@article {pmid39901476,
year = {2026},
author = {Clarissa, EM and Karmacharya, M and Choi, H and Kumar, S and Cho, YK},
title = {Nature Inspired Delivery Vehicles for CRISPR-Based Genome Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {16},
pages = {e2409353},
doi = {10.1002/smll.202409353},
pmid = {39901476},
issn = {1613-6829},
support = {IBS-R020-D1//Institute for Basic Science/ ; RS-2024-00508821//Korean government/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Nanoparticles/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; *Gene Transfer Techniques ; Liposomes/chemistry ; Extracellular Vesicles/metabolism ; },
abstract = {The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing technologies has opened up groundbreaking possibilities for treating a wide spectrum of genetic disorders and diseases. However, the success of these technologies relies heavily on the development of efficient and safe delivery systems. Among the most promising approaches are natural and synthetic nanocarrier-mediated delivery systems, including viral vectors, extracellular vesicles (EVs), engineered cellular membrane particles, liposomes, and various nanoparticles. These carriers enhance the efficacy of the CRISPR system by providing a unique combination of efficiency, specificity, and reduced immunogenicity. Synthetic carriers such as liposomes and nanoparticles facilitate CRISPR delivery with high reproducibility and customizable functions. Viral vectors, renowned for their high transduction efficiency and broad tropism, serve as powerful vehicles for delivering CRISPR components to various cell types. EVs, as natural carriers of RNA and proteins, offer a stealth mechanism to evade immune detection, allowing for the targeted delivery of genome editors with minimal off-target effects. Engineered cellular membrane particles further improve delivery by simulating the cellular environment, enhancing uptake, and minimizing immune response. This review explores the innovative integration of CRISPR genome editors with various nanocarrier systems, focusing on recent advancements, applications, and future directions in therapeutic genome editing.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
Nanoparticles/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Animals
*Gene Transfer Techniques
Liposomes/chemistry
Extracellular Vesicles/metabolism

@article {pmid40987370,
year = {2025},
author = {Sarwar, A and Shakeel, F and Fatima, T and Amin, R and Rizvi, SNB and Hussain, T and Afzal, A},
title = {DNA nanotechnology for next-generation biosensors: Principles, strategies, and challenges.},
journal = {International journal of biological macromolecules},
volume = {329},
number = {Pt 2},
pages = {147825},
doi = {10.1016/j.ijbiomac.2025.147825},
pmid = {40987370},
issn = {1879-0003},
mesh = {Humans ; Animals ; *Nanotechnology/methods ; *Biosensing Techniques/methods ; *DNA/chemistry ; Fluorescence ; Electrochemical Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {The unique structural and functional properties of nucleic acids make DNA nanotechnology a powerful platform for constructing programmable nanostructures with precise control over size, shape, and biorecognition. This review highlights how DNA-based biosensors achieve unparalleled sensitivity (e.g., attomolar detection via CRISPR systems) and multiplexing capabilities, overcoming limitations of conventional methods like PCR and ELISA. We critically evaluate design strategies, including aptamers, DNAzymes, DNA origami, and CRISPR integration, for detecting diverse analytes (nucleic acids, proteins, pathogens) in clinical, environmental, and food safety applications. While challenges such as nuclease susceptibility and scalability persist, emerging solutions like backbone modifications and microfluidic automation are discussed. By synthesizing recent advances, this review underscores DNA nanotechnology's transformative potential in next-generation diagnostics, offering a roadmap for bridging lab-scale innovations to real-world deployment.},
}

Humans
Animals
*Nanotechnology/methods
*Biosensing Techniques/methods
*DNA/chemistry
Fluorescence
Electrochemical Techniques/methods
CRISPR-Cas Systems

@article {pmid41135835,
year = {2026},
author = {Chiu, CN and Tam, BM and Burns, P and Moritz, OL},
title = {RPE65 knockout Xenopus laevis have a compromised but detectable electroretinogram and altered visual responses, without retinal degeneration or altered melanophore dispersion.},
journal = {Experimental eye research},
volume = {262},
number = {},
pages = {110699},
doi = {10.1016/j.exer.2025.110699},
pmid = {41135835},
issn = {1096-0007},
mesh = {Xenopus laevis/genetics ; Animals ; Gene Knockout Techniques ; *Retinoid Isomerohydrolase/genetics ; Disease Models, Animal ; *Rhodopsin ; Retinal Rod Photoreceptor Cells/pathology ; Electroretinography ; *Photophobia/physiopathology ; *Melanophores/physiology ; Retinal Cone Photoreceptor Cells ; CRISPR-Cas Systems ; Larva ; Behavior, Animal ; },
abstract = {The isomerohydrolase RPE65 is a critical element of the visual cycle, the series of enzymatic reactions by which the chromophore of the visual pigments is regenerated following light exposure. In humans, mutations in the RPE65 gene cause a severe form of blindness called Leber's congenital amaurosis. Studies of Rpe65[-/-] mice have shown dramatic depletion of 11-cis-retinal in the retina, resulting in a slow retinal degeneration. However, a number of studies suggest that RPE65 may not be necessary for the regeneration of photopigment in all photoreceptor types. Using CRISPR/Cas9 technology, we previously generated RPE65 knockout Xenopus laevis in order to test the involvement of rhodopsin chromophore in the cell death mechanisms associated with rhodopsin mutations and rhodopsin quality control. Here we further characterize the effects of RPE65 knockout in these animals, and show their rod photoreceptors have shortened outer segments that lack detectable rhodopsin photopigment. However, there is no progressive degeneration of rods or cones. Via electroretinography we found greatly reduced but significant responses to light under scotopic and photopic conditions. We also found reduced behavioral sensitivity to light, while light-induced melanophore dispersion was unaffected. RPE65 knockout X. laevis may be a useful system for examining RPE65-independent photosensation mechanisms in vertebrates.},
}

Xenopus laevis/genetics
Animals
Gene Knockout Techniques
*Retinoid Isomerohydrolase/genetics
Disease Models, Animal
*Rhodopsin
Retinal Rod Photoreceptor Cells/pathology
Electroretinography
*Photophobia/physiopathology
*Melanophores/physiology
Retinal Cone Photoreceptor Cells
CRISPR-Cas Systems
Larva
Behavior, Animal

@article {pmid41652229,
year = {2026},
author = {Xu, X and Fang, J and Chen, J and Yang, J and Yang, X and Hou, S and Sun, W and Champer, J},
title = {Assessing target genes for homing suppression gene drive.},
journal = {The EMBO journal},
volume = {45},
number = {6},
pages = {2074-2094},
pmid = {41652229},
issn = {1460-2075},
support = {32302455//MOST | National Natural Science Foundation of China (NSFC)/ ; 32270672//MOST | National Natural Science Foundation of China (NSFC)/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; Female ; *Gene Drive Technology/methods ; Male ; *Fertility/genetics ; CRISPR-Cas Systems ; Drosophila Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Gene drives are engineered alleles that bias their own inheritance in offspring, enabling the spread of specific traits throughout a population. Targeting female fertility genes in a gene drive can be an efficient strategy for population suppression. In this study, we investigated nine female fertility genes in Drosophila melanogaster using CRISPR-based homing gene drives. Employing a multiplexed gRNA approach to prevent the formation of functional resistance alleles, we aimed to maintain high drive-conversion efficiency with low fitness costs in female drive-carriers. Drive efficiency was assessed in individual crosses and had varied performance across different target genes. Notably, drives targeting the octopamine β2 receptor (oct) and stall (stl) genes exhibited the highest drive-conversion rates and were further tested in cages. A drive targeting stl successfully suppressed a cage population with a high release frequency, though suppression failed in another replicate cage with a lower initial release frequency. Fitness costs in female drive carriers were observed in test cages, impacting the overall efficiency of population suppression. Further tests on the fertility of these lines using individual crosses indicated that some fitness costs were due to maternal deposition of Cas9 combined with new gRNA expression, which would only occur in progeny of drive males when testing split drives with separate Cas9 (when mimicking cages with complete drives) but not for complete drive systems. This could enable success in complete drives with higher maternal Cas9 deposition, even if cage experiments in split drives fail. Overall, our findings identify oct and stl as promising fertility targets and demonstrate both the potential and the constraints of fertility-based suppression drives, providing empirical evidence to guide the design and assessment of more efficient population control strategies.},
}

Animals
*Drosophila melanogaster/genetics
Female
*Gene Drive Technology/methods
Male
*Fertility/genetics
CRISPR-Cas Systems
Drosophila Proteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41654520,
year = {2026},
author = {Zhang, J and Hołubowicz, R and Smidak, R and Hu, Y and Du, SW and Felgner, JH and Palczewska, G and Rodrigues Menezes, C and Risaliti, E and Dong, Z and Ma, X and Shayegan, MH and Chen, PZ and Xing, L and Hołubowicz, M and Li, B and Liu, DR and Felgner, PL and Tochtrop, GP and Palczewski, K},
title = {A combinatorial synthetic strategy for developing genome-editing protein-delivery agents targeting mouse retina.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41654520},
issn = {2041-1723},
support = {T32GM008620; F30EY033642//U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)/ ; R01EY009339; R01EY030873, P30EY034070//U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)/ ; CHE-1904530//National Science Foundation (NSF)/ ; P30CA062203//U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; P30 CA062203/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; Liposomes/chemistry ; Mice ; CRISPR-Cas Systems/genetics ; *Retina/metabolism ; Ribonucleoproteins/genetics/metabolism/administration & dosage ; Disease Models, Animal ; Rosaniline Dyes/chemistry ; Retinal Pigment Epithelium/metabolism ; },
abstract = {CRISPR/Cas9-based gene-editing technologies offer promise for treating inherited retinal diseases (IRDs), however safe and efficient ocular delivery of precision editors remains challenging. To address this challenge, we report a class of Coomassie brilliant blue (CBB)-derived lipidoids that bind and deliver proteins. Subretinal injection of Cre complexed with these lipidoids into mT/mG mice leads to robust recombination in the retinal pigment epithelium and photoreceptors. We employ the CBB-lipidoid platform to deliver adenine base editor (ABE) ribonucleoproteins (RNP). Incorporating CBB lipidoids into liposomes improves delivery efficiency. CBB11 stands out for facilitating precise in vivo ABE-mediated gene editing. Delivery of liposome-CBB11-RNP complexes results in a 120-fold increase in base editing compared to RNP alone and restores the scotopic ERG b-wave response in the rd12 mouse model. These results demonstrate the potential of CBB-augmented, liposome-RNP systems for therapeutic gene editing in the eye, paving the way for single-dose precision medicines to treat IRDs.},
}

Animals
*Gene Editing/methods
Liposomes/chemistry
Mice
CRISPR-Cas Systems/genetics
*Retina/metabolism
Ribonucleoproteins/genetics/metabolism/administration & dosage
Disease Models, Animal
Rosaniline Dyes/chemistry
Retinal Pigment Epithelium/metabolism

@article {pmid41700547,
year = {2026},
author = {Su, T and Zhu, D and Li, X and Qu, Z and Li, F and Zhao, D and Shao, G and Feng, Z and Su, S and Chao, J and Zuo, X and Fan, C and Wang, L},
title = {Accelerated CRISPR/Cas12a-Based Point-of-Care Diagnostics Through Critical Coupling Distance Control.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {16},
pages = {e23381},
doi = {10.1002/adma.202523381},
pmid = {41700547},
issn = {1521-4095},
support = {62071251//National Natural Science Foundation of China/ ; 62235008//National Natural Science Foundation of China/ ; 62227803//National Natural Science Foundation of China/ ; 62288102//National Natural Science Foundation of China/ ; BK20212012//Natural Science Foundation of Jiangsu Province-Major Project/ ; BZ2022011//"Belt and Road" Innovation Cooperation Project of Jiangsu/ ; SKT2306//Project of State Key Laboratory of Transducer Technology/ ; GZR2022010026//Project of State Key Laboratory of Organic Electronics and Information Displays, Nanjing University of Posts and Telecommunications/ ; BK20243057//Basic Research Program of Jiangsu/ ; BK20253006//Basic Research Program of Jiangsu/ ; },
mesh = {*CRISPR-Cas Systems ; *Point-of-Care Systems ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism ; *CRISPR-Associated Proteins/metabolism ; DNA/chemistry ; Humans ; },
abstract = {Prompt pathogen detection in resource-limited settings remains constrained by energy-intensive instrumentation and a shortage of trained personnel. The CRISPR/Cas12a-based diagnostic technology, despite its robustness as a promising tool, is constrained by suboptimal detection speed and sensitivity. Here we designed triblock DNA-mediated spherical nucleic acids (tSNA) that acts as a spatially confined reporter with critical coupling distances between substrates, enabling Cas12a protein to rapidly identify concentrated and stretched single-stranded substrates with size-matching intervals. Precise control of distances on tSNA of varying sizes revealed a direct correlation between trans-cleavage efficiency and coupling distance, indicating that only when the distance exceeds the protein size can it offer an appropriate reaction space. It demonstrates a rapid "scooting" reaction model on tSNA, resulting in a trans-cleavage rate of 10 nm A30-tSNA 12 times faster and a sensitivity that is two orders of magnitude higher than that in bulk solution. Furthermore, tSNA can serve as a novel recognition and colorimetric element in lateral-flow strips, thereby reducing the detection time for pathogen nucleic acids to just 3 min. This "size-matching" model of tSNA offers a new perspective on the regulation of Cas12a enzymatic activity, establishing a versatile platform to advance diagnostic development through ultrafast, CRISPR-powered POC systems.},
}

*CRISPR-Cas Systems
*Point-of-Care Systems
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism
*CRISPR-Associated Proteins/metabolism
DNA/chemistry
Humans

@article {pmid41766140,
year = {2026},
author = {Hartig, AM and Dai, W and Zhang, K and Rottinghaus, AG and Moon, TS and Parker, KM},
title = {Genetic Markers Remain Detectable in Genetically Engineered Microbes Biocontained with a CRISPR Kill Switch.},
journal = {Environmental science & technology},
volume = {60},
number = {10},
pages = {7983-7994},
doi = {10.1021/acs.est.6c00321},
pmid = {41766140},
issn = {1520-5851},
mesh = {Escherichia coli/genetics ; Genetic Markers ; *CRISPR-Cas Systems ; Genetic Engineering ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Microorganisms, Genetically-Modified/genetics ; },
abstract = {Biocontainment strategies, such as kill switches, have been developed to avoid the unintended proliferation of genetically engineered microbes (GEMs) intended for open-release environmental applications. However, the presence of GEM DNA after successful biocontainment presents new environmental risks and challenges for monitoring. In this study, we investigated whether biocontainment using a CRISPR-Cas9 kill switch, which causes double-strand breaks in target genes essential for GEM growth, could resolve this challenge in a model Escherichia coli GEM. Surprisingly, the escape rates of the GEM as determined by CRISPR-targeted gene abundances were as high as 10[-1.6] to 10[-1.0] in LB media, despite the escape rates measured by colony forming units (cfu) being only 10[-6.2] under the same condition. This discrepancy suggested that the CRISPR-Cas9 kill switch prevents colony growth while still leaving a large fraction of target genes intact for detection by molecular methods. Within 1 h after biocontainment, these target genes remained predominantly inside an intact cell membrane and were resistant to degradation by DNase, though degradation was observed in river water over multiple days. Overall, a detailed understanding of the impact of the biocontainment mechanism on both the GEM and its DNA is needed to minimize unintended environmental risks.},
}

Escherichia coli/genetics
Genetic Markers
*CRISPR-Cas Systems
Genetic Engineering
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Microorganisms, Genetically-Modified/genetics

@article {pmid41773916,
year = {2026},
author = {Sui, Z and Chen, B and Zhao, J and Deng, R and Xu, J},
title = {Pronounced Fluorescence Polarization Enhancement Driven by RPA-CRISPR/Cas12a Induced Nucleoprotein Assembly for Salmonella Analysis in Animal-Derived Food Matrices.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7822-7831},
doi = {10.1021/acs.analchem.5c08279},
pmid = {41773916},
issn = {1520-6882},
mesh = {*Fluorescence Polarization/methods ; *CRISPR-Cas Systems ; *Salmonella/isolation & purification/genetics ; Animals ; *Nucleoproteins/metabolism/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Food Microbiology ; *CRISPR-Associated Proteins/metabolism ; *Recombinases/metabolism ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins/metabolism/genetics ; },
abstract = {Salmonella is one of the most hazardous foodborne pathogens, posing a serious threat to public health and food safety worldwide. Conventional recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection assays predominantly rely on the trans-cleavage of fluorescent reporters; however, such signal-generation modes are inherently susceptible to photobleaching, signal drift, and fluctuation, thereby compromising quantitative accuracy and long-term signal stability in practical pathogen detections. To overcome these limitations, we developed a trans-cleavage-independent fluorescence polarization (FP) sensing platform for the rapid and quantitative detection of Salmonella. Unlike conventional reporter-cleavage-based readouts, the proposed system exploits target-induced nucleoprotein assembly to achieve direct, physical signal amplification. In this design, a FAM-labeled forward primer serves as an intrinsic molecular reporter, while exonuclease I (Exo I) selectively degrades unincorporated primers, effectively suppressing background interference. Upon recognition of Salmonella genomic DNA, RPA produces rigid double-stranded amplicons that restrict fluorophore rotational freedom, and subsequent crRNA-guided Cas12a binding further increases molecular size and hydrodynamic volume, resulting in a stepwise enhancement of FP signals. The assay exhibits excellent linearity over a concentration range of 3 × 10[1]-3 × 10[6] CFU mL[-1], with an ultralow detection limit of 5 CFU mL[-1]. In addition, it demonstrates outstanding photostability, reproducibility, and high specificity against nontarget bacteria. Importantly, reliable Salmonella detection was achieved in complex food matrices, including meat, eggs, and dairy products, with consistently high recoveries and strong tolerance to matrix interference, offering a promising alternative to conventional fluorescence-intensity-based CRISPR diagnostics in complex food systems.},
}

*Fluorescence Polarization/methods
*CRISPR-Cas Systems
*Salmonella/isolation & purification/genetics
Animals
*Nucleoproteins/metabolism/chemistry
*Nucleic Acid Amplification Techniques/methods
*Food Microbiology
*CRISPR-Associated Proteins/metabolism
*Recombinases/metabolism
*Endodeoxyribonucleases/metabolism
Bacterial Proteins/metabolism/genetics

@article {pmid41785043,
year = {2026},
author = {Xu, X and Zhang, R and Hai, G and Wang, Y and Zheng, H and Cui, P and Kou, B and Jin, X and Peng, J},
title = {DNA Logic-Gated CRISPR/Cas13a and PNTs-Hemin Biomimetic Nanozyme for Ratiometric Detection of BRCA1 and circROBO1.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7580-7589},
doi = {10.1021/acs.analchem.5c07475},
pmid = {41785043},
issn = {1520-6882},
mesh = {*Hemin/chemistry ; Humans ; *BRCA1 Protein/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Biomimetic Materials/chemistry ; *DNA/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Logic ; Electrochemical Techniques ; },
abstract = {Although nanozyme-based biosensors show great promise for the early diagnosis of cancer, their application is often limited by poor catalytic activity at neutral pH and susceptibility to matrix interference. This study involved the construction of a biomimetic nanozyme through coordination-driven self-assembly on peptide nanotubes. This rigid framework enables the periodic arrangement of histidine residues to achieve precise axial coordination with the iron center of the hemin molecule, effectively mimicking the active site and catalytic microenvironment of natural horseradish peroxidase. Consequently, PNTs-hemin exhibits peroxidase activity 2.7 times that of free hemin under near-physiological conditions. To ensure detection specificity, an AND logic gate design was integrated, triggering CRISPR/Cas13a-mediated trans-cleavage only when both targets (BRCA1 and circROBO1) are present simultaneously. Furthermore, combining the nanozyme with graphdiyne-supported gold nanoparticles resulted in the formation of a cascade catalytic system that produced a ratiometric electrochemical reading (IFc/Ihemin). This effectively corrects for environmental fluctuations and false positive signals. This study presents an effective strategy that combines the specificity of molecular logic gates with biomimetic catalysis. This opens up new avenues for the precise diagnosis of multiple targets in complex biological samples.},
}

*Hemin/chemistry
Humans
*BRCA1 Protein/analysis
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Biomimetic Materials/chemistry
*DNA/chemistry
Gold/chemistry
Metal Nanoparticles/chemistry
Logic
Electrochemical Techniques

@article {pmid41787951,
year = {2026},
author = {Chatla, K and Roper, B and Ayalew, L and Ko, P and Lippold, S and Doma, M and Camperi, J},
title = {Assessing mRNA and sgRNA Quality for Cell and Gene Therapy Applications Using Nanopore Direct RNA Sequencing.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7452-7461},
doi = {10.1021/acs.analchem.5c06819},
pmid = {41787951},
issn = {1520-6882},
mesh = {*RNA, Messenger/genetics/analysis ; Humans ; *Sequence Analysis, RNA/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/analysis ; *Genetic Therapy ; *Nanopore Sequencing/methods ; *Nanopores ; },
abstract = {Recent advances in RNA technology have enabled the development of diverse therapeutics spanning vaccines, immunotherapies, and genome-editing platforms. Ensuring clinical efficacy and safety requires precise characterization and control of RNA critical quality attributes (CQAs). Nanopore direct RNA sequencing (NDRS) has emerged as a powerful single-molecule analytical approach capable of simultaneously resolving sequence and structural features consistent with regulatory expectations. In this study, we establish NDRS as a comprehensive, multiattribute analytical platform by integrating novel strategies to assess key CQAs in a single assay. Following workflow optimization, NDRS accurately determined full-length mRNA sequences and evaluated transcript integrity. Notably, we developed the first sequencing-based method for quantifying 5' capping efficiency directly from native RNA molecules. Additionally, we demonstrated, for the first time, full-length sequencing of 100-nucleotide single-guide RNA (sgRNA) molecules by incorporating a 5' RNA oligo adapter, enabling complete identity verification. Quantitative results for poly(A) tail length, integrity, and capping efficiency were consistent with established orthogonal techniques, including chromatography and mass spectrometry. Moreover, functional correlation studies with Cas9 mRNA and sgRNA used in CRISPR-Cas9 editing revealed that increased mRNA degradation led to decreased knockout efficiency. Together, these findings position NDRS as a versatile and unified analytical platform for comprehensive characterization of mRNA and sgRNA, supporting quality assurance, comparability, and control in the development and manufacturing of next-generation RNA therapeutics.},
}

*RNA, Messenger/genetics/analysis
Humans
*Sequence Analysis, RNA/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/analysis
*Genetic Therapy
*Nanopore Sequencing/methods
*Nanopores

@article {pmid41824451,
year = {2026},
author = {Yuan, L and Liu, Q and Xiao, X and Xu, L and Liang, L and Guo, Y and Yao, Y and Wang, H and Feng, Y and Hua, X and Feng, Y},
title = {AlphaFold 3-powered discovery of phage proteins that inhibit bacterial transcription initiation.},
journal = {Cell reports},
volume = {45},
number = {3},
pages = {117082},
doi = {10.1016/j.celrep.2026.117082},
pmid = {41824451},
issn = {2211-1247},
abstract = {Many phages encode proteins that specifically inhibit host RNA polymerase activity, thereby sabotaging and, in some cases, hijacking the host transcription machinery to serve their needs. Traditional methods for identifying new phage proteins that inhibit bacterial transcription are labor intensive and require access to live phages. To overcome these limitations, we develop a highly efficient pipeline for AlphaFold 3-guided discovery of phage proteins that inhibit bacterial transcription initiation. Using this pipeline, three phage proteins are identified and characterized. Structural and biochemical analyses demonstrate that these phage proteins bind to distinct sites on RNA polymerase and inhibit transcription initiation via different mechanisms. This study showcases the power of AlphaFold 3 in discovering novel binders of large protein complexes, and the pipeline developed here could be readily adapted to screen modulators of other large targets, such as the ribosome, proteasome, and CRISPR-Cas systems.},
}

@article {pmid41824934,
year = {2026},
author = {Arif, MA and Mubashir, F and Raffay, A and Talha, M and Zohaib, M and Khan, AR and Naseer, MU and Khan, MH and Idrees, M and Rafique, S and Afzal, S and Amin, I and Shahid, M},
title = {Recent Advances in CRISPR-Cas Systems for Dengue Virus Detection.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {36},
number = {1},
pages = {19-35},
doi = {10.1615/CritRevEukaryotGeneExpr.2025062420},
pmid = {41824934},
issn = {2162-6502},
mesh = {*Dengue Virus/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Dengue/diagnosis/virology/genetics ; RNA, Viral/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Dengue virus (DENV) infections persist as a significant global health threat despite decades of surveillance and control efforts. The disease may progress to severe dengue, marked by hemorrhage, plasma leakage, and vital organ impairment, contributing to substantial worldwide morbidity and mortality. The rapidly escalating DENV burden demands rapid and innovative diagnostic approaches that move beyond conventional detection methods. CRISPR-Cas-based technologies have emerged as a revolutionary approach, offering next-generation solutions for DENV diagnostics. This review outlines the recent advances in the use of CRISPR based technologies for robust and more sensitive detection of dengue virus nucleic acids, critically evaluating their advantages over conventional diagnostics, current limitations, and future prospects. The roles of Cas12 and Cas13 in DENV-RNA detection are discussed in detail. Additional key areas mentioned include field-deployable and portable CRISPR-Cas technologies, serotype-specific detection, hybrid and isothermal amplification-based approaches, and a combination of CRISPR with electrochemical sensing techniques and nanotechnology. Collectively, these advances highlight the potential of CRISPR-based diagnostics in evolving future strategies for rapid and effective dengue virus detection and control of infections.},
}

*Dengue Virus/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
*Dengue/diagnosis/virology/genetics
RNA, Viral/genetics
Nucleic Acid Amplification Techniques/methods

@article {pmid41825677,
year = {2026},
author = {Xu, Z and Haghighat, M and Shafiabady, N and Wu, R and Sang, R and Deng, W},
title = {Nuclear-targeted CRISPR/Cas delivery by using aptamer-modified polymer lipid nanoparticles.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {74},
number = {},
pages = {102928},
doi = {10.1016/j.nano.2026.102928},
pmid = {41825677},
issn = {1549-9642},
abstract = {Efficient nuclear delivery of CRISPR/Cas ribonucleoproteins (RNP) remains a significant hurdle for non-viral systems. To address this, we developed a polymer-lipid hybrid nanoparticle functionalized with the AS1411 aptamer, targeting nucleolin to facilitate nucleus-directed delivery. Confocal imaging confirmed the accumulation of these aptamer-modified nanoparticles within the cell nuclei. For precise quantification, we utilized an AI-assisted segmentation approach based on deep convolutional neural networks (CNN) to analyse nanoparticle and DAPI colocalization. We further evaluated in vitro gene knockout efficiency of Cas9/sgRNA by using this nucleus-targeted system. Aptamer-functionalised nanoparticles reduced GFP-positive cells to 30.0%, compared with 40.8% for untargeted nanoparticles. Further evaluation targeting the Lcn2 gene demonstrated higher knockout efficacy and a more potent inhibition of breast cancer cell proliferation. These findings indicate that aptamer-mediated nuclear targeting enhances CRISPR/Cas9 editing efficacy and may offer the potential to advance the performance of non-viral gene therapies.},
}

@article {pmid41759757,
year = {2026},
author = {Schmidt, GE and Weaver, EA and Kim, TH},
title = {CRISPR-based functional analysis of chicken IRF9 reveals distinct modulation of dsRNA stimulated innate immune pathways.},
journal = {Developmental and comparative immunology},
volume = {177},
number = {},
pages = {105577},
doi = {10.1016/j.dci.2026.105577},
pmid = {41759757},
issn = {1879-0089},
mesh = {Animals ; *Chickens/immunology/genetics ; Immunity, Innate/genetics ; *Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics/metabolism ; *RNA, Double-Stranded/immunology ; *Avian Proteins/genetics/metabolism ; Interferon Type I/metabolism ; Signal Transduction ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation ; },
abstract = {The chicken immune system is distinct from mammalian models due to its reduced immune gene repertoire, yet it retains the ability to mount a highly effective immune response. In mammals, interferon regulatory factor 9 (IRF9) is a key transcriptional regulator of the type I interferon (IFN) pathway, stimulating the expression of hundreds of antiviral genes. Although IRF9 was previously thought to be absent in chickens, current chicken reference genome annotation (bGalGal1.mat.broiler.GRCg7b) lists a putative chicken IRF9. To investigate the function of this gene in chickens, we utilized a clustered regularly interspace short palindromic repeats (CRISPR) based transcriptional modulation platform to elucidate the role of the putative chicken IRF9 in the innate immune response. We analyzed the transcriptomes of IRF9 repressed cells stimulated with double stranded RNA at 0, 0.5, 1, and 6 h post-stimulation. Gene set enrichment analysis revealed that IRF9 repression resulted in the enrichment of pathways associated with regulating the type I IFN response, including the retinoic acid inducible gene I like (RIG-I like) receptor pathway and the Toll-like receptor pathway. Furthermore, concurrent transcriptional repression of type I IFN modulator IRF7 with transcriptional activation of IRF9 failed to rescue the expression of downstream IFN-stimulated genes. These results suggest chicken IRF9 plays a distinct regulatory role from canonical mammalian IRF9 in the type I IFN response and demonstrate a need for functional evidence-based classification of chicken IRFs.},
}

Animals
*Chickens/immunology/genetics
Immunity, Innate/genetics
*Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics/metabolism
*RNA, Double-Stranded/immunology
*Avian Proteins/genetics/metabolism
Interferon Type I/metabolism
Signal Transduction
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems
Gene Expression Regulation

@article {pmid41820207,
year = {2026},
author = {Porwal, S and Malviya, R and Belagodu Srighar, S and Shareef, J},
title = {Ribosome-targeted and Adjuvant Strategies to Combat Antibiotic Resistance.},
journal = {Infectious disorders drug targets},
volume = {},
number = {},
pages = {},
doi = {10.2174/0118715265396934250805055608},
pmid = {41820207},
issn = {2212-3989},
abstract = {INTRODUCTION: This study examines ribosome-targeted and adjuvant strategies to combat the growing threat of antimicrobial resistance (AMR), with a focus on novel therapeutic approaches, including phage therapy, monoclonal antibodies, CRISPR systems, and AI-driven drug discovery. The objective is to review current challenges and evaluate innovative strategies targeting bacterial ribosomes, a primary site for antibiotic action.

METHODS: A systematic literature review was conducted using databases, such as PubMed, ScienceDirect, Scopus, and Google Scholar.

RESULTS: The study indicates that bacteria evade ribosome-targeting antibiotics through various mechanisms, including porin modification, efflux pumps, ribosomal mutations, and enzymatic degradation. Innovative strategies, including AI-enabled virtual screening, phage-antibiotic synergy, ribosomal protein-targeted monoclonal antibodies and vaccines, and CRISPR-Cas systems, have shown potential in overcoming these mechanisms and restoring antibiotic efficacy.

DISCUSSION: These advanced strategies represent a significant shift from traditional approaches, as they directly target ribosomal functions or resistance genes. While promising, limitations such as phage specificity, challenges in CRISPR delivery, and regulatory concerns must be addressed to ensure clinical translation. The integration of AI with molecular techniques enhances therapeutic precision and development speed.

CONCLUSION: Ribosome-targeted therapies and adjunctive strategies, such as AI, phage therapy, monoclonal antibodies, and CRISPR, offer precise and innovative solutions to overcome antibiotic resistance.},
}

@article {pmid41820622,
year = {2026},
author = {Padureanu, T and Cocoș, R and Matache, IM and Bucur, O},
title = {Gene-sized editing for the therapy of genetic diseases.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {41820622},
issn = {1438-7948},
abstract = {Programmable genome editing technologies have reshaped the landscape of biomedical sciences, enabling the development of methods with great translational potential. CRISPR-Cas represents one of the most important and widely adopted genome editing tools, although its reliance on double-stranded DNA breaks implies inherent limitations on the precision and safety of genomic insertions. Thus, several research groups have focused on the development of new editing technologies, among which prime editing has emerged as a cutting-edge system. Ongoing advancements in prime editing, including protein engineering, have enhanced its efficiency and expanded its functionality. However, prime editing cannot achieve integration of large DNA sequences larger than 5 kilobases. To overcome this limitation, PASTE and PASSIGE methods were developed as novel genome editing methods that merge precise genome rewriting with efficient recombinase-mediated gene insertion. In this review, we investigate the mechanistic principles of these systems, compare their performance in cellular and animal models, and discuss the ongoing efforts to enhance system components and delivery. We extended our investigation to recent progress supporting their translational potential, assessing efficient delivery methods, genome site specificity, safety, and long-term efficacy, which are crucial for successful in vivo applications.},
}

@article {pmid41821244,
year = {2026},
author = {Elamin Eltom, A and Kareem, AK and Shaaban, Z and Sanaan Jabbar, H and Sharma, MK and Ghazi Al-Shawi, S and AlMohamadi, H and Abbas, Z and S Jabir, M and Ahmed AbdRabou, M},
title = {Progress of Magnetic Particles-Integrated CRISPR/Cas Biosensors for Pathogen Bacteria Detection: Design, Mechanism and Application.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10408347.2026.2636658},
pmid = {41821244},
issn = {1547-6510},
abstract = {Ensuring food safety through rapid, sensitive, and point-of-care (POC) detection of microbial pathogens is crucial for protecting public health and minimizing the socio-economic losses associated with foodborne diseases. Despite stringent regulatory measures, foodborne illnesses caused by microbial contamination continue to pose a significant global challenge. In this context, the emergence of CRISPR/Cas systems has significantly improved the performance of biosensors due to their programmability, high specificity, and precise recognition of target RNA and DNA sequences. Following target recognition, Cas proteins exhibit both cis- and trans-cleavage activities, enabling highly sensitive signal amplification. To achieve rapid analysis and low detection limits, recent studies have increasingly focused on integrating CRISPR/Cas system with magnetic particles (MPs). MPs offer key advantages, including superparamagnetism, biocompatibility, and facile surface functionalization, which enhance target enrichment, assay speed, and analytical sensitivity. Accordingly, substantial progress has been made in MP-conjugated CRISPR/Cas biosensors for the detection of diverse foodborne microbial pathogens. This review comprehensively summarizes recent advances in the integration strategies of magnetic particles with CRISPR/Cas-based biosensing platforms for the quantitative detection of microbial pathogens. Particular emphasis is placed on performance metrics, assay design, and the feasibility of these systems for POC applications, highlighting their potential to enhance food safety monitoring.},
}

@article {pmid41823840,
year = {2026},
author = {Loweree-Rivera, FD and Pérez-Álvarez, S and Castillo, AM and Vega Mares, JH and Leyva-Hernández, HA and Sánchez Chávez, E and Escobedo-Bonilla, CM and Uranga-Valencia, LP and Chávez Medina, JA},
title = {Pelecyphora chihuahuensis (Britton & Rose) D. Aquino & Dan. Sánchez: A Review on Its Taxonomy, Ecology and Conservation of an Endemic Mexican Cactus Species with Biotechnological Perspectives.},
journal = {Biology},
volume = {15},
number = {5},
pages = {},
pmid = {41823840},
issn = {2079-7737},
abstract = {The cactus Pelecyphora chihuahuensis is endemic to northern Mexico and represents an interesting subject on the integration of classical taxonomy with modern biotechnological tools to solve conservation issues. Because of its narrow ecological range and high ornamental value, the species is increasingly at risk from degradation of its habitats, climate change, and plant poaching. This review includes current knowledge on its taxonomic status, ecological distribution, and conservation needs, with a focus on biotechnological means to aid its preservation. Aspects such as molecular markers, next-generation sequencing, and previously reported GIS-based species distribution models provide valuable insights into its identity and ecological niche. Biotechnological tools for ex situ conservation include in vitro propagation and cryopreservation. Potential applications of CRISPR-Cas and synthetic biology in preserving rare cacti are also discussed. By uncovering gaps, this review opens a window of opportunity to urgently promote the sustainable management of P. chihuahuensis and related endangered cacti by merging biotechnology with ecology and taxonomy, the results presented here underscore the importance of integrating scientific findings into future research that supports the development and implementation of effective policies that prioritize the conservation and biocultural preservation of arid-land flora, ensuring that both ecological integrity and cultural values are maintained for these unique ecosystems.},
}

@article {pmid41824054,
year = {2026},
author = {Ren, C and Lin, Y and Mohamed, MSM and Liu, C and Liang, Z},
title = {A simplified RUBY reporter-enabled hairy-root system for rapid evaluation of genome editing and gene function in grapevine.},
journal = {Planta},
volume = {263},
number = {4},
pages = {},
pmid = {41824054},
issn = {1432-2048},
support = {YSBR-093//CAS Project for Young Scientists in Basic Research/ ; 32530090//National Natural Science Foundation of China/ ; 2022078//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; },
mesh = {*Vitis/genetics/metabolism ; *Gene Editing/methods ; *Plant Roots/genetics/metabolism ; Genes, Reporter ; CRISPR-Cas Systems ; Lignin/metabolism/biosynthesis ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; Glucosyltransferases/genetics ; Gene Expression Regulation, Plant ; },
abstract = {The simplified RUBY represents an efficient reporter for monitoring plant transformation and, when combined with hairy-root system in grapevine, can facilitate the application of novel CRISPR technologies and gene functional study. Monitoring successful transformation events is essential for plant transformation and genome editing. The development and application of the RUBY reporter enable effective selection of transformation events based solely on distinct red pigmentation. Here, we report that a simplified version of RUBY (siRUBY), lacking the glucosyltransferase gene, also functions effectively as a visual selection marker for plant transformation. Furthermore, the siRUBY-assisted hairy-root system was established as a rapid and efficient platform for evaluating activity of adenine base editor (ABE) in grapevine. Targeted A-to-G base editing was achieved using ABE8e, with an average efficiency of approximately 14%. Moreover, this platform is also suitable for functional investigation of genes of interest. Knockout of the MYB4a gene promoted lignin deposition, likely through upregulating key lignin biosynthesis genes while repressing transcription of downstream flavonoid biosynthesis genes. Collectively, these results demonstrate that siRUBY coupled with grapevine hairy roots provides a robust platform for rapid and efficient genome editing in grapevine.},
}

*Vitis/genetics/metabolism
*Gene Editing/methods
*Plant Roots/genetics/metabolism
Genes, Reporter
CRISPR-Cas Systems
Lignin/metabolism/biosynthesis
Plants, Genetically Modified
Plant Proteins/genetics/metabolism
Glucosyltransferases/genetics
Gene Expression Regulation, Plant

@article {pmid40588030,
year = {2026},
author = {Guo, Z and Tang, J and Ling, S and Zheng, C and Bass, C and He, S and Wan, H and Li, J and Ma, K},
title = {Upregulation of a cytochrome P450 gene, CYP6B50, confers multi-insecticide resistance in Spodoptera frugiperda.},
journal = {Journal of advanced research},
volume = {82},
number = {},
pages = {99-111},
doi = {10.1016/j.jare.2025.06.083},
pmid = {40588030},
issn = {2090-1224},
mesh = {Animals ; *Spodoptera/genetics/drug effects ; *Insecticide Resistance/genetics ; *Cytochrome P-450 Enzyme System/genetics/metabolism/chemistry ; Insecticides/pharmacology ; Up-Regulation ; *Insect Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; Larva/genetics/drug effects ; Molecular Docking Simulation ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Oxazines ; },
abstract = {INTRODUCTION: The fall armyworm, Spodoptera frugiperda, is a globally invasive pest that has developed resistance to numerous insecticides. Cytochrome P450 enzymes have been commonly implicated in insecticide resistance in insect pests, including in S. frugiperda.

OBJECTIVE: Our previous study revealed that the P450 gene CYP6B50 was significantly overexpressed in a multi-insecticide resistant strain of S. frugiperda. However, the functional role of CYP6B50 in conferring multiple resistance remains unclear.

METHODS: Here we used reverse genetic (RNAi, CRISPR/Cas9, and transgenic Drosophila melanogaster) and computational modelling approaches to investigate the role of CYP6B50 in resistance to insecticides.

RESULTS: Functional analysis revealed that CYP6B50 is highly expressed during the larval stage of S. frugiperda, particularly in key tissues associated with detoxification such as the fat body, midgut, hemolymph, and Malpighian tubules. The expression of CYP6B50 was significantly upregulated after exposure to the insecticides indoxacarb, chlorantraniliprole, lambda-cyhalothrin, tetrachlorantraniliprole, and deltamethrin. Functional validation of CYP6B50 confirmed its contribution to resistance against the five tested insecticides. Molecular docking revealed a strong binding affinity of CYP6B50 to these insecticides.

CONCLUSIONS: These results identify CYP6B50 as a key generalist detoxification P450 in S. frugiperda and its role in mediating resistance to multiple insecticides. These findings provide insights into the role of cytochrome P450s in the evolution of insecticide resistance and will inform the development of strategies to manage or overcome resistance in a globally distributed crop pest.},
}

Animals
*Spodoptera/genetics/drug effects
*Insecticide Resistance/genetics
*Cytochrome P-450 Enzyme System/genetics/metabolism/chemistry
Insecticides/pharmacology
Up-Regulation
*Insect Proteins/genetics/metabolism
Drosophila melanogaster/genetics
Larva/genetics/drug effects
Molecular Docking Simulation
Animals, Genetically Modified
CRISPR-Cas Systems
Oxazines

@article {pmid40712808,
year = {2026},
author = {Jin, M and Huang, S and Zhou, S and Shen, W and Cao, J and Song, S and Wei, Y and Kalds, P and Hua, J and Ma, B and Ross, PJ and Wang, X and Chen, Y},
title = {Efficient derivation of stable sheep embryonic stem cells opens a new avenue for agricultural and biomedical application.},
journal = {Journal of advanced research},
volume = {82},
number = {},
pages = {155-169},
doi = {10.1016/j.jare.2025.07.036},
pmid = {40712808},
issn = {2090-1224},
mesh = {Animals ; Sheep ; *Embryonic Stem Cells/cytology/metabolism ; Gene Editing/methods ; Cell Differentiation/genetics ; CRISPR-Cas Systems ; Agriculture/methods ; Cell Culture Techniques/methods ; Blastocyst/cytology ; Pluripotent Stem Cells/cytology ; },
abstract = {INTRODUCTION: Embryonic stem cells (ESCs), capable of generating all adult cell types, hold transformative potential for agriculture and biomedicine. However, stable sheep ESCs derivation remains challenging, limiting their application in further research.

OBJECTIVES: We aimed to establish stable sheep ESCs using a simplified protocol, validate their pluripotency, and demonstrate genome editing utility.

METHODS: Sheep ESCs were derived from blastocyst inner cell masses via an IWR-1-containing culture system (termed TePR). Pluripotency was assessed through long-term culture (>100 passages), trilineage differentiation, transcriptomic analysis (E0-E6 embryos), and cross-species comparisons. ATAC-seq and WGBS mapped chromatin accessibility and methylation patterns, respectively. Genome editing utilized PiggyBac transposition and CRISPR/Cas9.

RESULTS: Sheep ESCs derived under TePR condition (termed TePR-sESCs) exhibited stable morphology and trilineage differentiation. Transcriptomics showed TePR-sESCs resemble 8-cell/morula embryos, consistent with sheep genome activation timing. ATAC-seq revealed accessible chromatin at pluripotency loci (e.g., POU5F1, NANOG). WGBS identified hypomethylation in pluripotency-associated regions. Efficient mCherry integration and MSTN knockouts confirmed editing compatibility.

CONCLUSION: The TePR system enables robust derivation of sheep ESCs with embryonic-like pluripotency. TePR-sESCs' editing proficiency supports applications in livestock trait engineering and biomedical modeling, overcoming a major barrier in ungulate stem cell research.},
}

Animals
Sheep
*Embryonic Stem Cells/cytology/metabolism
Gene Editing/methods
Cell Differentiation/genetics
CRISPR-Cas Systems
Agriculture/methods
Cell Culture Techniques/methods
Blastocyst/cytology
Pluripotent Stem Cells/cytology

@article {pmid41544897,
year = {2026},
author = {Hossain, NS and Tasnim, N and Ferdoush, J and Roy, A and Saha, SC and Saha, A},
title = {Next-generation lung-cancer-on-a-chip: Toward personalized therapy, AI, and CRISPR-driven models.},
journal = {Drug discovery today},
volume = {31},
number = {2},
pages = {104604},
doi = {10.1016/j.drudis.2026.104604},
pmid = {41544897},
issn = {1878-5832},
mesh = {Humans ; *Lung Neoplasms/genetics/drug therapy/pathology/therapy ; *Precision Medicine/methods ; *Artificial Intelligence ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Animals ; CRISPR-Cas Systems ; *Lab-On-A-Chip Devices ; Tumor Microenvironment ; },
abstract = {Lung-cancer-on-a-chip (LCOC) technologies have advanced rapidly, yet most models evaluate mechanical strain, patient-derived tumors, multi-organ interactions, artificial intelligence (AI) analytics, and clustered regularly interspaced short palindromic repeats (CRISPR) editing in isolation. In this review, we uniquely integrate these emerging components into a unified framework centered on the breathing LCOC. We highlight how embedding patient-derived lung tumor fragments into cyclically stretched microenvironments, then linking them to downstream organ compartments, enables patient-specific mapping of metastatic routes under physiologically relevant mechanics. We further describe how continuous high-resolution imaging from these platforms can feed AI pipelines for automated drug-response prediction and metastatic trajectory simulation, and how on-chip CRISPR editing enables accurate investigation of metastatic drivers within dynamic, strain-modulated microenvironments. By synthesizing these technologies, we outline a next-generation, personalized multi-organ-on-chip architecture capable of predicting individual disease progression without direct patient risk. We also address practical barriers, including tumor fragility under strain, imaging domain shift, and gene-editing delivery challenges, and how to overcome such barriers.},
}

Humans
*Lung Neoplasms/genetics/drug therapy/pathology/therapy
*Precision Medicine/methods
*Artificial Intelligence
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Animals
CRISPR-Cas Systems
*Lab-On-A-Chip Devices
Tumor Microenvironment

@article {pmid41654238,
year = {2026},
author = {Jo, SJ and Park, SC and Kim, SG},
title = {CRISPR/Cas9-engineered Salmonella phage displaying antimicrobial peptide LL37 for enhanced antibacterial activity.},
journal = {International journal of antimicrobial agents},
volume = {67},
number = {4},
pages = {107734},
doi = {10.1016/j.ijantimicag.2026.107734},
pmid = {41654238},
issn = {1872-7913},
mesh = {Animals ; *Salmonella typhimurium/virology/drug effects ; *Cathelicidins/genetics/pharmacology ; *CRISPR-Cas Systems ; *Salmonella Phages/genetics ; *Anti-Bacterial Agents/pharmacology ; Moths/microbiology ; *Antimicrobial Cationic Peptides/genetics/pharmacology ; Epithelial Cells/microbiology ; Humans ; Larva/microbiology ; Phage Therapy ; *Antimicrobial Peptides/genetics/pharmacology ; Salmonella Infections/therapy ; Gene Editing ; },
abstract = {OBJECTIVES: The increasing prevalence of antibiotic-resistant Salmonella Typhimurium has highlighted the urgent need for alternative therapeutic strategies. This study engineered a lytic S. Typhimurium bacteriophage to present the antimicrobial peptide LL-37 on the virion surface, followed by evaluation of its enhanced antibacterial efficacy.

METHODS: A recombinant lytic bacteriophage displaying LL-37 on its capsid was constructed using CRISPR/Cas9-mediated genome editing. The engineered phage was characterized for structural stability, adsorption kinetics, and lytic activity. Antibacterial efficacy was evaluated through bacterial growth inhibition assays, assessment of phage resistance rates, and host-pathogen interaction studies using intestinal epithelial cells. Intracellular bacterial survival was assessed in vitro, and prophylactic efficacy was further examined in a Galleria mellonella infection model.

RESULTS: The engineered phage exhibited thermal and pH stability comparable to that of the wild-type phage, while demonstrating enhanced adsorption efficiency. In a cell lysis assay, the engineered phage sustained bacterial suppression for 24-72 h, whereas the wild-type phage permitted bacterial regrowth due to the emergence of phage-resistant mutants. The engineered phage significantly reduced bacterial attachment and intracellular survival in an intestinal epithelial cell model. Furthermore, it improved larval survival in a Galleria mellonella infection model in a dose-dependent manner, without inducing significant cytotoxicity.

CONCLUSIONS: LL-37-engineered bacteriophages demonstrated improved antibacterial activity and intracellular infection control against S. Typhimurium. These findings support antimicrobial peptide-armed phages as a promising strategy for enhancing phage therapy and mitigating antibiotic-resistant bacterial infections.},
}

Animals
*Salmonella typhimurium/virology/drug effects
*Cathelicidins/genetics/pharmacology
*CRISPR-Cas Systems
*Salmonella Phages/genetics
*Anti-Bacterial Agents/pharmacology
Moths/microbiology
*Antimicrobial Cationic Peptides/genetics/pharmacology
Epithelial Cells/microbiology
Humans
Larva/microbiology
Phage Therapy
*Antimicrobial Peptides/genetics/pharmacology
Salmonella Infections/therapy
Gene Editing

@article {pmid41672309,
year = {2026},
author = {Terakawa, Y and Koshi, D and Kawauchi, M and Nakazawa, T and An, GH and Honda, Y},
title = {Establishment of a CRISPR/Cas9 protocol by biolistic transformation of the filamentous basidiomycete Pleurotus ostreatus.},
journal = {Journal of microbiological methods},
volume = {243},
number = {},
pages = {107430},
doi = {10.1016/j.mimet.2026.107430},
pmid = {41672309},
issn = {1872-8359},
mesh = {*Pleurotus/genetics ; *CRISPR-Cas Systems ; *Transformation, Genetic ; *Gene Editing/methods ; *Biolistics/methods ; Genetic Vectors ; Genome, Fungal ; },
abstract = {Genome editing was successfully performed by introducing a CRISPR/Cas9 expression vector through biolistic transformation of Pleurotus ostreatus, an oyster mushroom. This method can be applied for genome editing in filamentous basidiomycetes without protoplasting. To our knowledge, this is the first report of genome editing with biolistic transformation in filamentous basidiomycetes.},
}

*Pleurotus/genetics
*CRISPR-Cas Systems
*Transformation, Genetic
*Gene Editing/methods
*Biolistics/methods
Genetic Vectors
Genome, Fungal

@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/genetics ; Mice ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Colorectal Neoplasms/therapy/immunology/genetics ; Humans ; *Phosphoinositide Phospholipase C/genetics/metabolism/antagonists & inhibitors ; Female ; Mice, Inbred BALB C ; },
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/genetics
Mice
*CRISPR-Cas Systems
Cell Line, Tumor
*Colorectal Neoplasms/therapy/immunology/genetics
Humans
*Phosphoinositide Phospholipase C/genetics/metabolism/antagonists & inhibitors
Female
Mice, Inbred BALB C

@article {pmid41813887,
year = {2026},
author = {Tou, CJ and Xie, K and Ferreira da Silva, J and Kalailingam, P and Amar-Lewis, E and Rufino-Ramos, D and Sawyer, W and Eller, ML and Starzyk, J and Majumdar, I and Wang, J and Lee, D and Yang, S and Meis, RJ and Dahl, GA and Li, J and Shan, R and Artzi, N and Musolino, PL and Wu, H and Kleinstiver, BP},
title = {Immune evasive DNA donors and recombinases license kilobase-scale writing.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41813887},
issn = {1476-4687},
abstract = {Genome-editing technologies that use recombinases to insert kilobase-scale DNA sequences into mammalian genomes canonically require large double-stranded DNA (dsDNA) donors[1,2]. However, dsDNA molecules evoke problematic and toxic innate immune responses, limiting integration efficiencies and generally constraining applicability to ex vivo or immune-deficient contexts. By harnessing mechanisms of integrative prokaryotic viruses and mobile genetic elements, here we demonstrate that recombinases are compatible with immune evasive circular single-stranded DNA molecules optimally bearing a partial-duplex region that reconstitutes the recombinase recognition sequence. This approach, which we term integration through nucleus-synthesized template addition of large lengths (INSTALL), is compatible with diverse protein and RNA-guided recombinases for high-fidelity kilobase-scale human genome writing. INSTALL minimizes innate immune responses in primary human cells and in mice, improving recombinase-mediated integration efficiencies and supporting systemic in vivo non-viral DNA delivery by substantially increasing tolerability and broadening the dosing range compared with lipid nanoparticle-delivered dsDNA molecules. Together, INSTALL overcomes fundamental challenges for DNA delivery and integration methods by synergizing immune-stealth nucleic acids with recombinases to enable kilobase-scale integration strategies without viral vectors.},
}

@article {pmid41816357,
year = {2026},
author = {Pan, Z and Xu, L and Fan, Z and Cao, Y and Ren, F},
title = {CRISPR-based diagnostics for infectious diseases: mechanisms, advancements and clinical transformation prospects.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1769226},
pmid = {41816357},
issn = {2235-2988},
mesh = {Humans ; *CRISPR-Cas Systems ; *Communicable Diseases/diagnosis ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Infectious diseases continue to pose significant global public health challenges, necessitating the development of rapid, sensitive, specific, and field-deployable diagnostic platforms. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) has revolutionized genome editing and concurrently enabled a new generation of molecular diagnostic tools. Leveraging the inherent trans-cleavage activities of Cas enzymes, platforms such as SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) have emerged, combining target recognition precision with reporter systems to achieve ultra-sensitive detection of pathogen-specific nucleic acids. This review systematically examines the mechanistic foundations of CRISPR diagnostics, synthesizes recent advancements in infectious disease applications, evaluates their advantages in sensitivity, specificity, operational simplicity, and multiplexing capacity, and critically analyzes current implementation barriers and future translational pathways.},
}

Humans
*CRISPR-Cas Systems
*Communicable Diseases/diagnosis
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity

@article {pmid41816914,
year = {2026},
author = {Zhao, H and Zhou, T and Zhang, M and Wang, C and Wang, R and Shu, X and Cheng, F and Xue, Q and Liu, C and Xu, J and Cao, X and Du, J and Wang, L and Liu, H and Li, M},
title = {Associate toxin-antitoxin with CRISPR-Cas to harness (ATTACH) engineered microbes.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41816914},
issn = {1362-4962},
support = {2024YFA0918500//National Key Research and Development Project/ ; XDB0810000//Chinese Academy of Sciences/ ; 32370090//National Natural Science Foundation of China/ ; 32150020//National Natural Science Foundation of China/ ; 32400063//National Natural Science Foundation of China/ ; 32270092//National Natural Science Foundation of China/ ; 32200057//National Natural Science Foundation of China/ ; 32370120//National Natural Science Foundation of China/ ; 2020090//Youth Innovation Promotion Association of CAS/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; Mice ; *Toxin-Antitoxin Systems/genetics ; Escherichia coli/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Gene Editing/methods ; Promoter Regions, Genetic ; },
abstract = {Robust biocontainment is essential for the safe use of engineered microbes, but existing strategies suffer from genetic instability and/or laborious construction. Here, we present ATTACH, a kill switch that associates toxin-antitoxin with CRISPR-Cas to harness engineered microbes. Our approach employs a CRISPR-repressed toxin-antitoxin (CreTA) module to make microbes addicted to the type I-F Cas effector proteins, and places both the Cas3 nuclease and the chromosome-targeting guide RNA under inducible promoters, thereby improving the genetic stability and stringency of the CRISPR-based suicidal program. Additionally, we have developed a single-plasmid, antibiotic-independent ATTACH device, which shows robust, stringent containment of a microbial chassis in murine gut, and negligible impacts on culture growth or lycopene production during batch fermentation. Our data highlight the potential of CreTA to stabilize CRISPR-based kill switches, advancing their development into more portable and reliable biocontainment tools for engineered microbes.},
}

*CRISPR-Cas Systems
Animals
Mice
*Toxin-Antitoxin Systems/genetics
Escherichia coli/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Plasmids/genetics
Gene Editing/methods
Promoter Regions, Genetic

@article {pmid41818662,
year = {2026},
author = {Wu, L and Chen, W and Huang, R and Zhou, F},
title = {EXAGO: An Argonaute-Based Primer-Free Exponential Amplification Strategy for Ultrasensitive Zero-Background Detection of Point Mutation.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c06961},
pmid = {41818662},
issn = {1520-6882},
abstract = {Ultrasensitive gene detection is crucial for precise molecular diagnostics. Conventional nucleic acid amplification methods frequently encounter nonspecific amplification triggered by exogenous primers, which limits their utility in ultrasensitive detection. Programmable gene editing tools, such as CRISPR/Cas and Argonaute (Ago), provide new avenues for developing next-generation detection technologies. Here, we develop an Ago-mediated exponential amplification strategy, EXAGO, for ultrasensitive detection of the epidermal growth factor receptor (EGFR) L858R mutation─a critical biomarker in nonsmall cell lung cancer. Leveraging the flexible programmability and single-base-resolution cleavage activity of Ago, EXAGO can specifically initiate DNA polymerase reactions at the target mutation site. The mechanism of dual-circuit operation allows the system to achieve an efficient exponential amplification under thermal cycling. By circumventing exogenous primers, it also prevents nonspecific amplification caused by primer misidentification at the source. Moreover, wild-type genes are entirely unable to trigger signal amplification, thereby underscoring their superior specificity and the potential for practical application. Experimental results demonstrated that EXAGO achieves femtomolar-level sensitivity and exhibits favorable recovery rates for detecting plasma-diluted samples. Moreover, the use of a thermostable enzyme allows direct compatibility with cell thermal lysis, enabling detection in cell lysates. In summary, EXAGO offers a robust and practical solution for accurate genetic mutation analysis in complex samples and promotes the application of Ago-based tools in molecular diagnostics.},
}

@article {pmid41819069,
year = {2026},
author = {Stephan, TL and Hoodless, PA},
title = {The ups and downs of maturing zonated hepatoctyes.},
journal = {Developmental cell},
volume = {61},
number = {3},
pages = {462-463},
doi = {10.1016/j.devcel.2026.02.009},
pmid = {41819069},
issn = {1878-1551},
mesh = {Animals ; *Hepatocytes/metabolism/cytology ; *Liver/metabolism/embryology/cytology ; Mice ; CRISPR-Cas Systems ; *Cell Differentiation ; },
abstract = {Current in vitro protocols differentiating hepatocytes fail to activate mature metabolic genes, induce zone-specific phenotypes, and suppress fetal liver signatures. In this issue, Taguchi, Magalhães et al.[1] used CRISPR-Cas9 screening in a mouse model of hepatic development to identify Nr1i3 and Nfix as regulators of hepatocyte maturation and zonation.},
}

Animals
*Hepatocytes/metabolism/cytology
*Liver/metabolism/embryology/cytology
Mice
CRISPR-Cas Systems
*Cell Differentiation

@article {pmid41819070,
year = {2026},
author = {Wang, J and Lai, L},
title = {Repurposing Cas13's collateral cleavage activity to mitigate host cell dominance in interspecies chimera formation.},
journal = {Developmental cell},
volume = {61},
number = {3},
pages = {464-465},
doi = {10.1016/j.devcel.2026.02.010},
pmid = {41819070},
issn = {1878-1551},
mesh = {Animals ; Humans ; Mice ; *Chimera ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {The insufficient contribution of human cells is a key obstacle to interspecies chimera. In this issue of Developmental Cell, He et al. harnessed the RNA collateral cleavage activity of Cas13 to diminish the competitive advantage of host cells, increasing integration ratio of human cells to 1% in host mice.},
}

Animals
Humans
Mice
*Chimera
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics

@article {pmid41819296,
year = {2026},
author = {Wang, C and Zhu, C and Liu, Q and Yang, L},
title = {Prokaryotic argonaute proteins: From ancient defense mechanisms to modern biosensing applications.},
journal = {Biotechnology advances},
volume = {89},
number = {},
pages = {108869},
doi = {10.1016/j.biotechadv.2026.108869},
pmid = {41819296},
issn = {1873-1899},
abstract = {Prokaryotic Argonaute (pAgo) proteins constitute an evolutionarily ancient nuclease family that is rapidly maturing into a versatile molecular toolkit rivaling CRISPR-Cas. This review synthesizes recent advances in pAgo biology and biotechnology, tracing their phylogeny across thermophilic, mesophilic, and psychrotolerant lineages and highlighting temperature-adapted catalytic signatures that diverge from eukaryotic Agos. In vivo studies reveal pAgo roles in gDNA-guided host defense, transcriptional silencing and recombination, all executed through programmable DNA- or RNA-guided nuclease activity. We detail how guide length, 5' nucleotide identity, divalent cations and accessory factors modulate cleavage efficiency, enabling rational optimization. The review then maps the explosion of pAgo-based biosensing platforms, including selective nucleic acid enrichment platforms, ultrasensitive pathogen detection methods, programmable DNA cloning systems, and high-resolution imaging techniques. Their independence from protospacer adjacent motifs (PAMs), stable DNA guides, multi-turnover kinetics, and broad thermal tolerance position pAgos as ideal complements to CRISPR systems. Finally, we outline current limitations and future directions, including the discovery and engineering of novel variants, elucidation of guide-generation mechanisms, and development of next-generation gene-editing tools, aiming to accelerate translation of these versatile enzymes into practical biotechnological and therapeutic translation.},
}

@article {pmid41813543,
year = {2026},
author = {Qiao, JH and Gao, Q and Wang, XB},
title = {Virus-induced genome editing: toward crop breeding applications.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2026.01.007},
pmid = {41813543},
issn = {1878-4372},
abstract = {CRISPR-Cas-based genome editing has revolutionized precise genome manipulation in plants, yet its practical application is still constrained by the inefficient delivery of editing reagents across different genotypes. Plant viruses are promising vehicles for delivering genome-editing components, bypassing plant transformation and/or tissue culture. Virus-induced genome editing (VIGE) has provided powerful tools for achieving heritable edits in model plants such as Arabidopsis thaliana and Nicotiana benthamiana. VIGE has now progressed from proof-of-concept to practical applications in agricultural crops. Notably, a recent breakthrough in VIGE in tiller has successfully achieved heritable genome editing in hexaploid wheat. This review outlines the latest advances in VIGE across diverse plant species, highlights its potential for crop improvement, and discusses future research directions.},
}

@article {pmid41811974,
year = {2026},
author = {Baba, S and Oncul, O and Aktas, Z},
title = {CRISPR-Cas Based Plasmid Design for Multidrug Resistant Klebsiella Pneumoniae Isolates.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnag026},
pmid = {41811974},
issn = {1574-6968},
abstract = {Antimicrobial resistance (AMR) is a major global health concern that requires innovative therapeutic strategies. This study aimed to address this challenge by designing Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein (CRISPR-Cas)-based plasmid systems for potential genome editing applications in multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates. Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem were determined according to EUCAST guidelines. All isolates (n = 5) were resistant, with MIC ranges of 4-128 μg/mL for imipenem, 8-64 μg/mL for meropenem, and 8-256 μg/mL for ertapenem. Resistance gene analysis revealed blaOXA-48-like and blaCTX-M-15 in all isolates, while blaNDM-1 was detected in one isolate. Two CRISPR-based plasmid systems, CRISPR-Cas9 and CRISPR-assisted cytidine deaminase, were designed. Target genes were amplified by PCR, and guide RNA (gRNA) sequences were designed from selected regions. Apramycin (50 μg/mL) was identified as a suitable selection marker. The pSGKP-AmpR(Pro)-ApmR plasmid was successfully constructed, whereas Cas9 and APOBEC constructs could not be cloned. Overall, this study highlights technical challenges in developing CRISPR-based tools for MDR K. pneumoniae and emphasizes the need for isolate-specific plasmid design and gRNA optimization.},
}

@article {pmid41811515,
year = {2026},
author = {Thiebaut, F and Urquiaga, MC and de Araújo, PM and de Carvalho Vivarini, A and Grativol, C},
title = {Small but big player: the important role of microRNAs in legume crops.},
journal = {Molecular genetics and genomics : MGG},
volume = {301},
number = {1},
pages = {},
pmid = {41811515},
issn = {1617-4623},
mesh = {*MicroRNAs/genetics ; *Crops, Agricultural/genetics/growth & development ; *Fabaceae/genetics/growth & development ; Gene Expression Regulation, Plant ; *RNA, Plant/genetics ; Symbiosis/genetics ; RNA Interference ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Legumes are essential components of global cropping systems due to their nutritional value and contribution to sustainable agriculture. Among the regulatory molecules, small RNAs (sRNAs), particularly microRNAs (miRNAs), play crucial roles in plant development and in responses to biotic and abiotic stresses. miRNAs regulate genes involved in diverse developmental processes, including nodule formation, which is fundamental for the nitrogen-fixing symbiosis that characterizes legumes. Functional studies have demonstrated that miRNAs are key modulators of plant defense, contributing to resistance against pathogens and environmental challenges. Moreover, miRNAs also participate in cross-kingdom communication, such as plant-bacteria interactions, influencing symbiotic efficiency. Advances in molecular biology have enabled the manipulation of miRNAs and their targets for crop improvement. Current approaches include the design of artificial miRNAs (amiRNA), modulation of miRNA expression through miRNA-encoded peptides, genome editing of non-coding genes using CRISPR/Cas9, and the application of RNA interference (RNAi) technology. Together, these strategies highlight the potential of miRNA-based tools in plant biotechnology. A deeper understanding of the molecular mechanisms governing miRNA-mediated gene silencing will provide powerful resources for optimizing legume productivity and resilience within sustainable agricultural systems.},
}

*MicroRNAs/genetics
*Crops, Agricultural/genetics/growth & development
*Fabaceae/genetics/growth & development
Gene Expression Regulation, Plant
*RNA, Plant/genetics
Symbiosis/genetics
RNA Interference
Gene Editing
CRISPR-Cas Systems

@article {pmid41811507,
year = {2026},
author = {Thakur, MK and Pandey, S and Singh, SK and Singh, SK and Singh, A},
title = {Hybrid seed production: new paradigms and challenges in the twenty-first century.},
journal = {Planta},
volume = {263},
number = {4},
pages = {},
pmid = {41811507},
issn = {1432-2048},
mesh = {*Seeds/genetics/growth & development ; *Plant Breeding/methods ; *Hybridization, Genetic ; *Crops, Agricultural/genetics ; Hybrid Vigor ; Genomics ; Climate Change ; },
abstract = {Hybrid seed technology future depends on integrating advanced genomics, AI-driven breeding, and enabling policies to sustainably delivery climate-resilient, high-performing hybrids with broad accessibility and equitable benefits worldwide. Hybrid seeds, which exploit heterosis, have driven agricultural productivity gains since the 1920s. Understanding the genetics and molecular biology of hybrid generation led to the development of modern hybrid systems. With time, modern hybrid systems integrated advanced genomic tools such as CRISPR/Cas, marker-assisted selection (MAS), and genomic selection (GS) with established technologies like cytoplasmic male sterility (CMS), restorer-of-fertility (Rf) systems, and chemical hybridizing agents (CHAs) for better hybrid production in a shorter time. Moreover, the integration of emerging approaches leveraging artificial intelligence and machine learning (AI/ML) for trait prediction, multi-parent populations to expand genetic diversity, and epigenetics to engineer climate-resilient hybrids with enhanced stress tolerance is also being explored. However, regulatory hurdles, such as divergent global policies for genetically modified (GM) hybrids, intellectual property (IP) disputes, and restricted germplasm exchange under access-and-benefit-sharing frameworks like the Nagoya Protocol, hinder innovation. Climate change exacerbates both biotic and abiotic stresses, disrupts production zones, and threatens pollinator-dependent crops, while socio-economic barriers limit the adoption of smallholder farming. Case studies of different crops demonstrate the success of hybrids, yet gaps in scalability and accessibility persist. Overall, realizing the potential of hybrid technology hinges on sustained collaboration across scientific, industrial, and policy domains to overcome technical, environmental, and socio-economic constraints. This review examines various techniques for hybrid production that incorporate genomics, future advancements, and synergies between synthetic biology, automation, and predictive breeding, as well as policies that strike a balance between intellectual property protection and germplasm accessibility for hybrid seed production.},
}

*Seeds/genetics/growth & development
*Plant Breeding/methods
*Hybridization, Genetic
*Crops, Agricultural/genetics
Hybrid Vigor
Genomics
Climate Change

@article {pmid41811196,
year = {2026},
author = {Vieira, CSD and Wang, W and Sanchez-Valdez, F and Lim, J and White, BE and Souza, CGS and Tarleton, RL and Paes, MC and Nogueira, NPA},
title = {Glycosomal Phosphoenolpyruvate Carboxykinase CRISPR/Cas9-Deletion and Its Role in Trypanosoma cruzi Metacyclogenesis and Infectivity in Mammalian Host.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {6},
pages = {e71672},
pmid = {41811196},
issn = {1530-6860},
support = {00x0ma614//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/ ; 88887.311601/2018-00-2629/2018//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/ ; E26/010.001706/2019//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)/ ; E26/010.100623/2018//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)/ ; E26/211.815/2021//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)/ ; 402419/2022-7//Conselho Nacional Pesquisa (CNPq) SWE (Sanduíche no Exterior)/ ; },
mesh = {Animals ; *Trypanosoma cruzi/pathogenicity/genetics/enzymology/growth & development ; *CRISPR-Cas Systems ; Mice ; *Chagas Disease/parasitology ; Glucose/metabolism ; *Phosphoenolpyruvate Carboxykinase (ATP)/genetics/metabolism ; *Microbodies/enzymology/metabolism ; Mitochondria/metabolism ; *Protozoan Proteins/genetics/metabolism ; },
abstract = {Trypanosoma cruzi, the causative agent of Chagas disease, possesses glycosomes-unique organelles that house key metabolic enzymes, several of which are promising therapeutic targets. Among them, phosphoenolpyruvate carboxykinase (PEPCK) plays a central role in succinic fermentation, the main pathway for NAD[+] regeneration within the organelle. Using CRISPR/Cas9 editing, the PEPCK gene was disrupted in T. cruzi, producing single-allele knockout epimastigotes (TcPEPCK-sKO) with reduced PEPCK expression and enzyme activity. In a high glucose environment, PEPCK disruption impaired glucose consumption and mitochondrial respiration, particularly oxidative phosphorylation, reducing dependence on mitochondrial ATP production when glucose was supplied. To compensate, pyruvate phosphate dikinase was upregulated, increasing alanine production, possibly to maintain redox balance in glycosomes. Despite this metabolic adaptation, the growth of TcPEPCK-sKO epimastigotes was partially reduced compared with non-deleted parasites. In contrast, under low glucose conditions, PEPCK activity was not critical for mitochondrial bioenergetics, ATP production, or proliferation. Although TcPEPCK-sKO epimastigotes exhibited a minor reduction in growth in high glucose medium, their differentiation (metacyclogenesis) and invasion were severely compromised. However, once inside the host cell, TcPEPCK-sKO amastigotes increased their replication, leading to enhanced trypomastigote production. The same was observed in in vivo infection, where TcPEPCK-sKO infection in IFNγ-deficient mice caused uncontrolled parasitemia and severe pathology, highlighting the critical role of PEPCK in host-pathogen interactions. However, an intact immune system effectively contained TcPEPCK-sKO infection. Taken together, our findings demonstrate that glycosomal PEPCK is crucial for coupling glycolysis to mitochondrial bioenergetics, enabling the parasite differentiation within the insect vector and controlling the infection of mammalian host cells.},
}

Animals
*Trypanosoma cruzi/pathogenicity/genetics/enzymology/growth & development
*CRISPR-Cas Systems
Mice
*Chagas Disease/parasitology
Glucose/metabolism
*Phosphoenolpyruvate Carboxykinase (ATP)/genetics/metabolism
*Microbodies/enzymology/metabolism
Mitochondria/metabolism
*Protozoan Proteins/genetics/metabolism

@article {pmid41811192,
year = {2026},
author = {Shen, Y and Yeung, AT and Ditchfield, P and Korn, E and Clements, R and Chen, X and Wang, B and Huang, Z and Sheen, M and Jarman, PA and Han, C},
title = {A genome-wide MAGIC kit for recombinase-independent mosaic analysis in Drosophila.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41811192},
issn = {2050-084X},
support = {R24OD031953//NIH Office of the Director/ ; },
mesh = {Animals ; *Mosaicism ; *Drosophila/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Crossing Over, Genetic ; CRISPR-Cas Systems ; *Genome, Insect ; },
abstract = {Mosaic analysis has been instrumental in advancing developmental and cell biology. Most current mosaic techniques rely on exogenous site-specific recombination sequences that need to be introduced into the genome, limiting their application. Mosaic analysis by gRNA-induced crossing-over (MAGIC) was recently developed in Drosophila to eliminate this requirement by inducing somatic recombination through CRISPR/Cas9-generated DNA double-strand breaks. However, MAGIC has not been widely adopted because gRNA markers, a required component for this technique, are not yet available for most chromosomes. Here, we present a complete, genome-wide gRNA-marker kit that incorporates optimized designs for enhanced clone induction and more effective clone labeling in both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). With this kit, we demonstrate clonal analysis in a broad range of Drosophila tissues, including cell types that have been difficult to analyze using recombinase-based systems. Notably, MAGIC enables clonal analysis of pericentromeric genes, deficiency chromosomes and in interspecific hybrid animals, opening new avenues for gene function study, rapid gene discovery, and understanding cellular basis of speciation. This MAGIC kit complements existing systems and makes mosaic analysis accessible to address a wider range of biological questions.},
}

Animals
*Mosaicism
*Drosophila/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Crossing Over, Genetic
CRISPR-Cas Systems
*Genome, Insect

@article {pmid41810550,
year = {2026},
author = {Rahmanian, M and Khoshandam, M and Mousazadeh, M and Yang, P and Soltaninejad, H and Karami Dehkordi, P and Sadeghizadeh, M and Hedayati Goudarzi, MT and Azimi, AH and Sheykhhasan, M},
title = {CRISPR in Medicine: A Systematic Review of Clinical Trials and Therapeutic Applications.},
journal = {Human gene therapy},
volume = {37},
number = {5-6},
pages = {170-182},
doi = {10.1177/10430342251409714},
pmid = {41810550},
issn = {1557-7422},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Clinical Trials as Topic ; Neoplasms/therapy/genetics ; *Hematologic Diseases/therapy/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9 (CRISPR/Cas9) technology has become a revolutionary tool in medicine, offering substantial potential for treating a wide range of diseases, including hematological disorders, cancers, genetic conditions, and ophthalmological diseases. This systematic review evaluates the efficacy, safety, and applicability of CRISPR/Cas9 in clinical trials. A comprehensive search of the PubMed, Scopus, Web of Science, and Cochrane databases was conducted. All studies, up to November 2024, meeting the eligibility criteria assessing the application of CRISPR for the treatment of diseases were included. A quality assessment of the included studies was conducted using the Cochrane risk of bias tool. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement for systematic reviews and meta-analyses was followed, and a total of 17 studies were included. This systematic review of CRISPR/Cas9 technology focused on its effectiveness and safety across various diseases. In nonmalignant hematological disorders, CRISPR successfully treated β-thalassemia and sickle cell disease, resulting in high transfusion independence and the elimination of disease crises. In malignant hematological disorders, B-cell acute lymphoblastic leukemia, CRISPR-engineered chimeric antigen receptor T (CAR-T) cells achieved an 83.3% complete remission rate. Furthermore, CRISPR-based CAR-T cells showed promising results in B-cell non-Hodgkin's lymphoma. In oncology, lung cancer and other solid tumors are among the diseases that have been safely engineered using CRISPR gene editing technology. For genetic disorders, CRISPR improved vision in retinal degeneration and reduced symptoms in hereditary angioedema and transthyretin amyloidosis with mild side effects. The results demonstrated CRISPR's potential across a wide range of conditions. In conclusion, the findings underscore the potential role of CRISPR/Cas9 technology across a wide range of diseases. However, challenges remain, including optimizing delivery systems, minimizing off-target effects, addressing immunogenicity concerns, and ethical considerations.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Therapy/methods
Clinical Trials as Topic
Neoplasms/therapy/genetics
*Hematologic Diseases/therapy/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41810315,
year = {2026},
author = {Guo, Z and Hu, R and Wang, J and Zhou, M and Zhu, K and Xu, Y},
title = {Research Progress on Point-of-Care Testing Technology for Mycoplasma Pneumonia.},
journal = {International journal of general medicine},
volume = {19},
number = {},
pages = {584824},
pmid = {41810315},
issn = {1178-7074},
abstract = {Mycoplasma pneumoniae (MP) is a significant respiratory pathogen in children, often causing refractory and severe pneumonia. Sensitive, rapid, and portable diagnostic tools are crucial for guiding clinical management. Although traditional methods like culture, ELISA, and PCR are widely used, they suffer from drawbacks such as lengthy turnaround times, complex procedures, or reliance on laboratory equipment. Consequently, Point-of-care testing (POCT) technologies, valued for their speed, portability, and ease of use, have emerged as a key research focus for MP diagnosis. This review systematically summarizes advancements in POCT platforms, covering: (1) immunological methods; (2) molecular biology methods; and (3) biosensor technologies. Their sensitivity, specificity, and clinical performance are comparatively analyzed. Colloidal gold immunochromatography delivers results within 15 minutes but exhibits limited sensitivity. Molecular methods like LAMP and CRISPR-coupled systems achieve single-copy detection limits via isothermal amplification and gene editing, with processing times under 1 hour. Biosensors enable high-sensitivity automated detection through integrated signal amplification and microfluidics. Despite these advantages, POCT development faces challenges including cost-sensitivity tradeoffs, standardization barriers, and sample matrix interference. Future directions encompass multi-modal detection, AI-assisted interpretation, multiplex pathogen screening, and dynamic drug resistance gene monitoring. These innovations will expand POCT device deployment in primary care and home settings. This will ultimately improve effectiveness in controlling respiratory infections.},
}

@article {pmid41656303,
year = {2026},
author = {Lee, CJ and Nam, Y and Rim, YA and Ju, JH},
title = {Harnessing induced pluripotent stem cells and organoids for disease modeling and precision medicine.},
journal = {Stem cell research & therapy},
volume = {17},
number = {1},
pages = {},
pmid = {41656303},
issn = {1757-6512},
support = {HI22C1314//Ministry of Health and Welfare/ ; },
mesh = {Humans ; *Organoids/metabolism/cytology ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Precision Medicine/methods ; Gene Editing ; CRISPR-Cas Systems ; Animals ; },
abstract = {The convergence of CRISPR genome editing, patient-derived organoids, and induced pluripotent stem cells (iPSCs) has reshaped in vitro disease modeling by enabling mechanistic investigations of human pathophysiology within genetically matched, tissue-relevant systems. Together, these technologies provide a synergistic platform for precise manipulation of disease-associated variants and support the generation of isogenic organoid models that reproduce key phenotypic and functional hallmarks across cancer, neurodegenerative, inflammatory, and monogenic disorders. In this review, we highlight how diverse CRISPR modalities-including knock-out, knock-in, CRISPRa/i, and genome-scale screening-have been applied to dissect gene function, model disease progression, and guide therapeutic development using iPSC- and organoid-based systems. We further discuss the application of these platforms in genotype- and phenotype-driven precision medicine, enabling patient stratification, drug-response prediction, and individualized treatment design. We illustrate these convergent applications with representative case studies spanning mechanistic research and early clinical translation. By combining the scalability of genome engineering with the physiological fidelity of organoids, CRISPR-integrated platforms are redefining the frontiers of experimental medicine. These approaches accelerate the discovery of disease mechanisms and actionable therapeutic targets while establishing individualized clinical strategies for complex human diseases. Collectively, they position CRISPR-enabled organoid systems as a foundational infrastructure that bridges genome editing to individualized therapy and supports next-generation precision medicine.},
}

Humans
*Organoids/metabolism/cytology
*Induced Pluripotent Stem Cells/metabolism/cytology
*Precision Medicine/methods
Gene Editing
CRISPR-Cas Systems
Animals

@article {pmid41810060,
year = {2026},
author = {Shen, Z and Liu, Y and Hao, Y and Bo, Y and Dai, X and Wang, S and Xia, T and Su, X and Liu, H},
title = {Advances in Double-Stranded DNA Targeting Technologies.},
journal = {Exploration (Beijing, China)},
volume = {6},
number = {1},
pages = {20250065},
pmid = {41810060},
issn = {2766-2098},
abstract = {Double-stranded DNA (dsDNA) serves as a fundamental repository of genetic information and plays a pivotal role in the diagnosis and therapeutic management of diseases. However, the inherent stability of the DNA double helix under physiological conditions presents a challenge in accessing internal bases. To address this, various molecular targeting technologies have been developed, offering high specificity while destabilizing the DNA structure. This review provides a comprehensive overview of current dsDNA targeting tools, such as hybridization probes, modified nucleic acid probes, zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system, Argonaute proteins (Agos), and the lambda exonuclease-pDNA system (λ Exo-pDNA), and some cutting-edge molecular tools. It delves into the mechanisms behind these technologies. It highlights their applications in diverse areas, including in vitro detection, in situ imaging, gene editing, and their integration with artificial intelligence (AI)-driven tools. Additionally, the review compares these techniques, discusses future technological opportunities, and identifies challenges in integrating these tools into diagnostic and therapeutic practices. By providing a holistic view of these rapidly evolving technologies, this review aims to fill a gap in the current literature and explore the future potential of dsDNA targeting innovations.},
}

@article {pmid41809894,
year = {2025},
author = {Yang, P and Khoshandam, M and Bhia, I and Raji, S and Soltaninejad, H and Hosseinkhani, S and Sani, M and Hamidieh, AA and Sheykhhasan, M},
title = {Integrating CRISPR/Cas technology with clinical trials: Principles, progress and challenges.},
journal = {Asian journal of pharmaceutical sciences},
volume = {20},
number = {6},
pages = {101068},
pmid = {41809894},
issn = {2221-285X},
abstract = {CRISPR represent a groundbreaking genome-editing technology that has revolutionized genetic modification. This innovative tool offers an unparalleled revolution in the future treatment of genetic disorders, neurological diseases, infectious diseases and cancer. Despite the rapid expansion of CRISPR applications, its clinical use in humans is still relatively limited, with only 69 active clinical trials and 6 completed studies reported so far. This review examined current clinical trials and their processes in addressing various diseases via the CRISPR/Cas system. While earlier literatures have focused mainly on delivery methods and materials for CRISPR/Cas9, our review emphasized innovative targeting conditions and approaches for novel and functional therapeutic designs. In addition, we reviewed recent research to increase the efficiency of CRISPR editing in the management of genetic disorders and cancer, while exploring their future challenges and potential. This review provided a unique perspective on the advancement of CRISPR technology. By addressing these aspects, we aim to contribute to ongoing efforts to improve CRISPR-based therapies and expand their clinical applications, ultimately striving to transform the future of medical treatment.},
}

@article {pmid41808396,
year = {2026},
author = {Zargul, A and Liu, H and Zhang, W and Wang, H and Liu, J and Chen, C},
title = {Advances in Pathogen Detection by Nanosensors: Biorecognition Strategies, Signal Amplification, and Platform Engineering.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.5c22148},
pmid = {41808396},
issn = {1936-086X},
abstract = {The escalating global threat of infectious diseases, compounded by antimicrobial resistance (AMR), calls for improved diagnostic strategies. Conventional pathogen detection techniques─culture, enzyme-linked immunosorbent assay (ELISA), and microscopy─remain hindered by prolonged turnaround times, suboptimal sensitivity for low-abundance analytes, and operational intricacy. Nanosensor technologies have emerged as powerful enablers of rapid, ultrasensitive, and field-deployable diagnostics. This review delineates the convergence of three transformative domains: (1) advanced biorecognition strategies─including monoclonal antibodies, aptamers, bacteriophages, antimicrobial peptides, molecularly imprinted polymers, and lectins─that confer high-fidelity molecular selectivity within complex biological matrices; (2) multimodal signal amplification technologies, encompassing nanomaterial-enhanced mechanisms, enzymatic cascades, and isothermal nucleic acid amplification that drive detection down to the single-cell and femtomolar regimes; and (3) integrated platform engineering, uniting clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems, artificial intelligence (AI), and microfluidics to achieve multiplexed, real-time, point-of-care deployment. Advances are critically evaluated through standardized performance metrics─limit of detection, assay time, specificity, and operational simplicity─to reveal both synergistic opportunities and enduring translational bottlenecks. Collectively, these developments define a strategic framework for next-generation nanosensor diagnostics poised to revolutionize infectious disease surveillance and enable precision-guided therapeutic intervention.},
}

@article {pmid41807728,
year = {2026},
author = {Godsil, M and Wei, N and Meeske, AJ},
title = {Conditional activation of Cas13 enforces lysogeny in a native type VI-A CRISPR host.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41807728},
issn = {2058-5276},
support = {R35 GM142460/GM/NIGMS NIH HHS/United States ; FAIN2235762//NSF | BIO | Division of Molecular and Cellular Biosciences (MCB)/ ; R35GM142460//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems present a barrier to prophage acquisition by restricting invading phages or by inducing autoimmune cleavage of integrated prophage DNA. The RNA-sensing type VI CRISPR nuclease Cas13 mediates non-specific RNA cleavage upon recognition of phage lytic transcripts, but how this system influences the temperate phage life cycle remains unknown. Here we report that the Listeria seeligeri type VI-A CRISPR system restricts the lytic cycle of temperate phages but tolerates prophage acquisition and interferes with prophage induction through a non-abortive mechanism. During attempts at induction, Cas13 activation forces prophage re-integration, thus maintaining lysogeny. We also find that during polylysogenic induction, Cas13 acts specifically, restricting only the targeted phage, in contrast to its behaviour during lytic replication. Our findings show that Cas13 elicits a unique response to each stage of the temperate phage life cycle, enabling type VI CRISPR hosts to acquire potentially beneficial prophages while mitigating lysis.},
}

@article {pmid41807051,
year = {2026},
author = {Gur Dedeoglu, B and Noyan, S and İlhan, KNK},
title = {Non-coding RNAs regulation in breast cancer pathogenesis.},
journal = {Epigenomics},
volume = {},
number = {},
pages = {1-20},
doi = {10.1080/17501911.2026.2642583},
pmid = {41807051},
issn = {1750-192X},
abstract = {Breast cancer represents a molecularly heterogeneous disease shaped by complex genetic, epigenetic, and transcriptional dysregulation. Non-coding RNAs (ncRNAs) including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) as well as small nucleolar RNAs (snoRNAs), piwi-interacting RNAs (piRNAs), and small nuclear RNAs (snRNAs), have emerged as key epigenetic regulators that integrate multiple layers of gene control. Through interactions with chromatin-modifying enzymes, RNA-binding proteins, and signaling effectors, ncRNAs modulate transcriptional activity, chromatin accessibility, and post-transcriptional stability of target genes. miRNAs predominantly act as post-transcriptional repressors, whereas lncRNAs and circRNAs exert transcriptional and epigenetic control via scaffolding, miRNA sponging, and chromatin remodeling; some circRNAs even encode functional peptides. Aberrant ncRNA expression contributes to proliferation, metastasis, metabolic reprogramming, immune evasion, and therapeutic resistance, with distinct expression signatures associated with triple-negative, HER2-positive, and hormone receptor - positive breast cancers. Owing to their stability and detectability in plasma and exosomes, ncRNAs hold promise as minimally invasive biomarkers for early detection and disease monitoring. Moreover, therapeutic strategies targeting ncRNAs, such as antisense oligonucleotides, RNA interference, CRISPR/Cas-based editing, and ncRNA-derived vaccines, are advancing toward clinical translation. Collectively, ncRNAs redefine the epigenetic landscape of breast cancer, offering a framework for integrated diagnostic and therapeutic approaches in precision oncology.},
}

@article {pmid41806830,
year = {2026},
author = {Escobar, M and Malik, SA and Srinivasa, MA and Mendez-Sosa, MA and Miller, JM and Lydon, SL and Luong, SN and Mathew, PR and Abouleisa, RRE and Chakravarty, S and Pathan, S and Mohamed, TMA and Ghanta, RK and Hilton, IB},
title = {CRISPR-Cas-based activation of PPARGC1A boosts endogenous mitochondria and enhances cardiac function after myocardial infarction.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2026.02.027},
pmid = {41806830},
issn = {1525-0024},
support = {R01 HL147921/HL/NHLBI NIH HHS/United States ; R01 HL178610/HL/NHLBI NIH HHS/United States ; R61 HL177472/HL/NHLBI NIH HHS/United States ; },
abstract = {Insufficient energy supply due to impaired mitochondria has emerged as a key pathological factor in the development of heart failure (HF) after myocardial infarction (MI). Unfortunately, no current therapeutic strategies directly augment myocardial energy production. While mitochondrial biogenesis is orchestrated by the activity of multiple genes, activation of PPARGC1A, a key regulator, can increase cellular mitochondria; however, supraphysiological levels of PPARGC1A result in adverse tissue remodeling and heart dysfunction. CRISPR activation (CRISPRa) technologies present a unique opportunity to address these shortcomings, as they enable tunable control over endogenous target gene expression. Here, we demonstrate that transcriptional activation of PPARGC1A using CRISPRa increases cellular mitochondria in human cell types. This effect is mediated through the activation of transcriptional programs driving mitochondrial biogenesis, mitochondrial function, and cellular bioenergetics. These activated transcriptional programs synergize to increase ATP production and reserve capacity in human cardiomyocytes. CRISPRa targeting of PPARGC1A in vivo increases cardiac mitochondria to recover heart ejection fraction in an acute MI model. Furthermore, CRISPRa acts on the adult human heart to increase PPARGC1A protein and cellular mitochondria, elevating mitochondrial function in both normal and HF-diagnosed hearts. These results provide the first proof of concept that endogenous gene activation via CRISPRa can improve heart function after MI.},
}

@article {pmid41713565,
year = {2026},
author = {Shashikala, T and Yogi, D and Akshay, K and Nagesh, SN and Manamohan, M and Venkataravanappa, V and Jha, GK and Ashok, K and Asokan, R},
title = {First report of CRISPR/Cas13a-based rapid detection of groundnut bud necrosis virus without amplification.},
journal = {Methods (San Diego, Calif.)},
volume = {249},
number = {},
pages = {9-22},
doi = {10.1016/j.ymeth.2026.02.010},
pmid = {41713565},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Plant Diseases/virology ; RNA, Viral/genetics/isolation & purification ; *Tospovirus/genetics/isolation & purification ; Vigna/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Globally, the groundnut bud necrosis virus (GBNV) (Bunyaviridae), pose a serious threat to solanaceous and leguminous crops causing serious crop loss. This requires a rapid and sensitive diagnostics for initially identifying at the earliest stage and further to initiate disease management. This study presents the first report of a CRISPR/Cas13a-based diagnostic assay for GBNV detection without amplification. GBNV was maintained in cowpea cv. C152 through mechanical inoculation, further viral RNA was isolated to clone the two target genes viz. nucleocapsid (NP) and movement protein (MP) genes. These genes were ligated to pTZ57R/T vector and sequenced. Similarly, the LshCas13a gene was cloned from pUC19 into pET28a, expressed in E. coli BL21, and purified using Ni-NTA affinity chromatography. Guide RNAs targeting conserved regions of NP and MP genes were synthesized by in vitro transcription and mixed with Cas13a protein to form ribonucleoprotein (RNP) complex. Target RNA, obtained either by in vitro-transcription or crude extract of infected tomato was used to detect GBNV, using a fluorescence-based reporter assay. This method found to be highly sensitive that could detect GBNV at as low as 0.01 ng. From the field perspective, GBNV could be detected from the crude extract of the GBNV infected tomato leaves using an alkaline PEG buffer. Thus CRISPR/Cas13a-based assay provides a rapid, amplification-free, and field-deployable diagnostic platform for GBNV. This lays the groundwork for a field adoptable CRISPR diagnostics for other plant RNA viruses also.},
}

*CRISPR-Cas Systems/genetics
*Plant Diseases/virology
RNA, Viral/genetics/isolation & purification
*Tospovirus/genetics/isolation & purification
Vigna/virology
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41678668,
year = {2026},
author = {Kim, J and Yoon, J and Chen, J and Lee, J and Lee, HJ and Whitworth, K and Redel, B and Prather, RS and Lee, K},
title = {Enhancing the specificity of gene editing outcomes by using Cas9 variants in porcine embryos.},
journal = {Journal of animal science},
volume = {104},
number = {},
pages = {},
doi = {10.1093/jas/skag030},
pmid = {41678668},
issn = {1525-3163},
mesh = {Animals ; *Gene Editing/veterinary/methods ; Swine/embryology/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Embryo, Mammalian ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 technology has improved the ability to introduce targeted modifications in cells and embryos in diverse species. The use of this technology enables the establishment of genetically modified livestock models to study human diseases or improve food production. However, one of the main concerns with employing this technology is the possibility of introducing unintended genome modifications induced by the Streptococcus pyogenes Cas9 (SpCas9), a commonly used Cas9 protein. Recent advancements in CRISPR/Cas9 technology offer Cas9 variants that are designed to improve gene editing specificity. Here, three high-fidelity SpCas9 variants (eSpCas9, HiFi Cas9, and LZ3 Cas9) were employed to examine their efficacy and specificity in pig embryos. To introduce targeted modifications, mRNA coding for each Cas9 variant was mixed with IGH single guide RNA (sgRNA) and were injected into fertilized pig zygotes. The frequency of on- and off-targeting was calculated by amplifying IGH, AR, and RBFOX1 regions from genomic DNA derived from the injected embryos at the blastocyst stage and sent for Sanger sequencing. The sgRNA targeting IGH locus resulted in a 100% on-target editing rate using SpCas9. However, SpCas9 introduced off-targeting events in AR and RBFOX1 at a high frequency (> 60%) in embryos. Injecting each Cas9 variant at 20 ng/µl could modify the target gene (IGH) at 100% efficiency except for LZ3 Cas9 (59.1%). Importantly, off-target events on AR and RBFOX1 were not detected in any Cas9 variant groups. Gradually reducing the concentration of Cas9 mRNAs lowered the efficacy of on-targeting in all groups; however, the reduction was more dramatic in HiFi Cas9 and LZ3 Cas9 injected embryos. No embryonic toxicity was identified in embryo injected with Cas9 variants and more embryos reached blastocyst stage when injected with either eSpCas9 or HiFiCas9 mRNA. In vivo competency of embryos receiving eSpCas9 was examined by embryo transfer and fetuses recovered from a pregnant sow presented 100% on-target editing efficiency without any detectable off-target events. In summary, among the Cas9 variants examined, eSpCas9 presented the highest specificity with no detectable off-target events and supported the development of gene-edited fetuses. Our findings indicate that the use of Cas9 variants can advance the field of gene editing in livestock models.},
}

Animals
*Gene Editing/veterinary/methods
Swine/embryology/genetics
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
*Embryo, Mammalian
Female
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41644661,
year = {2026},
author = {Davydova, S and Liu, J and Kandul, NP and Antoshechkin, I and Mann, J and Braswell, WE and Akbari, OS and Meccariello, A},
title = {Generating cisgenic sexing strains in insect pests.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41644661},
issn = {2399-3642},
support = {101059523//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; AP23PPQS&T00C108//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; },
mesh = {Animals ; Male ; Female ; *Ceratitis capitata/genetics ; CRISPR-Cas Systems ; *Pest Control, Biological/methods ; Gene Editing ; Introns ; },
abstract = {Insect pest population control via sterile insect technique markedly benefits from separation by sex prior to release. To simplify this process, traditional genetics has been deployed to develop genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic significance, although very few are applied in the field due to associated fitness costs and instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and relied on the sex-specifically spliced intron of its endogenous transformer gene, which we seamlessly inserted a copy into the pupal colouration white pupae gene. This minimal modification resulted in the generation of a homozygous strain we term IMPERIAL that was genetically and phenotypically stable where all female pupae are brown while male pupae are white with overall good fitness. By minimally editing the genome, our novel CGSS approach can be applied to other pests that may aid more efficient and economically suitable pest control.},
}

Animals
Male
Female
*Ceratitis capitata/genetics
CRISPR-Cas Systems
*Pest Control, Biological/methods
Gene Editing
Introns

@article {pmid41454745,
year = {2026},
author = {Mehnath, S},
title = {Engineering stimuli-responsive nanocarriers for CRISPR/Cas9 genome editing: next-generation cancer therapeutics.},
journal = {The Journal of pharmacy and pharmacology},
volume = {78},
number = {3},
pages = {},
doi = {10.1093/jpp/rgaf127},
pmid = {41454745},
issn = {2042-7158},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neoplasms/genetics/therapy ; Animals ; *Nanoparticles ; Drug Delivery Systems/methods ; Drug Carriers ; Genetic Therapy/methods ; },
abstract = {OBJECTIVES: To highlight recent developments in CRISPR/Cas9 genome-editing strategies for cancer therapy and to evaluate how nanocarrier-based delivery systems enable controlled, spatiotemporal manipulation of genetic information to overcome off-target effects, cytotoxicity, and limitations in clinical translation.

KEY FINDINGS: CRISPR/Cas9 has emerged as a simple and programmable tool for correcting cancer-associated mutations and regulating adaptive immune responses; however, challenges such as off-target effects, unintended mutations in healthy cells, and cytotoxicity hinder its clinical application. Nanocarriers address these limitations through refined spatiotemporal delivery of Cas9 nuclease and sgRNA using internal and external stimuli-responsive functional groups. These systems improve cancer-cell specificity by engineering guide RNAs, prevent premature clearance, enhance systemic circulation and intracellular delivery, enable nuclear targeting, and regulate Cas9 activity. Stimuli such as light, heat, ultrasound, magnetic fields, pH, redox conditions, glutathione, and oxygen play key roles in controlled activation and release.

SUMMARY: This review critically evaluates the structural design of nanocarriers, advanced spatiotemporal regulation strategies, and safety and efficacy concerns in CRISPR/Cas9-based cancer therapeutics. It discusses the role of cell-specific promoters, small-molecule stimulation, and stimuli-responsive delivery systems in improving genome-editing precision and therapeutic outcomes. The review also outlines future opportunities for exploiting CRISPR/Cas9 in advanced biomedical applications to enhance the effectiveness of next-generation cancer therapy.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Neoplasms/genetics/therapy
Animals
*Nanoparticles
Drug Delivery Systems/methods
Drug Carriers
Genetic Therapy/methods

@article {pmid41235827,
year = {2026},
author = {Chen, W and Zhang, X and Fan, R and Li, X and Guan, F and Wan, G and Kong, W and Qi, X and Pan, S and Shi, S and Su, Y and Gao, S and Huang, W and Xian, X and Liu, J and Wang, Y and Ma, Y},
title = {Generating golden Syrian hamsters with conditional alleles via zygote microinjection of CRISPR/Cas9.},
journal = {Animal models and experimental medicine},
volume = {9},
number = {2},
pages = {308-318},
doi = {10.1002/ame2.70107},
pmid = {41235827},
issn = {2576-2095},
support = {2060204//State Key Laboratory Special Fund/ ; Peking University, 202411//Open Research Project in State Key Laboratory of Vascular Homeostasis and Remodeling/ ; 2023-PT180-01//The Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences/ ; HH24KYZX0007//Haihe Laboratory of Cell Ecosystem Innovation Fund/ ; 2021-I2M-1-024//CAMS Innovation Fund for Medical Sciences/ ; 2022-I2M-1-020//CAMS Innovation Fund for Medical Sciences/ ; 2023-I2M-2-001//CAMS Innovation Fund for Medical Sciences/ ; 2021YFF0702802//the National Key Research and Development Program of China from the Ministry of Science and Technology/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Mesocricetus/genetics ; *Microinjections ; Cricetinae ; *Zygote ; Alleles ; Gene Knockout Techniques ; Female ; Male ; Animals, Genetically Modified ; },
abstract = {BACKGROUND: The golden Syrian hamster is a valuable animal model for studying carcinogenesis, metabolic disorders, cardiovascular diseases, and viral infections due to its biological and pathological similarities to humans. However, the development of genetically engineered hamsters has lagged behind that of mice and rats, largely because of an embryonic development block at the two-cell stage in vitro. Although CRISPR/Cas9-mediated gene knockout has been achieved in hamsters, precise DNA fragment insertion or conditional knockout (cKO) models have not previously been reported, likely due to technical limitations in embryo manipulation and insufficient efficiency of homology-directed repair (HDR).

METHODS: In this study, we generated conditional alleles of the ApoF gene in golden Syrian hamsters. A two-cut strategy was applied using Cas9 protein, two sgRNAs, and a single donor plasmid containing exon 2 flanked by loxP sites and two ~0.8 kb homology arms. A mixture of Cas9 protein, sgRNAs, and the donor plasmid was microinjected into the pronuclei of one-cell stage hamster embryos.

RESULTS: The efficiency of CRISPR/Cas9-mediated loxP knock-in reached up to 27%, and the genetically modified floxed alleles were successfully transmitted through the germline. The functionality of the inserted loxP sites was validated by in vivo Cre-mediated recombination following local administration of AAV vectors, including AAV-cTnT-Cre in the heart and AAV-CMV-Cre in the brain.

CONCLUSIONS: To our knowledge, this work represents the first successful establishment of a conditional knockout model in the golden Syrian hamster, providing a valuable tool for mechanistic studies of gene function and disease modeling.},
}

Animals
*CRISPR-Cas Systems/genetics
*Mesocricetus/genetics
*Microinjections
Cricetinae
*Zygote
Alleles
Gene Knockout Techniques
Female
Male
Animals, Genetically Modified

@article {pmid41191919,
year = {2026},
author = {Liu, C and Xu, F and Wu, Y and Li, J and Ni, M and Xia, S and Chen, L and Zhao, H and Yu, M and Zhou, Y and Zhang, M and Li, J and Wu, X and Huang, Y and Zhu, T and Chen, X},
title = {Genome-wide CRISPR-Cas9 screening identifies CLK1 inhibition as a strategy to restore PARP inhibitor sensitivity via ERCC1 isoform switching.},
journal = {Protein & cell},
volume = {17},
number = {3},
pages = {248-262},
doi = {10.1093/procel/pwaf091},
pmid = {41191919},
issn = {1674-8018},
support = {82403208//National Natural Science Foundation of China/ ; 82303660//National Natural Science Foundation of China/ ; 82272898//National Natural Science Foundation of China/ ; 82272682//National Natural Science Foundation of China/ ; 82503636//National Natural Science Foundation of China/ ; SACA-AX202215//Shanghai Anti-Cancer Association/ ; 22YF1408100//Science and Technology Commission of Shanghai Municipality/ ; 16411950200//Science and Technology Commission of Shanghai Municipality/ ; KW1711//Science and Technology Commission of Shanghai Municipality/ ; 17411963000//Science and Technology Commission of Shanghai Municipality/ ; 22YF1409100//Science and Technology Commission of Shanghai Municipality/ ; CORP-252-1//Chinese Anti-Cancer Association-Hengrui PARP Inhibitor Cancer Research Fund/ ; 2025ZD0545600//Noncommunicable Chronic Diseases-National Science and Technology Major/ ; LGF21H160008//Social Development Project of Public Welfare Technology Research in Zhejiang Province/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Female ; *Protein-Tyrosine Kinases/genetics/metabolism ; *Endonucleases/metabolism/genetics ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Ovarian Neoplasms/drug therapy/genetics/pathology/metabolism ; Animals ; *DNA-Binding Proteins/metabolism/genetics ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/antagonists & inhibitors/genetics/metabolism ; Mice ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Drug Resistance, Neoplasm/drug effects ; *Carcinoma, Ovarian Epithelial/drug therapy/genetics/pathology/metabolism ; Protein Isoforms/metabolism/genetics ; Apoptosis/drug effects ; },
abstract = {Epithelial ovarian cancer (EOC) is an aggressive malignancy with limited therapeutic options. Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown remarkable efficacy, especially in BRCA-mutant patients, and are approved as maintenance therapy to prevent recurrence after initial response to chemotherapy. However, the development of PARPi resistance poses a major clinical challenge. This study utilized a whole-genome CRISPR-Cas9 genetic screening to identify genes associated with PARPi sensitivity upon knockout. Based on the screening and validated through further experiments, we confirmed that CLK1 knockdown is synthetically lethal with PARPi in ovarian cancer. The combination of the PARPi Olaparib and CLK1 inhibitor TG003 exhibited potent anti-proliferative effects both in vitro and in vivo. Mechanistically, CLK1 inhibition downregulated the functional ERCC1-202 isoform, resulting in enhanced DNA damage and apoptosis. Our findings reveal a novel mechanism underlying PARPi sensitivity and suggest that targeting CLK1 in combination with PARPi may represent a promising therapeutic strategy for PARPi-resistant ovarian cancer.},
}

Humans
*CRISPR-Cas Systems
Female
*Protein-Tyrosine Kinases/genetics/metabolism
*Endonucleases/metabolism/genetics
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
*Ovarian Neoplasms/drug therapy/genetics/pathology/metabolism
Animals
*DNA-Binding Proteins/metabolism/genetics
Cell Line, Tumor
*Protein Serine-Threonine Kinases/antagonists & inhibitors/genetics/metabolism
Mice
Phthalazines/pharmacology
Piperazines/pharmacology
Drug Resistance, Neoplasm/drug effects
*Carcinoma, Ovarian Epithelial/drug therapy/genetics/pathology/metabolism
Protein Isoforms/metabolism/genetics
Apoptosis/drug effects

@article {pmid41806414,
year = {2026},
author = {Kim, H and Kim, D and Han, H and Lee, C and Roh, YH and Han, TS and Lim, EK and Park, J and Ahn, JK and Kang, T and Jung, J and Lee, CY},
title = {On-site microRNA detection with 'off-the-shelf' glucose meter empowered by chimeric probe connecting CRISPR/Cas13a activation to kinases-driven glucose phosphorylation.},
journal = {Biosensors & bioelectronics},
volume = {304},
number = {},
pages = {118568},
doi = {10.1016/j.bios.2026.118568},
pmid = {41806414},
issn = {1873-4235},
abstract = {MicroRNAs (miRNAs) are promising biomarkers for cancer diagnosis due to their stability in body fluids and disease-specific expression profiles. However, current detection methods suffer from limitations including cumbersome workflows, heavy instrumentation for signal readout, or vulnerability in minimizing instrumentation. To address these challenges, we describe a novel point-of-care miRNA detection platform executable with "off-the-shelf", personal glucose meter (PGM), termed 'KEY-FACT (Kinases Ensemble-driven glucose phosphorYlation upon Fuel-Aided CRISPR acTivation)'. Upon recognition of target miRNA, a fuel-assisted toehold-mediated strand displacement reactions liberate guide RNAs (gRNAs) to activate Cas13a to cleave a chimeric reporter probe, producing 2',3'-cyclic adenosine monophosphates (cAMP). Subsequent dephosphorylation and kinases ensemble-mediated phosphorylation/dephosphorylation cycles lead cAMP to consume a large amount of glucose. A user can immediately measure resulting glucose level change with PGM on the spot. This strategy allows sensitive, prompt detection of miR-135b, a gastric cancer (GC) biomarker, with a limit of detection (LOD) of 1.4 pM within 2 h. KEY-FACT is specific to the target miRNA and is applicable to body fluids such as human serum with dilution (95.2% < recovery rates <104.3%, coefficients of variation ≤13%). Owing to its simple probe design, KEY-FACT was readily expanded to detect another GC biomarker, miR-21, with comparable sensitivity (LOD = 1.5 pM). The proposed platform fulfills minimal instrumentation and thus enables cost-effective, field-deployable analysis, paving the way for practical, on-demand miRNA diagnostics.},
}

@article {pmid41806413,
year = {2026},
author = {Jeong, Y and Lee, J and Choi, S and Shin, D and Jang, S and Son, SU and Kang, T and Jung, J and Hwang, J and Lim, EK},
title = {On-site detection of airborne foodborne pathogens using a field-deployable recombinase polymerase amplification and CRISPR/Cas12a cleavage activity assay.},
journal = {Biosensors & bioelectronics},
volume = {304},
number = {},
pages = {118571},
doi = {10.1016/j.bios.2026.118571},
pmid = {41806413},
issn = {1873-4235},
abstract = {With the global increase in single-person households, the demand for meal kits is increasing, leading to the development of large-scale food production systems and complex supply chains. However, under the influence of global warming, these systems can be susceptible to food contamination, particularly by airborne foodborne bacteria. Conventional methods for detecting airborne bacteria involve complex, time-consuming, and labor-intensive processes, which limit their applicability for field use and rapid food hygiene surveillance. In the present study, we developed a field-deployable diagnostic platform by combining recombinase polymerase amplification with CRISPR/Cas12a cleaVage Activity (RCCVA assay) for the rapid and sensitive identification of airborne foodborne bacteria. Airborne bacteria were collected using a self-developed electrostatic air sampler and analyzed using a portable isothermal amplification device. The RCCVA assay was designed to detect four major foodborne pathogens: Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes, and Bacillus cereus. The limit of detection was measured as 274.9, 4.5, 9.5, and 28.5 culture-forming units (CFU)/mL, respectively, within 45 min. This platform enables early on-site detection of airborne pathogens within approximately 1 h (for the analytical phase) and shows potential for real-time monitoring in food processing environments, thereby contributing to improved public health and food safety.},
}

@article {pmid41805869,
year = {2026},
author = {Moghe, AS and Nandi, SS and Bhonde, RR and Kamyab, SS and Sawant, SA and Karandikar, MN},
title = {Engineering of cell line assembled enteric organoid for enterovirus infection.},
journal = {Archives of virology},
volume = {171},
number = {4},
pages = {},
pmid = {41805869},
issn = {1432-8798},
support = {5/3/8/57/2020-ITR Date:11.03.2021//Indian Council of Medical Research/ ; },
mesh = {Humans ; *Organoids/virology ; Virus Replication ; Cell Line ; *Enterovirus Infections/virology ; *Enterovirus A, Human/physiology ; Epithelial Cells/virology ; *Enterovirus/physiology ; CRISPR-Cas Systems ; Coculture Techniques ; },
abstract = {The non-polio-enteroviruses are ubiquitous pathogens infecting over a billion people in the world. An alarming number of enterovirus-associated acute flaccid paralysis, encephalitis, hand, foot, and mouth disease, conjunctivitis and diarrhoea cases are reported worldwide. Despite their clinical significance, vaccine development has been hindered due to lack of suitable in vitro models for preclinical investigations. The present study was undertaken to develop a cell line assembled organotypic model of human intestine for replication of enteroviruses. An enterovirus specific PSGL1 receptor was introduced in intestinal epithelial HCT-8 cell line employing CRISPR/cas9 gene editing. It was co-cultured with human colon (CCD-18) and endothelial (HUVEC) cell lines with peripheral blood mononuclear cells in hanging drops and rotating wall vessel bioreactor to yield three-dimensional organoids. Histological analysis of the organoids showed presence of columnar epithelium cells with prominent intracytoplasmic mucin, hyperchromatic nuclei and presence of CK, CK20, MUC 2 and Villin markers characteristic of epithelial cells. Infection with Enterovirus A71 (EV-A71) demonstrated significantly higher viral titre in organoids compared to individual cell lines. Collectively, these findings determine, for the first time, a cell line-derived enteric organoid model that supports robust enterovirus replication, offering a cost-effective and physiologically relevant system for virology research and preclinical applications.},
}

Humans
*Organoids/virology
Virus Replication
Cell Line
*Enterovirus Infections/virology
*Enterovirus A, Human/physiology
Epithelial Cells/virology
*Enterovirus/physiology
CRISPR-Cas Systems
Coculture Techniques

@article {pmid41805349,
year = {2026},
author = {Han, F and Xu, Y and Wang, W and Li, Z and Zhang, Z and Du, R and Xu, Q},
title = {Methylobacterium as a Dual-Function Platform: Advances in C1-Based Biomanufacturing and Plant-Associated Applications.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c08026},
pmid = {41805349},
issn = {1520-5118},
abstract = {One-carbon (C1) substrates are promising feedstocks for microbial bioproduction. Methylobacterium, known for its exceptional C1 utilization capacity, has emerged as a model microbial chassis for sustainable biomanufacturing. In this review, we first outline the C1 assimilation pathways in Methylobacterium and underscore its potential for producing valuable native metabolites. Furthermore, we then survey the genetic tools available for engineering this genus, including plasmid-based methods, transposon mutagenesis, homologous recombination, and CRISPR/Cas systems. Notably, recent advances in metabolic engineering have significantly expanded its biosynthetic scope, enabling the biosynthesis of diverse non-native compounds. Beyond its biomanufacturing potential, Methylobacterium also serves as a versatile plant growth-promoting bacterium, enhancing plant health and productivity through hormone synthesis, nutrient mobilization, stress mitigation, and induced systemic resistance. Collectively, this work highlights the dual potential of Methylobacterium as a sustainable microbial cell factory for biomanufacturing and a beneficial bioinoculant for agriculture.},
}

@article {pmid41805130,
year = {2026},
author = {Yu, W and Yuan, L and Zhou, W and He, L and Huang, X and Yu, J and Deng, J and Zhang, T and Hu, Y and Zhang, Y and Chen, S},
title = {Orn-mediated c-di-GMP regulates the CRISPR-Cas system to confer stress response in Mycobacterium tuberculosis.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41805130},
issn = {1362-4962},
support = {2021YFA1300901//National Key R&D Program of China/ ; 2022YFA1303500//National Key R&D Program of China/ ; GZNL2024A01024//Guangzhou National Laboratory/ ; //National Key Research and Development Program of China/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/metabolism/drug effects ; *CRISPR-Cas Systems/genetics ; *Cyclic GMP/analogs & derivatives/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Oxidative Stress/genetics ; *Stress, Physiological/genetics ; Promoter Regions, Genetic ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Mycobacterium tuberculosis (Mtb) possesses a type III-A CRISPR-Cas system and has anti-plasmid immune activity. However, whether this system exerts other additional functions remains to be characterized. Here, we investigated the in vivo roles of the Mtb CRISPR-Cas system. We show that this system is transcriptionally dependent and exhibits limited ability to counteract exogenous nucleic acids, primarily through the Csm6 protein rather than the Cas10 HD domain. We further demonstrate that this system plays a role in mitigating oxidative stress and antibiotic treatment, a function mainly mediated by the Cas10 HD domain. Importantly, through transposon library screening, we identified oligoribonuclease (Orn) as a regulatory protein of the Mtb CRISPR-Cas system. Deletion of the orn gene resulted in elevated c-di-GMP levels. A subsequent biotin-labeled c-di-GMP pull-down assay identified the transcriptional regulator Rv3058. Knockdown of rv3058 significantly increased cas6 promoter activity, and its transcriptional repressor function was directly modulated by c-di-GMP. This regulatory pathway enhances stress defense by activating multiple protective pathways, including DNA repair, cell envelope maintenance, and iron homeostasis regulation. Together, we conclude that the regulation of the CRISPR-Cas system by Orn-mediated c-di-GMP contributes to oxidative and antibiotic stress responses in Mtb.},
}

*Mycobacterium tuberculosis/genetics/metabolism/drug effects
*CRISPR-Cas Systems/genetics
*Cyclic GMP/analogs & derivatives/metabolism
Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Oxidative Stress/genetics
*Stress, Physiological/genetics
Promoter Regions, Genetic
CRISPR-Associated Proteins/genetics/metabolism

@article {pmid41804827,
year = {2026},
author = {Parada, F and Cabedo-Díaz, P and Cerda, A and Osorio-Navarro, C and Toledo, JA and Villalobos-González, L and Handford, M and Pimentel, P},
title = {CRISPR/dCas9-Mediated BRL3 Activation Enhances Growth and Metabolic Resilience Under Osmotic Stress in Nicotiana tabacum.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70816},
doi = {10.1111/ppl.70816},
pmid = {41804827},
issn = {1399-3054},
support = {3240290//ANID-FONDECYT Postdoctoral Project/ ; 3210631//ANID-FONDECYT Postdoctoral Project/ ; 1231417//ANID-FONDECYT Regular Project/ ; RF23F0002//ANID Fortalecimiento de Centros Regionales Project/ ; NCN2024_047//ANID-Millennium Science Initiative Program/ ; },
mesh = {*Nicotiana/genetics/growth & development/metabolism/physiology ; Osmotic Pressure/physiology ; Gene Expression Regulation, Plant ; *Plant Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic/genetics ; Brassinosteroids/metabolism ; Plant Leaves/genetics ; },
abstract = {Brassinosteroids (BRs) are crucial plant hormones that influence growth and stress adaptation. However, the specific function of the BR receptor BRL3 under osmotic stress remains largely unexplored outside Arabidopsis thaliana. In this study, we used a CRISPR/dCas9-based transcriptional activation (CRISPRa) system to upregulate the Nicotiana tabacum BRASSINOSTEROID INSENSITIVE-LIKE 3 receptor (NtBRL3) and assessed its impact on osmotic stress tolerance. Synthetic activation vectors were constructed using Loop Assembly, featuring dCas9-6TAL-VP128 modules driven by either a constitutive (CaMV35S) or ABA-inducible (SlAREB) promoter, paired with dual sgRNAs targeting the NtBRL3 promoter. Transient Agrobacterium-mediated transformation followed by PEG treatment was used to impose osmotic stress. RT-qPCR confirmed a 3- to 4-fold activation of NtBRL3 transcripts in CRISPRa-infiltrated leaves. The stress-inducible SlAREB promoter produced the strongest improvements, yielding nearly four-fold higher leaf biomass and a five-fold increase in root biomass relative to PEG-stressed controls. Both constructs reduced malondialdehyde (MDA) accumulation, indicating diminished oxidative damage, and modulated osmoprotectant balance, including reduced root proline and increased total soluble solids, particularly under SlAREB-driven activation. Histological segmentation revealed promoter-dependent anatomical remodeling, with NtBRL3-activated plants exhibiting a higher frequency of enlarged leaf cells and expanded tissue domains, consistent with brassinosteroid-mediated structural plasticity. Collectively, these findings demonstrate that CRISPR/dCas9-mediated transcriptional activation of NtBRL3 enhances osmotic stress resilience in tobacco through coordinated biomass recovery, oxidative stress mitigation, osmolyte homeostasis, and tissue remodeling. This transient, non-integrative CRISPRa approach provides a robust synthetic biology framework for dissecting BR signaling and engineering stress-tolerant crops.},
}

*Nicotiana/genetics/growth & development/metabolism/physiology
Osmotic Pressure/physiology
Gene Expression Regulation, Plant
*Plant Proteins/metabolism/genetics
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified
Promoter Regions, Genetic/genetics
Brassinosteroids/metabolism
Plant Leaves/genetics

@article {pmid41803497,
year = {2026},
author = {Perez Taboada, V and Wu, Y and Cassidy, R and Medvedev, KE and Loeff, L and Nemudraia, A and Nemudryi, A},
title = {Bacterial Schlafen proteins mediate phage defence.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41803497},
issn = {2058-5276},
support = {R00AI171893//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 1T32GM156737-01//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
abstract = {Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here we demonstrate that prokaryotic Schlafen nucleases are widespread antiviral effectors that protect bacteria from bacteriophages and are fused to a diverse array of phage-sensing domains. We expressed seven Enterobacterales Schlafen systems in Escherichia coli, identifying two that confer defence against coliphages. We focused on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrated that it recognizes tail assembly chaperones of T5-like phages. Upon activation, the Schlafen nuclease cleaves both E. coli and phage-encoded tRNAs and restricts T5 phage by reducing its burst size. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.},
}

@article {pmid41803127,
year = {2026},
author = {Zhou, R and Liu, Y and Zhang, Q and Yin, Z and Tong, J and Zhang, C and Zhang, L and Li, X and Zhao, Y and Zhang, S and Liu, Z and Chen, W and Ji, N and Zhang, H and Li, Z and Yin, H and Zuo, S and Wei, Y},
title = {Structural and mechanistic insights into the dual-nuclease defense protein Upx as an anti-phage system.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-70435-x},
pmid = {41803127},
issn = {2041-1723},
support = {2022YFC3400400//National Science Foundation of China | Key Programme/ ; 23ZYCGSY00750//Tianjin Science and Technology Committee (Tianjin Municipal Science and Technology Commission)/ ; },
abstract = {Nucleic acid degradation is a common strategy for prokaryotic anti-phage systems, as exemplified by the CRISPR-Cas system. The PD-(D/E)-XK nucleases constitute a widely distributed family in these defenses. Notably, most members exhibit a single nuclease domain, while variants containing dual nuclease domains within a single polypeptide remain underexplored, and their molecular mechanisms largely obscure. Here, we biochemically and functionally study a single-protein system containing an uncharacterized PD-(D/E)-XK defense protein (Upx). As revealed by single-particle electron cryo-microscopy (cryo-EM) structure, the C-terminal domain (CTD) harboring the conserved PD-(D/E)XK catalytic core is buttressed by the N-terminal domain (NTD) and the middle domain (MD). Functional assays demonstrate that the nucleic acid binding capability of the CTD is enhanced by the MD. The NTD also displays a noncanonical, basal exonuclease activity that is auto-inhibited by MD. IP-MS experiments identify Upx-interacting phage proteins, and substrate profiling defines its physiological preferences, collectively pointing to its potential physiological targets. Notably, the phage protein gp16 was found to relieve MD-mediated inhibition of the NTD, suggesting a virus-triggered mechanism for activating Upx's dual nuclease activity. Together, these findings establish Upx as a single-protein dual-nuclease anti-phage system, expanding our understanding of bacterial immunity and informing antiviral strategy development.},
}

@article {pmid41802999,
year = {2026},
author = {Hao, M and Zhou, M and Pan, F and Liu, T and Li, Y and Su, N and Ashfaq, A and Song, M and Wang, H and Wang, W and Liu, J and Li, C and Fu, L and He, P and Hu, Q and Mei, D and Cheng, H},
title = {Efficient CRISPR/Cas-SF01 genome editing tools with high editing efficiency in allotetraploid oilseed rape.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70221},
pmid = {41802999},
issn = {1744-7909},
support = {2025BEA003//the Major Program (JD) of Hubei Province/ ; CAAS-CSNCB-202303//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 2025AFB468//Hubei Provincial Natural Science Foundation of China/ ; CARS-12//Earmarked Fund for China Agriculture Research System/ ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.},
}

@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/isolation & purification/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; DNA/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; Limit of Detection ; *Bacterial Proteins/chemistry/genetics ; Nucleic Acid Hybridization ; Transcription, Genetic ; },
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/isolation & purification/analysis
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*CRISPR-Associated Proteins/genetics/chemistry
DNA/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics
Limit of Detection
*Bacterial Proteins/chemistry/genetics
Nucleic Acid Hybridization
Transcription, Genetic

@article {pmid41710969,
year = {2026},
author = {Xu, Z and Wei, M and Jiang, M and Wang, Y and He, K},
title = {Single-step duplex CRISPR coupled with lateral flow assay for point-of-care detection of human immunodeficiency virus and Treponema pallidum.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {10},
pages = {1996-2004},
doi = {10.1039/d5ay02168e},
pmid = {41710969},
issn = {1759-9679},
mesh = {Humans ; *Treponema pallidum/genetics/isolation & purification ; *Point-of-Care Systems ; *HIV Infections/diagnosis/virology ; *Syphilis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; *HIV/genetics/isolation & purification ; Point-of-Care Testing ; Sensitivity and Specificity ; },
abstract = {Continued acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) and syphilis caused by Treponema pallidum (TP) pose great challenges for global health, highlighting the need for rapid and sensitive diagnostics. Here, we introduced DIAL (Duplex Integrated All-in-one CRISPR Lateral flow assay), a simplified CRISPR-based diagnostic system for simultaneous detection of HIV and TP. We developed a single-step duplex CRISPR assay for dual-target recognition, which incorporated reverse transcription, multiplex recombinase polymerase amplification, transcription, CRISPR/Cas12a detection, and CRISPR/Cas13a detection in one pot. Then, we combined CRISPR detection assay with a "line-elimination" dual-line lateral flow assay for low-cost and equipment-free visual readout. We further developed reagent lyophilization and extraction-free sample lysis techniques to meet the point-of-care testing. The optimized DIAL system detected as low as 100 copies per µL of both HIV RNA and TP DNA within 45 minutes. In a clinical evaluation of 46 samples, it demonstrated 100% sensitivity and 97.5% specificity for HIV and 90% sensitivity and 100% specificity for TP, relative to quantitative PCR. The DIAL provided a scalable, accessible approach for decentralized screening of sexually transmitted infections or other pathogens in resource-limited settings.},
}

Humans
*Treponema pallidum/genetics/isolation & purification
*Point-of-Care Systems
*HIV Infections/diagnosis/virology
*Syphilis/diagnosis/microbiology
*CRISPR-Cas Systems/genetics
*HIV/genetics/isolation & purification
Point-of-Care Testing
Sensitivity and Specificity

@article {pmid41707429,
year = {2026},
author = {Nong, J and Pan, Z and Li, Y and Wei, J and Gong, Y and Li, J and Zhang, K and Liao, X},
title = {COF-confined CsPbBr3 nanocomposite with CRISPR/Cas12a-driven DNA walking for ultrasensitive electrochemiluminescent detection of circulating tumor DNA.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118536},
doi = {10.1016/j.bios.2026.118536},
pmid = {41707429},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; *Circulating Tumor DNA/blood/isolation & purification/genetics ; Endodeoxyribonucleases/chemistry ; DNA Probes/chemistry ; Metal-Organic Frameworks/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {A highly sensitive electrochemiluminescence biosensor was developed for circulating tumor DNA detection by integrating a covalent organic framework-confined CsPbBr3 nanocomposite with a CRISPR/Cas12a-driven amplification strategy. The covalent organic framework not only stabilizes CsPbBr3 nanocrystals in aqueous environments but also regulates interfacial charge transfer and enables programmable immobilization of DNA probes. Upon recognition of the target sequence, Cas12a is activated and progressively cleaves surface-tethered quencher-modified DNA strands, leading to stepwise restoration of electrochemiluminescence emission. This surface-confined signal amplification eliminates the need for polymerase chain reaction or isothermal preamplification. Under optimized conditions, the biosensor exhibited a linear response over a concentration range from 10 fM to 10 nM, with a detection limit of 5.4 fM. The method demonstrated good selectivity toward single-base mismatches and satisfactory performance in diluted serum and clinical plasma samples. These results highlight a synergistic material-enzyme strategy for sensitive and robust nucleic acid detection and provide a proof-of-concept platform for electrochemiluminescence-based circulating tumor DNA analysis.},
}

*Biosensing Techniques/methods
Humans
*Nanocomposites/chemistry
Luminescent Measurements/methods
CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
Limit of Detection
*Circulating Tumor DNA/blood/isolation & purification/genetics
Endodeoxyribonucleases/chemistry
DNA Probes/chemistry
Metal-Organic Frameworks/chemistry
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41707427,
year = {2026},
author = {Tu, Z and Wang, Y and Qian, X and Chen, J and Li, L and Wang, T and Chen, H and Wei, H and Yang, P and Pan, J and Wang, S and Rong, Z},
title = {Plasmonic magnetic nanoparticles-enabled universal enrichment, photothermal lysis, and duplex CRISPR detection of bacteria in urine samples.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118518},
doi = {10.1016/j.bios.2026.118518},
pmid = {41707427},
issn = {1873-4235},
mesh = {Humans ; *Escherichia coli/isolation & purification/genetics ; *Magnetite Nanoparticles/chemistry ; *Enterococcus faecalis/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Urinary Tract Infections/microbiology/urine/diagnosis ; CRISPR-Cas Systems ; Surface Plasmon Resonance/methods ; *Escherichia coli Infections/urine/microbiology/diagnosis ; Limit of Detection ; Boronic Acids/chemistry ; },
abstract = {Urinary tract infections (UTIs), exacerbated by antibiotic resistance and evolving pathogen diversity, demand rapid and sensitive diagnostics. This study introduces an integrated platform combining magnetic enrichment, photothermal lysis, and CRISPR-based detection (ME-CRISPR) for simultaneous identification of Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) in UTIs. We engineered plasmonic magnetic nanoparticles functionalized with 4-mercaptophenylboronic acid for broad-spectrum bacterial capture within 10 min. Subsequent near-infrared laser irradiation leveraged localized surface plasmon resonance to lyse captured bacteria in situ. The released nucleic acids were directly analyzed in a single-step duplex RPA-CRISPR/Cas12a-Cas13a assay. This 40-min workflow achieved a limit of detection of 10 CFU/mL for both pathogens. Validation with 90 clinical samples (39 E. coli, 26 E. faecalis, and 25 negative controls) demonstrated 100% sensitivity and specificity, matching qPCR performance while significantly reducing turnaround time. The platform overcomes limitations of centrifugation and Gram-class-dependent lysis, offering a rapid and ultrasensitive point-of-care testing tool to curb antibiotic misuse.},
}

Humans
*Escherichia coli/isolation & purification/genetics
*Magnetite Nanoparticles/chemistry
*Enterococcus faecalis/isolation & purification/genetics
*Biosensing Techniques/methods
*Urinary Tract Infections/microbiology/urine/diagnosis
CRISPR-Cas Systems
Surface Plasmon Resonance/methods
*Escherichia coli Infections/urine/microbiology/diagnosis
Limit of Detection
Boronic Acids/chemistry

@article {pmid41707424,
year = {2026},
author = {Zhao, L and Liu, Z and Ding, G and Zhu, Y and Wang, H and Liu, R and Qu, F and Ao, Q and Zhu, X and Zhang, Y and Yang, G and Wang, Z},
title = {Dual-readout aptasensor based on CRISPR/Cas12a and nanozyme for accurate detection of KIM-1 and its application in kidney transplant prognosis.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118533},
doi = {10.1016/j.bios.2026.118533},
pmid = {41707424},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis A Virus Cellular Receptor 1/isolation & purification ; *Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems ; *Kidney Transplantation ; Limit of Detection ; Prognosis ; Metal Nanoparticles/chemistry ; Silver/chemistry ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; SELEX Aptamer Technique ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Kidney injury molecule-1 (KIM-1) has emerged as a pivotal prognostic biomarker for renal allograft function. However, its detection remains challenging due to sensitivity and accuracy limitations. An innovative biosensing platform synergizing aptamer recognition, CRISPR trans-cleavage and nanozyme amplification for dual-readout KIM-1 detection has been presented in this work. This platform employs a meticulously selected high-affinity aptamer with capillary electrophoresis SELEX for specific target recognition, leverages the CRISPR/Cas12a system for signal transduction and cascade amplification, and utilizes engineered FeNi MOF@AgNPs nanozyme for dual-signal output. With the presence of KIM-1, whose binding with aptamer, effectively inhibits the trans-cleavage activity of the CRISPR/Cas12a system, and uninhibited Cas12a subsequently cleaves nanozyme-conjugated magnetic probes, releasing FeNi MOF@AgNPs nanozymes that catalyze a TMB-based reaction to generate intense colorimetric and fluorescent dual-readout signals. The as developed aptasensor demonstrates satisfied sensitivity achieving detection limits of 58.7 pg/mL (colorimetric) and 34.4 pg/mL (fluorometric), and dependable accuracy achieving average relative deviation of -2.7% (colorimetric) and 3.2% (fluorometric) with commercial ELISA kit in urine samples from patients with acute kidney injury. Moreover, longitudinally track the dynamic changes in urinary KIM-1 concentrations over the first 8 days following renal transplantation was successfully realized. This work not only provides a robust analytical tool for KIM-1 detection but also establishes a generic research approach for extending CRISPR-based systems to the precise detection of proteins.},
}

*Biosensing Techniques/methods
Humans
*Hepatitis A Virus Cellular Receptor 1/isolation & purification
*Aptamers, Nucleotide/chemistry
CRISPR-Cas Systems
*Kidney Transplantation
Limit of Detection
Prognosis
Metal Nanoparticles/chemistry
Silver/chemistry
Colorimetry/methods
Metal-Organic Frameworks/chemistry
SELEX Aptamer Technique
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41702192,
year = {2026},
author = {Lu, L and Zhang, Y and Liu, B and Zhou, N and Yu, DJ and Wang, Y},
title = {Magnetic Fe3O4-Au@UIO-66-NH2@toehold probe mediated fluorescent sensor for detecting ovarian cancer-specific circRNA coupled with hybridization chain reaction and the CRISPR-Cas12a system.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118535},
doi = {10.1016/j.bios.2026.118535},
pmid = {41702192},
issn = {1873-4235},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *RNA, Circular/genetics/isolation & purification ; *Biosensing Techniques/methods ; Female ; *Ovarian Neoplasms/genetics/diagnosis ; Gold/chemistry ; Fluorescent Dyes/chemistry ; Nucleic Acid Hybridization ; Limit of Detection ; Magnetite Nanoparticles/chemistry ; Ferrosoferric Oxide/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Circular RNAs (circRNAs) represent an emerging family of noncoding transcripts defined by closed-loop architecture, which are now established as key participants in the etiology of tumorigenesis and malignant progression. While circRNAs show potential as therapeutic targets and biomarkers, the accurate detection of circRNAs remains challenging due to interference from homologous linear RNAs. In this study, an ultrasensitive method for detecting circ_0051240 based on the sulfhydrated toehold capture probe-initiator assembly-mediated hybridization chain reaction (HCR) and the CRISPR-Cas12a system was developed. The capture probe (with a toehold domain)-initiator strand duplex was ingeniously fabricated to identify the back-splice junction (BSJ) of circRNA. After magnetic enrichment and separation by the Fe3O4-Au@UIO-66-NH2 nanocomposite, the initiator strand (H0) was delivered to trigger HCR. The HCR product dsDNA concatemers contained multiply repeated CRISPR-targetable DNA sites that were readily recognized by the CRISPR RNA (crRNA). This specific recognition and binding activated the CRISPR-Cas12a system's collateral endonuclease activity, leading to cleavage of the fluorophore-quencher (FQ) reporters and fluorescence emission at a characteristic wavelength. This design eliminated linear RNA-related interference and enhanced the detection of fluorescence intensity (FI). Under optimal conditions, the proposed HCR/CRISPR-Cas12a method exhibited a wide quantitative measurement range spanning from 45 pM up to 180 nM, achieving a notable limit of detection (LOD) of 0.03 pM. In this study, a novel circRNA sensing strategy capable of accurate and highly sensitive quantification of ovarian cancer-specific circRNA was reported. The proposed method exhibits acceptable performance when compared to present approaches.},
}

Humans
CRISPR-Cas Systems/genetics
*RNA, Circular/genetics/isolation & purification
*Biosensing Techniques/methods
Female
*Ovarian Neoplasms/genetics/diagnosis
Gold/chemistry
Fluorescent Dyes/chemistry
Nucleic Acid Hybridization
Limit of Detection
Magnetite Nanoparticles/chemistry
Ferrosoferric Oxide/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41677799,
year = {2026},
author = {Boubakri, H},
title = {CRISPR-Cas9-mediated genome editing in fungi: applications, challenges, and future directions.},
journal = {Journal of applied microbiology},
volume = {137},
number = {3},
pages = {},
doi = {10.1093/jambio/lxag046},
pmid = {41677799},
issn = {1365-2672},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems ; *Fungi/genetics ; *Genome, Fungal ; },
abstract = {The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been shown to be an effective genome-editing tool in many organisms, including fungi. It enables precise modifications to the DNA of fungal species, facilitating advancements in research, agriculture, and biotechnology. CRISPR-Cas9-edited non-pathogenic antagonists have emerged as a promising alternative for biocontrol. Several filamentous fungi have been engineered to produce secondary metabolites. Furthermore, the CRISPR-Cas9 system has been used to improve the quality of several edible fungi. However, the application of CRISPR-Cas9 technology for fungal genome editing is still facing some challenges that researchers must address. This review highlights the major approaches and applications of genome editing in fungi, as well as the associated challenges.},
}

*Gene Editing/methods/trends
*CRISPR-Cas Systems
*Fungi/genetics
*Genome, Fungal

@article {pmid41670347,
year = {2026},
author = {Sullivan, JR and Ferrara, KM and Barrick, R and Romano, KP and Warrier, T and Hung, DT},
title = {An inducible CRISPRi system for phenotypic analysis of essential genes in Pseudomonas aeruginosa.},
journal = {mBio},
volume = {17},
number = {3},
pages = {e0276725},
pmid = {41670347},
issn = {2150-7511},
support = {U19 AI142780/AI/NIAID NIH HHS/United States ; U19AI142780/NH/NIH HHS/United States ; },
mesh = {*Pseudomonas aeruginosa/genetics/drug effects/growth & development ; *Genes, Essential ; Phenotype ; *CRISPR-Cas Systems ; Plasmids ; Gene Expression Regulation, Bacterial ; Rhamnose/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Promoter Regions, Genetic ; },
abstract = {Precise and tunable genetic tools are essential for high-throughput functional genomics. To address this need in the important gram-negative pathogen Pseudomonas aeruginosa, we developed and characterized a tightly regulated CRISPR-interference (CRISPRi) system that enables precise and tunable repression of essential genes. The system utilizes a rhamnose-inducible promoter to control both the Streptococcus pasteurianus-derived dCas9 and gene-specific sgRNAs, each encoded on separate plasmids for modularity and efficiency. The combination of tight regulation and high conjugation efficiency facilitated the rapid and facile construction of strains with regulated depletion of 16 essential genes spanning diverse pathways. Comparison of phenotypes across the different genetically depleted strains, including growth rate, susceptibility to antibiotics, and changes in transcriptional programs, revealed novel aspects of gene function or small-molecule mechanism of action. Finally, the rhamnose-inducible CRISPRi system supports the generation and stable maintenance of pooled mutant libraries, thereby paving the way for future genome-wide, systematic assessment of individual gene vulnerabilities, which will provide critical insights for target prioritization in antibiotic discovery efforts against this recalcitrant pathogen.IMPORTANCECRISPR-interference (CRISPRi) has become an invaluable tool for studying genetics. In particular, the ability to knockdown (KD) genes enables the study of essential genes and their role in cell survival. However, a tightly regulated gene KD system is required to gain valuable insights into these vulnerable genes by virtue of their essentiality. We report a tightly regulated CRISPRi system to study the biology of essential gene perturbations in Pseudomonas aeruginosa, an important gram-negative pathogen that causes severe infections and is increasingly resistant to current antibiotics. This system enables characterization of both chemical genetic interactions between small molecules and specific gene depletions and the impact of genetic perturbations on transcriptional networks. Genetic perturbations using CRISPRi can thus further our understanding of basic biology with translation toward future antimicrobial development.},
}

*Pseudomonas aeruginosa/genetics/drug effects/growth & development
*Genes, Essential
Phenotype
*CRISPR-Cas Systems
Plasmids
Gene Expression Regulation, Bacterial
Rhamnose/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Promoter Regions, Genetic

@article {pmid41642947,
year = {2026},
author = {Pelea, O and Tálas, A and Carrera, JF and Mathis, N and van de Venn, L and Yeh, CD and Kulcsár, PI and Marquart, KF and Weber, Y and Gerecke, SE and Harvey-Seutcheu, IF and Mailänder, D and Pfleiderer, MM and Chanez, C and Corn, JE and Schwank, G and Jinek, M},
title = {Programmable genome editing in human cells using RNA-guided bridge recombinases.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6790},
pages = {eadz1884},
doi = {10.1126/science.adz1884},
pmid = {41642947},
issn = {1095-9203},
mesh = {Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Plasmids ; Recombination, Genetic ; Genome, Human ; *DNA Nucleotidyltransferases/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; DNA/genetics ; },
abstract = {Site-specific insertion of gene-sized DNA fragments remains an unmet need in the field of genome editing. IS110-family serine recombinases have recently been shown to mediate programmable DNA recombination in bacteria by using a bispecific RNA guide (bridge RNA) that simultaneously recognizes target and donor sites. In this work, we have shown that the bridge recombinase ISCro4 is highly active in human cells and provided structural insights into its enhanced activity. Using plasmid- or all-RNA-based delivery, ISCro4 supports programmable multikilobase excisions and inversions and facilitates donor DNA insertion at genomic sites with efficiencies that exceed 6%. Last, we assessed ISCro4 specificity and off-target activity. These results establish a framework for the development of bridge recombinases as next-generation tools for editing modalities that are beyond the capabilities of current technologies.},
}

Humans
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
Plasmids
Recombination, Genetic
Genome, Human
*DNA Nucleotidyltransferases/chemistry/metabolism/genetics
CRISPR-Cas Systems
DNA/genetics

@article {pmid41524478,
year = {2026},
author = {Kim, M and Kim, K and Lee, J and Lee, S and Choi, S and Park, SA and Jeong, E and Choi, SY and Park, HH and Park, TE and Kwon, T and Myung, K and Yoo, J and Cho, SW and Joo, J},
title = {FAST-CRISPR: Fusogenic Association and Secured Transfection of CRISPR/Cas9 Ribonucleoproteins Using Lipid-Silica Hybrid Nanoparticles for Therapeutic Genome Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {15},
pages = {e11362},
pmid = {41524478},
issn = {1613-6829},
support = {RS-2024-00512120//Korean ARPA-H Project through the KHIDI/ ; 22A0102L1-11//Korean Fund for Regenerative Medicine (KFRM)/ ; RS-2024-00509412//National Research Foundation (NRF)/ ; RS-2023-00209822//National Research Foundation (NRF)/ ; RS-2023-00207746//National Research Foundation (NRF)/ ; IBS-R022-D1//Institute for Basic Science/ ; 1.250006.01//UNIST research fund/ ; RS-2024-00403508//Korea Basic Science Institute/ ; 1.250006.01//Ulsan National Institute of Science and Technology/ ; },
mesh = {*Nanoparticles/chemistry ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/metabolism/genetics ; *Silicon Dioxide/chemistry ; Humans ; *Lipids/chemistry ; Animals ; *Transfection/methods ; Mice ; Cell Line, Tumor ; },
abstract = {Clinical translation of CRISPR/Cas9 therapeutics is challenged by inefficient cytosolic delivery and toxicity issues associated with viral vectors and nanoparticle-based carriers. To overcome these concerns, herein we report a lipid-silica hybrid nanoparticle platform for fusogenic association and secured transfection of CRISPR/Cas9 (FAST-CRISPR), designed for rapid cytosolic delivery of CRISPR/Cas9 ribonucleoproteins, followed by efficient gene editing. Through direct fusion with the plasma membrane and bypassing conventional endocytic barriers, FAST-CRISPR nanoparticles displayed superior intracellular delivery efficacy. Optimizing lipid compositions, we discovered that a 1:1 weight mixture of cationic DOTAP and ionizable DODMA lipids, combined with tailored large-pore silica nanoparticles, enables enhanced loading capacity, rapid cytosolic dispersion, and significant nuclear transport of Cas9/gRNA complexes. FAST-CRISPR nanoparticles efficiently delivered multiplex genome-targeting ribonucleoproteins to induce targeted double-strand DNA breaks, triggering apoptosis in cancer cells and significantly suppressing tumor growth in a mouse xenograft model without systemic toxicity. Our findings demonstrate the therapeutic efficacy and translational potential of FAST-CRISPR nanoparticles as a safe and versatile non-viral delivery platform for precision genome editing.},
}

*Nanoparticles/chemistry
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/metabolism/genetics
*Silicon Dioxide/chemistry
Humans
*Lipids/chemistry
Animals
*Transfection/methods
Mice
Cell Line, Tumor

@article {pmid41270742,
year = {2026},
author = {Sun, J and Atiş, IS and Empke, SLL and Khokha, MK and Breslow, DK},
title = {A microscopy-based CRISPR screening platform enables organellar functional genomics and illuminates ciliary biology.},
journal = {Developmental cell},
volume = {61},
number = {3},
pages = {687-705.e9},
pmid = {41270742},
issn = {1878-1551},
support = {S10 OD023651/OD/NIH HHS/United States ; R35 GM137956/GM/NIGMS NIH HHS/United States ; S10 OD018034/OD/NIH HHS/United States ; R01 HD102186/HD/NICHD NIH HHS/United States ; S10 OD030363/OD/NIH HHS/United States ; },
mesh = {*Cilia/metabolism/genetics ; Humans ; Animals ; *Genomics/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microscopy, Fluorescence/methods ; Xenopus ; },
abstract = {Microscopy offers an indispensable technique for visualizing biological processes and for defining cytological abnormalities characteristic of disease. However, combining microscopy with the power of pooled CRISPR screening presents considerable technical challenges, hindering application of systematic genetic analysis to imaging-defined phenotypes. Here, we establish a fluorescence microscopy-based CRISPR screening platform that combines ease of implementation with flexible analysis of live-cell or antibody-based molecular markers, including post-translational modifications. Applying this methodology, we systematically identify regulators of primary cilium structure and function in human cells through targeted and genome-wide screens. We further show that integration of screens focused on distinct ciliary phenotypes yields multi-dimensional profiles that delineate precise gene functions. Among the identified hits, TZMP1 (SMIM27) encodes a microprotein at the ciliary transition zone that is required for ciliogenesis in human cells and for ciliary function in Xenopus embryos. More broadly, our approach provides a technological and conceptual strategy for microscopy-based functional genomics.},
}

*Cilia/metabolism/genetics
Humans
Animals
*Genomics/methods
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Microscopy, Fluorescence/methods
Xenopus

@article {pmid37751223,
year = {2023},
author = {Ji, S and Wang, X and Wang, Y and Sun, Y and Su, Y and Lv, X and Song, X},
title = {Advances in Cas12a-Based Amplification-Free Nucleic Acid Detection.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {405-418},
doi = {10.1089/crispr.2023.0023},
pmid = {37751223},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; *Nucleic Acids/genetics/analysis ; Biosensing Techniques/methods ; *Bacterial Proteins/genetics ; Electrochemical Techniques/methods ; },
abstract = {In biomedicine, rapid and sensitive nucleic acid detection technology plays an important role in the early detection of infectious diseases. However, most traditional nucleic acid detection methods require the amplification of nucleic acids, resulting in problems such as long detection time, complex operation, and false-positive results. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) systems have been widely used in nucleic acid detection, especially the CRISPR-Cas12a system, which can trans cleave single-stranded DNA and can realize the detection of DNA targets. But, amplification of nucleic acids is still required to further improve detection sensitivity, which makes Cas12a-based amplification-free nucleic acid detection methods a great challenge. This article reviews the recent progress of Cas12a-based amplification-free detection methods for nucleic acids. These detection methods apply electrochemical detection methods, fluorescence detection methods, noble metal nanomaterial detection methods, and lateral flow assay. Under various optimization strategies, unamplified nucleic acids have the same sensitivity as amplified nucleic acids. At the same time, the article discusses the advantages and disadvantages of each method and further discusses the current challenges such as off-target effects and the ability to achieve high-throughput detection. Amplification-free nucleic acid detection technology based on CRISPR-Cas12a has great potential in the biomedical field.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/genetics/metabolism
Humans
*Endodeoxyribonucleases/genetics/metabolism
*Nucleic Acids/genetics/analysis
Biosensing Techniques/methods
*Bacterial Proteins/genetics
Electrochemical Techniques/methods

@article {pmid37739193,
year = {2024},
author = {Mori, AA and Malaquias, VB and Bonjour, K and Ferreira, GM and Bortolin, RH and Borges, JB and Oliveira, VF and Gonçalves, RM and Faludi, AA and Bastos, GM and Thurow, H and Sampaio, MF and Ciconelli, RM and Cury, AN and Fajardo, CM and Hirata, RDC and Hirata, MH},
title = {Effects of LDLR variants rs5928, rs750518671 and rs879254797 on protein structure and functional activity in HepG2 cells transfected with CRISPR/Cas9 constructs.},
journal = {Gene},
volume = {890},
number = {},
pages = {147821},
doi = {10.1016/j.gene.2023.147821},
pmid = {37739193},
issn = {1879-0038},
mesh = {Humans ; Hep G2 Cells ; *Receptors, LDL/genetics/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; Mutation, Missense ; *Hyperlipoproteinemia Type II/genetics ; Female ; Male ; Polymorphism, Single Nucleotide ; Transfection ; Middle Aged ; },
abstract = {Familial Hypercholesterolemia (FH) is a genetic disorder associated with premature atherosclerosis and increased risk of cardiovascular diseases. LDLR deleterious mutations are associated with FH, however the role of some missense variants in FH pathogenicity remains to be elucidated. This study explored the predictive impact of LDLR missense variants on protein structure and investigated their functional effects on LDLR expression in HepG2 cells transfected with CRISPR/Cas9 constructs. FH (n = 287) and non-FH patients (n = 45) were selected, and lipid profile was obtained from medical records. LDLR variants were identified using an exon-targeted gene sequencing strategy, considering its cost-effective to increase accuracy in the identification step of the most likely FH-related variants in a less laborious process. LDLR variants were selected based on conflicting pathogenicity results found in Clinvar, in silico prediction tools, affected LDLR domains, and less common variants considering minor allele frequency < 0.05. Molecular modeling studies were used to predict the effects of LDLR missense variants on protein structure. Recombinant LDLR variants were constructed using CRISPR/Cas9 system and were used to transfect HepG2 cells. Functional assays in transfected cells were performed to assess LDLR expression using flow cytometry and western blotting, and LDLR activity using flow cytometry and confocal microscopy. The variants rs121908039 (c.551G>A, p.C184Y), rs879254797 (c.1118G>A, p.G373D), rs28941776 (c.1646G>A, p.G549D), rs750518671 (c.2389G>C, p.V797L), rs5928 (c.2441G>A, p.R814Q) and rs137853964 (c.2479G>A, p.V827I) were selected for molecular docking analysis. The p.C184Y exhibited a favorable energy change for protein stability due to its interaction with EGF-A/EGF-B regions; p.G373D and p.G549D displayed intermediate energy changes; and p.R814Q and p.V827I showed smaller energy changes. The results of functional assays showed that p.G373D, p.V797L and p.R814Q reduced LDLR expression and activity (p < 0.05). Microscopic analysis of the p.V797L and p.G373D variants revealed altered lipid localization and accumulation in transfected HepG2 cells. Carriers of p.G549D, p.V797L and p.R814Q had higher LDL cholesterol levels than non-FH group, and (p < 0.05). p.G373D and p.G549D were associated with clinical manifestations of FH. In conclusion, the p.C184Y, p.G373D, p.G549D and p.R814Q variants alter protein stability and intramolecular interactions, while p.V797L has a minimal impact on protein stability, and p.V827I has no significant intramolecular interactions. p.G373D, p.V767L and p.R814Q are associated with impaired LDLR expression and activity.},
}

Humans
Hep G2 Cells
*Receptors, LDL/genetics/chemistry/metabolism
*CRISPR-Cas Systems/genetics
Mutation, Missense
*Hyperlipoproteinemia Type II/genetics
Female
Male
Polymorphism, Single Nucleotide
Transfection
Middle Aged

@article {pmid41802204,
year = {2026},
author = {de Paula, JA and de Araújo, MRB and Guimarães Sousa, E and Prates, FD and Castro, DLC and Fonseca, PAS and Brenig, B and Felice, AG and Pacheco, LGC and Viana, MVC and Azevedo, VAC and Soares, S},
title = {Clonal clusters of multidrug-resistant Brazilian Corynebacterium striatum strains reveal putative virulence traits.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxag070},
pmid = {41802204},
issn = {1365-2672},
abstract = {CORYNEBACTERIUM STRIATUM: Has been increasingly associated with nosocomial outbreaks and antimicrobial resistance.

OBJECTIVES: This study presents the comparative analysis of 26 multidrug-resistant (MDR) C. striatum strains isolated in Brazil.

METHODS: Additional genomes from international sources were incorporated. The analyses encompassed in vitro antimicrobial susceptibility testing and an in silico workflow for genomic similarity comparison, phylogenetic reconstruction, genomic clustering, pangenome analysis, mobilome content, virulence prediction, and functional annotation of unique proteins and putative virulence clusters.

RESULTS: Strong in silico evidence of clonality among several Brazilian isolates was obtained at the same time that some strains consistently indicated a divergent genomic profile. There are 196 unique coding sequences (CDSs) across the Brazilian IHPs. Of particular interest, strain IHP2030 carried an exclusive fimbria, sharing less than 50% similarity with other fimbriae in the dataset. Yet, structural predictions suggested conservation of key structural domains typically associated with fimbrial proteins. Mobilome content analysis revealed that IHPs strains were overall similar, differing primarily in the number of insertion sequences and in the presence or absence of CRISPR-Cas defense systems. Regarding virulence, an exclusive cluster in IHP2050 and IHP2060 suggests adaptive advantages associated with their respective environments of isolation.

CONCLUSION: This study reveals a complex genomic landscape among Brazilian MDR C. striatum strains, marked by clonal dissemination alongside strain-level genetic variation in accessory genomes, mobilome composition, and virulence-associated gene repertoires, providing genomic evidence of diversification within hospital-associated lineages.},
}

@article {pmid41799755,
year = {2026},
author = {Vadrot, N and Moulin, M and Ferreiro, A and Richard, P and Buendia, B},
title = {LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2.},
journal = {International journal of medical sciences},
volume = {23},
number = {3},
pages = {741-757},
pmid = {41799755},
issn = {1449-1907},
mesh = {Animals ; Cell Differentiation/genetics ; *Myocytes, Cardiac/metabolism ; Cell Proliferation/genetics ; CRISPR-Cas Systems/genetics ; Mice ; *Membrane Proteins/genetics/metabolism ; MEF2 Transcription Factors/genetics/metabolism ; Protein Isoforms/genetics/metabolism ; Rats ; *Aging/genetics ; Cell Line ; Gene Knockdown Techniques ; Myocardium/metabolism ; Humans ; Heart/growth & development/physiology ; DNA-Binding Proteins ; },
abstract = {Haploinsufficiency of Lap2 alpha (LAP2a), a nuclear partner of Lamins A/C, has been associated with cardiac disease in rare cases, but LAP2a function remains largely unknown. To investigate the functional role of LAP2a in cardiomyocytes, we generated clones of embryonic myocardium-derived H9C2 cells in which LAP2a expression was specifically reduced through gene editing of the LAP2a gene Tmpo by CRISPR-Cas9. Downregulation (+/-) and absence (-/-) of LAP2a expression led to a decreased proliferation capacity of cardiomyocytes in vitro. Upon differentiation, the expression of myocardial markers (alpha cardiac Actin 1/Actc1, cardiac Troponin T2/Tnnt2, Myosin-2/Myh2 and Myosin-7/Myh7) was higher in LAP2a -/- cells compared to LAP2a +/- or LAP2a +/+ cells, with consistently higher expression of their upstream regulator Mef2c in LAP2a-devoid cells. These results suggest that LAP2a promotes cardiomyocyte proliferation and negatively modulates cardiomyocyte differentiation, through mechanisms including Mef2c regulation. Accordingly, normal protein expression of LAP2a was downregulated upon cardiomyocyte differentiation, contrary to LAP2b and a LAP2b-related shorter isoform. The latter tended to increase upon differentiation in all cells, most significantly in the LAP2a -/- clone. In postnatal mouse hearts, LAP2a levels were higher in the right than in the left ventricle, and lowest in the septum. The LAP2a:LAP2b ratio was much lower in murine hearts than in H9C2 cells, and decreased significantly upon ageing, specifically in the left ventricle. Finally, our data show that expression of the nuclear envelope proteins LEMD2 and Lamin A might be influenced by LAP2a upon cardiac differentiation. Our results show that LAP2 expression is finely regulated upon cardiac differentiation in vitro and is dependent on age and heart compartment in vivo. They contribute to clarifying the potential impact of genetic LAP2a defects and their connection with heart disease, possibly including reduced cardiomyoblast proliferation, increased cardiomyocyte differentiation and altered nuclear envelope remodelling.},
}

Animals
Cell Differentiation/genetics
*Myocytes, Cardiac/metabolism
Cell Proliferation/genetics
CRISPR-Cas Systems/genetics
Mice
*Membrane Proteins/genetics/metabolism
MEF2 Transcription Factors/genetics/metabolism
Protein Isoforms/genetics/metabolism
Rats
*Aging/genetics
Cell Line
Gene Knockdown Techniques
Myocardium/metabolism
Humans
Heart/growth & development/physiology
DNA-Binding Proteins

@article {pmid41799190,
year = {2026},
author = {Zhao, P and Li, H and Cai, Z and Zhang, X and Wen, X and Liu, Z and Jiang, S and Jiang, X and Wang, J and Dang, Z and Liu, M and Xie, F and Ma, X},
title = {Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx.},
journal = {Theranostics},
volume = {16},
number = {9},
pages = {4843-4864},
pmid = {41799190},
issn = {1838-7640},
mesh = {Animals ; Mice ; *TRPC Cation Channels/metabolism/genetics ; *Calcium/metabolism ; Mice, Inbred C57BL ; *Calcium Signaling/drug effects ; *Hydrogen/metabolism/pharmacology ; Humans ; Cell Movement/drug effects ; Molecular Dynamics Simulation ; Molecular Docking Simulation ; Brain/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {RATIONALE: Hydrogen gas (H2) produces pleiotropic therapeutic actions, but the exact molecular targets and ion-channel-based signaling cascades that underlie these benefits remain elusive. H2 may regulate calcium ion (Ca[2+])-dependent processes, but the direct involvement of H2 in Ca[2+] signaling and its underlying molecular mechanisms are unknown. We propose that H2 functions as a gaseous messenger that selectively opens a plasma-membrane Ca[2+] channel to evoke Ca[2+] transients ([Ca[2+] i]t) while avoiding cytotoxic overload, thereby offering a mechanism for its diverse biological effects.

METHODS: This study employed real-time calcium imaging and CRISPR-Cas9 gene editing, with live-cell imaging to monitor real-time calcium signal intensity in living cells. Two-photon in vivo imaging was applied to detect real-time Ca[2+] signals in the brain and dorsal skin of C57BL/6 mice carrying adeno-associated virus-delivered calcium sensors. Live-cell F-actin staining and a wound healing (scratch) assay were used to assess the effects of H2 on cell motility. Protein-protein docking and molecular dynamics simulations were performed to analyze the interaction interface and binding forces between TRPC4 and TRPC4AP in three-dimensional space. Additionally, RNA sequencing was performed to validate downstream biological effects and transcriptional regulation triggered by H2.

RESULTS: H2 elicited rapid and reversible [Ca[2+] i]t across multiple cell types in a Ca[2+]- and concentration-dependent manner, an effect that was absent in TRPC4⁻/⁻ or TRPC4AP⁻/⁻ cells. In vivo imaging in mice expressing a genetically encoded Ca²⁺ sensor showed that H2 inhalation elevated Ca[2+] signals in the motor cortex (M1 region) and dorsal skin. Functionally, live-cell imaging and wound-healing assays confirmed that H2-induced Ca[2+] transients enhanced cell motility. Mechanistically, protein docking revealed a dual-arginine cluster within the CIRB domain of TRPC4; its interaction with TRPC4AP was essential for H2-evoked Ca[2+] influx. Mutating these arginines to alanine residues completely abolishing the response. H2 triggered proton efflux and increased intracellular pH. Molecular dynamics simulations indicated that altered pH modulates the binding force between TRPC4 Arg730/Arg731 and TRPC4AP. Transcriptomic analysis further demonstrated that H2 activates calcium-related channels and promotes cytoskeletal remodeling and cell migration.

CONCLUSIONS: This study identifies H2 as a novel gaseous signaling molecule that can regulate Ca[2+] channels via the TRPC4-TRPC4AP axis. The 730Arg-731Arg motif in TRPC4 serves as a critical H2-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic framework for developing gas-controlled H2 regenerative therapeutics.},
}

Animals
Mice
*TRPC Cation Channels/metabolism/genetics
*Calcium/metabolism
Mice, Inbred C57BL
*Calcium Signaling/drug effects
*Hydrogen/metabolism/pharmacology
Humans
Cell Movement/drug effects
Molecular Dynamics Simulation
Molecular Docking Simulation
Brain/metabolism
CRISPR-Cas Systems
HEK293 Cells

@article {pmid41797538,
year = {2026},
author = {Fang, M and Yap, J and Fei, M and Gong, M and Li, N and Lu, Y and Yu, M and Xu, Y and Wu, F and Gao, H and Sun, D},
title = {LysR-type regulator LrhA promotes CRISPR-Cas immunity in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41797538},
issn = {1362-4962},
support = {32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; 31930003//National Natural Science Foundation of China/ ; 2020C02031//Key Research and Development Program of Zhejiang Province/ ; LHDMY23H160003//Natural Science Foundation of China/ ; YS2022005//Natural Science Foundation of China/ ; 2026C02A1080//Zhejiang Lingyan Research and Development Program/ ; LMRY26H200010//Joint Funds of the Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/immunology/virology ; *CRISPR-Cas Systems/genetics ; *Escherichia coli Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Promoter Regions, Genetic ; *Transcription Factors/genetics/metabolism ; Operon ; Plasmids/genetics ; Trans-Activators/genetics ; },
abstract = {The CRISPR-Cas defense system safeguards prokaryotes against foreign genetic elements. Its activity is determined by the combined effects of adaptation and interference. However, the dynamic regulation of these two processes remains not fully understood. In this study, we identify the LysR-type transcriptional regulator LrhA, which is differentially expressed in various Escherichia coli strains, as a novel CRISPR-Cas activator that plays a critical role in modulating host defense levels. In a representative strain expressing a high level of LrhA, the regulator enhances CRISPR-Cas-mediated adaptive immunity against bacteriophage infection by promoting cas gene transcription through direct interaction with the promoter of the cas operon. Moderate activation of cas genes by weakly expressed LrhA in another representative strain efficiently accelerates the clearance of horizontally transferred CRISPR-targeted plasmids by enhancing spacer acquisition via interference-driven adaptation. This divergence, likely a result of genome evolution, suggests that adaptive immunity is optimized with intermediate transcription levels of cas genes by triggering positive feedback between adaptation and interference. Collectively, our findings highlight the crucial role of LrhA in fine-tuning host defense responses.},
}

*Escherichia coli/genetics/immunology/virology
*CRISPR-Cas Systems/genetics
*Escherichia coli Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Promoter Regions, Genetic
*Transcription Factors/genetics/metabolism
Operon
Plasmids/genetics
Trans-Activators/genetics

@article {pmid41796733,
year = {2026},
author = {Lin, J and Wang, Y and Zeng, B and Chen, Z and Lin, X and Zeng, T},
title = {CRISPR-Cas12a/Cas13a in cancer molecular diagnosis.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {120934},
doi = {10.1016/j.cca.2026.120934},
pmid = {41796733},
issn = {1873-3492},
abstract = {Cancer remains a leading cause of global mortality, with early diagnosis being pivotal for improving treatment outcomes. Traditional tissue biopsy is limited by its invasiveness, inability to capture tumor heterogeneity, and failure to support dynamic monitoring. Liquid biopsy has emerged as a non-invasive alternative, enabling the analysis of circulating tumor biomarkers (e.g., ctDNA, miRNAs, exosomes) in bodily fluids. However, current liquid biopsy technologies (e.g., NGS, ddPCR) suffer from high costs, complex workflows, poor standardization, and insufficient sensitivity for low-abundance biomarkers. The CRISPR-Cas systems, particularly Cas12a and Cas13a, have revolutionized molecular diagnostics due to their programmable sequence recognition, robust signal amplification via trans-cleavage/collateral cleavage activity, and compatibility with point-of-care testing (POCT). Cas12a targets DNA molecules, enabling sensitive detection of gene mutations and DNA methylation, while Cas13a specifically recognizes RNA, facilitating direct analysis of miRNAs and viral RNAs. Additionally, these systems have been extended to non-nucleic acid biomarkers (e.g., proteins, exosomes) through signal conversion strategies. This review summarizes the latest advances in CRISPR-Cas12a/Cas13a-based biosensors for cancer molecular diagnosis, including the detection of gene mutations, epigenetic modifications, miRNAs, tumor-associated viruses, and non-nucleic acid biomarkers. We critically analyze current challenges (e.g., PAM dependence, matrix interference, multiplexing limitations, clinical validation gaps) and discuss future perspectives, such as engineering PAM-less Cas variants, integrating nanotechnology, microfluidics, and artificial intelligence/artificial intelligence (AI), and advancing clinical standardization. This review aims to provide a comprehensive reference for the development and clinical translation of CRISPR-based cancer diagnostic technologies.},
}

@article {pmid41795439,
year = {2026},
author = {Zhu, L and Yang, C and Bernards, R and Wang, C},
title = {CLIM-TIME links genetic cancer drivers to immune landscapes.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1263-1265},
doi = {10.1016/j.cell.2026.01.014},
pmid = {41795439},
issn = {1097-4172},
mesh = {Humans ; *Neoplasms/genetics/immunology/therapy ; Immunotherapy ; Tumor Microenvironment/immunology/genetics ; Animals ; CRISPR-Cas Systems ; Genes, Tumor Suppressor ; },
abstract = {Immunotherapy resistance is associated with immune-privileged microenvironments, yet the interacting role of tumor-intrinsic genetics remains unclear. In this issue of Cell, Wang et al. introduce CLIM-TIME, a spatially resolved in vivo CRISPR screening platform linking loss of tumor suppressor genes to distinct metastatic immune architectures and divergent responses to immunotherapy.},
}

Humans
*Neoplasms/genetics/immunology/therapy
Immunotherapy
Tumor Microenvironment/immunology/genetics
Animals
CRISPR-Cas Systems
Genes, Tumor Suppressor

@article {pmid41794473,
year = {2026},
author = {Safenkova, IV and Kamionskaya, MV and Serchenya, TS and Sviridov, OV and Dzantiev, BB and Zherdev, AV},
title = {CRISPR/Cas12a and fork-shaped probe enhance LAMP-LFT integration for equipment-free detection of Listeria monocytogenes.},
journal = {Food research international (Ottawa, Ont.)},
volume = {230},
number = {},
pages = {118592},
doi = {10.1016/j.foodres.2026.118592},
pmid = {41794473},
issn = {1873-7145},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Listeria monocytogenes/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Food Microbiology/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Limit of Detection ; DNA, Bacterial/genetics ; Food Contamination/analysis ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Listeria monocytogenes is an important foodborne pathogen associated with high mortality rates, especially among vulnerable populations, and therefore requires diagnostic methods that are not only highly sensitive and rapid but also suitable for use in resource-limited settings. In this study, we developed an isothermal amplification assay integrated with a lateral flow test (LFT) for reliable detection of L.monocytogenes. Two assay formats were designed and compared: (1) loop-mediated isothermal amplification (LAMP) with LFT detection of fluorescein- and biotin-labeled amplicons, and (2) LAMP combined with CRISPR/Cas12a, using LFT to detect a cleaved fork-shaped enhanced probe labeled with three fluoresceins. Both LFT formats utilized a common conjugate of gold nanoparticles and anti-fluorescein antibodies (anti-FAM), but differed in the test zone immobilization strategy: streptavidin for LAMP, and anti-FAM for LAMP-CRISPR/Cas12a. Among 12 tested (primer - label) combinations, the most effective was identified, but the sensitivity of the LAMP-LFT format was limited by high signal variability. In contrast, the LAMP-CRISPR/Cas12a assay, targeting LAMP amplicons with guide RNA, achieved a detection limit of 0.9 copies/reaction-representing > 20,000-fold improvement in detectable DNA concentration compared with LAMP-LFT-and comparable to fluorescence-based detection techniques. The LAMP-CRISPR/Cas12a-LFT assay was first reported to detect L.monocytogenes cells following thermal lysis (10 min at 95 °C), with a single-cell detection limit (0.2 cells/reaction in buffer, 1 cells/reaction in spiked milk) and an analysis time of 80 min. These results demonstrate the potential of the approach for sensitive, equipment-free detection of foodborne pathogens in complex food matrices.},
}

*Nucleic Acid Amplification Techniques/methods
*Listeria monocytogenes/isolation & purification/genetics
*CRISPR-Cas Systems
*Food Microbiology/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Limit of Detection
DNA, Bacterial/genetics
Food Contamination/analysis
Gold/chemistry
Metal Nanoparticles/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41793913,
year = {2026},
author = {Li, X and Liu, L and Luo, C and Chen, Z and Shu, B},
title = {Efficient CRISPR/Cas9 system established via co-cultivation of plantlets and Agrobacterium tumefaciens for positive transgenic calluses generation and regeneration in cultivated strawberry (Fragaria × ananassa).},
journal = {Plant physiology and biochemistry : PPB},
volume = {232},
number = {},
pages = {111195},
doi = {10.1016/j.plaphy.2026.111195},
pmid = {41793913},
issn = {1873-2690},
abstract = {Recently, an Agrobacterium-mediated CRISPR/Cas9 editing system was successfully applied in a gene function analysis, highlighting its great value for improving strawberry genetics. However, the resulting low transformation rates and long regeneration cycles have limited its extensive application. Based on the biological characteristics of crown branching, an Agrobacterium tumefaciens-mediated CRISPR/Cas9 gene editing system was developed to increase the transformation rate and decrease the regeneration time of cultivated strawberry. Two single guide (sg)RNAs were designed for the strawberry anthracnose-related transcription factor, WRKY (FxaC_17g55530), and its alleles. These sgRNAs were inserted into pKSE401G using pCBC-DT1T2; sgRNAs for subtilisin-like protease (FxaC_22g21540) were designed and cloned in a similar manner. After 10 days of co-cultivating plantlets (without media supply of carbon) and GV3101, 65 (61.9%) and 72 (68.6%) GFP-positive calluses for the two genes were respectively obtained from the crown of 105 plantlets. The positive calluses were removed from the crown and placed on Murashige and Skoog media containing 3 mg/L thidiazuron and 0.2 mg/L indole-3-butyric acid. After 50-80 days, 3-5 positive shoots were obtained from different positive calluses for each gene. The three T0 lines for FxaC_17g55530 and FxaC_22g21540 were found to be successfully edited at the target sites of both sgRNA1 and sgRNA2 or either sgRNA1 or sgRNA2. Overall, a quick and effective CRISPR-Cas 9 gene editing system was developed for cultivated strawberry, highlighting the applicability of gene editing in breeding and gene function analysis.},
}

@article {pmid41791397,
year = {2026},
author = {Low, SJ and O'Neill, MT and Fernando, JA and Kerry, WJ and Prestedge, J and Wild, N and Chahal, S and Pollock, GL and Papadakis, G and Krysiak, M and Williams, E and Azzato, F and Tran, T and Fairley, C and Bradshaw, C and Chen, MY and Lim, CK and Williamson, DA and Pasricha, S},
title = {CRISPR-Cas-based diagnostics for point-of-care detection of sexually transmitted infections: a laboratory development and evaluation study.},
journal = {The Lancet. Microbe},
volume = {},
number = {},
pages = {101289},
doi = {10.1016/j.lanmic.2025.101289},
pmid = {41791397},
issn = {2666-5247},
abstract = {BACKGROUND: Timely, point-of-care diagnosis of sexually transmitted infections (STIs) is crucial for enabling prompt treatment and reducing transmission. We aimed to develop a portable, multiplexed, CRISPR-based assay panel for the detection of Neisseria gonorrhoeae (including the ciprofloxacin resistance marker gyrA S91F), Chlamydia trachomatis, Treponema pallidum, and herpes simplex virus (HSV).

METHODS: In this laboratory development and evaluation study, we developed and optimised four multiplexed, CRISPR-based, diagnostic STI assays for point-of-care use. The complete assay panel comprised a CRISPR TP-HSV (cTP-HSV) panel for the detection of T pallidum and pan-HSV, with reflex testing to distinguish HSV-1 from HSV-2, and a CRISPR NG-CT (cNG-CT) panel for the detection of N gonorrhoeae and C trachomatis, with reflex testing to detect N gonorrhoeae using two additional genome regions and to identify the gyrA S91F mutation. Each pathogen was targeted at two independent genomic regions by isothermal amplification and CRISPR-Cas reaction using Cas12a and Cas13a, each with distinct fluorescent reporters. Analytical specificity and limits of detection (LODs) were determined, and a retrospective, masked concordance study was conducted on genomic DNA from 900 clinical samples (400 for cTP-HSV and reflex testing and 500 for cNG-CT and reflex testing), using quantitative PCR as the reference standard. The diagnostic accuracy of the test was assessed by analysis of receiver operating characteristic curves.

FINDINGS: The overall sensitivity of the TP-HSV CRISPR assay was 82·5% (95% CI 74·0-88·7) for T pallidum and 94·4% (90·2-97·0) for pan-HSV; LODs were 6·2 copies per μL for T pallidum and 7·8 copies per μL for HSV. Reflex testing gave sensitivities of 97·0% (91·1-99·3) for HSV-1 and 96·0% (89·7-98·7) for HSV-2. The NG-CT CRISPR assay had an overall sensitivity of 80·0% (74·0-84·9) for N gonorrhoeae and 73·0% (65·5-79·3) for C trachomatis, with a LOD of 3·9 copies per μL for both pathogens. Reflex testing for the detection of the gyrA S91F mutation in N gonorrhoeae showed an overall sensitivity of 63·1% (55·1-70·4); however, this was dependent on sample type, with a sensitivity of 85·7% (46·7-99·5) in genital samples and 61·2% (52·8-68·9) in extragenital samples. For all pathogens, assay sensitivity was positively correlated with pathogen load. Area under the curve (AUC) values were 0·90 for T pallidum and 0·99 for pan-HSV in the TP-HSV assay, with values of 0·99 for HSV-1 and 0·97 for HSV-2 obtained in the reflex HSV-1-HSV-2 assay. For the cNG-CT assay, AUC values were 0·90 for N gonorrhoeae and 0·85 for C trachomatis, with a value of 0·72 obtained for gyrA S91F in the reflex cNG-gyrA assay.

INTERPRETATION: Our multiplexed, CRISPR-based, point-of-care platform achieved performance consistent with WHO target product profiles for N gonorrhoeae and T pallidum. Proof-of-concept detection of the gyrA S91F resistance marker highlights its potential for resistance-guided therapy. Although optimisation is required before large-scale deployment, this suite offers a promising approach for rapid, decentralised, and resistance-informed STI diagnosis, particularly in resource-limited settings.

FUNDING: Victorian Government Department of Health, Australian Government Department of Health, Disability and Ageing and Aged Care, and Australian Research Council.},
}

@article {pmid41789142,
year = {2026},
author = {Xu, C and Zeng, C and Wang, M and Wei, X and Song, M and Liu, X and Wang, W and Chen, Q and Ji, X and Luo, P and Ma, L and Sun, Y and Gou, H and Zhu, Z and Li, X and Lv, YX and Liu, P and Zhu, JK},
title = {mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo.},
journal = {Innovation (Cambridge (Mass.))},
volume = {7},
number = {3},
pages = {101151},
pmid = {41789142},
issn = {2666-6758},
abstract = {Programmable epigenetic editors (EEs) that achieve long-term gene expression modulation without altering the DNA sequence hold immense therapeutic potential. However, the clinical translation of current CRISPR-based epigenome editors is impeded by substantial challenges, particularly their large molecular size, which limits efficient in vivo delivery. Here, we report the rational design and engineering of compact, mRNA-delivered EEs (CRISPR OFF-EE) using Streptococcus pyogenes Cas9 (SpCas9), intein-split-SpCas9, or the smaller Cas-SF01 (a Cas12i3 variant). Combined with optimized mRNA architecture and lipid nanoparticle (LNP) delivery, a single intravenous LNP administration of the optimized OFF-EE V2 mRNA, along with selected guide RNAs (gRNAs) targeting Pcsk9 in mice, resulted in an ∼83.2% reduction in circulating PCSK9 levels and a corresponding ∼51.4% reduction in low-density lipoprotein cholesterol (LDL-C) levels, persisting for at least 180 days. SF01-based EEs showed higher specificity with fewer off-target methylation events than SpCas9-based counterparts. Our optimized LNP formulation also demonstrated a favorable safety profile with predominantly liver-tropic activity. These findings establish a robust and versatile platform for advancing in vivo therapeutics based on precise and durable epigenetic silencing using transiently delivered, engineered mRNA editors.},
}

@article {pmid41785880,
year = {2026},
author = {Petersen, AØ and Damholt, B and Grove, M and Hink, J and Marotte-Hurbon, T and Söderqvist, J and Troy, A and Zdravkovic, M and Bayer, L and Brunner, K and Bryde, T and Clube, J and Gencay, YE and Gram, A and Haaber, JK and Hallström, B and Jasinskytė, D and Pascal, R and Petersen, M and Semsey, S and Torio, AS and Turcu, IC and Smrekar, F and Taur, Y and Satlin, MJ and Sommer, MOA and van der Helm, E and Grøndahl, C},
title = {Safety, recovery, and pharmacodynamics of CRISPR-Cas therapeutic SNIPR001: a phase 1, randomised, double-blind, first-in-human, dose-escalation study.},
journal = {The Lancet. Microbe},
volume = {},
number = {},
pages = {101257},
doi = {10.1016/j.lanmic.2025.101257},
pmid = {41785880},
issn = {2666-5247},
abstract = {BACKGROUND: Patients with haematological cancer who receive stem-cell transplantation are at risk of bloodstream infections, often caused by multidrug resistant gut pathogens such as Escherichia coli. SNIPR001 is a cocktail of four CRISPR-Cas-armed bacteriophages that reduce colonisation of E coli in the gastrointestinal tract in animal models and is designed to not affect other members of the commensal microbiota. We aimed to investigate the safety and tolerability of SNIPR001 in healthy participants.

METHODS: In this randomised, placebo-controlled, double-blind, first-in-human, dose-escalation trial conducted at a single centre (Medpace Clinical Pharmacology Unit; Cincinnati, OH, USA), we sequentially enrolled healthy participants (aged 18-65 years) with more than 10[7]E coli colony-forming units per gram of stool into cohorts 1, 2, and 3, pending a safety review of the previous enrolment group where applicable. Participants in each cohort were randomly assigned to treatment or placebo using a unique three-digit participant identification number. Participants were orally administered 10[8] plaque-forming units (PFU) per dose (cohort 1), 10[10] PFU per dose (cohort 2), and 10[12] PFU per dose (cohort 3) of SNIPR001 or placebo (phosphate-buffered saline buffer), twice daily for 7 days. All personnel, except for a pharmacy staff member who prepared both SNIPR001 and placebo vials, were masked to the administered dose and assignment; masking was ensured by fully covering the surface of each vial. Participants were followed up to day 187. The primary outcome was the incidence and severity of adverse events and medically attended adverse events from the first administration of the study drug until 4 weeks after the last dose administration on day 35 of the study. Recovery and biodistribution of SNIPR001 in faeces, blood, and urine; pharmacodynamics, including the ability of SNIPR001 to reduce E coli levels in stool (assessed using a linear mixed-effects model); and microbiome composition (using Bray-Curtis dissimilarity) were secondary outcomes. Primary safety analyses were assessed per-protocol (ie, all enrolled participants who received at least one administration of the study drug). This trial was conducted under an Investigational New Drug application from the US Food and Drug Administration, is registered with ClinicalTrials.gov (NCT05277350), and is closed to new participants.

FINDINGS: The trial was carried out between March 24, 2022, and Nov 30, 2022. 36 eligible participants were randomly assigned to receive SNIPR001 or placebo in cohorts 1 (six assigned to 10[8] PFU per dose and two assigned to placebo), 2 (six to 10[10] PFU per dose and two to placebo), and 3 (12 to 10[12] PFU per dose and eight to placebo). The mean age of participants was 42·1 years (SD 13·8), with 14 (39%) female participants and 22 (61%) male participants. During the trial and 4-week follow-up period, only mild and moderate adverse events were observed, with most adverse events occurring in the placebo group (13, six, one, and nine for participants receiving either placebo or SNIPR001 at 10[8], 10[10], and 10[12] PFU twice a day, respectively). The number of participants who had adverse events was not significantly higher in treatment groups than in the placebo group (p=0·94, one-sided Fisher's exact test). The most frequently reported adverse events were headaches and diarrhoea. No grade 3-4 adverse events were reported and no serious adverse events were reported in the SNIPR001 dose groups. During and after the dosing period, the gut microbiota composition did not significantly differ between the treatment and placebo groups (p>0·05, two-sided Mann-Whitney U test of Bray-Curtis distances, false discovery rate [FDR]-corrected). Functional SNIPR001 was recovered from stool samples in concentrations proportional to the administered dose but was not meaningfully detected in plasma (only one sample) or urine (only one sample). SNIPR001 was undetected in all samples 6 months after the last dosing, which is a favourable pharmacokinetic property and meets regulatory expectations. We observed the largest reduction in E coli levels compared with placebo 2 weeks after treatment initiation at day 14 (78%; -0·65 log10 [SE 0·64] for 10[12] PFU SNIPR001 twice-daily group), according to a linear mixed-effects model for the highest dose population; however, this change was not statistically significant (p=0·811, linear mixed-effects model, FDR-corrected).

INTERPRETATION: This first-in-human study of SNIPR001 supported its safety, tolerability, and restriction to the gastrointestinal tract, while not systemically disrupting the gut microbiome. These results justify further clinical development of SNIPR001 in an ongoing phase 1b/2a trial.

FUNDING: Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) and SNIPR Biome.},
}

@article {pmid41721525,
year = {2026},
author = {Chen, N and Gao, M and Bai, Y and Wang, M and Liu, M and Xiong, W and Mo, B},
title = {5'UTR Editing of the ribosomal protein UL3Z gene unveils its critical roles in pre-rRNA processing and global mRNA translation dynamics.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
doi = {10.1093/plphys/kiag073},
pmid = {41721525},
issn = {1532-2548},
support = {32470592//National Natural Science Foundation of China/ ; 32270595//National Natural Science Foundation of China/ ; 2023B001//SZU 2035 Excellence Research Program/ ; },
mesh = {*5' Untranslated Regions/genetics ; *Ribosomal Proteins/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *RNA Precursors/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Protein Biosynthesis/genetics ; RNA, Messenger/metabolism/genetics ; Gene Expression Regulation, Plant ; *RNA Processing, Post-Transcriptional/genetics ; Gene Editing ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {The EMBRYO DEFECTIVE 2207 (EMB2207) gene, encoding ribosomal protein UL3Z, is critical for embryonic development in Arabidopsis, with loss of function resulting in embryo lethality. Despite its importance, the role of UL3Z in the complicated protein translation machinery in plants remains poorly understood due to the lack of viable hypomorphic alleles. In this study, we utilized CRISPR/Cas9 to edit the 5' untranslated region (5'UTR) of UL3Z, generating 5 ul3z mutants with varying degrees of reduced expression levels of UL3Z proteins. The mutant with the lowest expression exhibited the most severe developmental defects. In contrast, null mutants of its paralog UL3Y displayed no observable phenotypes. Interestingly, expression of UL3Y driven by the UL3Z/EMB2207 promoter successfully rescued the phenotypes of ul3z, demonstrating that these 2 paralogous ribosomal proteins actually possess functionally interchangeable roles. GUS staining results showed that UL3Z was constitutively expressed in all examined tissues, while UL3Y was only appreciably expressed in specific tissues. Molecular analysis further revealed the accumulation of ribosomal RNA (rRNA) maturation intermediates and increased polysome levels in ul3z mutants, indicating compromised pre-rRNA processing and disturbed global mRNA translation. Interestingly, 3' ends of many rRNA precursors in ul3z had higher frequency of non-encoded tails compared with Col-0. This study demonstrates that CRISPR/Cas9-mediated 5'UTR editing is an effective tool for generating viable hypomorphic alleles of lethal genes and uncovers the critical roles of UL3Z/EMB2207 in pre-rRNA processing and the maintenance of appropriate mRNA translation on ribosomes, underscoring its importance in plant development.},
}

*5' Untranslated Regions/genetics
*Ribosomal Proteins/genetics/metabolism
*Arabidopsis/genetics/metabolism
*RNA Precursors/metabolism/genetics
*Arabidopsis Proteins/genetics/metabolism
*Protein Biosynthesis/genetics
RNA, Messenger/metabolism/genetics
Gene Expression Regulation, Plant
*RNA Processing, Post-Transcriptional/genetics
Gene Editing
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid41650957,
year = {2026},
author = {Shi, B and Li, J and Wang, X and Liu, D and Xiang, J and Wang, H and Xu, C and Zou, X and Wang, Z and Huang, T and Min, Q and Wang, K and Yang, Y and Li, J and Wang, B and Zhao, C and Pei, D},
title = {Generating high-quality porcine iPSCs with the new medium cocktail LACID.},
journal = {Stem cell reports},
volume = {21},
number = {3},
pages = {102790},
doi = {10.1016/j.stemcr.2026.102790},
pmid = {41650957},
issn = {2213-6711},
mesh = {Animals ; *Induced Pluripotent Stem Cells/cytology/metabolism/drug effects ; Swine ; Cellular Reprogramming ; *Culture Media/pharmacology/chemistry ; Blastocyst/cytology/metabolism ; *Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Nuclear Transfer Techniques ; Cells, Cultured ; Cell Differentiation ; },
abstract = {Pigs are important for disease model generation, xenotransplantation, and interspecies organogenesis. Porcine induced pluripotent stem cells (piPSCs) should enable these efforts, but have not been generated to meet the attributes, such as feeder-free culture, robust development potential, and blastocyst generation through nuclear transfer. We report an improved strategy to generate such piPSCs. We show that chemically defined medium 3 promotes the formation of epithelium-like colonies in porcine reprogramming, which allows further reprogramming under the new medium cocktail LACID. The resulting piPSCs have key features, including flat morphology with feeder-free culture, generating robust teratoma and blastoids, forming chimeric blastocysts, and readily edited with CRISPR-Cas9. Lastly, nuclear transfer with piPSCs can develop into blastocysts. Despite maintaining a primed pluripotent state, our results suggest that the newly established LACID piPSCs may be ideal for applications in regenerative medicine. This method may be further improved to generate naive or totipotent stem cells.},
}

Animals
*Induced Pluripotent Stem Cells/cytology/metabolism/drug effects
Swine
Cellular Reprogramming
*Culture Media/pharmacology/chemistry
Blastocyst/cytology/metabolism
*Cell Culture Techniques/methods
CRISPR-Cas Systems
Nuclear Transfer Techniques
Cells, Cultured
Cell Differentiation

@article {pmid41517973,
year = {2026},
author = {Jin, G and Li, H and Yu, H and Gu, Z and Cui, H and Chen, J and Li, X},
title = {Asp-2078-Gly mutation in ACCase confers quizalofop-p-ethyl resistance in Eleusine indica and establishment of a LAMP-CRISPR/Cas12a visual genotyping assay for the target mutation.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3374-3383},
doi = {10.1002/ps.70468},
pmid = {41517973},
issn = {1526-4998},
support = {//the Key Project in Soybean Bio breeding and Commercialization/ ; //the China Agriculture Research System/ ; 2022ZD04021//the Sci-Tech Innovation 2030 Agenda/ ; },
mesh = {*Herbicide Resistance/genetics ; *Acetyl-CoA Carboxylase/genetics/metabolism ; *Eleusine/genetics/drug effects/enzymology ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; *Herbicides/pharmacology ; Mutation ; *Genotyping Techniques/methods ; *Plant Proteins/genetics/metabolism ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Eleusine indica is a widespread, competitive weed causing yield losses in major crops. Repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in cotton fields has led to the evolution of resistant populations, posing a growing threat to cotton production in China. This study aimed to elucidate the target-site resistance mechanism of E. indica to quizalofop-p-ethyl and establish a rapid visual detection method based on the identified mutation.

RESULTS: The NJC-R population showed resistance to quizalofop-p-ethyl (resistance index = 5.5). Gene sequencing revealed that an Asp-2078-Gly mutation in ACCase was one of the mechanisms underlying resistance. Loop-mediated isothermal amplification (LAMP) combined with the CRISPR/Cas12a system was developed to detect this mutation in E. indica. This method not only enabled genotype discrimination (wild-type, heterozygous, homozygous mutant), but also provided visual results within 70 min, exhibiting superior performance compared with the derived cleaved amplified polymorphic sequences assay. In addition, this method eliminated false positives from nonspecific LAMP amplification, was ~100-fold more sensitive than a polymerase chain reaction, and the assay results were 100% concordant with Sanger sequencing for the 50 samples tested.

CONCLUSION: This study confirmed that the Asp-2078-Gly mutation confers quizalofop-p-ethyl resistance in E. indica from the cotton field in China, and LAMP-CRISPR/Cas12a was first applied for detecting ACCase target-site mutations in E. indica. Given its rapidity and high accuracy, this technique has the potential to be applied for resistance monitoring and to guide rational herbicide application. © 2026 Society of Chemical Industry.},
}

*Herbicide Resistance/genetics
*Acetyl-CoA Carboxylase/genetics/metabolism
*Eleusine/genetics/drug effects/enzymology
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
*Herbicides/pharmacology
Mutation
*Genotyping Techniques/methods
*Plant Proteins/genetics/metabolism
Molecular Diagnostic Techniques

@article {pmid41445368,
year = {2026},
author = {Wei, J and Jiang, C and Chen, Y and Yang, X and Li, Q},
title = {Functional characterization of Hsk1 and Chit1 genes in the virulence of Metarhizium guizhouense Xct1 via CRISPR-Cas9-mediated gene editing.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3625-3639},
doi = {10.1002/ps.70484},
pmid = {41445368},
issn = {1526-4998},
support = {//SCCXTD-2024-04/ ; },
mesh = {*CRISPR-Cas Systems ; Virulence/genetics ; Gene Editing ; Animals ; *Metarhizium/genetics/pathogenicity ; *Fungal Proteins/genetics/metabolism ; Spodoptera/growth & development/microbiology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Larva/growth & development/microbiology ; Pest Control, Biological ; },
abstract = {BACKGROUND: The entomopathogenic fungus Metarhizium guizhouense Xct1 exhibits high virulence against early-instar Spodoptera frugiperda larvae (>90% mortality in preliminary studies), yet the molecular mechanisms, particularly the roles of key genes such as the chitin-degrading enzyme (Chit1) and serine/threonine kinase (Hsk1) are poorly understood. Functional studies using CRISPR-Cas9 are lacking, limiting its biocontrol application.

RESULTS: Chit1 and Hsk1 genes were amplified from M. guizhouense Xct1. Chit1 showed high homology to M. anisopliae, whereas Hsk1 exhibited greater genetic diversity. Expression analysis revealed peak Chit1 expression on Day (D)4 and peak Hsk1 expression on D2. A CRISPR-Cas9 system was established, and knockout of Chit1 resulted in thickened cell walls [119 nm versus 87 nm in wild-type (WT)] and reduced virulence [median lethal time (LT50) = 7.4 days versus 4.8 days in WT]. Overexpression of Chit1 improved virulence (LT50 = 3.3 days). Hsk1 knockout was lethal, confirming its essential role, while overexpression did not alter virulence (LT50 = 4.8 days).

CONCLUSIONS: Chit1 is a critical virulence factor, influencing cell-wall integrity and insecticidal activity, while Hsk1 is essential for fungal viability. This study presents the first CRISPR-Cas9-mediated functional analysis of these genes, revealing that Chit1 overexpression enhances biocontrol efficacy against S. frugiperda. © 2025 Society of Chemical Industry.},
}

*CRISPR-Cas Systems
Virulence/genetics
Gene Editing
Animals
*Metarhizium/genetics/pathogenicity
*Fungal Proteins/genetics/metabolism
Spodoptera/growth & development/microbiology
*Protein Serine-Threonine Kinases/genetics/metabolism
Larva/growth & development/microbiology
Pest Control, Biological

@article {pmid41057363,
year = {2025},
author = {Li, J and Pan, Z and Peng, X and Feng, Y and Wu, J and Liang, F and Feng, Q and Yu, X and Deng, H},
title = {Ecdysone signaling-induced dumpless1 expression controls nurse cell dumping in Drosophila oogenesis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8917},
pmid = {41057363},
issn = {2041-1723},
support = {32170494//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Oogenesis/genetics/physiology ; *Ecdysone/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; Animals ; Signal Transduction ; *Drosophila melanogaster/genetics/metabolism ; rho GTP-Binding Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Ovarian Follicle/cytology/metabolism ; Integrin beta Chains/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Oocytes/cytology/metabolism ; Female ; },
abstract = {Nurse cell (NC) dumping, a process essential for oocyte development, involves the rapid cytoplasmic transfer from germline-derived NCs into the oocyte. However, its regulatory mechanism remains unclear. Here, we report that ecdysone signaling in stretch follicle cells (SFCs) regulates NC dumping through dumpless1, a ZAD-C2H2 zinc finger transcription factor, in Drosophila. Ecdysone induced dumpless1 expression in SFCs, and CRISPR/Cas9-mediated knockout of dumpless1 or its functional domain ZAD suppresses NC dumping. Depletion of dumpless1 upregulates integrin βPS expression in SFC plasma membrane, while reducing cortical enrichment of Rho1 signaling-dependent phosphorylated myosin light chain (p-MLC) and disrupting actin cables organization in NCs. SFC-specific overexpression of integrin βPS reduces p-MLC enrichment in the NC cortex, whereas its knockdown in SFCs of dumpless1[-/-] mutants partially rescues NC dumping defect. Our findings identify dumpless1 as a critical effector of ecdysone signaling, bridging somatic-germline communication through the integrin βPS-Rho1-p-MLC axis, revealing a multicellular regulatory mechanism in Drosophila oogenesis.},
}

*Oogenesis/genetics/physiology
*Ecdysone/genetics/metabolism
*Drosophila Proteins/genetics/metabolism
Animals
Signal Transduction
*Drosophila melanogaster/genetics/metabolism
rho GTP-Binding Proteins/genetics/metabolism
CRISPR-Cas Systems
Ovarian Follicle/cytology/metabolism
Integrin beta Chains/genetics/metabolism
Transcription Factors/genetics/metabolism
Oocytes/cytology/metabolism
Female

@article {pmid37766223,
year = {2023},
author = {Zhang, H and Duan, K and Du, Y and Xiao, S and Fang, L and Zhou, Y},
title = {One-Step Assembly of a PRRSV Infectious cDNA Clone and a Convenient CRISPR/Cas9-Based Gene-Editing Technology for Manipulation of PRRSV Genome.},
journal = {Viruses},
volume = {15},
number = {9},
pages = {},
pmid = {37766223},
issn = {1999-4915},
support = {32130103//National Natural Science Foundation of China/ ; 32002279//National Natural Science Foundation of China/ ; },
mesh = {*Porcine respiratory and reproductive syndrome virus/genetics ; Animals ; *CRISPR-Cas Systems ; *Genome, Viral ; *Gene Editing/methods ; Swine ; *DNA, Complementary/genetics ; Porcine Reproductive and Respiratory Syndrome/virology ; Reverse Genetics ; Cell Line ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes.},
}

*Porcine respiratory and reproductive syndrome virus/genetics
Animals
*CRISPR-Cas Systems
*Genome, Viral
*Gene Editing/methods
Swine
*DNA, Complementary/genetics
Porcine Reproductive and Respiratory Syndrome/virology
Reverse Genetics
Cell Line

@article {pmid37722405,
year = {2023},
author = {Low, SJ and O'Neill, MT and Kerry, WJ and Krysiak, M and Papadakis, G and Whitehead, LW and Savic, I and Prestedge, J and Williams, L and Cooney, JP and Tran, T and Lim, CK and Caly, L and Towns, JM and Bradshaw, CS and Fairley, C and Chow, EPF and Chen, MY and Pellegrini, M and Pasricha, S and Williamson, DA},
title = {Rapid detection of monkeypox virus using a CRISPR-Cas12a mediated assay: a laboratory validation and evaluation study.},
journal = {The Lancet. Microbe},
volume = {4},
number = {10},
pages = {e800-e810},
doi = {10.1016/S2666-5247(23)00148-9},
pmid = {37722405},
issn = {2666-5247},
mesh = {*CRISPR-Cas Systems ; Humans ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Monkeypox virus/isolation & purification/genetics ; *Molecular Diagnostic Techniques/methods ; *Mpox, Monkeypox/diagnosis/virology ; Limit of Detection ; Proof of Concept Study ; Point-of-Care Systems ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The 2022 outbreak of mpox (formerly known as monkeypox) led to the spread of monkeypox virus (MPXV) in over 110 countries, demanding effective disease management and surveillance. As current diagnostics rely largely on centralised laboratory testing, our objective was to develop a simple rapid point-of-care assay to detect MPXV in clinical samples using isothermal amplification coupled with CRISPR and CRISPR-associated protein (Cas) technology.

METHODS: In this proof-of-concept study, we developed a portable isothermal amplification CRISPR-Cas12a-based assay for the detection of MPXV. We designed a panel of 22 primer-guide RNA sets using pangenome and gene-agnostic approaches, and subsequently shortlisted the three sets producing the strongest signals for evaluation of analytical sensitivity and specificity using a fluorescence-based readout. The set displaying 100% specificity and the lowest limit of detection (LOD) was selected for further assay validation using both a fluorescence-based and lateral-flow readout. Assay specificity was confirmed using a panel of viral and bacterial pathogens. Finally, we did a blind concordance study on genomic DNA extracted from 185 clinical samples, comparing assay results with a gold-standard quantitative PCR (qPCR) assay. We identified the optimal time to detection and analysed the performance of the assay relative to qPCR using receiver operating characteristic (ROC) curves. We also assessed the compatibility with lateral-flow strips, both visually and computationally, where strips were interpreted blinded to the fluorescence results on the basis of the presence or absence of test bands.

FINDINGS: With an optimal run duration of approximately 45 min from isothermal amplification to CRISPR-assay readout, the MPXV recombinase polymerase amplification CRISPR-Cas12a-based assay with the selected primer-guide set had an LOD of 1 copy per μL and 100% specificity against tested viral pathogens. Blinded concordance testing of 185 clinical samples resulted in 100% sensitivity (95% CI 89·3-100) and 99·3% specificity (95% CI 95·7-100) using the fluorescence readout. For optimal time to detection by fluorescence readout, we estimated the areas under the ROC curve to be 0·98 at 2 min and 0·99 at 4 min. Lateral-flow strips had 100% sensitivity (89·3-100) and 98·6% specificity (94·7-100) with both visual and computational assessment. Overall, lateral-flow results were highly concordant with fluorescence-based readouts (179 of 185 tests, 96·8% concordant), with discrepancies associated with low viral load samples.

INTERPRETATION: Our assay for the diagnosis of mpox displayed good performance characteristics compared with qPCR. Although optimisation of the assay will be required before deployment, its usability and versatility present a potential solution to MPXV detection in low-resource and remote settings, as well as a means of community-based, on-site testing.

FUNDING: Victorian Medical Research Accelerator Fund and the Australian Government Department of Health.},
}

*CRISPR-Cas Systems
Humans
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*Monkeypox virus/isolation & purification/genetics
*Molecular Diagnostic Techniques/methods
*Mpox, Monkeypox/diagnosis/virology
Limit of Detection
Proof of Concept Study
Point-of-Care Systems
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid37721041,
year = {2023},
author = {Gentili, HG and Pignataro, MF and Olmos, J and Pavan, MF and Ibañez, LI and Santos, J and Velazquez Duarte, F},
title = {CRISPR/Cas9-based edition of frataxin gene in Dictyostelium discoideum.},
journal = {The Biochemical journal},
volume = {480},
number = {19},
pages = {1533-1551},
doi = {10.1042/BCJ20230244},
pmid = {37721041},
issn = {1470-8728},
mesh = {*Dictyostelium/genetics/metabolism/growth & development ; Frataxin ; *Iron-Binding Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; *Protozoan Proteins/genetics/metabolism ; Mutation ; },
abstract = {In this paper, we describe the development of a Dictyostelium discoideum strain deficient in frataxin protein (FXN). We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the D. discoideum fxn locus and isolated a defective mutant, clone 8, which presents landmarks of frataxin deficiency, such as a decrease in Fe-S cluster-dependent enzymatic functions, growth rate reduction, and increased sensitivity to oxidative stress. In addition, the multicellular development is affected as well as growing on bacterial lawn. We also assessed the rescuing capacity of DdFXN-G122V, a version that mimics a human variant present in some FA patients. While the expression of DdFXN-G122V rescues growth and enzymatic activity defects, as DdFXN does, multicellular development defects were only partially rescued. The results of the study suggest that this new D. discoideum strain offers a wide range of possibilities to easily explore diverse FA FXN variants. This can facilitate the development of straightforward drug screenings to look for new therapeutic strategies.},
}

*Dictyostelium/genetics/metabolism/growth & development
Frataxin
*Iron-Binding Proteins/genetics/metabolism
*CRISPR-Cas Systems
Humans
*Gene Editing/methods
*Protozoan Proteins/genetics/metabolism
Mutation

@article {pmid37718190,
year = {2023},
author = {Kalinna, BH},
title = {Programming schistosomes - a crisper approach to transgenesis.},
journal = {Trends in parasitology},
volume = {39},
number = {11},
pages = {896-897},
doi = {10.1016/j.pt.2023.09.003},
pmid = {37718190},
issn = {1471-5007},
mesh = {Animals ; *Schistosoma mansoni/genetics ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; *Schistosoma/genetics ; Transgenes ; },
abstract = {Ittiprasert and colleagues identified genomic safe harbour (GSH) sites in Schistosoma mansoni using computational methods and inserted a transgene into one of the sites through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-assisted homology-directed repair. This study outlines a promising strategy for functional genomics to study this parasite that causes a debilitating and neglected tropical disease.},
}

Animals
*Schistosoma mansoni/genetics
*Gene Transfer Techniques
CRISPR-Cas Systems
*Schistosoma/genetics
Transgenes

@article {pmid37702151,
year = {2023},
author = {van Schie, JJM and de Lint, K and Molenaar, TM and Moronta Gines, M and Balk, JA and Rooimans, MA and Roohollahi, K and Pai, GM and Borghuis, L and Ramadhin, AR and Corazza, F and Dorsman, JC and Wendt, KS and Wolthuis, RMF and de Lange, J},
title = {CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin.},
journal = {Nucleic acids research},
volume = {51},
number = {18},
pages = {9594-9609},
pmid = {37702151},
issn = {1362-4962},
support = {10701//Dutch Cancer Society/ ; },
mesh = {Cohesins ; Humans ; *Cell Cycle Proteins/metabolism/genetics ; *Chromosomal Proteins, Non-Histone/metabolism/genetics ; *Chromatin/metabolism/genetics ; *Chromatids/genetics/metabolism ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/metabolism ; Mitosis/genetics ; DNA Replication/genetics ; Nuclear Proteins/genetics/metabolism ; Sister Chromatid Exchange/genetics ; Cell Line ; Acetyltransferases ; DNA Helicases ; DEAD-box RNA Helicases ; },
abstract = {The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.},
}

Cohesins
Humans
*Cell Cycle Proteins/metabolism/genetics
*Chromosomal Proteins, Non-Histone/metabolism/genetics
*Chromatin/metabolism/genetics
*Chromatids/genetics/metabolism
CRISPR-Cas Systems
DNA-Binding Proteins/genetics/metabolism
Mitosis/genetics
DNA Replication/genetics
Nuclear Proteins/genetics/metabolism
Sister Chromatid Exchange/genetics
Cell Line
Acetyltransferases
DNA Helicases
DEAD-box RNA Helicases

@article {pmid37695673,
year = {2023},
author = {Zhang, H and Li, X and Wang, Y and Liu, X and Guo, J and Wang, Z and Zhang, L and Xiong, S and Dong, C},
title = {Genome-Wide CRISPR/Cas9 Screening Identifies That Mitochondrial Solute Carrier SLC25A23 Attenuates Type I IFN Antiviral Immunity via Interfering with MAVS Aggregation.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {211},
number = {9},
pages = {1406-1417},
doi = {10.4049/jimmunol.2300187},
pmid = {37695673},
issn = {1550-6606},
mesh = {Animals ; Mice ; *Interferon Type I/immunology/metabolism ; CRISPR-Cas Systems/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/immunology/genetics ; Humans ; Ubiquitin-Protein Ligases/metabolism ; *RNA Virus Infections/immunology ; Signal Transduction ; Tripartite Motif Proteins/metabolism ; Ubiquitination ; Immunity, Innate ; HEK293 Cells ; Mice, Inbred C57BL ; *Mitochondrial Membrane Transport Proteins/genetics/metabolism ; Mitochondria/metabolism ; },
abstract = {Activation of the mitochondrial antiviral signaling (MAVS) adaptor, also known as IPS-1, VISA, or Cardif, is crucial for antiviral immunity in retinoic acid-inducible gene I (RIG-I)-like receptor signaling. Upon interacting with RIG-I, MAVS undergoes K63-linked polyubiquitination by the E3 ligase Trim31, and subsequently aggregates to activate downstream signaling effectors. However, the molecular mechanisms that modulate MAVS activation are not yet fully understood. In this study, the mitochondrial solute carrier SLC25A23 was found to attenuate type I IFN antiviral immunity using genome-wide CRISPR/Cas9 screening. SLC25A23 interacts with Trim31, interfering with its binding of Trim31 to MAVS. Indeed, SLC25A23 downregulation was found to increase K63-linked polyubiquitination and subsequent aggregation of MAVS, which promoted type I IFN production upon RNA virus infection. Consistently, mice with SLC25A23 knockdown were more resistant to RNA virus infection in vivo. These findings establish SLC25A23 as a novel regulator of MAVS posttranslational modifications and of type I antiviral immunity.},
}

Animals
Mice
*Interferon Type I/immunology/metabolism
CRISPR-Cas Systems/genetics
*Adaptor Proteins, Signal Transducing/metabolism/immunology/genetics
Humans
Ubiquitin-Protein Ligases/metabolism
*RNA Virus Infections/immunology
Signal Transduction
Tripartite Motif Proteins/metabolism
Ubiquitination
Immunity, Innate
HEK293 Cells
Mice, Inbred C57BL
*Mitochondrial Membrane Transport Proteins/genetics/metabolism
Mitochondria/metabolism

@article {pmid37689217,
year = {2023},
author = {Colaco, JC and Chandrasekaran, AP and Karapurkar, JK and Gowda, DAA and Birappa, G and Rajkumar, S and Suresh, B and Ko, N and Hong, SH and Oh, SJ and Kim, KS and Ramakrishna, S},
title = {E3 ubiquitin ligase APC/C[Cdh1] regulates SLC35F2 protein turnover and inhibits cancer progression in HeLa cells.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1867},
number = {11},
pages = {130454},
doi = {10.1016/j.bbagen.2023.130454},
pmid = {37689217},
issn = {1872-8006},
mesh = {Humans ; HeLa Cells ; Ubiquitination ; *Ubiquitin-Protein Ligases/metabolism/genetics ; Disease Progression ; *Neoplasms/metabolism/pathology/genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Adenomatous Polyposis Coli Protein ; },
abstract = {BACKGROUND: The solute carrier family 35 F2 (SLC35F2), belongs to membrane-bound carrier proteins that control various physiological functions and are activated in several cancers. However, the molecular mechanism regulating SLC35F2 protein turnover and its implication in cancer progression remains unexplored. Therefore, screening for E3 ligases that promote SLC35F2 protein degradation is essential during cancer progression.

METHODS: The immunoprecipitation and Duolink proximity ligation assays (PLA) were used to determine the interaction between APC/C[Cdh1] and SLC35F2 proteins. A CRISPR/Cas9-mediated knockdown and rescue experiment were used to validate the functional significance of APC/C[Cdh1] on SLC35F2 protein stabilization. The ubiquitination function of APC/C[Cdh1] on SLC35F2 protein was validated using in vitro ubiquitination assay and half-life analysis. The role of APC/C[Cdh1] regulating SLC35F2-mediated tumorigenesis was confirmed by in vitro oncogenic experiments in HeLa cells.

RESULTS: Based on the E3 ligase screen and in vitro biochemical experiments, we identified that APC/C[Cdh1] interacts with and reduces SLC35F2 protein level. APC/C[Cdh1] promotes SLC35F2 ubiquitination and decreases the half-life of SLC35F2 protein. On the other hand, the CRISPR/Cas9-mediated depletion of APC/C[Cdh1] increased SLC35F2 protein levels. The mRNA expression analysis revealed a negative correlation between APC/C[Cdh1] and SLC35F2 across a panel of cancer cell lines tested. Additionally, we demonstrated that depletion in APC/C[Cdh1] promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells.

CONCLUSION: Our study highlights that APC/C[Cdh1] is a critical regulator of SLC35F2 protein turnover and depletion of APC/C[Cdh1] promotes SLC35F2-mediated tumorigenesis. Thus, we envision that APC/C[Cdh1]-SLC35F2 axis might be a therapeutic target in cancer.},
}

Humans
HeLa Cells
Ubiquitination
*Ubiquitin-Protein Ligases/metabolism/genetics
Disease Progression
*Neoplasms/metabolism/pathology/genetics
CRISPR-Cas Systems
Cell Proliferation
Adenomatous Polyposis Coli Protein

@article {pmid37688599,
year = {2023},
author = {Minnaar, L and den Haan, R},
title = {Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks.},
journal = {Applied microbiology and biotechnology},
volume = {107},
number = {22},
pages = {7013-7028},
pmid = {37688599},
issn = {1432-0614},
support = {137967//National Research Foundation/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism/isolation & purification/growth & development ; *Cellulose/metabolism ; Ethanol/metabolism ; Fermentation ; *Cellulases/genetics/metabolism ; CRISPR-Cas Systems ; *Metabolic Engineering ; },
abstract = {Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4-4.5 g/L, representing 35-40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production. KEY POINTS: • Process-related fermentation stresses influence heterologous protein production. • Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP. • CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.},
}

*Saccharomyces cerevisiae/genetics/metabolism/isolation & purification/growth & development
*Cellulose/metabolism
Ethanol/metabolism
Fermentation
*Cellulases/genetics/metabolism
CRISPR-Cas Systems
*Metabolic Engineering

@article {pmid37683439,
year = {2024},
author = {Liu, B and Li, Y and Du, L and Zhang, F and Liu, Y and Sun, J and Zhang, Q and Li, C and Li, X and Xue, Q},
title = {"One-to-many" signal-output strategy-based CRISPR/Cas12a system for sensitive label-free fluorescence detection of HBV-DNA.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {304},
number = {},
pages = {123338},
doi = {10.1016/j.saa.2023.123338},
pmid = {37683439},
issn = {1873-3557},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA, Viral/analysis/genetics ; *Hepatitis B virus/genetics ; *Biosensing Techniques/methods ; Spectrometry, Fluorescence/methods ; Limit of Detection ; Dendrimers/chemistry ; CRISPR-Associated Proteins/metabolism ; Fluorescence ; Nucleic Acid Amplification Techniques ; Humans ; Benzothiazoles ; },
abstract = {Although CRISPR/Cas12a systems significantly enhance the analytical accuracy and flexibility of fluorescent biosensors, their sensitivity is limited by traditional "one-to-one" mediation types and ineffective signal-output turnover routes. Herein, we demonstrate a "one-to-many" signal-output strategy-based CRISPR/Cas12a systems resembling a "seaweed" to enhance the sensitivity. Based on dendrimer DNA from high-dimensional hybridization chain (HCR) of three hairpin-free DNA building blocks, the 3D magnetic DNA machine was created. The HBV-DNA initiates the rolling circle amplification (RCA) reaction and produces DNA nanowires to activate the CRISPR/Cas12a system. The trans-cleavage of the "seaweed root" by CRISPR/Cas12a system left dendrimer DNA in solution, thus, adding SYBR Green I (SG I) to the high-density DNA duplexes, achieving multiple-turnover label-free fluorescence signal output demonstrated and a low LOD (1.502 pM). However, in the absence of target, the blocked RCA failed to activate the CRISPR/Cas12a system, resulting in complete separation from substrate and negligible fluorescence signals. Moreover, the mandatory RCA-based pre-amplification of the DNA activator could efficiently trigger the multiple-turnover trans-cleavage activity of Cas12a. it can cleave one single-stranded linker of "seaweed-like" DNA machine, thereby releasing massive DNA duplex-enriched dendrimer DNA with a "one-to-many" signal-output turnover. By coupling the periodically extended Cas12a activator generated by RCA with hyperbranched DNA duplex by high-dimensional HCR, compact 3D extension structures were formed, achieving high-density fluorescence distribution in focal volume, avoiding signal dilution and ensuring high enhancement. Additionally, spiked recoveries in physiological media exceeded 95%, demonstrating the potential application of such platforms in clinical diagnosis.},
}

*CRISPR-Cas Systems/genetics
*DNA, Viral/analysis/genetics
*Hepatitis B virus/genetics
*Biosensing Techniques/methods
Spectrometry, Fluorescence/methods
Limit of Detection
Dendrimers/chemistry
CRISPR-Associated Proteins/metabolism
Fluorescence
Nucleic Acid Amplification Techniques
Humans
Benzothiazoles