identified three that induced hypertrophic morphology (txnipa, mmp14b, and foxp1b) and an additional candidate that altered total adiposity (kazna). For functional validation, we generated stable loss-of-function alleles for both zebrafish foxp1 paralogues. Spatial analysis along the anterior-posterior axis revealed that foxp1b mutants display developmental hypertrophy but profoundly blunted adaptive responses to high-fat diet (~68% reduction across all spatial zones), while foxp1a mutants show normal baseline morphology but disrupted spatial patterning of diet-induced hypertrophy. Together, these findings establish a scalable CRISPR-imaging platform for in vivo genetic screening of adipose morphology and reveal distinct roles for Foxp1 paralogues in developmental patterning and adaptive responses to dietary challenge in adipose tissue.},
}

Animals
*Zebrafish/genetics
*Adipose Tissue/metabolism/pathology
Hyperplasia/genetics
Humans
Hypertrophy/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Zebrafish Proteins/genetics/metabolism
Adipocytes
CRISPR-Cas Systems

@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 = {Animals ; *Chromatin/metabolism/genetics ; *Gene Editing/methods ; Mice ; Cell Differentiation ; *Neural Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Mouse Embryonic Stem Cells/metabolism/cytology ; },
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.},
}

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

@article {pmid41744735,
year = {2026},
author = {Hu, X and Su, J and Song, S},
title = {CRISPR/Cas System-Based Biosensors.},
journal = {Biosensors},
volume = {16},
number = {2},
pages = {},
pmid = {41744735},
issn = {2079-6374},
abstract = {Over the past decade, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, originally identified as adaptive immune systems in bacteria and archaea that defend against invading nucleic acids, have revolutionized biological research [...].},
}

@article {pmid41747377,
year = {2026},
author = {Pan, MX and Lv, MM and Nie, YG and Su, M and Zha, CJ and Mei, RY and Ying, ZM},
title = {Ultrasensitive miRNA detection via magnetic bead-confined catalytic hairpin assembly enabling transcription-driven crRNA assembly and CRISPR/Cas12a activation.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118559},
doi = {10.1016/j.bios.2026.118559},
pmid = {41747377},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/analysis/isolation & purification/blood ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; DNA/chemistry/genetics ; Nucleic Acid Hybridization ; *Endodeoxyribonucleases/chemistry/genetics ; Limit of Detection ; Inverted Repeat Sequences ; Transcription, Genetic ; Catalysis ; Bacterial Proteins ; },
abstract = {The integration of CRISPR/Cas12a with catalytic hairpin assembly (CHA), a strategy that predominantly relies on CHA to generate dsDNA activators for direct Cas12a activation, has emerged as a powerful tool in molecular diagnostics. However, two major challenges remain: the strict protospacer adjacent motif (PAM) dependence of the dsDNA and background leakage from hairpin hybridization. Herein, we report a bead-confined platform that transcription mediates crRNA reassembly and template activation of Cas12a for ultrasensitive miRNA detection. The target-triggered CHA assembly dynamically constructed a T7 transcription template from three initially locked hairpins (H1, H2, and H3), which not only transcribed scaffold RNA but also hybridized with its own product to form a DNA/RNA complex that activates Cas12a. The integration of the split T7 promoter with CHA effectively suppressed background suppression and enhanced detection sensitivity. Additionally, the magnetic beads increase local concentration and reaction kinetics, collectively contributing to a substantially enhanced detection sensitivity. Moreover, a crRNA assembly strategy designed for transcription-powered Cas12a not only circumvents the conventional PAM-dependent dsDNA activation pathway of Cas12a but also enables self-supplied crRNA without requiring additional activators. We demonstrated that the biosensor exhibits exceptional sensitivity for miRNA-21 detection, achieving a limit of 65.3 aM. Furthermore, the practicality of this method was preliminarily confirmed through accurately quantifying target levels in cell lines and human serum. Our method presents a viable solution with transformative potential, designed to address complex challenges in contemporary diagnostic applications.},
}

*MicroRNAs/genetics/analysis/isolation & purification/blood
Humans
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/chemistry/genetics
DNA/chemistry/genetics
Nucleic Acid Hybridization
*Endodeoxyribonucleases/chemistry/genetics
Limit of Detection
Inverted Repeat Sequences
Transcription, Genetic
Catalysis
Bacterial Proteins

@article {pmid41747626,
year = {2026},
author = {Yoo, DH and Bayarsaikhan, D and Lee, J and Im, YS and Bayarsaikhan, G and Kang, HA and Lee, B and Kim, YO},
title = {Generation and characterization of SOX17-specific EGFP expressing human induced pluripotent stem cell line, KSCBi017-A-4, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103943},
doi = {10.1016/j.scr.2026.103943},
pmid = {41747626},
issn = {1876-7753},
mesh = {Humans ; *SOXF Transcription Factors/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Endoderm/cytology/metabolism ; },
abstract = {We generated a human induced pluripotent stem cell (hiPSC) reporter line in which EGFP was inserted in-frame at the C-terminus of the endogenous SOX17 locus using CRISPR/Cas9-mediated homologous recombination. The targeted clone, KSCBi017-A-4, was isolated by puromycin selection and validated by PCR and Sanger sequencing. This SOX17-EGFP hiPSC line retains a normal karyotype and pluripotency and displays specific EGFP expression upon directed definitive endoderm differentiation. This reporter line provides a reliable tool for monitoring SOX17 expression during human endoderm specification.},
}

Humans
*SOXF Transcription Factors/metabolism/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
Cell Differentiation
Endoderm/cytology/metabolism

@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 = {*Water/chemistry ; *Gravitation ; *Waxes/chemistry ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; Microspheres ; },
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.},
}

*Water/chemistry
*Gravitation
*Waxes/chemistry
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems
Microspheres

@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 = {*Endopeptidases/genetics/pharmacology/metabolism ; *Probiotics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Saccharomyces boulardii/genetics/metabolism ; *Listeria monocytogenes/drug effects ; Bacteriophages/genetics/enzymology ; Listeriosis/prevention & control/microbiology ; 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.},
}

*Endopeptidases/genetics/pharmacology/metabolism
*Probiotics/metabolism
Humans
*CRISPR-Cas Systems
*Saccharomyces boulardii/genetics/metabolism
*Listeria monocytogenes/drug effects
Bacteriophages/genetics/enzymology
Listeriosis/prevention & control/microbiology
Saccharomyces cerevisiae/genetics

@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 = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Candida/genetics/pathogenicity ; Humans ; Candidiasis/microbiology ; },
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.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Candida/genetics/pathogenicity
Humans
Candidiasis/microbiology

@article {pmid41751014,
year = {2026},
author = {Wang, Q and Zheng, L and You, G and Dong, H and Chen, S and Wang, S and Chen, S},
title = {Navigating the Complexity: Advancing Diagnostic Strategies for Avian Reovirus in Chinese Poultry.},
journal = {Animals : an open access journal from MDPI},
volume = {16},
number = {4},
pages = {},
pmid = {41751014},
issn = {2076-2615},
support = {XTCXGC2021018, XTCXGC2021012//the '5511' Collaborative Innovation Project of Fujian Academy of Agricultural Sciences, China/ ; },
abstract = {Avian reovirus (ARV) infections pose a significant and evolving threat to China's poultry industry, the world's largest. Diverse farming systems-ranging from modern intensive operations to traditional waterfowl-poultry polyculture-foster a unique ecological niche for ARV, defined by complex serotypic and genotypic diversity, marked regional variations, potential interspecies transmission between chickens and waterfowl, and recurrent co-infections. Collectively, these factors undermine the efficacy of conventional diagnostic approaches. This review systematically outlines the current epidemic landscape of ARV in China, highlighting the molecular characteristics of prevailing strains (particularly those from waterfowl) and their roles in diagnostic evasion. We critically assess the performance and limitations of existing diagnostic techniques (virus isolation, ELISA, PCR/qPCR) within the Chinese epidemiological setting. Furthermore, we discuss innovative technologies-including multiplex qPCR, CRISPR-Cas systems, and next-generation sequencing (NGS)-that offer potential for developing next-generation diagnostics tailored to China's specific challenges. Finally, we propose future directions, with an emphasis on standardization, data sharing, and interdisciplinary collaboration to bridge the gap between cutting-edge innovation and on-farm application for precise ARV control.},
}

@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 ; *Fragile X Messenger Ribonucleoprotein 1/genetics ; *Disease Models, Animal ; Female ; Sheep/genetics ; CRISPR-Cas Systems ; Male ; Gene Knockout Techniques ; Gene Editing ; },
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
*Fragile X Messenger Ribonucleoprotein 1/genetics
*Disease Models, Animal
Female
Sheep/genetics
CRISPR-Cas Systems
Male
Gene Knockout Techniques
Gene Editing

@article {pmid41751548,
year = {2026},
author = {Machel Gica, NG and Gica, WT and La, H and Mi, Y and Zhou, Y},
title = {Precision Breeding for a Global Staple Food: A Systematic Review with a Strategic Framework for CRISPR-Cas Applications in Rice (Oryza sativa L.).},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751548},
issn = {2073-4425},
support = {2025GBJ002388//Chinese Government Scholarship Council/ ; },
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems ; *Plant Breeding/methods ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Genome, Plant ; Crops, Agricultural/genetics ; },
abstract = {Background: Rice is one of the world's main staple crops, and improving its productivity and resilience is important to achieving food security under varying climatic conditions. Objectives: This systematic review synthesizes the existing evidence on the application, technical limitations, and potential of the development of genome editing technologies (CRISPR-Cas) in rice (Oryza sativa L.), as well as presents a novel approach called the CRISPR Trait Prioritization and Readiness Framework (CTPRF). Methods: Peer-reviewed articles that reported applications of genome editing based on the CRISPR-Cas system in the genome of rice for trait improvement or functional genomics were identified through searches fromPubMed, Scopus, Web of Science, and Google Scholar with studies published between 2012 and 2025. Studies were screened on predefined inclusion criteria related to experimental validation, reporting of editing efficiency, and clear phenotypic results. Data on CRISPR systems, target genes, methods of delivery, traits modified, and phenotypic results were extracted and synthesized by comparative analysis. Results: A wide variety of different CRISPR systems have been used in rice, and our results indicate that NHEJ-mediated knockouts are effective in average genotypes with editing efficiencies in the range of 70-90%, but HDR and prime editing are still under 10%. The CTPRF is being introduced as a strategic decision support tool to evaluate traits from four dimensions: technical feasibility, phenotypic predictability, impact potential, and regulatory pathway. We use this framework for case studies in pioneering countries (USA, Japan, China) and show how it can be useful for guiding research investment and policy. Conclusions: CRISPR-Cas technologies have transformed rice breeding, but their introduction requires overcoming genotype-dependent barriers to transformation and negotiating patchwork regulatory environments. The CTPRF offers a roadmap for the acceleration of the development of climate-resilient and nutritious rice varieties for the action plan.},
}

*Oryza/genetics
*CRISPR-Cas Systems
*Plant Breeding/methods
*Gene Editing/methods
Plants, Genetically Modified/genetics
Genome, Plant
Crops, Agricultural/genetics

@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 ; *Vesicular Transport Proteins/genetics/deficiency/metabolism ; *Cell Proliferation/genetics ; HEK293 Cells ; Apoptosis/genetics ; Mitochondrial Dynamics/genetics ; Mitochondria/metabolism/genetics ; CRISPR-Cas Systems ; },
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
*Vesicular Transport Proteins/genetics/deficiency/metabolism
*Cell Proliferation/genetics
HEK293 Cells
Apoptosis/genetics
Mitochondrial Dynamics/genetics
Mitochondria/metabolism/genetics
CRISPR-Cas Systems

@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 = {Animals ; *Bombyx/genetics/growth & development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Insect ; *Agriculture/methods ; 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.},
}

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

@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 ; *Genomics/methods ; CRISPR-Cas Systems ; 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
*Genomics/methods
CRISPR-Cas Systems
Phenotype
Genotype
Animals

@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 = {*Hordeum/genetics/microbiology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Ascomycota/pathogenicity ; *Plant Proteins/genetics ; *Plant Diseases/microbiology/genetics ; Alleles ; *Microbiota/genetics ; Mutation ; Plant Roots/microbiology/genetics ; },
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.},
}

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

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

@article {pmid41752921,
year = {2026},
author = {Panov, J and Elbert, A and Rosenthal, DS and Levi, M and Chumakov, K and Andino, R and Brodsky, L and Kaphzan, H},
title = {Spacio-Linear Screening for Ligand-Docking Cavities in Protein Structures: SLAM Algorithm.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {2},
pages = {},
pmid = {41752921},
issn = {2075-1729},
support = {R01 DK127830/DK/NIDDK NIH HHS/United States ; N/A//Tauber Foundation/ ; },
abstract = {Identifying structurally similar ligand-binding sites in unrelated proteins can facilitate drug repurposing, reveal off-target effects, and deepen our understanding of protein function. A number of tools were developed for structural screening, but many of them suffer from limited sensitivity and scalability. Using a data bank of crystallized protein structures, we aimed to discover novel protein targets for a ligand by leveraging a known ligand-binding query protein with a resolved structure. Here, we present SLAM (Spacio-Linear Alignment of Macromolecules), a novel alignment-based algorithm that detects local 3D similarities between ligand-binding cavities or protein-exposed surfaces of query and target proteins. SLAM encodes spatial substructure neighborhoods into short linear sequences of physicochemically annotated atoms, then applies pairwise sequence alignment combined with distance-correlation scoring to identify high-fidelity structural matches. Benchmarking using the Kahraman-36 dataset demonstrated that SLAM outperforms the state-of-the-art ProBiS algorithm in true-positive rate for predicting ligand-docking compatibility. Furthermore, SLAM identifies candidate ligands that may inhibit functionally critical domains of CRISPR-Cas proteins and predicts novel binding partners of toxic per- and polyfluoroalkyl Substance (PFAS) compounds (PFOA, PFOS) with plausible mechanistic links to toxicity. In conclusion, SLAM is a robust computationally efficient and flexible structural screening tool capable of detecting subtle physicochemical compatibilities between protein surfaces, promising to accelerate target discovery in pharmacology and elucidate protein-ligand interactions in environmental toxicology.},
}

@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 {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 ; Virus Replication ; Mice ; Humans ; *Coxsackievirus Infections/virology/immunology ; Viral Load ; *Host-Pathogen Interactions ; *Enterovirus/physiology/genetics ; Cell Line, Tumor ; },
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
Virus Replication
Mice
Humans
*Coxsackievirus Infections/virology/immunology
Viral Load
*Host-Pathogen Interactions
*Enterovirus/physiology/genetics
Cell Line, Tumor

@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 ; Rapid Diagnostic Tests ; Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Ebolavirus/genetics/isolation & purification ; Point-of-Care Testing ; },
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
Rapid Diagnostic Tests
Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Ebolavirus/genetics/isolation & purification
Point-of-Care Testing

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

@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/ ; },
mesh = {*Polyadenylation ; Genes, Reporter ; Humans ; *CRISPR-Cas Systems ; *Poly(A)-Binding Protein I/metabolism/genetics ; *Luminescent Proteins/genetics/metabolism ; DEAD-box RNA Helicases/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; Cell Proliferation ; HEK293 Cells ; Heterogeneous-Nuclear Ribonucleoproteins/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
Genes, Reporter
Humans
*CRISPR-Cas Systems
*Poly(A)-Binding Protein I/metabolism/genetics
*Luminescent Proteins/genetics/metabolism
DEAD-box RNA Helicases/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics
Cell Proliferation
HEK293 Cells
Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics

@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 {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 ; *Erythrocytes/chemistry/cytology ; Animals ; *Genetic Therapy/methods ; *Extracellular Vesicles/chemistry ; *RNA/genetics ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; },
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
*Erythrocytes/chemistry/cytology
Animals
*Genetic Therapy/methods
*Extracellular Vesicles/chemistry
*RNA/genetics
RNA, Messenger/genetics
CRISPR-Cas Systems

@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 ; *Chlamydomonas reinhardtii/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; *Microalgae/genetics/metabolism ; Gene Knockout Techniques ; Mutation ; 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
*Chlamydomonas reinhardtii/genetics/metabolism
*Ribonucleoproteins/metabolism/genetics
*Microalgae/genetics/metabolism
Gene Knockout Techniques
Mutation
CRISPR-Cas Systems

@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 {pmid41758644,
year = {2026},
author = {Adami, A and Garza, R and Dorazehi, F and Douse, CH and Jakobsson, J},
title = {Protocol for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104398},
pmid = {41758644},
issn = {2666-1667},
mesh = {Humans ; *Gene Silencing ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retroelements/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism ; },
abstract = {Here, we present a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). We describe steps for designing guide RNAs (gRNAs) to target TE families or unique TE loci. We also detail procedures for validating the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach combining bulk RNA sequencing, CUT&RUN epigenetic profiling, and proteomics. This framework optimizes the performance and interpretation of in vitro functional studies based on transcriptional manipulation of TEs in hiPSC models. For complete details on the use and execution of this protocol, please refer to Adami et al.[1].},
}

Humans
*Gene Silencing
*Induced Pluripotent Stem Cells/metabolism/cytology
*Retroelements/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Pluripotent Stem Cells/metabolism

@article {pmid41759295,
year = {2026},
author = {Molina, CE 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 = {23},
number = {2},
pages = {},
doi = {10.1088/1478-3975/ae4b7f},
pmid = {41759295},
issn = {1478-3975},
support = {MCB 2143760//NSF/ ; },
mesh = {Thermodynamics ; *CRISPR-Cas Systems ; Kinetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; *DNA/chemistry/metabolism ; *Endodeoxyribonucleases/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {Clustered regularly interspaced short palindromic repeat-associated proteins (CRISPR-Cas) biochemistry has been leveraged for 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 the DNA cleavage mechanism. In this review, we examine the thermodynamic and kinetic 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.},
}

Thermodynamics
*CRISPR-Cas Systems
Kinetics
*CRISPR-Associated Proteins/metabolism/chemistry
*DNA/chemistry/metabolism
*Endodeoxyribonucleases/metabolism/chemistry
*Bacterial Proteins/metabolism/chemistry

@article {pmid41759376,
year = {2026},
author = {Xu, Y and Wu, Y and Zheng, H and Zhao, J and Chen, J and Liu, S and Han, M and Li, F and Zhou, F and Zhang, X and Cao, Y and Zhang, H and Zhang, C},
title = {CRISPR-based metabolic screening identifies PLCE1 as a pivotal regulator of oncolytic viral antitumor immunity via tumor immune microenvironment remodeling.},
journal = {Biochemical and biophysical research communications},
volume = {810},
number = {},
pages = {153505},
doi = {10.1016/j.bbrc.2026.153505},
pmid = {41759376},
issn = {1090-2104},
mesh = {*Tumor Microenvironment/immunology ; Animals ; *Oncolytic Virotherapy/methods ; *Oncolytic Viruses/immunology/physiology ; Humans ; Cell Line, Tumor ; Mice ; *Colorectal Neoplasms/therapy/immunology/genetics ; *CRISPR-Cas Systems ; },
abstract = {As a promising cancer immunotherapeutic agent, oncolytic viruses (OV) can specifically kill tumor cells and elicit systemic antitumor immune responses. However, the intrinsic resistance of tumors to oncolytic virotherapy severely limits its therapeutic efficacy. This study identified phospholipase C epsilon 1 (PLCE1) as a key negative regulator of OV antitumor effects via CRISPR-Cas9 metabolic gene screening in MC38 colorectal cancer model. PLCE1 inhibitor U-73122 enhanced OV infection efficiency and immunogenic cell death in vitro. In vivo, U-73122 combined with OV synergistically reduced tumor volume and prolonged survival. The combination therapy has been shown to remodel the tumor immune microenvironment, leading to an increase in CD45[+] immune cells and CD8[+] T cells, including naïve subsets, and a decrease in FOXP3[+] Treg cells. This shift promotes T cell activation by modulating relevant genes and signaling pathways. This study provides a novel target for optimizing OV immunotherapy.},
}

*Tumor Microenvironment/immunology
Animals
*Oncolytic Virotherapy/methods
*Oncolytic Viruses/immunology/physiology
Humans
Cell Line, Tumor
Mice
*Colorectal Neoplasms/therapy/immunology/genetics
*CRISPR-Cas Systems

@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},
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/metabolism/drug therapy ; Animals ; *CRISPR-Cas Systems ; *Genomics/methods ; Mice ; Mutation ; Single-Cell Gene Expression Analysis ; Gene Expression Regulation, Leukemic ; Gene Regulatory Networks ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
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/metabolism/drug therapy
Animals
*CRISPR-Cas Systems
*Genomics/methods
Mice
Mutation
Single-Cell Gene Expression Analysis
Gene Expression Regulation, Leukemic
Gene Regulatory Networks
Clustered Regularly Interspaced Short Palindromic Repeats