@article {pmid41058711,
year = {2025},
author = {Barraclough, A and Bär, I and van Duijl, T and Fijnvandraat, K and Eikenboom, JCJ and Leebeek, FWG and Bierings, R and Voorberg, J and Trasanidou, D},
title = {Rewriting the script: gene therapy and genome editing for von Willebrand Disease.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1620438},
pmid = {41058711},
issn = {2673-3439},
abstract = {In recent years gene therapy has emerged as a powerful technology for treatment of a large variety of inherited disorders. With the FDA approval of in vivo gene therapy of hemophilia A and B using AAV-mediated transgene delivery to hepatocytes, the path towards a new treatment era seemed paved. Also, CRISPR-Cas based approaches have reached the clinic, as in the ex vivo treatment of hematopoietic stem cells for sickle cell disease and thalassemia patients. The question arises whether these innovative strategies will also be suitable for patients with von Willebrand Disease (VWD). Whilst in and ex vivo delivery to endothelial cells (ECs) has been demonstrated, and CRISPR-Cas9 gene editing has been successful in ECs, there are currently no gene therapy options available for VWD. The wide variety of pathogenic VWF mutations makes development of broadly applicable, cost-effective gene therapies challenging. While delivery of von Willebrand factor (VWF) as a therapeutic transgene would be optimal, the size of VWF challenges efficient delivery. Therefore, treatment of VWD requires targeted, personalized gene therapy; for instance by using the newest CRISPR-Cas technologies which can be tailored to facilitate alteration and restoration of various pathogenic VWD variants. This review describes the inherited bleeding disorder VWD and potential gene therapy approaches for management of the disease. Thereby we are exploring different CRISPR-Cas technologies and recent developments in the field. Moreover, we will discuss the ongoing advances of in vivo delivery systems, all with the scope on ECs.},
}

@article {pmid41057395,
year = {2025},
author = {Osgood, JA and Brown, AC and Burnham, KL and Mielczarek, O and Migliorini, G and Tay, C and Zhang, P and Palmer, MH and Davies, B and Cowley, SA and Knight, JC},
title = {Evidence for enhancer activity in intron 1 of TNFRSF1A using CRISPR/Cas9 in human induced pluripotent stem cell-derived macrophages.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {34885},
pmid = {41057395},
issn = {2045-2322},
support = {20773/VAC_/Versus Arthritis/United Kingdom ; 204969/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 2018-I2M-2-002//Chinese Academy of Medical Sciences Innovation 537 Fund for Medical Science/ ; LC0910-004//Oxford Martin School, University of Oxford/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Macrophages/metabolism/cytology ; *Introns/genetics ; *Enhancer Elements, Genetic ; *Receptors, Tumor Necrosis Factor, Type I/genetics ; Gene Editing ; Cell Differentiation ; Genome-Wide Association Study ; Gene Expression Regulation ; },
abstract = {TNFα is a common drug target in the treatment of autoimmune diseases, with pro-inflammatory functions that are primarily mediated through its receptor, TNFRSF1A. TNFRSF1A has been genetically associated with many immune-mediated diseases including ankylosing spondylitis, multiple sclerosis, and inflammatory bowel disease. Many of the genetic variants within or near TNFRSF1A that have been associated with disease through genome-wide association studies (GWAS) lie in non-coding regions of the genome. Understanding the functional consequences of these genetic variants is limited by incomplete understanding of TNFRSF1A gene regulation, including for specific cellular contexts relevant to inflammation and immunity such as macrophages. This work used CRISPR/Cas9 in human induced pluripotent stem cells followed by differentiation into macrophages to investigate putative regulatory elements in the TNFRSF1A gene locus. Through gene editing, with functional genomic readouts including the assay for transposase-accessible chromatin using sequencing (ATAC-Seq), chromatin immunoprecipitation with sequencing (ChIP-Seq), and RNA-Seq to assess the consequences of these edits, we present evidence for an enhancer of TNFRSF1A contained within an intron of the gene. Understanding gene regulation and the genomic context in which GWAS variants lie could bring us closer to deconvoluting the genetic basis of common disease aetiology and uncover effective drug targets.},
}

Humans
*CRISPR-Cas Systems
*Induced Pluripotent Stem Cells/cytology/metabolism
*Macrophages/metabolism/cytology
*Introns/genetics
*Enhancer Elements, Genetic
*Receptors, Tumor Necrosis Factor, Type I/genetics
Gene Editing
Cell Differentiation
Genome-Wide Association Study
Gene Expression Regulation

@article {pmid41057263,
year = {2025},
author = {Yang, B and Wang, K},
title = {CRISPR-Cas-Directed Genome Editing in Maize.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.top108448},
pmid = {41057263},
issn = {1559-6095},
abstract = {Genetic engineering techniques are essential for both plant science and agricultural biotechnology, enabling functional genomics studies, dissection of complex traits, and targeted crop improvement. Among the various genetic tools currently in use, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based genome editing has emerged as a transformative technology due to its precision, versatility, and ease of use. In particular, CRISPR-Cas9 has become the most widely adopted platform for genome manipulation in plant systems, including maize, owing to its high editing efficiency, multiplexing capabilities, and scalability for diverse applications. This review highlights the biological significance and technical considerations necessary to implement CRISPR-Cas9 in maize. We discuss critical components for successful editing, including the selection of strong and tissue-appropriate promoters for Cas gene and guide RNA expression, codon optimization of Cas nuclease genes, effective guide RNA design, and multiplexing strategies using RNA polymerase III (Pol III)- or Pol II-dependent promoter-driven polycistronic expression systems. Additionally, we provide insights into vector construction methodologies and reliable genotyping techniques to detect and validate genome edits. Together, these elements constitute a practical framework for deploying genome editing in maize research and breeding. By optimizing these parameters, researchers can enhance the efficiency and accuracy of CRISPR-mediated genome modifications, accelerating functional genomic discovery and the development of improved maize varieties tailored to meet future agricultural demands.},
}

@article {pmid41056201,
year = {2025},
author = {Weber, VJ and Reschigna, A and Gerhardt, MJ and Heigl, T and Hinrichsmeyer, KS and van den Engel, S and Otify, DY and Gavrilov, Z and Blaser, F and Meneau, I and Betz, C and Bolz, HJ and Biel, M and Michalakis, S and Becirovic, E},
title = {CRISPR/Cas-mediated activation of genes associated with inherited retinal dystrophies in human cells for diagnostic purposes.},
journal = {JCI insight},
volume = {},
number = {},
pages = {},
doi = {10.1172/jci.insight.189615},
pmid = {41056201},
issn = {2379-3708},
abstract = {Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.},
}

@article {pmid41055650,
year = {2025},
author = {Che, X and Wei, Y and Wang, X and Wang, X and Wu, Z and Deng, J and Ge, S and Liu, X and Cai, Z and Zhang, H and He, L and Xu, J},
title = {Lipoxygenase ZmLOX3 Enhances Salt Tolerance of Maize Under the Regulation of ZmNAC032.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c02349},
pmid = {41055650},
issn = {1520-5118},
abstract = {Lipoxygenase (LOX) plays a critical role in plant biotic and abiotic stress responses by mediating lipid peroxidation and the production of jasmonic acid (JA). In this study, maize ZmLOX3 was identified as a positive regulator in salt stress tolerance. Overexpression of ZmLOX3 enhanced the salt tolerance of Arabidopsis. When maize seedlings were subjected to salt stress, the ZmLOX3[OE] lines exhibited a better growth phenotype than the control (B104) and the zmlox3[CR] (CRISPR/Cas) knockout mutants. Overexpression of ZmLOX3 improved ROS scavenging, Na[+]/K[+] homeostasis, and cell membrane stability. Transcriptome analyses revealed that ZmLOX3[OE] triggered the expression of genes involved in both the JA synthesis and signaling pathways. A transcription factor ZmNAC032 was identified via Y1H screening and was able to bind to the C[A/G]CG[T/G] sequence in the ZmLOX3 promoter and activate its expression. These findings are helpful for deciphering the function and regulatory status of ZmLOX in improving salt tolerance.},
}

@article {pmid41054568,
year = {2025},
author = {Xia, B and Wang, Z and Fei, T and Ma, Y and Guo, Y and Fei, D and Shu, X and Zhao, G and Ma, M and Yuan, H},
title = {Development and application of a CRISPR/Cas12a-based reverse transcription-recombinase polymerase amplification assay with lateral flow dipstick and fluorescence detection for Getah virus.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e20119},
pmid = {41054568},
issn = {2167-8359},
mesh = {*CRISPR-Cas Systems ; *Alphavirus/isolation & purification/genetics ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Fluorescence ; *Alphavirus Infections/diagnosis/virology/veterinary ; Swine ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Getah virus (GETV), a mosquito-borne alphavirus classified as a zoonotic disease, primarily infects livestock, particularly pigs and horses. In recent years, it has re-emerged in multiple Asian countries, posing a potential threat to animal husbandry and public health. In this study, we developed a rapid and sensitive GETV detection method based on reverse transcription-recombinase polymerase amplification (RT-RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system combined with a lateral flow dipstick (LFD) for visual readout. By leveraging sequence conservation in the GETV E2 envelope protein-coding regions, we engineered matched crRNA guides and amplification primers to develop a rapid CRISPR-Cas12a diagnostic workflow. The optimized platform combines RT-RPA (42 °C/20 min) with Cas12a's trans-nuclease activity, permitting multiplex detection via real-time fluorescence quantification or immunochromatographic strip visualization. Analytical evaluation demonstrated a detection capability of 10 copies/µL and exclusive specificity against four pathogen controls, including Japanese encephalitis virus and pseudorabies virus. Validation performed using simulated clinical samples revealed 100% concordance between the results of RT-RPA-CRISPR/Cas12a-LFD and quantitative polymerase chain reaction (PCR), while reducing the total detection time to 50 minutes. This approach eliminated the need for advanced instrumentation owing to its simplified operational design, enabling field-deployable rapid detection capabilities that establish essential technical infrastructure for initiating timely GETV containment measures. This approach has broad application potential in the fields of food safety, clinical diagnostics, and environmental science.},
}

*CRISPR-Cas Systems
*Alphavirus/isolation & purification/genetics
Animals
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Fluorescence
*Alphavirus Infections/diagnosis/virology/veterinary
Swine
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41026092,
year = {2025},
author = {Toyonishi, G and Nakazawa, T and Koshi, D and Horii, M and An, GH and Kawauchi, M and Honda, Y},
title = {CRISPR/Cas9-directed disruption of wc-2 leads to the absence of fruiting body development in Pleurotus ostreatus.},
journal = {FEMS microbiology letters},
volume = {372},
number = {},
pages = {},
doi = {10.1093/femsle/fnaf104},
pmid = {41026092},
issn = {1574-6968},
support = {18KK0178//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; 22KK0090//Japan Society for the Promotion of Science/ ; PJ0175072025//Rural Development Administration/ ; },
mesh = {*Pleurotus/genetics/growth & development/radiation effects ; *Fruiting Bodies, Fungal/growth & development/genetics/radiation effects ; *CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; Light ; },
abstract = {Light, particularly blue light, is a key environmental factor that induces fruiting in certain agaricomycetes. In this study, we characterized mutant strains of Pleurotus ostreatus with disrupted wc-2, which encodes one of the white-collar proteins, Wc-2, to investigate the role of light in fruiting in P. ostreatus. We introduced two different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting wc-2 separately into the dikaryotic P. ostreatus strain PC9×#64. Among the 11 dikaryotic hygromycin-resistant transformants, six strains did not form fruiting bodies. Genomic PCR followed by sequencing analysis suggested that all six fruitless strains were dikaryotic wc-2 disruptants. Small aggregate structures were not observed in the dikaryotic wc-2 disruptants grown under light conditions, as in PC9×#64 grown in a red box. These results suggest that Wc-2 is essential for the initiation of blue light-induced fruiting in P. ostreatus.},
}

*Pleurotus/genetics/growth & development/radiation effects
*Fruiting Bodies, Fungal/growth & development/genetics/radiation effects
*CRISPR-Cas Systems
*Fungal Proteins/genetics/metabolism
Light

@article {pmid41002250,
year = {2025},
author = {Gillmore, JD and Gane, E and Täubel, J and Pilebro, B and Echaniz-Laguna, A and Kao, J and Litchy, W and Shahda, S and Haagensen, A and Walsh, L and Smith, D and Kachadourian, J and Ward, JH and Lebwohl, D and Zhu, P and Xu, Y and Leung, A and Sonderfan, A and Gutstein, DE and Manvelian, G and Adams, D},
title = {Nexiguran Ziclumeran Gene Editing in Hereditary ATTR with Polyneuropathy.},
journal = {The New England journal of medicine},
volume = {393},
number = {14},
pages = {1375-1386},
doi = {10.1056/NEJMoa2510209},
pmid = {41002250},
issn = {1533-4406},
mesh = {Humans ; *Amyloid Neuropathies, Familial/therapy/genetics ; Male ; Female ; Middle Aged ; *Prealbumin/genetics/metabolism/antagonists & inhibitors ; Adult ; Aged ; *Gene Editing ; *Genetic Therapy/adverse effects/methods ; CRISPR-Cas Systems ; Neurofilament Proteins/blood ; Body Mass Index ; },
abstract = {BACKGROUND: Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) is a rare, multisystem, progressive, debilitating, and fatal disease characterized by tissue deposition of misfolded transthyretin (TTR) in peripheral nerves. Nexiguran ziclumeran (nex-z) is an investigational in vivo therapy based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease) that is designed to reduce serum TTR levels through selective inactivation of TTR in the liver.

METHODS: In this phase 1, open-label study, we administered one infusion of nex-z to patients with ATTRv-PN. Primary objectives included assessment of the safety and pharmacodynamics of nex-z. Secondary end points included changes in the familial amyloid polyneuropathy stage, polyneuropathy disability score, serum neurofilament light chain (NfL) level, modified body-mass index (modified BMI, defined as the conventional BMI [weight in kilograms divided by square of height in meters] multiplied by the albumin level in grams per liter), and modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment).

RESULTS: A total of 36 patients received nex-z; the mean follow-up was 27 months. The mean percent change from baseline in the serum TTR level was -90% at day 28, which was sustained through month 24 (-92%). Treatment-related adverse events included transient infusion-related reactions (in 21 patients), decreased thyroxine level without hypothyroidism or elevated thyrotropin level (in 8), and headache (in 4). One participant died from cardiac amyloidosis, and one withdrew owing to progressive decline in motor function. Serious adverse events were reported in 11 patients. At month 24, the familial amyloid polyneuropathy stage and polyneuropathy disability score remained stable in 29 and 27 patients, respectively; improved in 2 and 5, respectively; and worsened in 2 and 2, respectively. The mean change in the serum NfL level was -9.0 pg per milliliter, and the change in the modified BMI was 24.7. The mean change from baseline in the mNIS+7 was -8.5.

CONCLUSIONS: A single administration of nex-z in patients with ATTRv-PN was associated with rapid, deep, and durable reductions in serum TTR levels. The results support further investigation of nex-z to treat ATTRv-PN. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).},
}

Humans
*Amyloid Neuropathies, Familial/therapy/genetics
Male
Female
Middle Aged
*Prealbumin/genetics/metabolism/antagonists & inhibitors
Adult
Aged
*Gene Editing
*Genetic Therapy/adverse effects/methods
CRISPR-Cas Systems
Neurofilament Proteins/blood
Body Mass Index

@article {pmid40990297,
year = {2025},
author = {Sheng, J and Dong, Y and Sun, S and Zhang, Y and Li, C and Xu, X and Wang, H},
title = {Construction of a Sensing Platform Integrated with a CRISPR/Cas12a-Triggered Colorimetric Strategy for the Quantitative Detection of Meat Freshness.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25604-25614},
doi = {10.1021/acs.jafc.5c04851},
pmid = {40990297},
issn = {1520-5118},
mesh = {*Colorimetry/methods/instrumentation ; *Meat/analysis/microbiology ; CRISPR-Cas Systems ; Animals ; *Pseudomonas/genetics/isolation & purification/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/instrumentation/methods ; *Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins ; },
abstract = {Monitoring microbial determinants, such as Pseudomonas spp., is thus essential for assessing meat freshness. Here, a novel colorimetric sensing platform based on magnetic enzyme-labeled nanoparticles combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a without nucleic acid molecule preamplification was developed for detecting meat freshness. Under optimal conditions, a high-specificity crRNA was systematically verified, and the colorimetric sensor could accurately quantify Pseudomonas spp. loads with levels ranging from 1 × 10[3.7] to 1 × 10[8.7] CFU/mL, with a color change from colorless to yellow. A smart colorimetric platform, including a self-designed image acquisition device and self-programmed image analysis software, was developed and applied to the integrated determination of meat freshness by using the B-value in the RGB channel. The platform has been applied to both consumers and producers and has been validated by 48 actual samples of chilled meat. These findings provide new insights into the exploration of reliable tools for monitoring meat freshness.},
}

*Colorimetry/methods/instrumentation
*Meat/analysis/microbiology
CRISPR-Cas Systems
Animals
*Pseudomonas/genetics/isolation & purification/metabolism
*Bacterial Proteins/genetics/metabolism
*Biosensing Techniques/instrumentation/methods
*Endodeoxyribonucleases/genetics/metabolism
CRISPR-Associated Proteins

@article {pmid40985907,
year = {2025},
author = {Fei, S and Zhang, C and Zhang, X and Xie, Y and Fu, S and Wu, J},
title = {An Ultrasensitive Immunocapture (IC)-RPA-CRISPR/Cas12a Assay with Three Readout Modes for Detecting Xanthomonas oryzae pv. oryzicola of Rice Bacterial Leaf Streak.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25664-25675},
doi = {10.1021/acs.jafc.5c04360},
pmid = {40985907},
issn = {1520-5118},
mesh = {*Oryza/microbiology ; *Xanthomonas/genetics/isolation & purification ; *Plant Diseases/microbiology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Plant Leaves/microbiology ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Xanthomonas oryzae pv oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS) and causes enormous losses of rice yields in many countries every year. Development of sensitive diagnostic techniques is crucial for its prevention and control. Here, we developed an ultrasensitive IC-RPA-CRISPR/Cas12a assay with three readout modes [qPCR machine, UV lamp, and lateral flow strip (LFS)] for Xoc detection in rice, which combined advantages of immunocapture, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based cleavage. Especially, the immunocapture step allows to capture and enrich Xoc from samples, which minimizes the interference from rice debris to benefit nucleic acid release and amplification and enhances the specificity and sensitivity of this assay. The detection limits of its three readout modes for Xoc bacterial suspension is 2, 6, and 60 CFU/mL, respectively. Collectively, this study provides a specific, ultrasensitive, practical approach for quarantine and detection of Xoc that will benefit the prevention and control of BLS.},
}

*Oryza/microbiology
*Xanthomonas/genetics/isolation & purification
*Plant Diseases/microbiology
CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods/instrumentation
Plant Leaves/microbiology
Bacterial Proteins/genetics/metabolism

@article {pmid40970715,
year = {2025},
author = {Zhao, C and Li, G and Shen, C and Xie, Y and Chen, Y and Ying, X and Chen, Y and Zhang, C},
title = {An extraction-free and one-pot two-temperature CRISPR/Cas12b system for visual detection of Group B Streptococcus by integrating with RPA.},
journal = {Journal of clinical microbiology},
volume = {63},
number = {10},
pages = {e0081925},
doi = {10.1128/jcm.00819-25},
pmid = {40970715},
issn = {1098-660X},
support = {2024KY1444//Medical and Health Research Project of Zhejiang Province/ ; LTGC23H200004//Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Streptococcus agalactiae/isolation & purification/genetics ; Humans ; *Streptococcal Infections/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Female ; Recombinases/metabolism ; Temperature ; Vagina/microbiology ; },
abstract = {UNLABELLED: Group B Streptococcus (GBS) is a major cause of neonatal infections, and rapid detection is essential for timely clinical intervention. In this study, we developed an extraction-free, one-pot CRISPR/Cas12b assay for visual detection of GBS by combining with isothermal amplification, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). The results showed that LAMP-CRISPR/Cas12b outperformed RPA-CRISPR/Cas12b system across all template concentrations, especially in low-copy template (30 and 10 copies/test) detection. To enhance the detection performance of RPA-CRISPR/Cas12b, we introduced a two-temperature protocol, with RPA reaction at 39°C followed by Cas12b activation at 62°C. Through the two-temperature approach, the detection rate of RPA-CRISPR/Cas12b system was significantly improved even in low-copy samples, achieving a sensitivity of 10 copies/test (1 copy/μL). Clinical validation using 60 vaginal-rectal swab samples showed 96.7% and 98.3% of concordance when compared to culture and qPCR methods, respectively. This assay offers a rapid (<1 h), highly sensitive, and user-friendly solution without requiring nucleic acid extraction or sophisticated instruments. Its compatibility with visual signal detection makes it ideal for point-of-care testing, especially in low-resource or time-sensitive settings. The platform can be adapted for broader pathogen detection in future field diagnostics.

IMPORTANCE: This study presents a rapid, convenient, and highly accurate method for Group B Streptococcus (GBS) detection by integrating the CRISPR/Cas12b system with recombinase polymerase amplification, an isothermal nucleic acid amplification technique. To streamline the workflow, we established a one-pot, extraction-free assay that significantly reduces the detection time. Through the systematic optimization of the dual-temperature conditions, we enhanced the amplification efficiency of target DNA, thereby improving the sensitivity of the CRISPR/Cas12b system. Additionally, the incorporation of a UV-visible detection system enables visual readout, facilitating instrument-free testing suitable for point-of-care (POC) applications.},
}

*Streptococcus agalactiae/isolation & purification/genetics
Humans
*Streptococcal Infections/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Female
Recombinases/metabolism
Temperature
Vagina/microbiology

@article {pmid40899880,
year = {2025},
author = {Liu, P and Zhang, J and Gong, Y and Liu, W and Xiao, G and Liang, J and Wang, X and Bi, J and Zhang, G},
title = {Application of engineered CRISPR/Cas12a variants with altered protospacer adjacent motif specificities for the detection of isoniazid resistance mutations in Mycobacterium tuberculosis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0016525},
pmid = {40899880},
issn = {2165-0497},
mesh = {*Isoniazid/pharmacology ; *Mycobacterium tuberculosis/genetics/drug effects/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Antitubercular Agents/pharmacology ; Bacterial Proteins/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Mutation ; Tuberculosis, Multidrug-Resistant/microbiology/diagnosis ; *Endodeoxyribonucleases/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Catalase/genetics ; Sensitivity and Specificity ; CRISPR-Associated Proteins/genetics ; Microbial Sensitivity Tests ; },
abstract = {UNLABELLED: Drug-resistant tuberculosis (TB) is a major global public health concern. Although isoniazid is currently considered one of the most effective first-line drugs for TB treatment, its efficacy is limited by the emergence of resistance. Therefore, it is imperative to develop new methods for detecting drug-resistant TB. In this study, we developed a nucleic acid detection system based on the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a_RR protein. The system combines recombinase polymerase amplification with an engineered CRISPR/Cas12a_RR protein to enable rapid and specific detection of the katG G944C mutation in isoniazid-resistant Mycobacterium tuberculosis (Mtb). It could detect the target DNA at concentrations as low as 1% in a mixed sample. Compared with TaqMan quantitative polymerase chain reaction and DNA sequencing, the CRISPR/Cas12a_RR system demonstrated superior detection performance in terms of sensitivity, specificity, and cost-effectiveness. Furthermore, it effectively differentiated between drug-resistant Mtb strains from wild-type Mtb strains in clinically isolated samples, with the entire detection process completed in 60 min. In conclusion, the CRISPR/Cas12a_RR detection system offers a novel, rapid, simple, sensitive, and specific approach for identifying isoniazid-resistant Mtb, with significant potential for clinical application, particularly in resource-limited settings.

IMPORTANCE: This study presents a novel method for detecting isoniazid-resistant Mycobacterium tuberculosis (Mtb) using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a mutants, offering rapid detection, cost-effectiveness, and high specificity, and thereby providing a promising new avenue for detecting isoniazid-resistant Mtb.},
}

*Isoniazid/pharmacology
*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification
*CRISPR-Cas Systems/genetics
*Antitubercular Agents/pharmacology
Bacterial Proteins/genetics
Humans
*Drug Resistance, Bacterial/genetics
Mutation
Tuberculosis, Multidrug-Resistant/microbiology/diagnosis
*Endodeoxyribonucleases/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Catalase/genetics
Sensitivity and Specificity
CRISPR-Associated Proteins/genetics
Microbial Sensitivity Tests

@article {pmid40866699,
year = {2025},
author = {Pacesa, M and Nickel, L and Schellhaas, C and Schmidt, J and Pyatova, E and Kissling, L and Barendse, P and Choudhury, J and Kapoor, S and Alcaraz-Serna, A and Cho, Y and Ghamary, KH and Vinué, L and Yachnin, BJ and Wollacott, AM and Buckley, S and Westphal, AH and Lindhoud, S and Georgeon, S and Goverde, CA and Hatzopoulos, GN and Gönczy, P and Muller, YD and Schwank, G and Swarts, DC and Vecchio, AJ and Schneider, BL and Ovchinnikov, S and Correia, BE},
title = {One-shot design of functional protein binders with BindCraft.},
journal = {Nature},
volume = {646},
number = {8084},
pages = {483-492},
pmid = {40866699},
issn = {1476-4687},
support = {R35 GM138368/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Gene Editing ; Protein Binding ; CRISPR-Cas Systems/genetics ; Allergens/immunology/metabolism/chemistry ; Immunoglobulin E/immunology/metabolism ; *Protein Engineering/methods ; *Proteins/metabolism/chemistry ; Receptors, Cell Surface/metabolism/chemistry ; Binding Sites ; Models, Molecular ; CRISPR-Associated Protein 9/metabolism ; Animals ; },
abstract = {Protein-protein interactions are at the core of all key biological processes. However, the complexity of the structural features that determine protein-protein interactions makes their design challenging. Here we present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold2 (ref. [1]) to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Last, we use cell-surface-receptor-specific binders to redirect adeno-associated virus capsids for targeted gene delivery. This work represents a significant advancement towards a 'one design-one binder' approach in computational design, with immense potential in therapeutics, diagnostics and biotechnology.},
}

Humans
Gene Editing
Protein Binding
CRISPR-Cas Systems/genetics
Allergens/immunology/metabolism/chemistry
Immunoglobulin E/immunology/metabolism
*Protein Engineering/methods
*Proteins/metabolism/chemistry
Receptors, Cell Surface/metabolism/chemistry
Binding Sites
Models, Molecular
CRISPR-Associated Protein 9/metabolism
Animals

@article {pmid40862592,
year = {2025},
author = {Shen, Y and Yi, C and Wang, H and Tang, Y and Li, J},
title = {Development of a rapid and sensitive RPA-CRISPR/Cas12a-based assay for the detection of Brucella melitensis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0099825},
pmid = {40862592},
issn = {2165-0497},
mesh = {*Brucellosis/diagnosis/microbiology ; *Brucella melitensis/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; Reproducibility of Results ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Brucellosis, a zoonotic disease caused by Brucella species, presents significant public health challenges due to its complex diagnosis and the limited availability of rapid detection methods. To address these challenges, we developed a novel detection method that integrates recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, enabling dual readout through fluorescence (FL) and lateral flow strip (LFS) detection. The RPA-CRISPR/Cas12a-FL assay demonstrated an impressive detection limit of 1 copy/μL, which is 10 times more sensitive than quantitative polymerase chain reaction, while the RPA-CRISPR/Cas12a-LFS method achieved a detection limit of 10 copies/μL, comparable to nested PCR. Specificity testing confirmed the robustness of the assay, as it produced strong signals exclusively for Brucella without cross-reactivity with other bacterial species. Clinical validation using serum samples from 24 confirmed brucellosis patients and six healthy controls demonstrated a 100% concordance with serological results, underscoring the reliability of this method for clinical applications. This assay provides a rapid, sensitive, and specific tool for Brucella detection, suitable for both laboratory and field settings, and holds significant potential for enhancing the diagnosis and control of brucellosis.IMPORTANCEBrucellosis is a significant zoonotic disease, and rapid and accurate diagnosis is crucial for its treatment and control. To address this need, we developed a novel detection method that combines recombinant enzyme polymerase amplification with a CRISPR/Cas12a system, achieving dual readout through fluorescence and lateral flow strips. The test demonstrates excellent sensitivity and specificity, with clinical validation confirming complete concordance with serological results. This approach offers a fast, reliable, and field-deployable solution for brucellosis diagnosis, significantly enhancing disease management and public health outcomes.},
}

*Brucellosis/diagnosis/microbiology
*Brucella melitensis/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Recombinases/genetics/metabolism
*Molecular Diagnostic Techniques/methods
Limit of Detection
Reproducibility of Results
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40856920,
year = {2025},
author = {Thiel, J and Sürün, D and Brändle, DC and Teichert, M and Künzel, SR and Friedrich, U and Dahl, A and Schubert, K and Rzagalinski, I and Shevchenko, A and Traikov, S and Mirtschink, P and Wagenführ, L and Buchholz, F and Hölig, K and Tonn, T and Kronstein-Wiedemann, R},
title = {Knock Out of miRNA-30a-5p and Reconstitution of the Actin Network Dynamics Partly Restores the Impaired Terminal Erythroid Differentiation during Blood Pharming.},
journal = {Stem cell reviews and reports},
volume = {21},
number = {8},
pages = {2637-2653},
pmid = {40856920},
issn = {2629-3277},
support = {530364326//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Cell Differentiation/genetics ; *Actins/metabolism ; *Erythroid Cells/metabolism/cytology ; *Erythropoiesis/genetics ; Proto-Oncogene Mas ; Hematopoietic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Erythrocytes/metabolism/cytology ; bcl-X Protein/metabolism/genetics ; Gene Knockout Techniques ; Cell Line ; },
abstract = {In vitro red blood cell (RBC) production offers a promising complement to conventional blood donation, particularly for patients with rare blood types. Previously, we developed imBMEP-A, the first erythroid cell line derived from reticulocyte progenitors, which maintains robust hemoglobin expression and erythroid differentiation in the presence of erythropoietin (EPO) despite its immortalized state. However, clinical translation remains hindered by the inability to scale up production due to impaired in vitro enucleation of RBC progenitor cell lines. Enhancing enucleation efficiency in imBMEP-A cells involved CRISPR/Cas9-mediated knockout (K.O.) of miR-30a-5p, a key enucleation inhibitor, moderately increasing rates to 3.3 ± 0.4%- 8.9 ± 1.7%. Further investigation of enucleation inefficiencies led to transcriptome and proteome comparisons between imBMEP-miR30a-K.O. cells and hematopoietic stem cells (HSCs). These analyses revealed altered gene expression and protein abundances linked to metabolic transitions, apoptosis promotion, and cytoskeletal regulation. Notably, forced expression of the proto-oncogene c-Myc, required for cell immortalization, emerged as a key driver of these physiological changes. Counteracting these effects required optimization of imBMEP-A cells by activating BCL-XL transcription and knocking out SCIN, which encodes the actin-severing protein scinderin. While BCL-XL is upregulated in normal erythropoiesis, it is downregulated in imBMEP-A. Conversely, SCIN, typically absent in erythroid cells, is highly expressed in imBMEP-A, disrupting actin organization. These interventions improved viability, restored actin network formation, and increased terminal erythropoiesis, yielding 22.1 ± 1.7% more orthochromatic erythroblasts. These findings establish a foundation for optimizing imBMEP-A cells for therapeutic use and advancing the understanding the pathophysiology of erythroleukemia.},
}

*MicroRNAs/genetics/metabolism
Humans
*Cell Differentiation/genetics
*Actins/metabolism
*Erythroid Cells/metabolism/cytology
*Erythropoiesis/genetics
Proto-Oncogene Mas
Hematopoietic Stem Cells/metabolism/cytology
CRISPR-Cas Systems
Erythrocytes/metabolism/cytology
bcl-X Protein/metabolism/genetics
Gene Knockout Techniques
Cell Line

@article {pmid40833103,
year = {2025},
author = {Omachi, R and Imai, K and Sato, A and Tanaka, M and Mizushina, H and Takeuchi, K and Maeda, T},
title = {Development and clinical evaluation of a novel SHERLOCK test for Mycoplasma genitalium.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0044525},
pmid = {40833103},
issn = {2165-0497},
support = {24K10540//KAKEN/ ; 2024 Research grant//Charitable Trust Laboratory Medicine Research Foundation of Japan/ ; Grant-in-Aid for Young Researchers//Saitama Medical University Hospital/ ; },
mesh = {*Mycoplasma genitalium/genetics/isolation & purification ; Humans ; *Mycoplasma Infections/diagnosis/microbiology/urine ; Male ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Urethritis/microbiology/diagnosis ; *Molecular Diagnostic Techniques/methods ; Japan ; Point-of-Care Testing ; DNA, Bacterial/genetics ; CRISPR-Cas Systems ; },
abstract = {Mycoplasma genitalium (MG) is a sexually transmitted pathogen associated with urethritis. Nucleic acid amplification tests are the gold standard for its diagnosis but often require specialized equipment, which limits their use in point-of-care testing. This study aimed to develop a rapid, sensitive detection method for MG using a specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) test, which combines isothermal recombinase polymerase amplification and a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a reaction. We developed a novel SHERLOCK test targeting the Mg219 gene in MG. The SHERLOCK method was evaluated using 128 first-void urine samples collected from male patients who were suspected of MG urethritis in Japan. The results of SHERLOCK were compared to those of the cobas TV/MG test and in-house quantitative PCR. SHERLOCK was optimized for use with crude DNA extracted from clinical urine samples. The results were detected via a lateral flow assay, allowing for visual interpretation within 60 min. The method demonstrated a limit of detection of 10 copies/reaction and showed no cross-reactivity with other pathogens. In clinical evaluations of 128 urine samples, SHERLOCK showed an overall agreement rate of 91.4% with the cobas TV/MG PCR test; the positive and negative agreement rates were 79.6 and 100%, respectively. SHERLOCK showed superior performance to quantitative PCR. This study demonstrates that the novel SHERLOCK assay for MG has potential as a point-of-care test in the clinical setting. Further evaluation in prospective studies is needed to confirm its clinical value.IMPORTANCEMycoplasma genitalium (MG) is a causative agent of sexually transmitted infections and is associated with urethritis and prostatitis in men. To prevent the transmission of MG, it is essential to identify infected individuals through diagnostic testing and provide appropriate treatment. Nucleic acid amplification tests are commonly used for MG diagnosis in the clinical setting, but the point-of-care testing (POCT) for MG remains limited. In this study, we developed a novel nucleic acid amplification test-specific high-sensitivity enzymatic reporter unlocking (SHERLOCK)-for MG, combining crude DNA extraction with a lateral flow assay. Our SHERLOCK assay successfully detected MG in approximately 1 h, with a detection limit of 10 copies/reaction. Clinical evaluations using urine samples showed a high agreement rate with the cobas TV/MG test. SHERLOCK is expected to be a useful tool for POCT for MG.},
}

*Mycoplasma genitalium/genetics/isolation & purification
Humans
*Mycoplasma Infections/diagnosis/microbiology/urine
Male
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Urethritis/microbiology/diagnosis
*Molecular Diagnostic Techniques/methods
Japan
Point-of-Care Testing
DNA, Bacterial/genetics
CRISPR-Cas Systems

@article {pmid40819286,
year = {2025},
author = {Beck, CW and Reily-Bell, M and Bicknell, LS},
title = {Unilateral loss of recql4 function in Xenopus laevis tadpoles leads to ipsilateral ablation of the forelimb, hypoplastic Meckel's cartilage, and vascular defects.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {10},
pages = {},
doi = {10.1093/g3journal/jkaf179},
pmid = {40819286},
issn = {2160-1836},
support = {PRG1732//Neurological Foundation of New Zealand/ ; },
mesh = {Animals ; *Xenopus laevis/genetics ; Larva/genetics ; *RecQ Helicases/genetics/metabolism ; *Forelimb/abnormalities ; *Cartilage/abnormalities/metabolism ; *Xenopus Proteins/genetics/metabolism ; Gene Knockdown Techniques ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {RECQL4 encodes a RecQ helicase, one of a family of DNA unwinding enzymes with roles in DNA replication, double-strand break repair, and genomic stability. Pathogenic variants in RECQL4 are clinically associated with 3 rare autosomal recessive conditions: Rothmund-Thomson syndrome type II, Baller-Gerold syndrome, and RAPADILINO syndrome. These 3 syndromes show overlapping growth retardation, low bone density, and skeletal defects affecting the arms and hands. Here, we take advantage of the ability to generate one-sided CRISPR knockdowns of recql4 in Xenopus laevis tadpoles. Tadpoles develop normally until feeding starts, after which growth slows on the edited side, leading to a curved posture, smaller eyes (microphthalmia), and reduced head size (microcephaly). Forelimb buds fail to develop, leading to complete absence of the forelimb on the edited side. Additionally, Meckel's cartilage (lower jaw) ossification is absent or reduced and the hyoid cartilage is smaller, but this is not due to deficiencies in cranial neural crest migration on the edited side. Knockdown of recql4 also results in hypoplastic vasculature, with reduced branching from the aorta on the edited side. Taken together, our results clearly show the utility of unilateral CRISPR editing in Xenopus for understanding the specific phenotypic developmental effects of mutations affecting cell proliferation.},
}

Animals
*Xenopus laevis/genetics
Larva/genetics
*RecQ Helicases/genetics/metabolism
*Forelimb/abnormalities
*Cartilage/abnormalities/metabolism
*Xenopus Proteins/genetics/metabolism
Gene Knockdown Techniques
Phenotype
CRISPR-Cas Systems

@article {pmid40794772,
year = {2025},
author = {Després, PC and Gervais, NC and Fogal, M and Rogers, RKJ and Cuomo, CA and Shapiro, RS},
title = {Targeted loss of heterozygosity in Candida albicans using CRISPR-Cas9 reveals the functional impact of allelic variation.},
journal = {Genetics},
volume = {231},
number = {2},
pages = {},
doi = {10.1093/genetics/iyaf154},
pmid = {40794772},
issn = {1943-2631},
support = {//FRQS/ ; //NSERC/ ; //N.C.G./ ; //M.F./ ; PJT 162195//CIHR/ ; //R.S.S./ ; //CIFAR/ ; //Fungal Kingdom/ ; //C.A.C./ ; U19 AI110818/AI/NIAID NIH HHS/United States ; //Canada Research Chair/ ; },
mesh = {*Candida albicans/genetics/drug effects ; *Loss of Heterozygosity ; *CRISPR-Cas Systems ; Alleles ; Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Drug Resistance, Fungal/genetics ; Gene Editing ; Genetic Variation ; Antifungal Agents/pharmacology ; },
abstract = {The diploid genome of the fungal pathogen Candida albicans is highly heterozygous, with most allele pairs diverging at either the coding or regulatory level. When faced with selection pressure like antifungal exposure, this hidden genetic diversity can provide a reservoir of adaptive mutations through loss of heterozygosity (LOH) events. Validating the potential phenotypic impact of LOH events observed in clinical or experimentally evolved strains can be difficult due to the challenge of precisely targeting one allele over the other. Here, we show that a CRISPR-Cas9 system can be used to overcome this challenge. By designing allele-specific guide RNA sequences, we can induce targeted, directed LOH events, which we validate by whole-genome long-read sequencing. Using this approach, we efficiently recapitulate a recently described LOH event that increases resistance to the antifungal fluconazole. Additionally, we find that the recombination tracts of these induced LOH events have similar lengths to those observed naturally. To facilitate future use of this method, we provide a database of allele-specific sgRNA sequences for Cas9 that provide near genome-wide coverage of heterozygous sites through either direct or indirect targeting. This approach will be useful in probing the adaptive role of LOH events in this important human pathogen.},
}

*Candida albicans/genetics/drug effects
*Loss of Heterozygosity
*CRISPR-Cas Systems
Alleles
Genome, Fungal
RNA, Guide, CRISPR-Cas Systems/genetics
Drug Resistance, Fungal/genetics
Gene Editing
Genetic Variation
Antifungal Agents/pharmacology

@article {pmid40758833,
year = {2025},
author = {Stuecker, TN and Hood, SE and Molina Pineda, J and Lenaduwe, S and Winter, J and Sadhu, MJ and Lewis, JA},
title = {Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {10},
pages = {},
doi = {10.1093/g3journal/jkaf175},
pmid = {40758833},
issn = {2160-1836},
support = {P20 GM103429/GM/NIGMS NIH HHS/United States ; MCB-1941824//National Science Foundation/ ; /HG/NHGRI NIH HHS/United States ; 1ZIAHG200401/GF/NIH HHS/United States ; //Arkansas IDeA Network of Biomedical Research Excellence/ ; //Summer Research Fellowship/ ; P20 GM103429/GM/NIGMS NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Vectors/genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal ; Escherichia coli/genetics ; },
abstract = {In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas "cutting" combined with homologous recombination of a "repair" template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains and rely on relatively low-efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an Escherichia coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo, which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic (HIS3, URA3) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a β-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide on how to use this system.},
}

*Saccharomyces cerevisiae/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Vectors/genetics
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Genome, Fungal
Escherichia coli/genetics

@article {pmid40495695,
year = {2025},
author = {Wang, H and Zhan, H and Pan, B and Zeng, L and Chen, Z and Liu, S and Zhang, Q and Hong, X and Lu, J and Lin, X and Zhao, X and Lai, J and Jie, K and Li, Y and Zhong, J and Peng, S and Chen, S and Chen, C and Zhong, W and Wu, S and Pan, Y and Lin, T and Chen, X},
title = {Engineering CRISPR System-Based Bacterial Outer Membrane Vesicle Potentiates T Cell Immunity for Enhanced Cancer Immunotherapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {39},
pages = {e2501565},
doi = {10.1002/adma.202501565},
pmid = {40495695},
issn = {1521-4095},
support = {2018YFA0902800//National Key Research and Development Program of China/ ; 82322056//National Natural Science Foundation of China/ ; 82341018//National Natural Science Foundation of China/ ; 82072827//National Natural Science Foundation of China/ ; 82273421//National Natural Science Foundation of China/ ; 82303405//National Natural Science Foundation of China/ ; 82472137//National Natural Science Foundation of China/ ; 2023A03J0718//Science and Technology Program of Guangzhou/ ; 2024B03J1234//Science and Technology Program of Guangzhou/ ; 2024A04J6558//Science and Technology Program of Guangzhou/ ; //Fundamental Research Funds for the Central Universities/ ; 23ykbj002//Sun Yat-sen University/ ; 2020B1111170006//Guangdong Provincial Clinical Research Centre for Urological Diseases/ ; 2020B1212060018//Guangdong Science and Technology Department/ ; 2018B030317001//Guangdong Science and Technology Department/ ; 2017B030314026//Guangdong Science and Technology Department/ ; },
mesh = {Animals ; Mice ; *Immunotherapy/methods ; *T-Lymphocytes/immunology ; Humans ; Cell Line, Tumor ; *Bacterial Outer Membrane/metabolism ; *CRISPR-Cas Systems ; *Neoplasms/therapy/immunology ; Escherichia coli/genetics ; Interleukin-12/genetics ; Chemokine CXCL9/genetics ; Genetic Engineering ; },
abstract = {Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment but only benefits a subset of patients because of insufficient infiltration and inactivation of effector T cells. Bacterial outer membrane vesicles (OMVs) can activate immunity and deliver therapeutic agents for immunotherapy. However, efficiently targeting and packaging therapeutic molecules into OMVs remains challenging. Here, the engineered E. coli BL21-derived OMVs enable the packaging of multiple genes, resulting in a 7-fold increase in DNA enrichment efficiency and gene silencing in vitro. Moreover, the engineered OMVs carrying genes encoding CXCL9 and IL12 (OMV-C9I12) reprogram tumor cells to secrete these factors, significantly enhancing T-cell chemotaxis and activation. More importantly, this system markedly inhibits tumors, extends survival, and synergizes with anti-PD-1/PD-L1 therapy in murine MB49 and B16F10 tumor models. Single-cell RNA sequencing (scRNA-seq) further reveals significant upregulation of T-cell chemotaxis and activation-related pathways following OMV-C9I12 treatment. Finally, OMV-C9I12 potentiates T cell-mediated immunotherapy and suppresses the growth of bladder and breast cancer tumors in humanized mouse models. These findings highlight the potential of this engineered OMV platform for cancer gene therapy and provide novel strategies to overcome resistance to immunotherapy.},
}

Animals
Mice
*Immunotherapy/methods
*T-Lymphocytes/immunology
Humans
Cell Line, Tumor
*Bacterial Outer Membrane/metabolism
*CRISPR-Cas Systems
*Neoplasms/therapy/immunology
Escherichia coli/genetics
Interleukin-12/genetics
Chemokine CXCL9/genetics
Genetic Engineering

@article {pmid40436751,
year = {2025},
author = {Bold-Erdene, A and Miura, K and Yamasaki, N and Miura, S and Ogata, S and Sasatani, M and Yamamoto, T and Kaminuma, O},
title = {Effect of gamma-ray exposure on the genome-editing efficiency of improved genome-editing via oviductal nucleic acids delivery (i-GONAD).},
journal = {Experimental animals},
volume = {74},
number = {4},
pages = {457-462},
doi = {10.1538/expanim.25-0036},
pmid = {40436751},
issn = {1881-7122},
mesh = {Animals ; Female ; *Gamma Rays/adverse effects ; *Gene Editing/methods ; Pregnancy ; Mice ; *Oviducts ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded/radiation effects ; *Nucleic Acids/administration & dosage ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {DNA double-strand breaks (DSBs) are among the most hazardous cellular damages, potentially leading to cell death or oncogenesis if unrepaired. Genome editing methods, such as the CRISPR/Cas9 system, induce DSBs and utilize these repair pathways for gene knockout and knock-in. Although ionizing radiation also induces DSBs, it is not clear whether the efficiency of genome editing is affected by ionizing radiation. This study investigated the impact of gamma-ray exposure on the genome editing efficiency of the improved genome editing via oviductal nucleic acid delivery (i-GONAD) method. Gamma-rays were exposed to pregnant mice receiving i-GONAD targeting the Hr gene, whose mutation causes hair loss in mice. The exposure on the fertilization day (Day 0) decreased natural delivery rates and litter sizes, with notable effects at 0.3 Gy or higher. Although the proportions of hairless offspring obtained by i-GONAD differed greatly between single-guide RNAs (sgRNAs) used, total mutation rates, including hairless, mosaic, and indel, were equivalent. Gamma-ray exposure on Day 0 and the day after fertilization (Day 1) similarly and almost dose-dependently enhanced the genome editing efficiency evaluated by the total mutation rate. This study suggests the improvement of genome editing efficiency by gamma-ray exposure, at least in i-GONAD method, potentially facilitating the creation of diverse experimental animal models.},
}

Animals
Female
*Gamma Rays/adverse effects
*Gene Editing/methods
Pregnancy
Mice
*Oviducts
CRISPR-Cas Systems
DNA Breaks, Double-Stranded/radiation effects
*Nucleic Acids/administration & dosage
RNA, Guide, CRISPR-Cas Systems

@article {pmid40029006,
year = {2025},
author = {Mattoscio, D and Baeza, LA and Bai, H and Colangelo, T and Castagnozzi, S and Marzotto, M and Cufaro, MC and Lotti, V and Yuan, YC and Mucci, M and Si, L and Zuccarini, M and Tredicine, M and D'Orazio, S and Pieragostino, D and Del Boccio, P and Sorio, C and Trerotola, M and Romano, M and Plebani, R},
title = {Inflammation and epithelial-mesenchymal transition in a CFTR-depleted human bronchial epithelial cell line revealed by proteomics and human organ-on-a-chip.},
journal = {The FEBS journal},
volume = {292},
number = {19},
pages = {5086-5104},
doi = {10.1111/febs.70050},
pmid = {40029006},
issn = {1742-4658},
support = {AT2021 to Roberto Plebani//Italian Ministry of Health (fund ex60%)/ ; AT2022 to Roberto Plebani//Italian Ministry of Health (fund ex60%)/ ; AIRC; #19548 to Tommaso Colangelo//Associazione Italiana per la Ricerca sul Cancro/ ; MFAG 2022 - ID. 27060 to Domenico Mattoscio//Associazione Italiana per la Ricerca sul Cancro/ ; //Fondazione Umberto Veronesi/ ; GMSG#01/2023 to Roberto Plebani//Fondazione per la Ricerca sulla Fibrosi Cistica/ ; FFC#11/2022 to Domenico Mattoscio//Fondazione per la Ricerca sulla Fibrosi Cistica/ ; },
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; Proteomics/methods ; *Epithelial-Mesenchymal Transition/genetics ; *Epithelial Cells/metabolism/pathology ; *Inflammation/pathology/genetics/metabolism ; *Cystic Fibrosis/pathology/genetics/metabolism ; *Bronchi/pathology/metabolism/cytology ; Cell Line ; Cell Movement/genetics ; Lab-On-A-Chip Devices ; CRISPR-Cas Systems ; Neutrophils/metabolism/pathology ; Signal Transduction ; Microphysiological Systems ; },
abstract = {Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, leading to chronic, unresolved inflammation of the airways due to uncontrolled recruitment of polymorphonuclear leukocytes (PMNs). Evidence indicates that CFTR loss-of-function, in addition to promoting a pro-inflammatory phenotype, is associated with an increased risk of developing cancer, suggesting that CFTR can exert tumor-suppressor functions. Three-dimensional (3D) in vitro culture models, such as the CF lung airway-on-a-chip, can be suitable for studying PMN recruitment, as well as events of cancerogenesis, that is epithelial cell invasion and migration, in CF. To gather insight into the pathobiology of CFTR loss-of-function, we generated CFTR-knockout (KO) clones of the 16HBE14o- human bronchial cell line by CRISPR/Cas9 gene editing, and performed a comparative proteomic analysis of these clones with their wild-type (WT) counterparts. Systematic signaling pathway analysis of CFTR-KO clones revealed modulation of inflammation, PMN recruitment, epithelial cell migration, and epithelial-mesenchymal transition. Using a latest-generation organ-on-a-chip microfluidic platform, we confirmed that CFTR-KO enhanced PMN recruitment and epithelial cell invasion of the endothelial layer. Thus, a dysfunctional CFTR affects multiple pathways in the airway epithelium that ultimately contribute to sustained inflammation and cancerogenesis in CF.},
}

Humans
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism
Proteomics/methods
*Epithelial-Mesenchymal Transition/genetics
*Epithelial Cells/metabolism/pathology
*Inflammation/pathology/genetics/metabolism
*Cystic Fibrosis/pathology/genetics/metabolism
*Bronchi/pathology/metabolism/cytology
Cell Line
Cell Movement/genetics
Lab-On-A-Chip Devices
CRISPR-Cas Systems
Neutrophils/metabolism/pathology
Signal Transduction
Microphysiological Systems

@article {pmid41053552,
year = {2025},
author = {Qin, Z and Surnido, W and Mizuta, H and Uji, T},
title = {Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1323},
pmid = {41053552},
issn = {1471-2229},
support = {JPMJOP1851//Japan Science and Technology Agency/ ; },
mesh = {*Ulva/genetics/drug effects ; *Gene Knock-In Techniques/methods ; *Transgenes/genetics ; Kanamycin Kinase/genetics ; CRISPR-Cas Systems ; Hygromycin B/pharmacology ; Anti-Bacterial Agents/pharmacology ; Edible Seaweeds ; },
abstract = {Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene (aph7"). Integration of aph7" in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7". The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7" cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase (UpAPT). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources.},
}

*Ulva/genetics/drug effects
*Gene Knock-In Techniques/methods
*Transgenes/genetics
Kanamycin Kinase/genetics
CRISPR-Cas Systems
Hygromycin B/pharmacology
Anti-Bacterial Agents/pharmacology
Edible Seaweeds

@article {pmid41053297,
year = {2025},
author = {Vanderperre, B and Muraleedharan, A and Dorion, MF and Larroquette, F and Del Cid Pellitero, E and Rajakulendran, N and Chen, CX and Larivière, R and Michaud-Tardif, C and Goiran, T and Chidiac, R and Lipuma, D and MacLeod, G and Thomas, R and Wang, Z and Reintsch, WE and Luo, W and Shlaifer, I and Zhang, F and Xia, K and Steinhart, Z and Linhardt, RJ and Trempe, JF and Liu, J and Durcan, TM and Angers, S and Fon, EA},
title = {Novel regulators of heparan sulfate proteoglycans modulate cellular uptake of α-synuclein fibrils.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1426},
pmid = {41053297},
issn = {2399-3642},
support = {021129//Michael J. Fox Foundation for Parkinson's Research (Michael J. Fox Foundation)/ ; MFE-152571//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/ ; Canada Research Chair (Tier 1) in Parkinson's disease//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/ ; },
mesh = {*alpha-Synuclein/metabolism/genetics ; Humans ; *Heparan Sulfate Proteoglycans/metabolism ; Golgi Apparatus/metabolism ; Animals ; CRISPR-Cas Systems ; Cation Transport Proteins/genetics/metabolism ; Mice ; },
abstract = {Synucleinopathies are characterized by the accumulation and propagation of α-synuclein (α-syn) aggregates throughout the brain, leading to neuronal dysfunction and death. In this study, we used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout screening to identify genes that regulate the entry and accumulation of α-syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in α-syn PFFs intracellular accumulation, including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. All confirmed hits affected heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including α-syn and tau. Intriguingly, deletion of SLC39A9 and C3orf58 genes, encoding respectively a Golgi-localized exporter of Zn[2+], and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of α-syn PFFs, by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains in SLC39A9[-/-] and C3orf58[-/-] cells indicated major defects in HS homeostasis. Additionally, Golgi accumulation of NDST1, a prime HSPG biosynthetic enzyme, was detected in C3orf58[-/-] cells. Interestingly, C3orf58[-/-] human iPSC-derived microglia and dopaminergic neurons exhibited a strong reduction in their ability to internalize α-syn PFFs. Altogether, our data identifies new modulators of HSPGs that regulate α-syn PFFs cell surface binding and uptake.},
}

*alpha-Synuclein/metabolism/genetics
Humans
*Heparan Sulfate Proteoglycans/metabolism
Golgi Apparatus/metabolism
Animals
CRISPR-Cas Systems
Cation Transport Proteins/genetics/metabolism
Mice

@article {pmid41051826,
year = {2025},
author = {McCallum, GE and Ho, SFS and Cummins, EA and Wildsmith, AJ and McInnes, RS and Weigel, C and Tong, LYS and Quick, J and van Schaik, W and Moran, RA},
title = {The Kocurious case of Noodlococcus: genomic insights into Kocuria rhizophila from characterisation of a laboratory contaminant.},
journal = {Microbial genomics},
volume = {11},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001526},
pmid = {41051826},
issn = {2057-5858},
mesh = {*Genome, Bacterial ; Phylogeny ; Genomics ; *Micrococcaceae/genetics/classification/isolation & purification ; Whole Genome Sequencing ; Sequence Analysis, DNA ; },
abstract = {The laboratory contaminant strain Noodlococcus was named for its coccoid cells and unusual colony morphology, which resembled a pile of noodles. Along with laboratory characterisation and electron microscopy, we generated a complete Noodlococcus genome sequence using Illumina and Oxford Nanopore data. The genome consisted of a single, circular, 2,732,108 bp chromosome that shared 97.5% average nucleotide identity (ANI) with the Kocuria rhizophila type strain TA68. We identified genomic features involved in replication (oriC), carotenoid synthesis (crt) and genome defence (CRISPR-Cas) and discovered four novel mobile elements (ISKrh4-7). Despite its environmental ubiquity and relevance to food production, bioremediation and human medicine, there have been few genomic studies of the Kocuria genus. We conducted a comparative, phylogenetic and pangenomic examination of all 257 publicly available Kocuria genomes, with a particular focus on the 56 that were identified as K. rhizophila. We found that there are two phylogenetically distinct clades of K. rhizophila, with within-clade ANI values of 96.7-100.0% and between-clade values of 89.5-90.4%. The second clade, which we refer to as Kocuria pseudorhizophila, exhibited ANI values of <95% relative to TA68 and should constitute a separate species. Delineation of the two clades would be consistent with the rest of the genus, where all other species satisfy the 95% ANI threshold criteria. Differences in the K. rhizophila and K. pseudorhizophila pangenomes likely reflect phenotypic as well as evolutionary divergence. This distinction is relevant to clinical and industrial settings, as strains and genomes from both clades are currently used interchangeably, which may lead to reproducibility issues and phenotype-genotype discordance. Investigating an innocuous laboratory contaminant has therefore provided useful insights into the understudied species K. rhizophila, prompting an unexpected reassessment of its taxonomy.},
}

*Genome, Bacterial
Phylogeny
Genomics
*Micrococcaceae/genetics/classification/isolation & purification
Whole Genome Sequencing
Sequence Analysis, DNA

@article {pmid41051673,
year = {2026},
author = {Kumar, P and Verma, V and Irfan, M},
title = {Target-Specific Single Guide RNA (sgRNA) Design and In Vitro Validation of Target-Specific sgRNAs for CRISPR/Cas9-Mediated Editing of a Plant Stress Memory-Associated Gene.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2988},
number = {},
pages = {145-156},
pmid = {41051673},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Arabidopsis/genetics ; *Stress, Physiological/genetics ; Arabidopsis Proteins/genetics ; Transcription Factors/genetics ; },
abstract = {CRISPR/Cas9 genome editing has emerged as a transformative tool in plant biology, enabling precise manipulation of genes involved in stress responses. In the context of plant stress memory, where prior exposure to environmental stress enhances subsequent stress tolerance. CRISPR-based approaches offer a powerful means to dissect and engineer underlying regulatory genes. A critical factor determining the success of CRISPR/Cas9 editing is the careful design and validation of single guide RNAs (sgRNAs), which guide the Cas9 nuclease to specific genomic targets. This chapter provides a detailed, step-by-step protocol for the design, in vitro transcription, and in vitro cleavage assay to check efficiency of target-specific sgRNAs for plant genome editing applications. As a case study, we describe the design and validation of sgRNAs targeting the Arabidopsis thaliana DREB2A gene, a key transcription factor associated with drought stress memory. Emphasis is placed on strategies to maximize on-target efficiency, minimize off-target effects, and assess sgRNA functionality in vitro prior to in planta applications. This chapter serves as a practical guide for researchers aiming to functionally characterize stress memory-associated genes using CRISPR/Cas9 technology.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*Arabidopsis/genetics
*Stress, Physiological/genetics
Arabidopsis Proteins/genetics
Transcription Factors/genetics

@article {pmid41051363,
year = {2025},
author = {Zhytnik, L and Ventura, L and Sclocco, A and Verhage, M and Bakker, AD and Shim, JH and Beaino, W and Pereira, PM and Hoogeland, ME and Heine, VM and Maas, H and Jaspers, RT and Niehoff, A and Zaucke, F and de Waard, V and Eekhoff, EMW and Micha, D},
title = {New Lens On Congenital Mild Bone Fragility: a Novel Col1a1 Knockout Mouse Model for Osteogenesis Imperfecta Type 1.},
journal = {Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research},
volume = {},
number = {},
pages = {},
doi = {10.1093/jbmr/zjaf138},
pmid = {41051363},
issn = {1523-4681},
abstract = {Osteogenesis imperfecta (OI) is a genetic disorder characterized by bone fragility. It is one of the most prevalent rare skeletal dysplasias. The mildest form, OI type 1, predominantly results from collagen type I haploinsufficiency due to pathogenic variants in the COL1A1 gene, leading to reduced collagen type I. Despite OI type 1 representing approximately half of the OI population, the lack of an effective mouse model has hindered research and therapy development(1). To address this gap, we developed a genetically engineered mouse model harbouring a heterozygous deletion of the Col1a1 allele using the CRISPR/Cas system. The bone phenotype was characterised in 8- and 24-week-old mice, assessing transcriptomics and serum markers for bone formation (procollagen type I N-terminal propeptide) and resorption (tartrate-resistant acid phosphatase 5b). Bone volume, microarchitecture, and strength were evaluated by micro-computed tomography, histomorphometry and three-point bending test. We showed that the decreased Col1a1 to Col1a2 mRNA ratio determines reduced collagen type I production in OI mice bones as the underlying mechanism of haploinsufficient OI. This was supported by COL1A1 to COL1A2 mRNA ratio findings in human OI cell models, including fibroblasts and induced mesenchymal stem cells, as well as in induced pluripotent and mesenchymal stem cell models that were edited to carry a heterozygous COL1A1 allele. Our findings indicate for the first time that reduced bone volume and altered bone microarchitecture in haploinsufficient OI depends on the Col1a1 to Col1a2 mRNA ratio regulation. This novel mouse model faithfully recapitulates OI type 1 and provides a vital tool for investigating the disease mechanism and developing targeted therapeutic strategies for this large neglected OI patient population.},
}

@article {pmid41048399,
year = {2025},
author = {Huang, S and Qin, H and Dai, B and Liu, M and Shen, J},
title = {Establishment and evaluation of a circAdpgk-0001 knockdown method using CRISPR-Cas13d RNA-targeting technology.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e20123},
pmid = {41048399},
issn = {2167-8359},
mesh = {*RNA, Circular/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; RNA, Small Interfering/genetics ; Humans ; Cell Line ; Actins/genetics/metabolism ; },
abstract = {BACKGROUND: The small interfering RNA (siRNA) method has been used to knock down circular RNAs (circRNAs). However, issues such as low efficiency and off-target effects have become increasingly recognized. Recent studies have demonstrated that CRISPR-Cas13 can specifically target and cleave RNA. In this study, we established a CRISPR-Cas13d-based RNA-targeting method to specifically knock down circRNAs, such as circAdpgk-0001, and compared its performance with the siRNA method.

METHODS: Four clustered regularly interspaced short palindromic repeats (CRISPR) RNAs (crRNAs) of different nucleotide lengths spanning the back-splicing junction (BSJ) of circAdpgk-0001 were designed. A CRISPR-RfxCas13d plasmid capable of specifically cleaving circAdpgk-0001 was constructed and transfected into the JS-1 cell line. Knockdown efficiency was assessed using quantitative real-time PCR (qRT-PCR) and compared with that of the siRNA method. The expression of activation-related factors alpha-smooth muscle actin (α-SMA) and collagen I in JS-1 cells was further evaluated using qRT-PCR and Western blot.

RESULTS: CRISPR-Cas13d with a 24-nucleotide crRNA showed the highest knockdown efficiency (∼50%). After further optimization, the knockdown efficiency of CRISPR-Cas13d reached 70%, significantly higher than that of the siRNA method (40%). Knockdown of circAdpgk-0001 using Cas13d reduced the expression of collagen I and α-SMA by approximately 40%, which was greater than the reduction achieved by siRNA-mediated knockdown.

CONCLUSION: CRISPR-Cas13d demonstrated higher efficiency than the siRNA method in knocking down circRNAs, providing a promising tool for investigating circRNA functions.},
}

*RNA, Circular/genetics
*CRISPR-Cas Systems/genetics
*Gene Knockdown Techniques/methods
RNA, Small Interfering/genetics
Humans
Cell Line
Actins/genetics/metabolism

@article {pmid41020566,
year = {2025},
author = {Xue, J and Mao, K and Tang, Z and Hu, J and Zhang, H},
title = {Machine-Learning-Assisted CRISPR/Cas12a Biosensors for Monitoring Organophosphorus Pesticide Degradation.},
journal = {Analytical chemistry},
volume = {97},
number = {39},
pages = {21491-21501},
doi = {10.1021/acs.analchem.5c03596},
pmid = {41020566},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Pesticides/analysis/metabolism ; *CRISPR-Cas Systems ; *Organophosphorus Compounds/analysis/metabolism ; *Machine Learning ; *Water Pollutants, Chemical/analysis/metabolism ; Smartphone ; Acetylcholinesterase/metabolism ; Manganese Compounds/chemistry ; Oxides/chemistry ; Bacterial Proteins/metabolism/genetics ; Limit of Detection ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Owing to the severe environmental and health issues posed by organophosphorus pesticides (OPs), a dual-enzyme cascade biosensing platform based on manganese dioxide (MnO2) and CRISPR/Cas12a was developed in this study. Smartphones were innovatively integrated with a stacked ensemble learning (SEL) model for ultrasensitive detection and dynamic monitoring of OPs in environmental water samples. A dual-enzyme cascade signal amplification strategy was used to construct this sensing platform. Acetylcholinesterase (AChE) catalyzes the generation of thiocholine (TCh), which, in turn, regulates the degradation of MnO2 nanosheets, releasing Cas12a activators and generating a fluorescence signal. Owing to the irreversible inhibition of AChE activity by OPs, dichlorvos (DDVP) was successfully detected, with a detection limit as low as 4.62 pg/mL. Additionally, the SEL model, integrated into the smartphone biosensing platform and incorporating random forest (RF), XGBoost, and ridge regression algorithms, exhibited strong performance in detecting OPs after optimization (R[2] = 0.9985). In real water samples, the SEL model achieved a recovery rate of 93.1-103.1%, and the degradation kinetics of DDVP were successfully monitored over 24 h, revealing significant differences in DDVP degradation rates across various water matrices. This study is the first to report the integration of CRISPR/Cas12a biosensing technology with an SEL model-driven smartphone detection platform, providing a novel approach for sensitive, portable, and intelligent monitoring of OPs and offering new insights for water quality monitoring and early detection of environmental risks.},
}

*Biosensing Techniques/methods
*Pesticides/analysis/metabolism
*CRISPR-Cas Systems
*Organophosphorus Compounds/analysis/metabolism
*Machine Learning
*Water Pollutants, Chemical/analysis/metabolism
Smartphone
Acetylcholinesterase/metabolism
Manganese Compounds/chemistry
Oxides/chemistry
Bacterial Proteins/metabolism/genetics
Limit of Detection
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40923283,
year = {2025},
author = {Man, Y and Posey, RR and Bai, H and Jiang, A and Dosta, P and Ocampo-Alvarado, D and Plebani, R and Ji, J and Belgur, C and Artzi, N and Ingber, DE},
title = {Preclinical assessment of pan-influenza A virus CRISPR RNA therapeutics in a human lung alveolus chip.},
journal = {Lab on a chip},
volume = {25},
number = {20},
pages = {5240-5254},
doi = {10.1039/d5lc00156k},
pmid = {40923283},
issn = {1473-0189},
mesh = {Humans ; *RNA, Viral/genetics ; *Pulmonary Alveoli/virology/cytology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Influenza A virus/genetics ; *Influenza A Virus, H3N2 Subtype/genetics ; *Lab-On-A-Chip Devices ; Influenza, Human/therapy ; },
abstract = {CRISPR technology offers an entirely new approach to therapeutic development because it can target specific nucleotide sequences with high specificity, however, preclinical animal models are not useful for evaluation of their efficacy and potential off-target effects because of high gene sequence variations between animals and humans. Here, we explored the potential of using the CRISPR effector Cas13 to develop a new therapeutic approach for influenza A virus (IAV) infections based on its ability to specifically and robustly cleave single-strand viral RNA using a complementary CRISPR RNA (crRNA). We engineered crRNAs to target highly conserved regions in the IAV genome to create a potential pan-viral treatment strategy. A human lung alveolus chip (Lung Chip) lined by human primary alveolar epithelial cells interfaced with human primary pulmonary microvascular endothelial cells and infected with a pandemic IAV H3N2 strain was used to evaluate the on-target and off-target effects of these antiviral crRNA therapeutics. Our data show that the crRNAs targeting highly conserved regions in the IAV genome potently reduced viral replication in the alveolar airspace in the Lung Chip, and this was accompanied by suppression of the human host inflammatory response as indicated by a significant reduction in cytokine production and recruitment of immune cells. Importantly, only minimal off-target effects were observed based on transcriptomic analyses. As these crRNAs inhibit replication of influenza H1N1 and H3N2 in A549 cells as well as H3N2 in Lung Chips, these findings support use of CRISPR-Cas13 as a potentially viable approach to develop pan-IAV therapeutics for combating future influenza pandemics. The results also demonstrate that human Organ Chips be useful as more clinically relevant preclinical models for testing the efficacy and safety of crRNA therapeutics.},
}

Humans
*RNA, Viral/genetics
*Pulmonary Alveoli/virology/cytology
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Influenza A virus/genetics
*Influenza A Virus, H3N2 Subtype/genetics
*Lab-On-A-Chip Devices
Influenza, Human/therapy

@article {pmid40910953,
year = {2025},
author = {Feng, D and Guo, J and Yan, J and Chen, J and Ding, L and Zhu, X and Chen, Z and Hu, Y and Zhang, M and Liu, J and Zhu, C and Liu, M and Zhao, C and Zhang, X and Xu, J},
title = {COG6 is an essential host factor for influenza A virus infection.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0136225},
doi = {10.1128/spectrum.01362-25},
pmid = {40910953},
issn = {2165-0497},
support = {2022YFC2604100//National Key Research and Development Program of China/ ; 2023YFC2605602//National Key Research and Development Program of China/ ; 82072273//National Natural Science Foundation of China/ ; },
mesh = {*Influenza A virus/physiology/genetics ; Humans ; Virus Replication ; Golgi Apparatus/metabolism ; Lysosomes/metabolism ; Animals ; *Influenza, Human/virology/metabolism ; *Host-Pathogen Interactions ; *Adaptor Proteins, Vesicular Transport/metabolism/genetics ; Viral Proteins/metabolism ; CRISPR-Cas Systems ; Madin Darby Canine Kidney Cells ; Dogs ; HEK293 Cells ; A549 Cells ; Virus Internalization ; },
abstract = {Influenza A virus (IAV) relies on the host cellular machinery to support its replication. Understanding these host dependencies can inform the development of novel antiviral strategies. In this study, we identified conserved oligomeric Golgi complex subunit 6 (COG6) as a novel host factor critical for IAV replication through a genome-wide clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) knockout screen. Disruption of COG6 significantly impaired viral replication. Mechanistically, COG6 supports IAV replication via two distinct means. First, consistent with the role of the COG complex in Golgi homeostasis, COG6 is required for the proper presentation of surface sialic acids, the primary receptor for IAV entry. Second, COG6 deficiency unexpectedly led to lysosome-dependent degradation of viral proteins. Notably, lysosomal activity was also upregulated in IAV-infected wild-type cells, albeit to a lesser extent than in COG6-deficient cells. Treatment with lysosomal inhibitors rescued viral protein stability in COG6 knockout cells. Protein interaction analysis further demonstrated that COG6-mediated stabilization of viral proteins did not rely on viral protein-COG6 interaction, refuting the hypothesis that COG6 acts as a shield factor to protect viral protein from lysosomal degradation. Moreover, knockout of other COG subunits produced similar antiviral effects, suggesting that an intact COG complex is required for IAV replication. Together, these findings uncover a critical role of the COG complex in regulating IAV replication and highlight a previously unappreciated functional link between the Golgi and lysosomes that could be exploited for treating IAV infections.IMPORTANCEDespite advances in virology, numerous host determinants facilitating influenza A virus (IAV) pathogenesis remain uncharacterized. Our study establishes conserved oligomeric Golgi complex subunit 6 (COG6) as a critical host factor promoting IAV infection through complementary mechanisms: receptor modulation and viral protein stabilization. This represents the first demonstration that the COG complex regulates viral pathogenesis through proteostasis mechanisms, fundamentally expanding our understanding of host-virus interactions at the organelle interface. These findings not only provide new perspectives on viral exploitation of Golgi trafficking networks but also identify potential therapeutic targets against evolving influenza strains.},
}

*Influenza A virus/physiology/genetics
Humans
Virus Replication
Golgi Apparatus/metabolism
Lysosomes/metabolism
Animals
*Influenza, Human/virology/metabolism
*Host-Pathogen Interactions
*Adaptor Proteins, Vesicular Transport/metabolism/genetics
Viral Proteins/metabolism
CRISPR-Cas Systems
Madin Darby Canine Kidney Cells
Dogs
HEK293 Cells
A549 Cells
Virus Internalization

@article {pmid40865226,
year = {2025},
author = {Zhang, X and Zhao, J and Dong, Y and Zhang, C and Hu, X and Wang, S and Chen, Y},
title = {Magnetic relaxation switching biosensor based on CRISPR-mediated cascade reaction for the amplification-free detection of Salmonella.},
journal = {Journal of hazardous materials},
volume = {497},
number = {},
pages = {139664},
doi = {10.1016/j.jhazmat.2025.139664},
pmid = {40865226},
issn = {1873-3336},
mesh = {*Biosensing Techniques/methods ; *Salmonella typhimurium/isolation & purification/genetics ; Alkaline Phosphatase/chemistry ; *CRISPR-Cas Systems ; Food Microbiology ; Limit of Detection ; Magnetite Nanoparticles/chemistry ; DNA, Single-Stranded ; DNA, Bacterial/analysis ; Food Contamination/analysis ; },
abstract = {Sensitive and accurate detection of foodborne pathogens is crucial for preventing foodborne outbreaks and ensuring public health safety, but challenged by extremely low infectious doses of many foodborne pathogens and matrix interference effects. In this study, we developed a magnetic relaxation switching biosensor boosted by CRISPR/Cas12a-mediated enzymatic cascade reaction (CMCR-MRS) for amplification-free detection of Salmonella typhimurium (S. typhimurium). CRISPR/Cas12a was designed to precisely target the pathogen-specific DNA and efficiently cleaved single-stranded DNA (ssDNA) immobilized on the magnetic nanoparticle-alkaline phosphatase (MNP-ALP) probes through trans-cleavage activity. Subsequently, the released ALP facilitated the conversion of paramagnetic Mn(VII) to Mn(II), which resulted in the change of transverse relaxation time (T2), achieving a high signal-to-background ratio with superior biocompatibility and minimal background interference, making them highly advantageous for sensitive detection in complex biological samples. Our assay showed a broad dynamic range from 40 to 10[7] CFU/mL and a limit of detection (LOD) of 10 CFU/mL for S. typhimurium without amplification. Furthermore, it has been successfully validated in real food samples, demonstrating strong consistency (R[2] = 0.989) with the quantitative real-time polymerase chain reaction (qPCR) test. CMCR-MRS can serve as a highly effective and reliable strategy for achieving sensitive and accurate pathogen detection.},
}

*Biosensing Techniques/methods
*Salmonella typhimurium/isolation & purification/genetics
Alkaline Phosphatase/chemistry
*CRISPR-Cas Systems
Food Microbiology
Limit of Detection
Magnetite Nanoparticles/chemistry
DNA, Single-Stranded
DNA, Bacterial/analysis
Food Contamination/analysis

@article {pmid40843899,
year = {2025},
author = {Stuible, M and Alpuche-Lazcano, SP and Gervais, C and Ouimet, M and Lippens, J and Pagé, M and Morasse, A and Moraitis, AN and Durocher, Y},
title = {Endogenous Retrovirus-Like Particle-Deficient CHO Cells Can be Generated by CRISPR or shRNA and Enriched Based on Cell-Surface Expression of Retroviral Envelope Protein.},
journal = {Biotechnology and bioengineering},
volume = {122},
number = {11},
pages = {3192-3204},
doi = {10.1002/bit.70043},
pmid = {40843899},
issn = {1097-0290},
support = {//The authors received no specific funding for this work./ ; },
mesh = {CHO Cells ; Cricetulus ; Animals ; *Viral Envelope Proteins/genetics/metabolism ; *RNA, Small Interfering/genetics/metabolism ; *Endogenous Retroviruses/genetics ; *CRISPR-Cas Systems ; Cricetinae ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Despite evidence that they are not functional or infective, retrovirus-like particles (RVLPs), originating from endogenous proviral sequences in Chinese hamster ovary (CHO) cells, present a safety risk for biotherapeutics manufactured using this cell line due to their resemblance to other mammalian leukemia viruses. Here, we demonstrate that CRISPR- and shRNA-based cell engineering strategies can be used to disrupt RVLP production by targeting the RVLP nucleotide sequences. Additionally, specific antibodies were generated to monitor RVLP protein expression, including RVLP envelope (Env) protein localized on the surface of CHO cells, greatly facilitating selection of RVLP-deficient clones. These modified CHO cells showed reduced RVLP production while maintaining or enhancing the ability to produce recombinant virus-like particles (VLPs), highlighting their potential application in biomanufacturing, especially for complex biologics that are incompatible with standard RVLP mitigation procedures, namely viral inactivation and nanofiltration.},
}

CHO Cells
Cricetulus
Animals
*Viral Envelope Proteins/genetics/metabolism
*RNA, Small Interfering/genetics/metabolism
*Endogenous Retroviruses/genetics
*CRISPR-Cas Systems
Cricetinae
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40829398,
year = {2025},
author = {Song, D and Xu, C and Sang, P and Liu, Y and Huang, X},
title = {Rapid and contamination-free detection of cucumber green mottle mosaic virus as a viral indicator in wastewater via UDG-RT-LAMP combined with CRISPR/Cas12a.},
journal = {Journal of hazardous materials},
volume = {497},
number = {},
pages = {139571},
doi = {10.1016/j.jhazmat.2025.139571},
pmid = {40829398},
issn = {1873-3336},
mesh = {*Wastewater/virology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Tobamovirus/isolation & purification/genetics ; Uracil-DNA Glycosidase/genetics ; Biosensing Techniques ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; Molecular Diagnostic Techniques ; },
abstract = {The removal of viruses by wastewater treatment plants plays a pivotal role in ensuring water environment safety, where precise evaluation of elimination efficiency is essential for controlling viral dissemination. Current bacterial indicators exhibit limited correlations with virological safety parameters, whereas conventional viral detection methods face practical constraints such as high instrumentation requirements and long detection cycles. To overcome these limitations, this study presents an on-site detection method for cucumber green mottle mosaic virus (CGMMV) in wastewater as a viral indicator, integrating uracil-DNA glycosylase (UDG)-reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 12a)-mediated biosensing system. The developed method achieved effective prevention of aerosol contamination from residual amplicons via the dUTP-UDG system, rapid amplification via RT-LAMP, and improved sensitivity and visualization by CRISPR/Cas12a-mediated biosensing system. The potential of CGMMV as a viral indicator in wastewater treatment process was demonstrated, and the rapid detection was realized using the proposed method. This integrated approach achieves sensitive CGMMV detection (limit of detection of 1.13 copies/μL) within 35 min, demonstrating field applicability through equipment independence, contamination resistance, and rapid operation. The proposed assay offers a promising tool for rapid viral monitoring in wastewater treatment system for resource-limited settings.},
}

*Wastewater/virology
CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Tobamovirus/isolation & purification/genetics
Uracil-DNA Glycosidase/genetics
Biosensing Techniques
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases
Molecular Diagnostic Techniques

@article {pmid40810567,
year = {2025},
author = {Ji, C and Ru, L and Han, T and Mai, G and Zheng, L and Jiang, Y},
title = {VGRCOT: a one-tube visual detection method for group B Streptococcus combining RPA and CRISPR/Cas12a for point-of-care testing in reproductive health.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0139525},
doi = {10.1128/spectrum.01395-25},
pmid = {40810567},
issn = {2165-0497},
support = {2025ZNSFSC1561//Natural Science Foundation of Sichuan Province/ ; 2024SZY001//Key Research and Developmet Guidance Projects of Deyang City/ ; },
mesh = {Humans ; *Streptococcal Infections/diagnosis/microbiology ; *Streptococcus agalactiae/genetics/isolation & purification ; *Point-of-Care Testing ; *CRISPR-Cas Systems ; Female ; Pregnancy ; Sensitivity and Specificity ; Pregnancy Complications, Infectious/diagnosis/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Group B Streptococcus (GBS) is a significant pathogen that causes perinatal infections, seriously threatening the health of pregnant women and newborns. Prophylactic antibiotic treatment for pregnant women who screen positive for GBS can notably reduce the incidence and fatality of neonatal infections. Herein, we developed a visual nucleic acid method for GBS that integrates RPA and CRISPR/Cas12a in a one-tube setup, termed VGRCOT. The VGRCOT method achieved one-tube detection by adding the appropriate reagents to the bottom and lid of the EP tube, respectively. By rigorous optimization of ssDNA-FQ reporter concentration, crRNA concentration, RPA reaction time, and CRISPR/Cas12a cleavage time, VGRCOT can exhibit fluorescence under ultraviolet light, enabling visual detection. Under optimal conditions, VGRCOT has a satisfactory selectivity, and the detection limit was determined as 10[1] copies/reaction. Finally, VGRCOT also showed good performance comparable to qPCR in the actual detection of clinical specimens. Due to its ease of operation and convenient signal acquisition, VGRCOT shows promise for point-of-care testing in reproductive health.IMPORTANCEThis study presents a convenient, sensitive, and accurate visual detection method (VGRCOT) for GBS, combining RPA and CRISPR/Cas12a in a single reaction vessel. Through optimization of experimental conditions, VGRCOT enables detection within 60 min, with a minimum detection limit of 10[1] copies per reaction. VGRCOT offers several advantages by adding the appropriate reagents to the bottom and lid of the EP tube. The one-tube visualization method effectively prevents aerosol contamination, simplifies procedures, and enables visual detection without complex instruments, making it ideal for resource-limited environments. Additionally, its editable crRNA and the use of commonly available laboratory reagents allow for easy reprogramming to detect various pathogens, supporting scalable and low-cost batch production.},
}

Humans
*Streptococcal Infections/diagnosis/microbiology
*Streptococcus agalactiae/genetics/isolation & purification
*Point-of-Care Testing
*CRISPR-Cas Systems
Female
Pregnancy
Sensitivity and Specificity
Pregnancy Complications, Infectious/diagnosis/microbiology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40645879,
year = {2025},
author = {Liang, Y and Tong, S and Zhang, J and Tan, GY and Zhang, L and Lee, SY and Tong, Y},
title = {Expanding horizons of CRISPR applications beyond genome editing.},
journal = {Trends in genetics : TIG},
volume = {41},
number = {10},
pages = {934-953},
doi = {10.1016/j.tig.2025.06.003},
pmid = {40645879},
issn = {0168-9525},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Biosensing Techniques ; Single-Cell Analysis ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technologies have rapidly evolved beyond genome editing, transforming fields such as molecular diagnostics, biosensing, transcriptional regulation, molecular imaging, protein interaction mapping, and single-cell analysis. Emerging CRISPR-based diagnostics harness the collateral cleavage activity of CRISPR-associated (Cas) enzymes for rapid nucleic acid detection. Advanced biosensors extend CRISPR's capabilities to detect ions, metabolites, and proteins by integrating synthetic biology components. Catalytically inactive Cas proteins enable precise gene regulation and live-cell imaging of nucleic acids, whereas CRISPR-guided proximity labeling has revolutionized the mapping of biomolecular interactions. Recent single-cell CRISPR screens provide unprecedented resolution of cellular heterogeneity. Future research will focus on overcoming current limitations. The integration of CRISPR technologies with artificial intelligence (AI), spatial omics, and microfluidics is expected to further amplify their impact.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Biosensing Techniques
Single-Cell Analysis

@article {pmid40032999,
year = {2025},
author = {Sahu, SU and Castro, M and Muldoon, JJ and Asija, K and Wyman, SK and Krishnappa, N and de Oñate, L and Eyquem, J and Nguyen, DN and Wilson, RC},
title = {Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.},
journal = {Nature protocols},
volume = {20},
number = {10},
pages = {2735-2770},
pmid = {40032999},
issn = {1750-2799},
support = {K08 AI153767/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; K08AI153767//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; UG3AI150552//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; L40AI140341//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Peptides/chemistry ; T-Lymphocytes/metabolism ; Cells, Cultured ; },
abstract = {Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in and base editing. The protocol involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) homology-directed repair template (HDRT) DNA may be included. In contrast to electroporation, PERC is appealing because it needs no dedicated hardware and has less impact on cell phenotype and viability. Because of the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Editing efficiencies can surpass 90% when using either Cas9 or Cas12a in primary T cells or HSPCs. After 3 h dedicated to reagent preparation, the PERC delivery step can be completed in 1 h, with the associated cell culture steps taking 3-7 d total. Because the protocol calls for only three readily available reagents (protein, RNA and peptide) and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes the protocol appealing for rapid deployment in research and clinical settings.},
}

Humans
*Hematopoietic Stem Cells/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/metabolism
*Peptides/chemistry
T-Lymphocytes/metabolism
Cells, Cultured

@article {pmid41046988,
year = {2025},
author = {Chen, Y and Qi, ZD and Ji, R and Shi, N and Chen, H and Wei, DX},
title = {Synthetic biology for scalable production of medical polyhydroxyalkanoates: Advances and applications.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108722},
doi = {10.1016/j.biotechadv.2025.108722},
pmid = {41046988},
issn = {1873-1899},
abstract = {Polyhydroxyalkanoates (PHAs), characterized by their biodegradability and biocompatibility, present a promising, sustainable alternative to conventional synthetic polymers for biomedical applications. This study highlights the diversity of PHA monomers and structures, controllable biodegradability, and excellent biocompatibility, emphasizing their suitability for tissue engineering (bone, skin, cardiovascular, oral), anti-hair loss treatments, and drug delivery systems. Significant advancements in synthetic biology, encompassing CRISPR/Cas genome editing, promoter engineering, ribosome binding site optimization, metabolic pathway fine-tuning, and morphology engineering, have led to substantial improvements in PHA production efficiency and a reduction in associated costs. The adoption of next-generation industrial biotechnology (NGIB) using halophiles further enhances economic viability and simplifies the production process. The current commercial landscape and the future prospects of medical-grade PHAs, poised to become mainstream biodegradable materials, are also critically discussed.},
}

@article {pmid41046905,
year = {2025},
author = {Jaballah, SA and Ali, LM and Jehad, MA and Akhlaq, S and Rizvi, TA and Mustafa, F},
title = {Retroviral Vector Technology for Gene Therapy: History, Current Landscape, and Future Prospects.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {169473},
doi = {10.1016/j.jmb.2025.169473},
pmid = {41046905},
issn = {1089-8638},
abstract = {The concept of gene therapy and its practice has been prevalent for over five decades. The first successful retroviral vector-based gene therapy trial took place ∼35 years ago, followed by several setbacks. However, recent years have seen a surge in successes, offering new hope to patients with genetic and other disorders once deemed untreatable. Over the past decade, rapid advancements in molecular biology have led to the development of safer and more effective gene therapy strategies with various gene delivery systems now in use. Among these, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses are the most widely employed in both research and clinical settings. This is due to their natural efficiency in delivering genetic material into target cells. Among these viral vectors, retroviruses stand out for their unique ability to reverse-transcribe and integrate their genetic material into the host genome, ensuring stable and long-term gene expression. This review highlights advances in retroviral vector development, examining both their therapeutic potential and associated challenges. It also explores strategies for vector production, including transient and stable systems tailored to meet clinical and regulatory demands. Significant progress is discussed in mitigating insertional mutagenesis and vector silencing. As a result, next-generation retroviral vectors with improved safety and efficacy have made it past regulatory-approval and are commercially available. Current innovations include replication-competent, non-integrating, integration-re-targeted, and hybrid CRISPR/Cas-expressing retroviral vectors undergoing pre-clinical and clinical investigations. This reflects a new era in gene therapy, with retroviral vectors reimagined for greater precision, control, and therapeutic impact.},
}

@article {pmid41045024,
year = {2025},
author = {Thomson, G and Mermaz, B and Sagawa, CHD and Lin, CI and Tachev, M and Joly, V and Irish, VF and Jacob, Y},
title = {Enzymatic depletion of transposable elements in sequencing libraries and its application for genotyping multiplexed CRISPR-edited plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70501},
doi = {10.1111/tpj.70501},
pmid = {41045024},
issn = {1365-313X},
support = {2023-70029-41277//U.S. Department of Agriculture/ ; },
mesh = {*DNA Transposable Elements/genetics ; Zea mays/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; DNA Methylation ; *Citrus/genetics ; Gene Library ; CRISPR-Cas Systems/genetics ; *Genotyping Techniques/methods ; Genotype ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; },
abstract = {Whole-genome sequencing has become a common strategy to genotype individual plants of interest. Although a limited number of genomic regions usually need to be surveyed with this strategy, excess sequencing information is almost always generated at an appreciable financial cost. Repetitive sequences (e.g., transposons), which can account for more than 80% of the genome of some plants, are often not required in these genotyping projects. Therefore, strategies that enrich DNA coding for the protein-coding genes prior to sequencing can lower the cost to obtain sufficient sequence information. Here, we present the development and application of methylation-sensitive reduced representation sequencing (MsRR-Seq), which relies on the cytosine methylation-sensitive restriction enzyme MspJI to deplete constitutive heterochromatic DNA before library construction. By applying MsRR-Seq to citrus and maize, we show that protein-coding genes can be enriched in sequencing datasets. We then describe the application of MsRR-Seq to facilitate the identification of complex mutants from populations of citrus plants resulting from multiplex CRISPR/Cas9 editing of four genes. Overall, this work demonstrates an easy and low-cost method to enrich non-repetitive DNA in high-throughput sequencing libraries, an approach that is especially useful for large plant genomes with an excessively high proportion of methylated repetitive sequences.},
}

*DNA Transposable Elements/genetics
Zea mays/genetics
*Gene Editing/methods
Genome, Plant/genetics
DNA Methylation
*Citrus/genetics
Gene Library
CRISPR-Cas Systems/genetics
*Genotyping Techniques/methods
Genotype
High-Throughput Nucleotide Sequencing/methods
Sequence Analysis, DNA/methods

@article {pmid41044600,
year = {2025},
author = {Conery, M and Pippin, JA and Wagley, Y and Trang, K and Pahl, MC and Villani, DA and Favazzo, LJ and Ackert-Bicknell, CL and Zuscik, MJ and Katsevich, E and Wells, AD and Zemel, BS and Voight, BF and Hankenson, KD and Chesi, A and Grant, SFA},
title = {GWAS-informed data integration and non-coding CRISPRi screen illuminate genetic etiology of bone mineral density.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {331},
pmid = {41044600},
issn = {1474-760X},
support = {Gates Grubstake Award//University of Colorado/ ; DMS 2113072//National Science Foundation, United States/ ; R01DK122586//National Institute of Diabetes and Digestive and Kidney Diseases,United States/ ; UM1 DK126194/DK/NIDDK NIH HHS/United States ; R01AI154773//National Institute of Allergy and Infectious Diseases/ ; R01 HD100406/HD/NICHD NIH HHS/United States ; UL1 TR001878/TR/NCATS NIH HHS/United States ; Henry Ruppenthal Family Professorship for Bioengineering and Orthopaedic Surgery//University of Michigan/ ; R01 AG072705/AG/NIA NIH HHS/United States ; Daniel B. Burke Endowed Chair for Diabetes Research//Children's Hospital of Philadelphia/ ; },
mesh = {Humans ; *Bone Density/genetics ; *Genome-Wide Association Study ; Osteoblasts/metabolism ; *CRISPR-Cas Systems ; Single-Cell Analysis ; Quantitative Trait Loci ; },
abstract = {BACKGROUND: Over 1100 independent signals have been identified with genome-wide association studies (GWAS) for bone mineral density (BMD), a key risk factor for mortality-increasing fragility fractures; however, the effector gene(s) for most remain unknown.

RESULTS: We execute a CRISPRi screen in human fetal osteoblasts (hFOBs) with single-cell RNA-seq read-out for 89 non-coding elements predicted to regulate osteoblast gene expression at BMD GWAS loci. The BMD relevance of hFOBs is supported by heritability enrichment from stratified LD-score regression involving 98 cell types grouped into 15 tissues. Twenty-three genes show perturbation in the screen, with four (ARID5B, CC2D1B, EIF4G2, and NCOA3) exhibiting consistent effects upon siRNA knockdown on three measures of osteoblast maturation and mineralization. Lastly, additional heritability enrichments, genetic correlations, and multi-trait fine-mapping unexpectedly reveal that many BMD GWAS signals are pleiotropic and likely mediate their effects via non-bone tissues.

CONCLUSIONS: Our results provide a roadmap for how single-cell CRISPRi screens may be applied to the challenging task of resolving effector gene identities at all BMD GWAS loci. Extending our CRISPRi screening approach to other tissues could play a key role in fully elucidating the etiology of BMD.},
}

Humans
*Bone Density/genetics
*Genome-Wide Association Study
Osteoblasts/metabolism
*CRISPR-Cas Systems
Single-Cell Analysis
Quantitative Trait Loci

@article {pmid41041610,
year = {2025},
author = {de Mello Fiallos, N and Irfan, M and Solbiati, J and R Walker, A and Frias-Lopez, J and Gibson, FC},
title = {CRISPR cas7 influences the host-pathogen interaction of Porphyromonas gingivalis.},
journal = {Journal of oral microbiology},
volume = {17},
number = {1},
pages = {2561790},
pmid = {41041610},
issn = {2000-2297},
abstract = {INTRODUCTION: Porphyromonas gingivalis, a Gram-negative anaerobe, is a key contributor to periodontal disease. Emerging evidence suggests a role for the P. gingivalis CRISPR-Cas system in disease progression, although the specific roles of its components remain unclear.

OBJECTIVES: Here we investigate the role of cas7, a Class 1 type I-B CRISPR-Cas system component, in P. gingivalis physiology and host interaction.

METHODS: We compared P. gingivalis wild-type and ∆cas7 strains for growth, biofilm formation, oxidative stress resistance, and hemagglutination. Host interactions were assessed using THP-1 macrophage-like cells to evaluate intracellular survival and cytokine response. Dual RNA-seq enabled host and microbe transcriptomic profiling during cellular infection, and Galleria mellonella was used to assess virulence.

RESULTS: The ∆cas7 mutant showed similar planktonic growth and biofilm formation compared to wild-type but was more sensitive to oxidative stress and had reduced hemagglutination. Although intracellular survival was unaffected, ∆cas7 altered the host cytokine production profile. Transcriptomic analysis revealed differential gene expression linked to oxidative stress and disease progression. In vivo, ∆cas7 infection led to a trend of increased larval mortality.

CONCLUSION: These findings reveal a previously unrecognized role for cas7 in modulating P. gingivalis virulence, offering new insights into CRISPR-Cas system functions in bacterial pathogenesis.},
}

@article {pmid40974079,
year = {2025},
author = {Mariki, A and Kohlmeier, KA and Mousavi, SM and Shabani, M},
title = {CRISPR and Myelin regeneration: a systematic review of applications in demyelinating CNS Disorders, with a focus on MS.},
journal = {Regenerative medicine},
volume = {20},
number = {9},
pages = {431-443},
doi = {10.1080/17460751.2025.2561451},
pmid = {40974079},
issn = {1746-076X},
mesh = {Humans ; Animals ; *Myelin Sheath/metabolism ; *Multiple Sclerosis/therapy/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Demyelinating Diseases/therapy/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Regeneration/genetics ; },
abstract = {AIMS: Current treatments for demyelinating disorders focus on slowing progression but fail to repair damaged myelin. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -based technology has the potential to address key challenges in myelin repair by targeting genetic dysfunctions, modulating immune responses, and promoting oligodendrocyte differentiation. This systematic review aimed to evaluate CRISPR applications for myelin regeneration.

METHODS: A comprehensive search of PubMed, Scopus, and other databases identified 48 studies. The included studies employed CRISPR in diverse experimental models, targeting genes associated with immune regulation and astrocyte activity, as well as correcting RNA splicing dysfunctions linked to neurodegeneration.

RESULTS: CRISPR-edited stem cells showed significant potential in promoting myelin regeneration, with enhanced functional recovery in animal models of multiple sclerosis (MS). While most research focused on MS, promising applications were also observed in neuromyelitis optica spectrum disorder (NMOSD), such as reducing astrocytic damage via AQP4 targeting, and in progressive multifocal leukoencephalopathy (PML), where CRISPR disrupted JC polyomavirus replication.

CONCLUSIONS: Despite its promise, challenges remain. Future research should prioritize optimizing CRISPR delivery systems, expanding applications to underexplored disorders, and conducting long-term safety assessments. Early results are encouraging, but further studies are essential to translate preclinical success into clinical therapies.},
}

Humans
Animals
*Myelin Sheath/metabolism
*Multiple Sclerosis/therapy/genetics
*CRISPR-Cas Systems
Gene Editing
*Demyelinating Diseases/therapy/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Regeneration/genetics

@article {pmid40972450,
year = {2026},
author = {Eom, KH and Yum, SY and Gim, GM and Kim, YC and Moon, B and Jang, G},
title = {SpCas9-mediated gene editing in bovine embryo via single adeno-associated virus infection using a novel micro-sized promoter.},
journal = {Theriogenology},
volume = {249},
number = {},
pages = {117676},
doi = {10.1016/j.theriogenology.2025.117676},
pmid = {40972450},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology/genetics ; *Gene Editing/veterinary/methods ; *Promoter Regions, Genetic ; *Dependovirus/genetics ; Genetic Vectors ; Embryo, Mammalian ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; },
abstract = {Genome editing in livestock offers practical solutions to address challenges related to land use, climate change, and food production. However, conventional delivery methods such as electroporation and microinjection impose physical stress on embryos, limiting scalability. This study aimed to develop a simplified, non-invasive, and scalable genome editing system for bovine embryos by designing an all-in-one adeno-associated virus (AAV) vector. A novel micro-sized promoter (50 bp), derived from the core regulatory region upstream of the bovine MSTN gene, was constructed to enable expression of Streptococcus pyogenes Cas9 (spCas9) within the AAV packaging limit (∼4.7 kb). This promoter was incorporated into an AAV cassette containing spCas9, a polyadenylation signal, a U6 promoter, and a single-guide RNA (sgRNA) targeting the bovine ALB gene. After confirming editing activity in bovine fibroblasts, the AAV6 vector was added directly to in vitro fertilization (IVF) cultures without physical manipulation. Genome editing was successfully induced, with insertion/deletion (indel) mutations detected in 33.8 ± 23.2 % of the blastocysts. Although blastocyst development was moderately reduced, gene editing was achieved without invasive techniques. These results demonstrate that a micro-promoter-based AAV system can support spCas9-mediated genome editing in bovine embryos through a single-vector infection strategy. The system presents a promising platform for producing gene-edited livestock and may contribute to more efficient and less labor-intensive applications in animal biotechnology.},
}

Animals
Cattle/embryology/genetics
*Gene Editing/veterinary/methods
*Promoter Regions, Genetic
*Dependovirus/genetics
Genetic Vectors
Embryo, Mammalian
*CRISPR-Associated Protein 9/genetics/metabolism
CRISPR-Cas Systems
Fertilization in Vitro/veterinary

@article {pmid40650655,
year = {2025},
author = {Li, Y and Zhang, Y and Li, C and Chen, G and Muhammad, P and Yao, Y and Gao, L and Liu, Z and Wang, Y},
title = {Advanced Cancer Immunotherapy via SMARCAL1 Blockade Using a Glucose-Responsive CRISPR Nanovaccine.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {37},
pages = {e02929},
doi = {10.1002/advs.202502929},
pmid = {40650655},
issn = {2198-3844},
support = {81901684//National Natural Science Foundation of China/ ; },
mesh = {*Immunotherapy/methods ; Animals ; Mice ; *Glucose/metabolism ; Humans ; *Cancer Vaccines/immunology/genetics ; *Neoplasms/therapy/immunology ; CRISPR-Cas Systems/genetics ; Glucose Oxidase ; Gene Editing/methods ; Membrane Proteins/metabolism ; Nanovaccines ; },
abstract = {Cancer immunotherapy that activates the stimulator of interferon genes (STING) signaling pathway to resist tumors has recently attracted considerable attention. However, STING activation can induce opposing interferon functions that contribute to T-cell exhaustion via programmed death-ligand 1 (PD-L1). In particular, effectively using the immune system to combat tumors remains a substantial challenge due to tumor immunosuppressive factors such as SMARCAL1. Here, a glucose-responsive CRISPR nanovaccine is developed for enhancing STING signaling while inhibiting interferon-mediated immunosuppressive feedback. The formulation encapsulates a bimetallic zeolitic imidazolate framework with glucose oxidase (GOx) and CRISPR-mediated SMARCAL1 gene-editing plasmids. The dual enzyme-driven cascade reactions of peroxidase and GOx generate reactive oxygen species (ROS) and gluconic acid, which release and activate the genome-editing system. The silencing of SMARCAL1 enhances STING activity and inhibits PD-L1 expression, resulting in the termination of PD-L1-mediated opposing functions of interferon. Zinc ions and double-stranded DNA formed via ROS further activate the STING pathway, effectively inducing dendritic cell maturation and immune system activation. This is a critical report of in situ CRISPR nanovaccination driven by dual enzymes. The work highlights the potential of glucose-responsive CRISPR nanovaccination in bolstering antitumor immunity and extends the implementation of gene editing in cancer immunotherapy.},
}

*Immunotherapy/methods
Animals
Mice
*Glucose/metabolism
Humans
*Cancer Vaccines/immunology/genetics
*Neoplasms/therapy/immunology
CRISPR-Cas Systems/genetics
Glucose Oxidase
Gene Editing/methods
Membrane Proteins/metabolism
Nanovaccines

@article {pmid40642954,
year = {2025},
author = {Fu, X and Wang, N and Li, L and Qiao, D and Qi, X and Liu, C and Gao, Z and Xie, C and Zhu, J},
title = {Development of cytosine and adenine base editors for maize precision breeding.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {10},
pages = {2731-2743},
doi = {10.1111/jipb.13964},
pmid = {40642954},
issn = {1744-7909},
support = {No. 2023YFD1202901//National Key Research and Development Program of China Stem Cell and Translational Research/ ; 241111112300//Henan Province key research and development project/ ; //Xinjiang "Leading the Charge with Open Competition" project/ ; No. 2022ZD04006//Science and Technology Innovation 2030 Major projects/ ; },
mesh = {*Zea mays/genetics/drug effects ; *Gene Editing/methods ; *Adenine/metabolism ; *Cytosine/metabolism ; *Plant Breeding/methods ; Plants, Genetically Modified ; Mutation/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Base editing technologies can improve crops, but their efficiency in maize remains suboptimal. This study attempts to overcome these limitations by examining optimized cytosine and adenine base editors (CBEs and ABEs), namely evoAPOBEC1, evoFERNY, evoCDA1, TadA8.20, and TadA8e, for precise genome editing in transient and stable expression maize cells. Employing a seed fluorescence reporter (SFR) system for rapid screening of BE transformants and transgene-free progenies, we enhanced editing efficiencies and heritability. Notably, TadA8.20 and evoCDA1 attained multiplexed editing efficiencies of up to 100.0% and 79.0% at the tested loci, respectively, with some homozygous and bi-allelic mutants exceeding 72.4% and 73.7%. Precise editing of ZmACC1/2 (acetyl-CoA carboxylase) improved herbicide resistance, with ZmACC2 mutants displaying improved performance. This study advances crop genetic engineering by facilitating robust, multi-locus modifications without altered agronomic performance, enhancing herbicide tolerance in maize. The successful utilization of these BE is a significant step forward in agricultural biotechnology and precision breeding.},
}

*Zea mays/genetics/drug effects
*Gene Editing/methods
*Adenine/metabolism
*Cytosine/metabolism
*Plant Breeding/methods
Plants, Genetically Modified
Mutation/genetics
CRISPR-Cas Systems/genetics

@article {pmid39994413,
year = {2025},
author = {Oliynyk, RT and Church, GM},
title = {Circular Vectors as an efficient, fully synthetic, cell-free approach for preparing small circular DNA as a plasmid substitute for guide RNA expression in CRISPR-Cas9 genome editing.},
journal = {Nature protocols},
volume = {20},
number = {10},
pages = {2942-2959},
pmid = {39994413},
issn = {1750-2799},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Plasmids/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *DNA, Circular/genetics ; Cell-Free System ; Humans ; },
abstract = {Robust expression of guide RNA (gRNA) is essential for successful implementation of CRISPR-Cas9 genome-editing methods. The gRNA components, such as an RNA polymerase promoter followed by the gRNA coding sequence and an RNA polymerase terminator sequence, and the Cas9 protein are expressed either via an all-in-one plasmid or separate dedicated plasmids. The preparation of such plasmids involves a laborious multi-day process of DNA assembly, bacterial cloning, validation, purification and sequencing. Our Circular Vector (CV) protocol introduces an efficient, fully synthetic, cell-free approach for preparing gRNA expression templates suitable for transfection, marking a significant advancement over traditional plasmid-based approaches. This protocol consists of the circularization and purification of linear double-stranded DNA (dsDNA) containing gRNA expression elements into compact, bacterial-backbone-free circular DNA expression vectors in as little as 3 h. We provide a guide to the design of the dsDNA template coding for gRNA elements for CRISPR-Cas9 base and prime editing, along with step-by-step instructions for the efficient preparation of gRNA-expressing CVs. In addition to rapid preparation, CVs created via this protocol offer several key advantages: a compact size, absence of a bacterial backbone, absence of bacterial endotoxins and no contamination by bacterial RNA or DNA fragments. These features make gRNA-expressing CVs a superior choice over plasmid-based gRNA expression templates.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
*Plasmids/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Vectors/genetics
*DNA, Circular/genetics
Cell-Free System
Humans

@article {pmid41039221,
year = {2025},
author = {Ilmi, AFN and Kaewsapsak, P and Rotcheewaphan, S},
title = {Repression of mab_1999 impairs growth and alters cellular morphology of Mycobacterium abscessus.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {599},
pmid = {41039221},
issn = {1471-2180},
mesh = {*Mycobacterium abscessus/growth & development/genetics/drug effects/cytology/metabolism ; *Bacterial Proteins/genetics/metabolism ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Cell Division/genetics ; Gene Expression Regulation, Bacterial ; Mycobacterium smegmatis/genetics ; Gene Knockdown Techniques ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Cell division is essential for bacterial survival and represents a promising target for the development of novel antibiotics, particularly in mycobacteria. The role of the division protein FtsL in Mycobacterium abscessus remains poorly understood. This study investigated the effects of MAB_1999, a predicted homolog of FtsL, on the growth and cell division of M. abscessus.

METHOD: To investigate the function of mab_1999, a knockdown mutant was generated via CRISPR interference (CRISPRi). The phenotypic impact of mab_1999 suppression was evaluated, with a focus on its effects on M. abscessus growth, cellular morphology, and antibiotic susceptibility.

RESULTS: The putative homolog of FtsL in M. abscessus (MAB_1999) shares 54% amino acid sequence identity with FtsL from M. smegmatis (MSMEG_4234). CRISPRi-mediated repression of mab_1999 expression resulted in cell elongation and growth defects, although complete growth arrest was not observed. Furthermore, reduced mab_1999 expression increased the susceptibility of M. abscessus to β-lactam antibiotics, including ceftriaxone and imipenem.

CONCLUSIONS: Our findings suggest that mab_1999 is involved in cell division and cell wall integrity in M. abscessus. However, further investigation is necessary to confirm its identity as FtsL and to fully elucidate its role in the cell division process and cell wall synthesis.},
}

*Mycobacterium abscessus/growth & development/genetics/drug effects/cytology/metabolism
*Bacterial Proteins/genetics/metabolism
Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests
Cell Division/genetics
Gene Expression Regulation, Bacterial
Mycobacterium smegmatis/genetics
Gene Knockdown Techniques
CRISPR-Cas Systems

@article {pmid41039127,
year = {2025},
author = {Safenkova, IV and Kamionskaya, MV and Ivanov, AV and Zherdev, AV and Dzantiev, BB},
title = {A novel tripod probe and lateral flow test to improve CRISPR/Cas12a assay: benefits of branched probe based on trebler phosphoramidite modification.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {711},
pmid = {41039127},
issn = {1436-5073},
support = {23-46-10011//Russian Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA Probes/chemistry ; *Biosensing Techniques/methods ; *Organophosphorus Compounds/chemistry ; Fluorescent Dyes/chemistry ; Limit of Detection ; Gold/chemistry ; *CRISPR-Associated Proteins/metabolism ; Metal Nanoparticles/chemistry ; *Bacterial Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; },
abstract = {CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of the probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 min. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin-streptavidin capture within 5 min. The CRISPR/Cas12a-tripod-LFT strategy demonstrated excellent sensitivity without preamplification, with a detection Limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.},
}

*CRISPR-Cas Systems
*DNA Probes/chemistry
*Biosensing Techniques/methods
*Organophosphorus Compounds/chemistry
Fluorescent Dyes/chemistry
Limit of Detection
Gold/chemistry
*CRISPR-Associated Proteins/metabolism
Metal Nanoparticles/chemistry
*Bacterial Proteins/metabolism
*Endodeoxyribonucleases/metabolism

@article {pmid40847019,
year = {2025},
author = {Ramani, B and Rose, IVL and Teyssier, N and Pan, A and Danner-Bocks, S and Sanghal, T and Yadanar, L and Tian, R and Ma, K and Palop, JJ and Kampmann, M},
title = {CRISPR screening by AAV episome-sequencing (CrAAVe-seq): a scalable cell-type-specific in vivo platform uncovers neuronal essential genes.},
journal = {Nature neuroscience},
volume = {28},
number = {10},
pages = {2129-2140},
pmid = {40847019},
issn = {1546-1726},
support = {R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; EDUC4-12812//California Institute for Regenerative Medicine (CIRM)/ ; UCSF Hillblom/BARI Graduate Fellowship Award//Larry L. Hillblom Foundation (Larry L. Hillblom Foundation, Inc.)/ ; R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Dependovirus/genetics ; *Neurons/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; *Genes, Essential/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice, Inbred C57BL ; *Genetic Testing/methods ; Humans ; },
abstract = {There is a substantial need for scalable CRISPR-based genetic screening methods that can be applied in mammalian tissues in vivo while enabling cell-type-specific analysis. Here we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in mouse tissues. We used this approach to screen two large sgRNA libraries, which collectively target over 5,000 genes, in mouse brains and uncovered genes essential for neuronal survival, of which we validated Rabggta and Hspa5. We highlight the reproducibility and scalability of the platform and show that it is sufficiently sensitive for screening in a restricted subset of neurons. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and coinfection rate on screen performance to establish general guidelines for large-scale in vivo screens.},
}

Animals
*Dependovirus/genetics
*Neurons/metabolism
Mice
*CRISPR-Cas Systems/genetics
*Genes, Essential/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mice, Inbred C57BL
*Genetic Testing/methods
Humans

@article {pmid41038958,
year = {2025},
author = {Chen, J and Huang, H and Chen, C and Xia, G and Huang, H and Xiong, Y and Luo, P and Chen, Y and Li, J and Wen, L and Li, L and Lin, J and Xu, G and Ji, C and Tian, W and Zhou, J and Wei, P and Shen, C and Wang, X},
title = {ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1414},
pmid = {41038958},
issn = {2399-3642},
support = {8217051361//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Receptors, LDL/metabolism/genetics ; Animals ; *Liver/metabolism ; Mice ; *Cholesterol, LDL/blood/metabolism ; *Multidrug Resistance-Associated Proteins/metabolism/genetics ; Humans ; Male ; Proprotein Convertase 9/metabolism/genetics ; Hepatocytes/metabolism ; Mice, Inbred C57BL ; Hypercholesterolemia/metabolism/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Hep G2 Cells ; },
abstract = {Low expression level of low-density lipoprotein receptor (LDLR) in hepatocytes leads to hypercholesterolemia and eventually contributes to atherosclerotic cardiovascular disease (ASCVD). Here, we report that inhibition of hepatocyte ABCC4, identified as a top hit from large-scale CRISPR/Cas9 screens, significantly increases hepatic LDLR abundance and enhances LDL cholesterol clearance. As a hepatic transporter for cAMP efflux, ABCC4 silencing alters its intracellular distribution and activates the downstream Epac2/Rap1a signaling pathway, which ultimately blocks PCSK9 protein expression, thereby preventing lysosomal degradation of LDLR. Furthermore, in both male mice and cell models, we demonstrate that liver-specific disruption and pharmacological inhibition of ABCC4 elevate hepatic plasma membrane LDLR levels and reduce plasma LDL cholesterol through ABCC4-cAMP-PCSK9 pathway. Collectively, our genome-wide CRISPR screening offers a valuable resource for identifying LDLR modifiers, providing potential insights for therapeutic strategies in hypercholesterolemia and atherosclerosis.},
}

*Receptors, LDL/metabolism/genetics
Animals
*Liver/metabolism
Mice
*Cholesterol, LDL/blood/metabolism
*Multidrug Resistance-Associated Proteins/metabolism/genetics
Humans
Male
Proprotein Convertase 9/metabolism/genetics
Hepatocytes/metabolism
Mice, Inbred C57BL
Hypercholesterolemia/metabolism/genetics
CRISPR-Cas Systems
Signal Transduction
Hep G2 Cells

@article {pmid41038616,
year = {2025},
author = {Aliciaslan, M and Erbasan, E and Erendor, F and Sanlioglu, S},
title = {Prime Editing: The Next Frontier in Precision Gene Therapy.},
journal = {The journal of gene medicine},
volume = {27},
number = {10},
pages = {e70040},
doi = {10.1002/jgm.70040},
pmid = {41038616},
issn = {1521-2254},
support = {//Akdeniz Üniversitesi/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Precision Medicine/methods ; CRISPR-Cas Systems ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing (PE) represents a significant advancement in genome editing, offering high precision for diverse genetic modifications without inducing double-strand breaks or requiring exogenous donor DNA templates. This "search-and-replace" technology employs a Cas9 nickase-reverse transcriptase fusion protein, guided by a PE guide RNA (pegRNA), to directly install specified edits including all 12 base-to-base conversions and targeted insertions/deletions with high fidelity. Since its introduction, PE systems have undergone rapid evolution (e.g., PE2-PE6, PEmax), markedly improving editing efficiency, product purity, and targeting scope. Although PE efficacy is context dependent, influenced by pegRNA design, cellular milieu, and DNA repair pathway engagement, ongoing research focuses on comprehensive system optimization. These efforts include engineering the Cas9 nickase and reverse transcriptase components for enhanced performance and processivity, alongside developing improved pegRNA architectures and chemical modifications to increase their stability and editing efficiency. Furthermore, strategies to modulate the cellular environment, such as transiently altering DNA repair pathway activities, particularly mismatch repair, are being explored to boost the accuracy and yield of precise edits. PE holds substantial promise for basic research, including precise disease modeling, and has demonstrated successful correction of pathogenic mutations in preclinical models of various genetic disorders like sickle cell disease, cystic fibrosis, and inherited retinal diseases. A significant milestone was the US Food and Drug Administration's granting of Investigational New Drug (IND) clearance for the first clinical trial of PM359, a therapeutic based on PE. This agent employs an ex vivo strategy, correcting the NCF1 gene in patient-derived hematopoietic stem cells for the treatment of chronic granulomatous disease. Despite considerable progress, unlocking the complete therapeutic promise of PE requires overcoming significant hurdles, particularly in developing effective in vivo delivery systems for its sizable components, with ongoing research actively investigating diverse viral and nonviral approaches. The translation of this versatile platform into transformative precision gene therapies is critically dependent upon its continued responsible advancement under robust ethical and regulatory oversight.},
}

*Gene Editing/methods
Humans
*Genetic Therapy/methods
*Precision Medicine/methods
CRISPR-Cas Systems
Animals
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41037600,
year = {2025},
author = {de Alba, EL and Salguero, I and Giménez-Llorente, D and Montes-Torres, J and Fernández-Sanromán, Á and Casajús-Pelegay, E and Terrón-Bautista, J and Barroso-González, J and Bernal, JA and Macintyre, G and Fernández-Leiro, R and Losada, A and Cortés-Ledesma, F},
title = {A comprehensive genetic catalog of human double-strand break repair.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6768},
pages = {eadr5048},
doi = {10.1126/science.adr5048},
pmid = {41037600},
issn = {1095-9203},
mesh = {Humans ; *DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; *DNA Repair/genetics ; Von Hippel-Lindau Tumor Suppressor Protein/genetics ; INDEL Mutation ; Gene Editing ; DNA End-Joining Repair/genetics ; Kidney Neoplasms/genetics ; DNA-Binding Proteins/genetics ; Transcription Factors/genetics ; CRISPR-Associated Protein 9 ; Genome, Human ; Gene Knockout Techniques ; Carcinoma, Renal Cell/genetics ; },
abstract = {The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. Using parallel in-pool profiling of Cas9-induced insertions and deletions (indels) within a genome-wide knockout library, we present a comprehensive catalog that assesses the influence of nearly every human gene on DSB repair outcomes. This REPAIRome resource uncovers uncharacterized mechanisms, pathways, and factors involved in DSB repair, including opposing roles for XLF and PAXX, a molecular explanation for Cas9-induced multinucleotide insertions, HLTF functions in Cas9-induced DSB repair, the involvement of the SAGA complex in microhomology-mediated end joining, and an indel mutational signature linked to VHL loss, renal carcinoma, and hypoxia. These results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.},
}

Humans
*DNA Breaks, Double-Stranded
CRISPR-Cas Systems
*DNA Repair/genetics
Von Hippel-Lindau Tumor Suppressor Protein/genetics
INDEL Mutation
Gene Editing
DNA End-Joining Repair/genetics
Kidney Neoplasms/genetics
DNA-Binding Proteins/genetics
Transcription Factors/genetics
CRISPR-Associated Protein 9
Genome, Human
Gene Knockout Techniques
Carcinoma, Renal Cell/genetics

@article {pmid41035653,
year = {2025},
author = {Naumovas, D and Rojas-Araya, B and Polanco, CM and Andrade, V and Čekauskienė, R and Valatkaitė-Rakštienė, B and Laurinaitytė, I and Jakubauskas, A and Stoškus, M and Griškevičius, L and Nalvarte, I and Inzunza, J and Baltriukienė, D and Arias, J},
title = {Identification of HLA-A, HLA-B, and HLA-C triple homozygous and double homozygous donors: a path toward synthetic superdonor advanced therapeutic medicinal products.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1626787},
pmid = {41035653},
issn = {1664-3224},
mesh = {Humans ; *Homozygote ; *HLA-B Antigens/genetics ; *HLA-C Antigens/genetics ; *HLA-A Antigens/genetics ; Alleles ; Gene Frequency ; Induced Pluripotent Stem Cells/immunology ; *Tissue Donors ; Genotype ; CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Human-induced pluripotent stem cells with broad immune compatibility are highly desirable for regenerative medicine applications. Human leukocyte antigen (HLA) class I homozygous cell sources are ideal for immune compatibility modeling. Here, we profile HLA-A, HLA-B, and HLA-C alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of HLA-A, 43.2% of HLA-B, and 59.2% of HLA-C, with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of European-American and British populations. We identified 153 double homozygotes and 51 triple homozygotes for HLA-A, HLA-B, and HLA-C. Compatibility modeling showed that triple homozygous profiles match 60.5% of Lithuanians, 13.4% of the British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt HLA-A or HLA-B while preserving HLA-C, producing edited profiles matching over 97.9% of Lithuanians, 95.7% of European-Americans, and 95.5% of the British population. Finally, we established 15 fibroblast lines from triple homozygotes as a bioresource for the derivation of human-induced pluripotent stem cells and immune compatibility studies.},
}

Humans
*Homozygote
*HLA-B Antigens/genetics
*HLA-C Antigens/genetics
*HLA-A Antigens/genetics
Alleles
Gene Frequency
Induced Pluripotent Stem Cells/immunology
*Tissue Donors
Genotype
CRISPR-Cas Systems
Female
Male

@article {pmid41034738,
year = {2025},
author = {Devkar, V and Ghose, K and D'Agostino, L and Patil, GB},
title = {Exonuclease-fused CRISPR-cas system enhances targeted genome editing for functional genomics in soybean.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1283},
pmid = {41034738},
issn = {1471-2229},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Glycine max/genetics ; *Genome, Plant ; *Exonucleases/genetics/metabolism ; Genomics/methods ; },
abstract = {CRISPR/Cas technologies have revolutionized plant genome editing, yet their inherent bias toward small insertions or deletions (indels) limits their utility for dissecting regulatory elements and generating impactful allelic variants. Here, we report the development and systematic evaluation of exonuclease-fused CRISPR/Cas systems in soybean to overcome this limitation. We engineered fusions of Cas9 and Cas12a with bacteriophage T5 exonuclease and human TREX2 and assessed their editing performance at the GmWOX5 locus using Agrobacterium rhizogenes-mediated transformation and deep amplicon sequencing. While native Cas9 and Cas12a predominantly generated micro-size deletions (1-10 bp), T5-Exo fusions shifted the mutation spectrum, producing a high frequency of moderate (26-50 bp) and large (> 50 bp) deletions. TREX2 fusions preferentially enhanced the generation of small (11-25 bp) to moderate deletions (26-50 bp). Fusion of exonucleases to Cas9 substantially reduced insertion frequencies and promoted more precise deletion patterns, as observed in T5-Exo-Cas9 and TREX2-Cas9. Deletions from both exonuclease fusions were biased toward the PAM-proximal region, reflecting altered repair outcomes likely driven by directional exonuclease activity and enhanced end resection. These results demonstrate that exonuclease fusions effectively expand the CRISPR toolkit by enabling efficient, targeted generation of larger deletions, which are often required for targeting cis-regulatory elements and microRNAs.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Glycine max/genetics
*Genome, Plant
*Exonucleases/genetics/metabolism
Genomics/methods

@article {pmid40974621,
year = {2025},
author = {Zheng, X and Yao, S and Yin, C and Zhao, H and Wang, J and Su, T and Li, H and Wang, J and Zhao, C},
title = {CRISPR-integrated nanoconfined interparticle catalytic hairpin assembly for enhanced dual-mode SARS-CoV-2 detection in wastewater.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {118008},
doi = {10.1016/j.bios.2025.118008},
pmid = {40974621},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation & purification/genetics ; *Wastewater/virology ; *Biosensing Techniques/methods/instrumentation ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; Limit of Detection ; Humans ; *RNA, Viral/analysis/genetics/isolation & purification ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Accurate monitoring of pathogenic viruses in wastewater is critical for early outbreak and risk assessment. This study presented a novel biosensing platform that combined an interparticle magnetic covalent organic framework (MCOF)-assisted mismatched catalytic hairpin assembly (iMMCHA) with CRISPR/Cas12a-activated colorimetric-photothermal dual-mode detection of SARS-CoV-2 RNA. The system strategically immobilized CHA reactants (H1 and mismatched H2) on separate MCOF nanoparticles, creating a spatially confined and collision-enhanced interparticle MCHA that achieved 270-fold higher local reactant concentration and 20-min faster kinetics than solution-phase CHA. Upon target recognition, the iMMCHA system generated dsDNA activators that triggered Cas12a-mediated cleavage of ssDNA linkers on magnetic bead-glucose oxidase conjugates. This cleavage event reduced the TMB-oxidizing activity of the magnetically isolated integrated enzyme system, producing inversely correlated colorimetric and photothermal signals. This iMMCHA-CRISPR dual-mode assay allowed for the rapid and sensitive detection of SARS-CoV-2 pseudovirus in sanitary wastewater samples, with detection limits of 100 and 120 copies/μL (colorimetric mode) and 100 and 140 copies/μL (photothermal mode) for S and N genes, respectively. This work established a powerful platform for aqueous environmental virus monitoring that combined the specificity of CRISPR with the signal enhancement and kinetics acceleration of nanoconfined interparticle CHA and the reliability of dual-mode detection.},
}

*SARS-CoV-2/isolation & purification/genetics
*Wastewater/virology
*Biosensing Techniques/methods/instrumentation
*COVID-19/diagnosis/virology
*CRISPR-Cas Systems
Limit of Detection
Humans
*RNA, Viral/analysis/genetics/isolation & purification
Colorimetry/methods
Metal-Organic Frameworks/chemistry
Nanoparticles/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40961645,
year = {2025},
author = {He, Y and Zhang, Y and Xiang, H and Ren, K and Yin, Y and Gao, Y and Yang, Y and Zhang, W and Liu, L and Han, H and Wang, W},
title = {Magnetic bead-assisted one-pot RCA-activated CRISPR/Cas12a electrochemiluminescence biosensor for the detection of citrus Huanglongbing pathogen.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117986},
doi = {10.1016/j.bios.2025.117986},
pmid = {40961645},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Citrus/microbiology ; *Plant Diseases/microbiology ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; *Liberibacter/isolation & purification/genetics/pathogenicity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Huanglongbing (HLB) poses a catastrophic threat to the global citrus industry, necessitating early detection of pathogen for disease control and minimize economic losses. Herein, we reported a one-pot electrochemiluminescence (ECL) biosensor for integrating rolling circle amplification (RCA)-activated CRISPR/Cas12a dual cleavage activity, and engineered magnetic beads-based quenched ECL emitter. Target-initiated RCA generated amplicons that activated Cas12a, simultaneously leveraging cis-cleavage for template recycling and trans-cleavage to degrade single stranded DNA attached on Ru(bpy)3[2+]-loaded magnetic beads. This dual-amplification strategy restored ECL signals, enabling ultrasensitive detection of Candidatus Liberibacter asiaticus (CLas) ribonucleotide-diphosphate reductase subunit beta gene fragments with high specificity. A linear range 10 fM-1 nM with the detection limit of 2 fM was obtained. The integrated platform eliminated multi-step incubations, and exhibited satisfactory performance in citrus leaf samples, offering a powerful tool for HLB diagnostics.},
}

*Biosensing Techniques/methods
*Citrus/microbiology
*Plant Diseases/microbiology
Luminescent Measurements/methods
CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
Limit of Detection
Nucleic Acid Amplification Techniques/methods
*Liberibacter/isolation & purification/genetics/pathogenicity
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40945113,
year = {2025},
author = {Che, R and Tang, D and Fu, B and Wen, T and Wang, Z and Feng, D and Huang, KJ and Xu, J},
title = {Smartphone-integrated tri-mode RCA-CRISPR/Cas12a biosensor with Fe3O4@Au nanozyme for on-site detection of sugarcane smut at attomolar level.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117985},
doi = {10.1016/j.bios.2025.117985},
pmid = {40945113},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Saccharum/microbiology ; Smartphone ; Gold/chemistry ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Limit of Detection ; *Plant Diseases/microbiology ; Colorimetry ; Electrochemical Techniques ; Metal Nanoparticles/chemistry ; },
abstract = {The devastating sugarcane smut causes up to 70 % sugar yield loss and secondary infections, but field-deployable diagnostics remain challenging due to the limitations of lab-dependent methods. Herein, we report a portable CRISPR/Cas12a-powered biosensor integrated with tri-functional Fe3O4@Au nanozymes and triple-modal signal readout for precise and on-site pathogen detection. By synergizing rolling circle amplification (RCA) with CRISPR/Cas12a trans-cleavage activity, the system achieves ultrasensitive target recognition (detection limit: 32.11 aM for electrochemical mode). The Fe3O4@Au@GOD bioconjugates simultaneously enables magnetic separation, optimizes GOD-mediated colorimetric signals (visual LOD: 49.28 fM), and enhances photothermal responses (LOD: 42.17 fM) via precise biocatalyst-catalyzed TMB oxidation. A smartphone-coupled 3D-printed device integrates electrochemical, colorimetric, and photothermal detection modes, providing cross-validated results that eliminate false positives in complex matrices (recovery: 98-104 %). This field-portable platform completes detection within 2.5-4.5 h (80 % cost reduction vs. qPCR) and demonstrates high specificity against non-target pathogens. The fusion of nanozyme engineering, CRISPR amplification, and multi-modal sensing offers a transformative tool for precision agriculture.},
}

*Biosensing Techniques/instrumentation
*Saccharum/microbiology
Smartphone
Gold/chemistry
CRISPR-Cas Systems
Nucleic Acid Amplification Techniques
Limit of Detection
*Plant Diseases/microbiology
Colorimetry
Electrochemical Techniques
Metal Nanoparticles/chemistry

@article {pmid40939269,
year = {2025},
author = {Zhou, C and Jiang, F and Chen, W and Nugen, SR and Huang, C},
title = {Synthetic biology meets diagnostics: Engineering biosensing platforms for rapid and accurate pathogen and viral detection.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117946},
doi = {10.1016/j.bios.2025.117946},
pmid = {40939269},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Synthetic Biology/methods/instrumentation ; Humans ; *Viruses/isolation & purification/genetics/pathogenicity ; *Virus Diseases/diagnosis/virology ; Nanotechnology/methods ; CRISPR-Cas Systems ; },
abstract = {The integration of synthetic biology with biosensor technologies has catalyzed a paradigm shift in the development of programmable, field-deployable diagnostic systems for precision detection of pathogens and viral threats. This review provides a comprehensive overview of current synthetic biology toolkits, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-based) systems, argonaute proteins, and modular genetic circuits. These tools were integrated into biosensors and in vitro diagnostic devices. The applications of cell-free systems, modular genetic circuits, and nanomaterial-enhanced platforms have further expanded the versatility of these tools, which include infectious disease diagnostics, public health monitoring, and food safety. Recent studies integrate synthetic biology with artificial intelligence (AI) and nanotechnology, enabling the development of automated, low-cost, and high-throughput diagnostic systems. This review provides a comprehensive overview of current technologies, emerging trends, future directions, and challenges, which offers valuable insights for advancing pathogen detection and in vitro diagnostics through synthetic biology.},
}

*Biosensing Techniques/methods/instrumentation
*Synthetic Biology/methods/instrumentation
Humans
*Viruses/isolation & purification/genetics/pathogenicity
*Virus Diseases/diagnosis/virology
Nanotechnology/methods
CRISPR-Cas Systems

@article {pmid40914019,
year = {2025},
author = {Ding, Y and Zhang, J and Li, K and Wang, X and Shi, X and Zhao, C and Dai, J and Wang, Q and Yao, S and Wang, J},
title = {A cascade amplification platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a for microRNA-21 and Listeria monocytogenes detection.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117947},
doi = {10.1016/j.bios.2025.117947},
pmid = {40914019},
issn = {1873-4235},
mesh = {*Listeria monocytogenes/isolation & purification/genetics ; CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics/isolation & purification ; *Biosensing Techniques/methods ; Entropy ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Animals ; DNA/chemistry ; Swine ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {As one of enzyme-free amplification strategies, entropy-driven catalytic (EDC) based on toehold-mediated strand displacement reaction could achieve efficient amplification without cumbersome temperature changing and expensive enzymes, which shows great potential in biological sensing. However, the limitations in reaction velocity and sensitivity need to be further improved. Herein, a cascade platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a was proposed. Benefiting from the increased local concentration of DNA on AuNPs, the reaction velocity was enhanced 2-fold compared to solution-based EDC efficiently and the signal was cascade amplified through specific recognition by the designed CRISPR/Cas12a with high sensitivity and selectivity. Impressively, utilizing the flexible design capabilities of DNA molecules, the proposed method achieved both nucleic acid and non-nucleic acid targets detection. The platform achieved a low limit of 6.1pM for microRNA-21 detection and 6 CFU/mL for Listeria monocytogenes detection. Moreover, it showed good performance in 10-fold diluted serum with 98.6-102.3 % recovery. And it has achieved good consistency with traditional plate culture methods in pork. Here, a rapid and sensitive platform based on entropy-driven DNA nanomachine coupled with CRISPR/Cas12a is proposed with great potential of application for early disease detection and food safety screening.},
}

*Listeria monocytogenes/isolation & purification/genetics
CRISPR-Cas Systems/genetics
*MicroRNAs/genetics/isolation & purification
*Biosensing Techniques/methods
Entropy
Nucleic Acid Amplification Techniques/methods
Limit of Detection
Metal Nanoparticles/chemistry
Gold/chemistry
Animals
DNA/chemistry
Swine
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40914018,
year = {2025},
author = {Deng, Z and Mao, X and Yang, Y and Wu, G and Meng, H and Hou, J and Yu, XF and Zhou, W and Mao, G and Ma, Y},
title = {Amplification-free CRISPR/Cas12a biosensor integrating AuNPs-mediated surface plasmon resonance for human papillomavirus detection and genotyping.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117960},
doi = {10.1016/j.bios.2025.117960},
pmid = {40914018},
issn = {1873-4235},
mesh = {Humans ; Gold/chemistry ; *Surface Plasmon Resonance/methods ; CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/virology/diagnosis ; Metal Nanoparticles/chemistry ; *Papillomaviridae/genetics/isolation & purification ; *DNA, Viral/genetics/isolation & purification ; *Biosensing Techniques ; Limit of Detection ; Genotyping Techniques ; Female ; Alkaline Phosphatase/chemistry ; Genotype ; Human Papillomavirus Viruses ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Screening for high-risk human papillomavirus (hrHPV) infection is essential for cervical cancer prevention. However, developing a simple, portable, and low-cost hrHPV genotyping method remains challenging, particularly in resource-limited settings. Herein, we present an innovative amplification-free, point-of-care hrHPV genotyping platform integrating CRISPR/Cas12a with alkaline phosphatase (ALP)-mediated surface plasmon effect. The platform detects HPV DNA through the Cas12a-crRNA complex recognition, activating the cleavage of ALP-labeled oligonucleotides within microwells and releasing ALP. The output signal is generated by changes in surface plasmon resonance of gold nanoparticles (AuNPs) induced by the ALP-mediated reaction of AuNPs with p-aminophenyl phosphate. This ALP-integrated CRISPR/Cas12a biosensing strategy enhances sensitivity by 10,000-fold compared to Cas12a-based detection integrating ALP-mediated p-nitrophenyl phosphate (p-NPP) hydrolysis. This approach allows the sensitive detection of HPV DNA with a detection limit of 300 aM. Moreover, integration with microplate separation allows specifically screen for the nine HPV subtypes targeted by the nine-valent HPV vaccine within 2.5 h. The platform's performance is validated using cervical swab samples, confirming its accuracy for HPV genotyping. Overall, this strategy provides a simple, portable, and cost-effective solution for multiplex nucleic acid targets detection without preamplification or instrumentation.},
}

Humans
Gold/chemistry
*Surface Plasmon Resonance/methods
CRISPR-Cas Systems/genetics
*Papillomavirus Infections/virology/diagnosis
Metal Nanoparticles/chemistry
*Papillomaviridae/genetics/isolation & purification
*DNA, Viral/genetics/isolation & purification
*Biosensing Techniques
Limit of Detection
Genotyping Techniques
Female
Alkaline Phosphatase/chemistry
Genotype
Human Papillomavirus Viruses
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40907452,
year = {2025},
author = {Hu, D and Lin, K and Xu, X and Chen, P and Wang, G and Parvin, R and Chen, X and Wang, D and Zhang, L and Ye, F},
title = {SEE-phAST: Spatially encapsulated emulsions for phenotypic antibiotic susceptibility testing via sequential digital RAA-CRISPR.},
journal = {Biosensors & bioelectronics},
volume = {290},
number = {},
pages = {117937},
doi = {10.1016/j.bios.2025.117937},
pmid = {40907452},
issn = {1873-4235},
mesh = {*Anti-Bacterial Agents/pharmacology ; *Biosensing Techniques ; Emulsions/chemistry ; Microbial Sensitivity Tests ; Humans ; DNA Copy Number Variations/genetics ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/drug effects ; Pseudomonas aeruginosa/genetics/drug effects ; Nucleic Acid Amplification Techniques ; Recombinases/chemistry ; Klebsiella pneumoniae/genetics/drug effects ; DNA, Bacterial/genetics ; },
abstract = {The escalating threat of antimicrobial resistance is exacerbated by delayed diagnostics and improper antibiotics use, underscoring an urgent demand for rapid, versatile AST tools to support evidence-based prescribing. In this study, we present an innovative, generalizable phenotypic AST approach by quantifying bacterial gDNA copy number variations (CNVs) following 0.5-h-brief culturing with antibiotic exposure, termed spatially encapsulated emulsions (SEE)-phAST. It employed gelatin-PEG biomimetic phase separation and microfluidic technology to one-step fabricate spatial-confined microcarriers that enabled on-demand separation and mixing of recombinase-aided amplification (RAA) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) reactions in a sequentially orchestrated manner. Importantly, it retained the superiority of droplet-based digitalization framework to identify 3-4 folds CNVs while synergistically enhancing signal-to-noise ratio and detection speed through RAA pre-amplification. By utilizing a phase diagram for precise separation, coupled with channel design and rate regulation, we controllably synthesized 180-210 μm microcarriers with 90-100 μm cores, achieving a 30-min RAA amplification that boosted sensitivity from 10[11] to 10[1] aM. The artificial urinary infection samples were prepared by introducing common bacteria, specifically Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa, into urinary samples obtained from healthy individuals. We demonstrated the system's capability to classify susceptibility and resistance to three specific drugs following a 30-min pre-culture, with epigenetic changes quantified as reduced to 0.5 for inhibition and maintained at 2 for unaffected growth. This strategy presents valuable potential for AST diagnosis and versatility in other sequence recognition scenarios.},
}

*Anti-Bacterial Agents/pharmacology
*Biosensing Techniques
Emulsions/chemistry
Microbial Sensitivity Tests
Humans
DNA Copy Number Variations/genetics
CRISPR-Cas Systems/genetics
Escherichia coli/genetics/drug effects
Pseudomonas aeruginosa/genetics/drug effects
Nucleic Acid Amplification Techniques
Recombinases/chemistry
Klebsiella pneumoniae/genetics/drug effects
DNA, Bacterial/genetics

@article {pmid40902593,
year = {2025},
author = {Chan, BKC and Zhang, C and Poon, CH and Lee, MHY and Chu, HY and Wang, B and Chen, SG and Yan, HHN and Leung, SY and Wong, ASL},
title = {A combined enteric neuron-gastric tumor organoid reveals metabolic vulnerabilities in gastric cancer.},
journal = {Cell stem cell},
volume = {32},
number = {10},
pages = {1595-1613.e10},
doi = {10.1016/j.stem.2025.08.006},
pmid = {40902593},
issn = {1875-9777},
mesh = {*Organoids/metabolism/pathology/drug effects ; *Stomach Neoplasms/metabolism/pathology ; Humans ; Animals ; *Neurons/metabolism/pathology/drug effects ; Mice ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Lipid Metabolism/drug effects ; Acetyl-CoA Carboxylase/antagonists & inhibitors/metabolism ; },
abstract = {The discrepancy between organoid and immortalized cell line cultures for cancer target discovery remains unclear. Here, our multi-tiered clustered regularly interspaced short palindromic repeats (CRISPR) screens reveal in vivo-relevant metabolic dependencies and synthetic lethal pairs that can be uncovered with tumor organoids but not cell lines or even three-dimensional (3D) spheroids. These screens identify lanosterol synthase and acetyl-coenzyme A (CoA) carboxylase inhibitors as effective treatments that impede xenografted tumor growth in mice. These lipid metabolic inhibitors exhibit nanomolar half-maximal inhibitory concentration (IC50) values across diverse human gastric cancer organoids resistant to first-line treatments. Mechanistically, gastric cancer organoids and in vivo tumors exhibit lipid metabolic adaptations not seen in two-dimensional (2D) in vitro cultures. Additionally, enteric neurons modulate lipid metabolism in tumor organoids, altering drug sensitivity by up to two orders of magnitude. A neuron-cocultured CRISPR screen further reveals that acetyl-CoA carboxylase expression determines lanosterol synthase inhibitor efficacy. These findings highlight the critical roles of organoid environment and neuronal interaction in cancer lipid reliance.},
}

*Organoids/metabolism/pathology/drug effects
*Stomach Neoplasms/metabolism/pathology
Humans
Animals
*Neurons/metabolism/pathology/drug effects
Mice
Cell Line, Tumor
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Lipid Metabolism/drug effects
Acetyl-CoA Carboxylase/antagonists & inhibitors/metabolism

@article {pmid40674704,
year = {2025},
author = {Yamaguchi, K and Koya, J and Mizuno, K and Mizukami, Y and Yoshifuji, K and Saito, Y and Tabata, M and Shingaki, S and Yuasa, M and Ito, Y and Nakashima, K and Dreval, K and Morin, RD and Chiba, K and Okada, A and Shiraishi, Y and Murakami, K and Kogure, Y and Ohshima, K and Kataoka, K},
title = {In vivo CRISPR screening reveals cooperation of KMT2D and TP53 deficiencies in B-cell lymphomagenesis.},
journal = {Blood advances},
volume = {9},
number = {19},
pages = {5040-5055},
doi = {10.1182/bloodadvances.2024015519},
pmid = {40674704},
issn = {2473-9537},
mesh = {*Tumor Suppressor Protein p53/genetics/deficiency ; Humans ; Animals ; Mice ; *Lymphoma, Large B-Cell, Diffuse/genetics/pathology ; *DNA-Binding Proteins/genetics/deficiency ; *CRISPR-Cas Systems ; *Lymphoma, B-Cell/genetics ; *Neoplasm Proteins/genetics/deficiency ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; },
abstract = {Although recent genetic studies have identified numerous genetic alterations in diffuse large B-cell lymphoma (DLBCL), their biological relevance remains elusive. Here, we performed in vivo CRISPR loss-of-function screening targeting 86 genes recurrently altered in DLBCL to examine oncogenicity of single-guide RNA (sgRNA)-targeted genes, association between genotype and lineage, occurrence of second-hit alterations, and cooperability among sgRNA-targeted genes and second-hit alterations. Transplantation of the CRISPR library-transduced hematopoietic stem/progenitor cells induces various hematologic malignancies, including B-cell lymphomas in mice. Enrichment analysis of sgRNA-targeted genes demonstrates significant overrepresentation of Kmt2d, Pax5, and Trp53 in B-cell lymphomas. Whole-exome sequencing identifies recurrent second-hit driver alterations, showing significant enrichment of Trp53 alterations in sgKmt2d-targeted B-cell lymphomas. Importantly, KMT2D and TP53 mutations are found to be the most prevalent co-occurring combination in human DLBCL, which is more prominent in relapsed/refractory DLBCL. Moreover, this combination confers significantly worse prognosis independent of clinical factors. Transcriptomic sequencing identifies overexpression of Yap1, the Hippo pathway component, in double sgKmt2d-targeted/Trp53-altered B-cell lymphomas. Furthermore, chromatin accessibility analysis demonstrates enrichment of transcriptional enhanced associate domain 1 binding motifs in regions that gained accessibility and increased expression of their nearest genes in these B-cell lymphomas. Most importantly, genetic and pharmacological inhibition of YAP1 suppresses in vitro cell proliferation and in vivo tumor growth of a human KMT2D/TP53-altered DLBCL cell line and prolongs survival of mice transplanted with double sgKmt2d-targeted/Trp53-altered B-cell lymphoma cells. Our findings demonstrate the utility of in vivo CRISPR screening to integrate human cancer genomics with mouse modeling and highlight the functional interplay between KMT2D and TP53 aberrations, providing insights into therapeutic strategies in DLBCL.},
}

*Tumor Suppressor Protein p53/genetics/deficiency
Humans
Animals
Mice
*Lymphoma, Large B-Cell, Diffuse/genetics/pathology
*DNA-Binding Proteins/genetics/deficiency
*CRISPR-Cas Systems
*Lymphoma, B-Cell/genetics
*Neoplasm Proteins/genetics/deficiency
*Clustered Regularly Interspaced Short Palindromic Repeats
Cell Line, Tumor

@article {pmid41033726,
year = {2025},
author = {Gupta, Y and Chosdol, K},
title = {Practical approaches to advanced molecular biology techniques.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {73-101},
doi = {10.1016/bs.mcb.2025.02.022},
pmid = {41033726},
issn = {0091-679X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Molecular Biology/methods ; Single-Cell Analysis/methods ; Animals ; Chromatin Immunoprecipitation/methods ; Proteomics/methods ; },
abstract = {The field of molecular biology has undergone tremendous advancements in recent years, with the development of powerful techniques that allow for in-depth exploration of cellular processes at the molecular level. This chapter, "Advanced Molecular Biology Techniques," provides a detailed protocol of the molecular techniques. We begin with CRISPR-Cas9 genome editing, a transformative tool for precise and efficient gene manipulation, enabling targeted mutations and gene knockouts in various organisms. Gene amplification via Real-Time PCR is then discussed, highlighting its ability to quantify gene expression and detect rare genetic variants with high sensitivity. Flowcytometry follows, offering a robust platform for analyzing cellular populations based on specific markers, enabling the study of immune cells, cancer diagnostics, and cell cycle analysis. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) is explored as a method for mapping protein-DNA interactions, providing insights into gene regulation and epigenetic modifications. The chapter also covers Single-cell RNA sequencing (scRNA-Seq), a groundbreaking technique for profiling gene expression at the single-cell level, allowing for the discovery of cell heterogeneity and complex biological processes. Next, we explore into proteomics through Mass Spectrometry-Based Analysis, which offers detailed proteome characterization and biomarker discovery by identifying and quantifying proteins in complex samples. Finally, Fluorescence In Situ Hybridization (FISH) is discussed as a method for visualizing the spatial localization of specific nucleic acid sequences within intact cells or tissues. Together, these advanced molecular biology techniques offer unparalleled precision and insight into the molecular mechanisms underlying health, disease, and cellular function.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Molecular Biology/methods
Single-Cell Analysis/methods
Animals
Chromatin Immunoprecipitation/methods
Proteomics/methods

@article {pmid41033723,
year = {2025},
author = {Hasan, N and Palungan, J and Ullah, M},
title = {Gene editing techniques in cancer research.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {287-312},
doi = {10.1016/bs.mcb.2025.03.002},
pmid = {41033723},
issn = {0091-679X},
mesh = {*Gene Editing/methods ; Humans ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Zinc Finger Nucleases/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Animals ; },
abstract = {The process of editing genes has emerged as a game-changing instrument in the field of cancer research. It has the potential to provide a whole new understanding of the biology of tumors and to facilitate the creation of tailored medicines. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system are the three basic methods of gene editing techniques that are discussed in this chapter. We investigate the protocol modifications that are specific to each approach, focusing on high-prevalence tumors, and we investigate the utility, efficiency, and application issues that are associated with each technique in oncology. In addition, we describe current developments in improving these methods to successfully target oncogenes and tumor suppressor genes, with the goal of driving forward advances in precision cancer therapy.},
}

*Gene Editing/methods
Humans
*Neoplasms/genetics/therapy
*CRISPR-Cas Systems/genetics
Zinc Finger Nucleases/genetics
Transcription Activator-Like Effector Nucleases/genetics
Animals

@article {pmid41033719,
year = {2025},
author = {Tiwari, PC and Chaudhary, MJ and Pal, R and Nath, R},
title = {In vivo cancer modeling using mouse models.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {221-250},
doi = {10.1016/bs.mcb.2025.02.013},
pmid = {41033719},
issn = {0091-679X},
mesh = {Animals ; Mice ; *Disease Models, Animal ; *Neoplasms/pathology/genetics ; Humans ; Tumor Microenvironment ; Xenograft Model Antitumor Assays/methods ; CRISPR-Cas Systems ; },
abstract = {Mouse models have contributed to a better understanding of cancer biology and the development of new treatments. This chapter elaborates on the various types of mouse models applied in cancer research, such as xenograft, syngeneic, and humanized models, together with the state-of-the-art techniques of genetic engineering involved in their generation. We described the methodologies of tumor induction and engraftment procedures and these model applications in drug development, efficacy testing, and studies on immuno-oncology. Further, the chapter covers ethical considerations and regulatory requirements on the use of animals in research, essentially aligned with international guidelines and those in India. The chapter illustrates that mouse models will not become outdated in preclinical testing any time soon but continue to be relevant for the study of tumor biology and the tumor microenvironment besides their use for investigating genetic and molecular pathways in cancer. Emerging technologies, such as CRISPR/Cas9 and organoid integration, are also highlighted for their work in improving the accuracy and translational potential of models. These developments combined with initiatives on collaborative and open science that enable the sharing of data and resources, hold great promise for the future of in vivo cancer modeling. The mouse models will continue to be one of the prime movers in advancing cancer research and formulating individual medication strategies that lead to improved patient outcomes through their integration of classical approaches with modern technologies.},
}

Animals
Mice
*Disease Models, Animal
*Neoplasms/pathology/genetics
Humans
Tumor Microenvironment
Xenograft Model Antitumor Assays/methods
CRISPR-Cas Systems

@article {pmid41032194,
year = {2025},
author = {Naderian, R and Alibabaei, F and Paraandavaji, E and Dehghan, P and Eslami, M},
title = {Phage-Microbiota Interactions in the Gut: Implications for Health and Therapeutic Strategies.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41032194},
issn = {1867-1314},
abstract = {The diversified ecology of microorganisms, including bacteria, archaea, fungi, protozoa, and viruses known collectively as the gut microbiota, which includes bacteriophages, is crucial to human health because it affects functions like immune system regulation, vitamin production, and pathogen protection. Bacteriophages are viruses that infect bacteria and are increasingly recognized as a viable treatment option for antibiotic-resistant strains, owing to their high host specificity, which enables precise targeting of drug-resistant bacteria while sparing commensal microbiota. The complex relationships between bacteriophages and gut microbiota are examined, with emphasis on their roles in maintaining health and contributing to disease. Gut microbiota homeostasis is influenced by a number of factors, including age, nutrition, and drugs. Bacteriophages, via lytic cycles and lysogenic conversion, influence the gut microbiota composition and microbial community structure. Gaining an understanding of these processes is crucial to appreciating their contribution to the stability and variety of microbes. Recent research highlights the gut phageome's potential for therapeutic interventions by demonstrating its substantial influence on immunological responses and metabolic problems. The study of phage-microbiota interactions has been transformed by cutting-edge technologies, including high-throughput sequencing, CRISPR-Cas systems, and viral metagenomics, which allow for thorough research and the creation of new therapeutics. Even though tailored medicine and pathogen management hold great potential, obstacles such as regulatory difficulties and bacterial resistance call for additional investigation. Phage-based therapeutic strategies are rapidly advancing, ranging from genetically engineered phages and phages with modified capsid proteins designed to enhance efficacy to phage cocktails that target multiple bacterial strains.},
}

@article {pmid40803956,
year = {2025},
author = {Murata, S and Kushiyama, N and Yabu, Y and Watanabe, K and Fujii, T and Yasui, R and Nojima, D and Maeda, Y and Yoshino, T and Matsuda, Y and Tanaka, T},
title = {Establishment of genome editing techniques in the marine oleaginous diatom Fistulifera solaris for improved oil accumulation.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {5},
pages = {271-276},
doi = {10.1016/j.jbiosc.2025.07.008},
pmid = {40803956},
issn = {1347-4421},
mesh = {*Diatoms/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Biofuels ; Lipase/genetics/metabolism ; Gene Knockout Techniques ; *Oils/metabolism ; Microalgae/genetics/metabolism ; Triglycerides/metabolism ; *Lipid Metabolism/genetics ; },
abstract = {Biofuel production using microalgae has attracted considerable attention owing to high growth rate and lipid accumulation properties. However, further enhancement in lipid productivity is required to render this economically feasible. CRISPR/Cas9, which is one of the powerful genome editing tools, is an essential technique that may solve this problem. The marine diatom Fistulifera solaris JPCC DA0580 is a promising candidate of the biofuel production, since it accumulates significant amount of lipids. However, genome editing techniques have not yet been established for F. solaris, which prevent the construction of valuable strains. In this study, CRISPR/Cas9-mediated specific gene knockout technique was established in F. solaris, through targeting adenine phosphoribosyl transferase gene (apt) and triacylglycerol (TAG) lipase gene (tgl1). Mutations in the target sequence were detected in apt- and tgl1-edited mutants. Moreover, the mutants showed distinct phenotypes, such as suppression of TAG degradation and resistance to 2-fluoroadenine. These results indicate the successful demonstration of CRISPR/Cas9-mediated genome editing in the oleaginous marine diatom F. solaris. Furthermore, oil degradation was successfully suppressed by knocking-out tgl1. The CRISPR/Cas9-mediated genome editing established in this study provides key molecular tools for both the basic biology and the future biotechnological applications of F. solaris, such as biofuel production.},
}

*Diatoms/genetics/metabolism
*Gene Editing/methods
CRISPR-Cas Systems
Biofuels
Lipase/genetics/metabolism
Gene Knockout Techniques
*Oils/metabolism
Microalgae/genetics/metabolism
Triglycerides/metabolism
*Lipid Metabolism/genetics

@article {pmid40774862,
year = {2025},
author = {Karashima, T and Oda, K and Futagami, T and Hokazono, H and Takashita, H},
title = {Ribonucleoprotein-based CRISPR/Cas9 genome co-editing in Aspergillus luchuensis mut. kawachii.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {5},
pages = {298-305},
doi = {10.1016/j.jbiosc.2025.07.006},
pmid = {40774862},
issn = {1347-4421},
mesh = {*CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Gene Editing/methods ; *Aspergillus/genetics/metabolism ; Gene Knockout Techniques ; *Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sulfate Adenylyltransferase/genetics ; },
abstract = {In this study, we established a ribonucleoprotein-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome co-editing method for the white koji fungus, Aspergillus luchuensis mut. kawachii. To introduce the single guide RNA-Cas9 ribonucleoprotein complex into protoplast cells of A. luchuensis mut. kawachii, we investigated the conditions for protoplast preparation using Yatalase -Plus-. Subsequently, we employed the ribonucleoprotein-based method to knockout the ATP sulfurylase-encoding sC gene, which imparts selenate resistance in the model strain NBRC 4308 and the industrial strain No. 8046. Furthermore, we explored genome co-editing by simultaneously targeting sC along with either the orotidine 5'-phosphate decarboxylase-encoding pyrG gene or the transcriptional activator of protease genes-encoding prtR gene in NBRC 4308. The transformants were selected in medium containing selenate, resulting in the successful generation of pyrG- and prtR-knockout strains. Similarly, transformants were selected on medium containing selenate, resulting in the successful generation of prtR-knockout strain in No. 8046. These results demonstrate that the ribonucleoprotein-based genome co-editing method is applicable not only to the model strain but also to industrial strains, making it a promising approach for manipulating A. luchuensis mut. kawachii.},
}

*CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/metabolism
*Gene Editing/methods
*Aspergillus/genetics/metabolism
Gene Knockout Techniques
*Genome, Fungal
RNA, Guide, CRISPR-Cas Systems/genetics
Sulfate Adenylyltransferase/genetics

@article {pmid40634141,
year = {2025},
author = {Shan, L and Verstrepen, KJ and Wang, Q and Dai, Z},
title = {A homologous recombination-proficient Yarrowia lipolytica chassis for multiplex genome manipulation.},
journal = {Trends in biotechnology},
volume = {43},
number = {10},
pages = {2627-2645},
doi = {10.1016/j.tibtech.2025.06.009},
pmid = {40634141},
issn = {1879-3096},
mesh = {*Yarrowia/genetics ; *Homologous Recombination/genetics ; *Gene Editing/methods ; DNA End-Joining Repair/genetics ; *Genome, Fungal/genetics ; CRISPR-Cas Systems ; },
abstract = {Homologous recombination (HR) greatly facilitates precise genome editing. However, most organisms prefer error-prone non-homologous end joining (NHEJ) for DNA double-strand break (DSB) repair. Here, the NHEJ-proficient Yarrowia lipolytica was transformed into a HR-proficient strain by systematic engineering of recombination machinery, regulating the multiinvasion-induced rearrangement (MIR) process, and expressing cognate single-stranded DNA-annealing protein (SSAP)-single-stranded DNA-binding protein (SSB) pairs. These strategies improved HR efficiency by 38.9, 1.6, and 1.2-fold compared with the NHEJ-deficient strain for multifragment multisite integration, and multi- and single-fragment single-site integration, respectively. Moreover, HR efficiency remained high at 58% even with 50-base pair (bp) homology arms (HAs) and reached 11% for simultaneously integrating two mega-DNA fragments (18.0 kb and 13.5 kb) at two genome sites. This strain also enabled simultaneous editing, repression, and activation of multiple genes, while cellular robustness parameters showed marked increases over the NHEJ-deficient strain. Our work provides a HR-proficient Y. lipolytica chassis allowing efficient and precise genome editing of this increasingly important microbe.},
}

*Yarrowia/genetics
*Homologous Recombination/genetics
*Gene Editing/methods
DNA End-Joining Repair/genetics
*Genome, Fungal/genetics
CRISPR-Cas Systems

@article {pmid40605271,
year = {2025},
author = {Stigzelius, V and Cavallo, AL and Chandode, RK and Nitsch, R},
title = {Peeling back the layers of immunogenicity in Cas9-based genomic medicine.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {10},
pages = {4714-4730},
doi = {10.1016/j.ymthe.2025.06.045},
pmid = {40605271},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems/immunology ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; *CRISPR-Associated Protein 9/immunology/genetics ; *Genomics/methods ; },
abstract = {The CRISPR-Cas9 genome editing system is rewriting the treatment of genetic disorders, offering unprecedented potential for detrimental and previously untreatable diseases. As this technology advances toward wider utilization in clinical applications, the immunogenicity of Cas9 nuclease has emerged as a potential challenge for in vivo therapies. Immune recognition of CRISPR-Cas9 components can trigger both innate and adaptive responses. The complex interactions between Cas9, delivery vectors, and host immune reactivity play a crucial role in determining the safety and efficacy of CRISPR-based treatments. Recent advances in mitigating Cas9 immunogenicity include epitope engineering, optimized delivery systems, and nucleic acid modifications. These strategies, explored across various tissue contexts and delivery methods, show promise in enhancing the tolerability of CRISPR-based therapies. However, pre-existing immunity to Cas9 and the potential for long-term adaptive immune responses remain important considerations. Addressing these immunological challenges requires an integrated approach, combining insights from immunology with innovative engineering solutions. As the field progresses, overcoming Cas9 immunogenicity will be crucial for realizing the full therapeutic potential of the CRISPR-Cas9 system in diverse clinical applications.},
}

*CRISPR-Cas Systems/immunology
Humans
*Gene Editing/methods
*Genetic Therapy/methods
Animals
*CRISPR-Associated Protein 9/immunology/genetics
*Genomics/methods

@article {pmid40368676,
year = {2025},
author = {Zhang, S and Xu, D and Li, F and Wang, J},
title = {CRISPR-based non-nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {43},
number = {10},
pages = {2494-2508},
doi = {10.1016/j.tibtech.2025.04.012},
pmid = {40368676},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acids/genetics/analysis ; Gene Editing/methods ; Humans ; },
abstract = {Characterization of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) trans-cleavage activities has initiated the era of next-generation CRISPR diagnostics. By using the trans-cleavage reaction for signal output, CRISPR systems have been engineered to detect non-nucleic acids (NNAs), including ions, inorganic small molecules, organic compounds, proteins, and bacteria. Diverse strategies are being used to specifically recognize NNAs and regulate Cas trans-cleavage activities, via generation or depletion of output signals. In this review, we introduce the principles and advantages of CRISPR-based NNA detection. We then classify CRISPR-based NNA detection strategies into three classes: the generation or depletion of free activators, synthesis of crRNAs, and reconstruction of active Cas effectors. Finally, we discuss the challenges and potential strategies to advance both clinical and nonclinical applications of CRISPR-based NNA detection.},
}

*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Nucleic Acids/genetics/analysis
Gene Editing/methods
Humans

@article {pmid41032193,
year = {2025},
author = {Yetiman, AE and Horzum, M and Kanbur, E and Çadir, M and Bahar, D and Gürbüz, Ş and Karaman, MZ and Fidan, Ö and Kaya, M and Yetiman, S and Doğan, M and Akbulut, M},
title = {Pangenome Analysis and Genome-Guided Probiotic Evaluation of Cyclic Dipeptides Producing Levilactobacillus brevis DY55bre Strain from a Lactic Acid Fermented Shalgam to Assess Its Metabolic, Probiotic Potentials, and Cytotoxic Effects on Colorectal Cancer Cells.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41032193},
issn = {1867-1314},
support = {FBA-2025-14779//Bilimsel Araştırma Projeleri, Erciyes Üniversitesi/ ; },
abstract = {This study investigates the genetic, metabolic, and probiotic characteristics of Levilactobacillus brevis DY55bre, a strain isolated from the traditional Turkish fermented beverage, shalgam. Whole-genome sequencing revealed a circular genome of 2.485 Mb with a GC content of 45.72%, predicted 2791 genes, and multiple CRISPR-Cas systems. Pangenome analysis demonstrated an open structure, with 18.9% core genes and 103 strain-specific genes, highlighting its genetic diversity. The DY55bre exhibits heterofermentative carbohydrate metabolism due to the presence of the araBAD operon and the lack of 1-phosphofructokinase (pfK) and fructose-1,6-bisphosphate aldolase enzymes. Probiotic evaluation revealed firm survival under simulated gastrointestinal conditions, including resistance to acidic pH (as low as 3.0) and bile salts (up to 1%), along with significant adhesion to intestinal epithelial cell lines (HT29;59.3%, Caco-2;87%, and DLD-1;60.8%). The strain exhibited high auto-aggregation (84.55%) and cell surface hydrophobicity (56.69%), essential for gut colonization. Safety assessments confirmed its non-hemolytic nature and absence of horizontally acquired antibiotic resistance genes. Notably, GC-MS analysis identified bioactive cyclic dipeptides, Cyclo(D-Phe-L-Pro) and Cyclo(L-Leu-L-Pro), which demonstrated cytotoxic effects against colorectal cancer cell lines, with IC50 values of 7.71 mg/mL for HT29 and 3.19 mg/mL for DLD-1. The cell-free supernatant exhibited antimicrobial activity against pathogens, likely due to the synergistic effects of cyclic dipeptides, organic acids, and other metabolites. Antioxidant assays revealed significant ABTS[+] (76.63%) and DPPH (34.25%) radical scavenging activities, while cholesterol assimilation tests showed a 27.29% reduction. These findings position the DY55bre as a promising candidate for functional foods, nutraceuticals, and therapeutic applications, warranting further in vivo validation.},
}

@article {pmid41032156,
year = {2025},
author = {Meshram, V and Jadhav, SK and Chandrawanshi, NK},
title = {Strain improvement of Cordyceps militaris for optimized bioactive metabolite biosynthesis: current progress and prospective approaches.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {11},
pages = {162},
pmid = {41032156},
issn = {1572-9699},
support = {201610136180//University Grants Commission/ ; },
mesh = {*Cordyceps/metabolism/genetics ; *Metabolic Engineering/methods ; Gene Editing ; CRISPR-Cas Systems ; Biosynthetic Pathways ; },
abstract = {Cordyceps militaris is a rare and highly valued medicinal fungus that has attracted considerable attention due to its production of diverse bioactive compounds, including nucleosides such as cordycepin, polysaccharides, lovastatin, carotenoids, etc., all of which exhibit significant nutritional and therapeutic potential. However, the large-scale utilization of C. militaris is constrained by several critical challenges. A major limitation is the progressive degeneration of strains over successive subcultures, which adversely affects fruiting body formation and metabolite biosynthesis. Moreover, genetic instability during long-term culture, contamination risks in large-scale production, and the lack of standardized cultivation and extraction protocols often result in variable product quality. The absence of efficient genetic transformation systems and the low success rate of genome editing approaches further complicate efforts in molecular strain improvement. This review provides a comprehensive overview of the principal bioactive compounds produced by C. militaris and critically evaluates the current challenges and limitations associated with both conventional and advanced strain improvement strategies. These include conventional approaches such as mutagenesis and protoplast fusion, as well as genome-editing technologies like CRISPR/Cas9, which are employed to enhance the biosynthesis of target metabolites. Moreover, the integration of metabolic engineering frameworks offers significant potential for rational strain design, optimization of bioprocesses, and the discovery of novel therapeutic agents.},
}

*Cordyceps/metabolism/genetics
*Metabolic Engineering/methods
Gene Editing
CRISPR-Cas Systems
Biosynthetic Pathways

@article {pmid41032013,
year = {2025},
author = {Huang, X and Li, H and Du, J and Xie, W and Liu, Y and Lin, Y and Xing, H},
title = {HUH endonuclease-mediated DNA-protein conjugates: sequence-specific tools and cellular applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc04628a},
pmid = {41032013},
issn = {1364-548X},
abstract = {This highlight review article summarizes recent advances in employing HUH endonucleases as self-labeling protein tags for the sequence-specific covalent conjugation of unmodified ssDNA and examines their applications in cellular studies via engineered DNA-protein conjugates. We outline the structural basis and catalytic mechanism of the conserved HUH and Y motifs, which enable high selectivity, bioorthogonality, and robust conjugation under physiological conditions. Recent applications demonstrate the versatility of HUH-based DNA-protein conjugates in programmable cellular interface engineering, targeted therapeutic delivery, and enhancement of genome editing systems such as CRISPR-Cas. In the perspective section, we further highlight two emerging directions: computational tools such as the HUHgle platform for predictive substrate design, and directed evolution strategies extending HUH reactivity toward RNA substrates. Together, these advancements establish HUH endonucleases as powerful, programmable tools for generating DNA-protein conjugates that enable innovations in chemical biology, synthetic biology, and therapeutics.},
}

@article {pmid41030550,
year = {2025},
author = {Yang, ZQ and Li, MJ and Ahmad, F and Jin, CZ and Li, T and Jin, FJ and Shin, KS and Jin, L},
title = {Application of the transposon-associated TnpB system of CRISPR-Cas in bacteria: Deinococcus.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1604032},
pmid = {41030550},
issn = {1664-302X},
abstract = {Deinococcus radiodurans is one of the most radioresistant organisms found on Earth to date, showing extreme resistance to damage factors such as UV, drought, and mutagens, and is of great interest to scientists around the world. It was determined that the TnpB protein from D. radiodurans ISDra2 functions as an RNA-guided endonuclease, serving as a functional ancestor for the widely used CRISPR-Cas endonucleases. The CRISPR-Cas system is an "acquired immune system" found in most Bacteria and Archaea, and used in a wide range of biological and medical research fields. Cas12f is the smallest RNA-directed nuclease that is currently known and possesses unique characteristics. There has been extensive research conducted on the origin, classification, and mechanism of action of CRISPR-Cas12f, as well as its application in the field of gene editing. TnpB, as the protein closest to Cas12f in the evolutionary tree, has the potential to be used as a new micro-editing tool. Systematic studies have been conducted on it to develop smaller volumes of precision gene editing and treatment tools. In this review, the research progress, mechanism, and application of TnpB protein in D. radiodurans were reviewed. In addition, the classification of CRISPR-Cas system and the application and function of CRISPR-Cas12f in gene editing are also introduced and summarized.},
}

@article {pmid41028589,
year = {2026},
author = {Bhargava, CN and Karuppannasamy, A and Ramasamy, A},
title = {CRISPR/Cas9-Mediated Genome Editing in the Management of Oriental Fruit Fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2966},
number = {},
pages = {259-270},
pmid = {41028589},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Tephritidae/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Insect ; Pest Control, Biological/methods ; },
abstract = {The oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive polyphagous pest that causes significant damage to horticultural crops of global importance. Traditional management practices have not been effective in controlling this pest, and therefore, there is a need for alternative management strategies. CRISPR/Cas9-driven genome editing has been successfully used in a wide range of insects to induce site-specific, off-target minimized mutations that result in loss of function. This technique can be used to develop precision-guided sterile insect technique (pgSIT) and gene drive programs, which can be used for area-wide suppression of the pest. This chapter provides a brief overview of the workflow for RNP-based genome editing, which can be used to validate and establish gene function for large-scale gene drive programs aimed at combating this pest. The RNP, or ribonucleoprotein complex, comprises the sgRNA and Cas9 protein, which are microinjected into the G0 stage embryos for heritable editing of the target gene(s).},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Tephritidae/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Insect
Pest Control, Biological/methods

@article {pmid41028581,
year = {2026},
author = {Ashokkumar, S and Ponnurangan, V and Krish, KK and Loganathan, A and Eswaran, K and Vaikuntavasan, P and Duraialagaraja, S and Shanmugam, V},
title = {CRISPR-Mediated Gene Editing for Inducing Thermosensitive Genic Male Sterility and Sheath Blight Resistance in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2966},
number = {},
pages = {127-140},
pmid = {41028581},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Oryza/genetics/microbiology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Infertility/genetics ; *Plant Diseases/genetics/microbiology ; Plants, Genetically Modified/genetics ; },
abstract = {Recent advances in genome editing enable the researchers to focus more and more on the ability to manipulate genomes at specific sites. Efficient methods for genome editing further promote gene discovery and functional gene analyses in model plants as well as the introduction of novel desired agricultural traits in important species. CRISPR/Cas9 technology enables precise genetic modification through the creation of double-strand breaks in a target region and the generation of desired alterations during the repair process. In this chapter, we describe the cloning strategy, transformation protocols, triparental mating procedure, and characterization of genome-edited genetic male sterile mutants and sheath blight disease-resistant mutant plants.},
}

*Gene Editing/methods
*Oryza/genetics/microbiology
*CRISPR-Cas Systems
*Disease Resistance/genetics
*Plant Infertility/genetics
*Plant Diseases/genetics/microbiology
Plants, Genetically Modified/genetics

@article {pmid41028476,
year = {2026},
author = {Raschmanová, H and Weninger, A and Kovar, K},
title = {Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {361-371},
pmid = {41028476},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Saccharomycetales/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; *Pichia/genetics ; Genome, Fungal ; },
abstract = {The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Saccharomycetales/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Metabolic Engineering/methods
*Pichia/genetics
Genome, Fungal

@article {pmid41028475,
year = {2026},
author = {Smirnov, K and Rieder, L and Glieder, A},
title = {High-Throughput Generation of Pichia pastoris Knock-Out Strains by Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {345-360},
pmid = {41028475},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; *Gene Knockout Techniques/methods ; *Saccharomycetales/genetics ; Genetic Vectors/genetics ; },
abstract = {The CRISPR/Cas9 (CRISPR is an acronym for clustered regularly interspaced short palindromic repeats) system is a powerful molecular biological tool simplifying the process of genome engineering. Originally demonstrated to function in human and mouse cells, the portfolio of organisms that can be engineered by the new and groundbreaking technology was soon expanded. In the past years, CRISPR/Cas9 tools for use in Komagataella phaffii were reported to allow the generation of K. phaffii mutant strains in less than 2 weeks. In addition, the K. phaffii tailored system uses episomal vectors for the expression of the CRISPR/Cas9 elements, which allows the recycling of the plasmid after the CRISPR editing to obtain empty mutant strains. This means that the engineered strains do not carry the expression cassette of the resistance marker and CRISPR/Cas9 plasmid in their genome and are therefore a superb starting point for further investigations.In this chapter, we describe a pipeline for the high-throughput generation of K. phaffii mutant strains with interrupted open reading frames of genes, by using the CRISPR/Cas9 system in combination with error-prone repair of the double-strand break by NHEJ. The pipeline we developed consists of four steps: (a) CRISPR/Cas9 plasmids assembly, (b) transformation of K. phaffii, (c) screening for mutant strains, and (d) plasmid elimination and is due to the detailed description of every step being easily reproducible. To intensify and simplify the research work, most of the described procedures can be performed in a 96-well format.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Plasmids/genetics
*Gene Knockout Techniques/methods
*Saccharomycetales/genetics
Genetic Vectors/genetics

@article {pmid41028463,
year = {2026},
author = {Pichler, C and Weiss, F and Glieder, A},
title = {Autonomously Replicating Sequence-Bearing Plasmids Utilized in Pichia pastoris.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {191-203},
pmid = {41028463},
issn = {1940-6029},
mesh = {*Plasmids/genetics ; *Saccharomycetales/genetics ; *DNA Replication ; Transformation, Genetic ; Saccharomyces cerevisiae/genetics ; },
abstract = {Plasmids are a common tool in biotechnology to deliver recombinant DNA into microbial cells for the production of enzymes, pharmaceutical proteins, chemicals, or metabolites. Therefore, a stable plasmid system that provides reliable gene expression over generations is essential for the successful utilization of single-cell organisms in research and production applications. Most Komagataella phaffii expression clones are generated by the integration of linear plasmids into the genome, as circular episomal plasmids are not stable under non-selective conditions. The low rate of homology-directed specific integration and the large variation among transformants of random integration limit the organism's application in enzyme engineering approaches or comparative studies where high transformation rates and uniform expression levels are desired. In the yeast Saccharomyces cerevisiae, the problem of circular plasmid stability and partition to the daughter cells during mitosis has been solved by combining centromeric sequences or elements of the 2-micron plasmid with an autonomously replicating sequence (ARS) that serves as an origin of replication. Similar attempts have not yet been successful or widely adapted in K. phaffii; hence, permanent selection pressure is required to maintain episomal plasmids in K. phaffii. There are no reports so far about functional 2-micron plasmids for P. pastoris, and CEN/ARS plasmids for P. pastoris are usually rather large and do not provide the high transformation rates as known for episomal plasmids of S. cerevisiae expression systems. However, the availability of a broad set of resistance, auxotrophic, and carbon source utilization markers facilitates reliable plasmid selection in small-scale screening applications and recently also proved to be successful for bioreactor-scale expression. This allows the combined advantages of high transformation rates and low clonal variability of ARS plasmids to be exploited. This article describes the successful utilization of ARS1-containing plasmids in K. phaffii, including antibiotic-free selection, complementation of knockout strains, or even for the application of CRISPR/Cas by transient gRNA and CAS9 gene expression in K. phaffii.},
}

*Plasmids/genetics
*Saccharomycetales/genetics
*DNA Replication
Transformation, Genetic
Saccharomyces cerevisiae/genetics

@article {pmid41028441,
year = {2025},
author = {Kannan, S and Tennyson, J},
title = {Regeneration of Transgenic Nicotiana benthamiana Raised from the Genome-Edited Protoplast.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2973},
number = {},
pages = {261-274},
pmid = {41028441},
issn = {1940-6029},
mesh = {*Nicotiana/genetics/growth & development ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; *Genome, Plant ; Transfection/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Regeneration/genetics ; },
abstract = {Plant genome editing is an emerging technique that has revolutionized plant genome engineering which helps to edit the plant genome precisely for the development of traits in many crops. Specifically, with clustered regularly interspaced short palindromic sequence (CRISPR)-CRISPR-associated protein (Cas) system, a progressive improvement in genome editing has been achieved with protoplast. Though protoplast isolation, transfection, and regeneration are available for many plants, regeneration of protoplast for many plants remains major challenge. In this methodology chapter, we outlined the construction of sgRNA for genome editing, transfection, and regeneration of transgenic N. benthamiana from the genome-edited protoplast and assay for gene targeting.},
}

*Nicotiana/genetics/growth & development
*Protoplasts/metabolism
*Gene Editing/methods
*Plants, Genetically Modified/genetics
CRISPR-Cas Systems
*Genome, Plant
Transfection/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Regeneration/genetics

@article {pmid41028420,
year = {2025},
author = {Andrew-Peter-Leon, MT and Pillai, MA and Kumar, KK and Sumithra, V},
title = {Agrobacterium-Mediated Genetic Transformation and Genome Editing Using CRISPR-Cas9 Constructs in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2973},
number = {},
pages = {27-49},
pmid = {41028420},
issn = {1940-6029},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; *Agrobacterium/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 has become an essential tool in every molecular plant breeding laboratory. CRISPR-Cas9 causes highly precise knock-out mutations in plants that can be exploited in crop improvement programmes. However, poor response to Agrobacterium-mediated genetic transformation in recalcitrant rice genotypes is a major limiting factor. This protocol describes a detailed procedure for genome editing with CRISPR-Cas9 in recalcitrant rice genotypes that otherwise show a poor response to tissue culture. With this method, high transformation efficiency can be achieved in relatively a short period.},
}

*Oryza/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Transformation, Genetic
*Agrobacterium/genetics
Plants, Genetically Modified/genetics
*Genome, Plant

@article {pmid41028383,
year = {2026},
author = {Yang, H and Bao, A and Tran, LP and Cao, D},
title = {CRISPR/Cas9-Based Gene Editing in Soybean.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2977},
number = {},
pages = {251-267},
pmid = {41028383},
issn = {1940-6029},
mesh = {*Glycine max/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Agrobacterium/genetics ; Transformation, Genetic ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean (Glycine max), a number of laboratories have obtained mutants by the CRISPR/Cas9 system; however, there has not yet been a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single-guide RNA) transgenes.},
}

*Glycine max/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Plasmids/genetics
Agrobacterium/genetics
Transformation, Genetic

@article {pmid41028112,
year = {2025},
author = {Najar, IN and Sharma, P and Das, R and Mondal, K and Singh, AK and Radha, A and Sharma, V and Sharma, S and Thakur, N and Gandhi, SG and Kumar, V},
title = {Unveiling the probiotic potential of the genus Geobacillus through comparative genomics and in silico analysis.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {33748},
pmid = {41028112},
issn = {2045-2322},
mesh = {*Probiotics ; *Geobacillus/genetics ; *Genome, Bacterial ; *Genomics/methods ; Computer Simulation ; CRISPR-Cas Systems ; Humans ; Prophages/genetics ; },
abstract = {Pursuing new probiotic targets has surged, driven by next-generation sequencing, facilitating a thorough exploration of bacterial traits. The genus Geobacillus stands out as a promising candidate for probiotics. The study explored the genetic attributes of the genus Geobacillus for their resilience to gastrointestinal conditions, nutrient production, and immunomodulatory compound creation, revealing potential probiotic traits. Predictive analyses of genomic elements like prophages, CRISPR-Cas systems, insertion sequences, genomic islands, antibiotic resistance genes, and CAZymes were conducted to assess safety. Comparative genomic analysis was performed using 18 published Geobacillus genomes and a few Lactobacillus and Bifidobacterium genomes as controls. Genes associated with probiotic traits, such as adhesion, stress tolerance (acid/bile, osmotic, oxidative), immune modulation, and molecular chaperones, were uniformly detected in Geobacillus. Mobile genetic elements (such as plasmids, prophages, and insertion sequences), virulence factors, toxins, and antibiotic resistance genes were absent, while CRISPR-Cas systems and CAZymes were present. The pan-genome comprised 25,284 protein-coding genes. Comparative genomic analysis revealed an open pan-genome for Geobacillus. Pan-genome exhibited variability, particularly in genes linked to environmental interaction and secondary metabolite synthesis. Geobacillus appears potentially safe and well-suited for the gut habitat. However, further in vitro studies are essential to confirm its probiotic potential.},
}

*Probiotics
*Geobacillus/genetics
*Genome, Bacterial
*Genomics/methods
Computer Simulation
CRISPR-Cas Systems
Humans
Prophages/genetics

@article {pmid41027434,
year = {2025},
author = {Jin, S and Zhu, Z and Li, Y and Zhang, S and Liu, Y and Li, D and Li, Y and Luo, Y and Cheng, Z and Zhao, KT and Gao, Q and Yang, G and Li, H and Liang, R and Zhang, R and Qiu, JL and Zhang, YE and Liu, JG and Gao, C},
title = {Functional RNA splitting drove the evolutionary emergence of type V CRISPR-Cas systems from transposons.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2025.09.004},
pmid = {41027434},
issn = {1097-4172},
abstract = {Transposon-encoded TnpB nucleases gave rise to type V CRISPR-Cas12 effectors through multiple independent domestication events. These systems use different RNA molecules as guides for DNA targeting: transposon-derived right-end RNAs (reRNAs or omega RNAs) for TnpB and CRISPR RNAs for type V CRISPR-Cas systems. However, the molecular mechanisms bridging transposon activity and CRISPR immunity remain unclear. We identify TranCs (transposon-CRISPR intermediates) derived from distinct IS605- or IS607-TnpB lineages. TranCs utilize both CRISPR RNAs and reRNAs to direct DNA cleavage. The cryoelectron microscopy (cryo-EM) structure of LaTranC from Lawsonibacter sp. closely resembles that of the ISDra2 TnpB complex; however, unlike a single-molecule reRNA, the LaTranC guide RNA is functionally split into a tracrRNA and crRNA. An engineered RNA split of ISDra2 TnpB enabled activity with a CRISPR array. These findings indicate that functional RNA splitting was the primary molecular event driving the emergence of diverse type V CRISPR-Cas systems from transposons.},
}

@article {pmid41026506,
year = {2025},
author = {Demissie, HA and Das, S and Thompson, JR and Lucks, JB},
title = {An Integrated Nucleic Acid Sequence-Based Amplification (NASBA) and CRISPR-Cas13a-Based Platform for Accurate and Sensitive Detection of Cucumber Mosaic Virus.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00406},
pmid = {41026506},
issn = {2161-5063},
abstract = {Cucumber mosaic virus (CMV) is a highly prevalent ssRNA viral crop pathogen that contributes to substantial losses in agricultural productivity worldwide. The first step in managing the impact of this pathogen is an accurate and timely diagnosis. However, current sensing strategies are hampered by several limitations, including insufficient sensitivity, off-target effects, and the need for complex instrumentation. To address these challenges, we refined a highly specific and sensitive system that pairs nucleic acid sequence-based amplification (NASBA) with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a to selectively amplify and detect crop pathogens. To configure this system for CMV biosensing, we first screened guide RNAs and successfully validated designs that detect attomolar concentrations of purified CMV fragments. We then developed a simplified reaction assembly workflow toward optimizing the system for downstream point-of-use utility. Using this workflow, we demonstrated minimal matrix effects when detecting purified CMV fragments in a range of plant lysate backgrounds and showed high test specificity to CMV in the presence of common nontarget viral crop pathogens. We also showed that the NASBA-Cas13a system effectively detects the viral target in infected plant samples, as validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Finally, we optimized the system for lyophilization and long-term storage, toward preparing it for point-of-use settings. This work expands the suite of CMV diagnostic tools, offering a sensitive, specific, and user-friendly biosensing strategy. Through modular design, this assay has the potential to be reconfigured for the detection of a range of crop viruses, enhancing viral surveillance and improving infection management.},
}

@article {pmid41026406,
year = {2025},
author = {Rahman, MU and Shah, JA and Khan, MN and Bilal, H and Zhu, D and Du, Z and Mu, DS},
title = {Innovative Approaches to Combat Antimicrobial Resistance: A Review of Emerging Therapies and Technologies.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41026406},
issn = {1867-1314},
abstract = {The threat of antimicrobial resistance (AMR) presents a challenge in infectious diseases, leading to higher illness and deaths worldwide. No new antibiotic has been introduced, leaving healthcare systems vulnerable to resistant pathogens. Researchers are exploring innovative approaches to overcome this growing resistance crisis. One promising strategy is synergistic therapy using combined drugs to enhance efficacy and reduce resistance. Other approaches focus on targeting the specific enzymes or proteins responsible for resistance mechanisms, thereby neutralizing the defense strategies of microorganisms. Advances in drug delivery systems have also shown promise in improving the effectiveness of existing antimicrobial agents. Biotechnological breakthroughs, such as bacteriophages and antibodies, have seen partial clinical implementation, while newer approaches like antimicrobial peptides (AMPs), lysins, and probiotics are still under development. Emerging technologies such as CRISPR-Cas and engineered phages demonstrate significant potential in preclinical studies, offering precision targeting of resistance genes and pathogen-specific lysis, respectively. However, their translational success hinges on overcoming delivery challenges, scalability, and regulatory hurdles. Additionally, physicochemical methods that disrupt microbial activity are being explored as alternative treatments. While innovative therapies like phage-derived lysins and CRISPR-Cas systems show promise in preclinical models, their clinical impact remains to be validated through large-scale trials. Their integration into mainstream medicine will depend on addressing practical challenges such as manufacturing consistency, cost considerations, and real-world efficacy assessments. These efforts are crucial for addressing the growing threat of AMR and advancing more effective, sustainable infection control strategies in clinical settings.},
}

@article {pmid41025805,
year = {2025},
author = {Nadar, S and Brown, JC and Coe, LSY and Koukoulidis, NM and Czyż, EM and Czyż, DM},
title = {Antimicrobial resistance and One Health in the high school biology curriculum.},
journal = {Journal of microbiology & biology education},
volume = {},
number = {},
pages = {e0014525},
doi = {10.1128/jmbe.00145-25},
pmid = {41025805},
issn = {1935-7877},
abstract = {Antimicrobial resistance (AMR) is the ability of a microbial organism to resist treatment designed to kill it. It poses a significant global threat to public health, affecting humans, animals, and the environment, in a concept collectively referred to as One Health. While one of the major mitigators of this pressing issue is education, the high school curriculum in the United States does not cover any aspects of AMR. As such, to address this challenge, we developed and delivered a one-week-long unit on AMR within a One Health framework into a high school biology curriculum. The unit aimed to enhance students' understanding of AMR and its implications across the One Health sectors. A survey was designed and administered to measure current knowledge, awareness, interest, and motivation. Through a combination of lectures developed using Universal Design of Learning principles, interactive discussions using team-based learning (TBL) with the help of content experts, hands-on laboratory exercise, and poster presentations, biology students explored the mechanisms of resistance and novel mitigation strategies. Pre- and post-assessments revealed a marked improvement in students' knowledge and comprehension of AMR and therapeutic strategies, such as silver nanoparticles, bacteriocins, bacteriophages, CRISPR-Cas, and immunotherapy. This research study provides a detailed overview of the curriculum design, instructional strategies, and assessment outcomes, offering a replicable model for broadly integrating AMR education into high school curricula. We found that the AMR mitigation strategies lesson, delivered through TBL, significantly enhanced students' understanding of novel therapeutic strategies and fostered high levels of engagement throughout the AMR and One Health unit.},
}

@article {pmid41025245,
year = {2025},
author = {Shin, K and Kim, ET},
title = {Efficient CRISPR-based genome editing for inducible degron systems to enable temporal control of protein function in large double-stranded DNA virus genomes.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {9},
pages = {e2504008},
doi = {10.71150/jm.2504008},
pmid = {41025245},
issn = {1976-3794},
support = {RS-2024-00352590//Ministry of Science and ICT/ ; RS-2023-00270936//Ministry of Education/ ; //Korea Health Industry Development Institute/ ; RS-2022-KH129726//Ministry of Health and Welfare/ ; RS-2024-00438990//Ministry of Health and Welfare/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Cytomegalovirus/genetics ; *Genome, Viral ; *Viral Proteins/genetics/metabolism ; Indoleacetic Acids/metabolism/pharmacology ; Green Fluorescent Proteins/genetics ; Recombinational DNA Repair ; Degrons ; },
abstract = {CRISPR-Cas9-based gene editing enables precise genetic modifications. However, its application to human cytomegalovirus (HCMV) remains challenging due to the large size of the viral genome and the essential roles of key regulatory genes. Here, we establish an optimized CRISPR-Cas9 system for precise labeling and functional analysis of HCMV immediate early (IE) genes. By integrating a multifunctional cassette encoding an auxin-inducible degron (AID), a self-cleaving peptide (P2A), and GFP into the viral genome via homology-directed repair (HDR), we achieved efficient knock-ins without reliance on bacterial artificial chromosome (BAC) cloning, a labor-intensive and time-consuming approach. We optimized delivery strategies, donor template designs, and component ratios to enhance HDR efficiency, significantly improving knock-in success rates. This system enables real-time fluorescent tracking and inducible protein degradation, allowing temporal control of essential viral proteins through auxin-mediated depletion. Our approach provides a powerful tool for dissecting the dynamic roles of viral proteins throughout the HCMV life cycle, facilitating a deeper understanding of viral pathogenesis and potential therapeutic targets.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
*Cytomegalovirus/genetics
*Genome, Viral
*Viral Proteins/genetics/metabolism
Indoleacetic Acids/metabolism/pharmacology
Green Fluorescent Proteins/genetics
Recombinational DNA Repair
Degrons

@article {pmid41025215,
year = {2025},
author = {Park, J and Sipe, GO and Tang, X and Ojha, P and Fernandes, G and Leow, YN and Zhang, C and Osako, Y and Natesan, A and Drummond, GT and Jaenisch, R and Sur, M},
title = {Astrocytic modulation of population encoding in mouse visual cortex via GABA transporter 3 revealed by multiplexed CRISPR/Cas9 gene editing.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41025215},
issn = {2050-084X},
support = {R01DA049005/NH/NIH HHS/United States ; R01MH126351/NH/NIH HHS/United States ; R01NS130361/NH/NIH HHS/United States ; R01MH133066/NH/NIH HHS/United States ; Multidisciplinary University Research Initiative W911NF2110328//United States Department of Defense/ ; F32EY022264/NH/NIH HHS/United States ; Autism Research Initiative Bridge to Independence award//Simons Foundation/ ; Picower Postdoctoral Fellowship//JPB Foundation/ ; },
mesh = {Animals ; *Visual Cortex/physiology ; *Astrocytes/physiology/metabolism ; *GABA Plasma Membrane Transport Proteins/metabolism/genetics ; Mice ; *CRISPR-Cas Systems ; *Gene Editing ; Mice, Knockout ; Neurons/physiology ; },
abstract = {Astrocytes, which are increasingly recognized as pivotal constituents of brain circuits governing a wide range of functions, express GABA transporter 3 (Gat3), an astrocyte-specific GABA transporter responsible for maintenance of extra-synaptic GABA levels. Here, we examined the functional role of Gat3 in astrocyte-mediated modulation of neuronal activity and information encoding. First, we developed a multiplexed CRISPR construct applicable for effective genetic ablation of Gat3 in the visual cortex of adult mice. Using in vivo two-photon calcium imaging of visual cortex neurons in Gat3 knockout mice, we observed changes in spontaneous and visually driven single neuronal response properties such as response magnitudes and trial-to-trial variability. Gat3 knockout exerted a pronounced influence on population-level neuronal activity, altering the response dynamics of neuronal populations and impairing their ability to accurately represent stimulus information. These findings demonstrate that Gat3 in astrocytes profoundly shapes the sensory information encoding capacity of neurons and networks within the visual cortex.},
}

Animals
*Visual Cortex/physiology
*Astrocytes/physiology/metabolism
*GABA Plasma Membrane Transport Proteins/metabolism/genetics
Mice
*CRISPR-Cas Systems
*Gene Editing
Mice, Knockout
Neurons/physiology

@article {pmid41024337,
year = {2025},
author = {Zhang, L and Fu, J and Long, T and Zhang, C and Fan, F and Lang, Z and Zhu, JK},
title = {A Modular and Customizable CRISPR/Cas Toolkit for Epigenome Editing of Cis-regulatory Modules.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e03917},
doi = {10.1002/advs.202503917},
pmid = {41024337},
issn = {2198-3844},
support = {KQTD20240729102038044//Shenzhen Science and Technology Program/ ; 32188102//National Natural Science Foundation of China/ ; },
abstract = {Epigenome and cis-regulome, comprising cis-regulatory elements (CREs) and modules (CRMs), jointly define the architecture of gene regulation. However, the causal mechanisms by which epigenetic marks influence CRM function remain elusive. To address this, modular epigenome editing frameworks, exemplified by dead Cas9-coupled DNA demethylation (dCd) and DNA methylation (dCm) platforms, are developed for programmable dissection and engineering of CRM activity. The dCd system modulates methylation levels and transcriptional output at CRMs in situ or ex situ, in accordance with CRM-specific methylation responsiveness, and alters co-transcriptional RNA processing to yield predictable phenotypic outcomes in plants. These findings underscore the reliability of targeted DNA demethylation. In parallel, the dCm system reconstitutes methylation-dependent and -sensitive CRMs of diverse origins in Saccharomyces cerevisiae, a species devoid of native DNA methylation, enabling causal dissection of epigenetic regulation and revealing cross-species portability. This system further uncovers crosstalk between DNA methylation and chromatin modifications, and enables logic-gated control of endogenous genes through CRM engineering. Incorporation of optogenetic and temperature-sensitive anti-CRISPR inhibitors confers tunable, reversible regulation, proposing dCm as a foundation for input-responsive synthetic epigenome editors. Together, these frameworks provide a versatile platform to decode and reprogram cis-regulatory epigenetic logic, with broad applications in trait design and synthetic biology.},
}

@article {pmid41023727,
year = {2025},
author = {Zhao, XY and Gao, C and Zhao, WW and Zhou, ZH and Zhuang, TC and Guo, C and Ji, MH},
title = {Development of a single-tube, dual-target CRISPR Cas12a/Cas13a system for rapid screening of coinfection with respiratory syncytial virus and rhinovirus.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {311},
pmid = {41023727},
issn = {1743-422X},
support = {Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; },
mesh = {Humans ; *Coinfection/diagnosis/virology ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; *Rhinovirus/genetics/isolation & purification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Respiratory Syncytial Virus, Human/genetics/isolation & purification ; *Picornaviridae Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Respiratory Tract Infections/diagnosis/virology ; CRISPR-Associated Proteins/genetics ; Infant ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: Respiratory syncytial virus (RSV) and human rhinovirus (HRV) are leading causes of respiratory infections in children, with increasing reports of coinfections leading to severe complications. Current CRISPR-based detection systems, such as Cas12a and Cas13a, are limited in multiplex detection due to the lack of specific reporter cleavage mechanisms. This study aims to develop a rapid, sensitive, and single-tube dual-gene detection method for RSV and HRV using the orthogonal trans-cleavage activities of CRISPR-Cas12a/13a combined with reverse transcription-recombinase polymerase amplification (RT-RPA).

METHODS: We designed a novel detection system leveraging RT-RPA for amplification and the distinct cleavage activities of Cas12a and Cas13a for simultaneous dual-gene detection.

RESULTS: The reaction components were optimized to complete detection within 30 min, achieving sensitivities of 10 copies/µL for RSV and 10[2] copies/µL for HRV. Clinical validation was performed on 543 respiratory infection samples, confirming high accuracy and specificity.

CONCLUSIONS: The RT-RPA-CRISPR-Cas12a/13a system provides a rapid, sensitive, and efficient solution for RSV and HRV coinfection detection. This method supports early diagnosis and improved clinical management, offering significant potential for public health applications in preventing severe respiratory complications in children.},
}

Humans
*Coinfection/diagnosis/virology
*Respiratory Syncytial Virus Infections/diagnosis/virology
*Rhinovirus/genetics/isolation & purification
*CRISPR-Cas Systems
Sensitivity and Specificity
*Respiratory Syncytial Virus, Human/genetics/isolation & purification
*Picornaviridae Infections/diagnosis/virology
*Molecular Diagnostic Techniques/methods
*Respiratory Tract Infections/diagnosis/virology
CRISPR-Associated Proteins/genetics
Infant
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41023259,
year = {2025},
author = {Ravenel, K and Poirier, W and Razafimandimby, B and Bouchara, JP and Gastebois, A and Giraud, S},
title = {Optimization of the Genome Editing CRISPR-Cas9 Technology in Scedosporium apiospermum.},
journal = {Mycopathologia},
volume = {190},
number = {6},
pages = {94},
pmid = {41023259},
issn = {1573-0832},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Scedosporium/genetics ; },
abstract = {Scedosporium species are opportunistic pathogens causing a large variety of human infections. To date, there is limited information on the pathogenic mechanisms of these fungi, partly because of the limited number of genetic tools available. Here, the CRISPR-Cas9 technology, which provided promising results for functional genomic studies in filamentous fungi, was optimized for Scedosporium species using in vitro assembled Cas9 ribonucleoprotein (RNP) complexes. In these fungi, functional genomic studies are particularly complex in a wild-type strain, because of the high frequency of non-homologous recombination. Prior disruption of the KU70 gene encoding one of the components of the non-homologous end joining system is required, which necessitates the use of a first selection marker. The cleavage of the target gene at each end using a dual RNA-guided Cas9 complex, followed by recombination with a repair template containing the hygromycin resistance gene, allowed disruption of the target gene in the ΔKU70 mutant. Four genes encoding dioxygenases, catalyzing the critical ring-opening step in aromatic hydrocarbons, were successfully disrupted, and the optimum efficiency was observed using 5 μg of the HygR repair cassette. Alternatively, in the wild-type strain, the exclusive use of two Cas9 RNP complexes was enough to achieve an efficient deletion method; one dioxygenase gene was successfully deleted in up to 20% of the obtained colonies. These last experimental conditions path the way to multiple gene deletions and complementation experiments, which cannot be reached using our first procedure since only two selection markers are available for Scedosporium species.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Scedosporium/genetics

@article {pmid41022734,
year = {2025},
author = {Fu, C and Saddawi-Konefka, R and Chinai, JM and Kim, SY and Kammula, AV and Perera, JJ and Jiang, A and Tiwari, P and Kistler, EN and Tang, S and Luna, SM and Colvin, KJ and Dubrot, J and Anderson, S and Fetterman, RA and Chuong, CL and Lane-Reticker, SK and Cheruiyot, CK and Muscato, AJ and Alipour, Z and Adkins, DR and Griffin, GK and Bernstein, BE and Egloff, AM and Yates, KB and Chernock, RD and Gutkind, JS and Uppaluri, R and Manguso, RT},
title = {In vivo CRISPR screening in head and neck cancer reveals Uchl5 as an immunotherapy target.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8572},
pmid = {41022734},
issn = {2041-1723},
support = {U01DE029188//U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)/ ; },
mesh = {Animals ; Humans ; *Ubiquitin Thiolesterase/genetics/metabolism/immunology ; Mice ; *Head and Neck Neoplasms/genetics/immunology/therapy/pathology ; *Immunotherapy/methods ; *Squamous Cell Carcinoma of Head and Neck/genetics/immunology/therapy/pathology ; Epithelial-Mesenchymal Transition/genetics/immunology ; Cell Line, Tumor ; CD8-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Extracellular Matrix/metabolism ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Tumor Escape/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Neoplastic ; Female ; },
abstract = {Recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with a significant unmet need for enhancing immunotherapy response given current modest efficacy. Here, we perform an in vivo CRISPR screen in an HNSCC mouse model to identify immune evasion genes. We identify several regulators of immune checkpoint blockade (ICB) response, including the ubiquitin C-terminal hydrolase 5 (UCHL5). Loss of Uchl5 in tumors increases CD8[+] T cell infiltration and improved ICB responses. Uchl5 deficiency attenuates extracellular matrix (ECM) production and epithelial-mesenchymal-transition (EMT) transcriptional programs, which contribute to stromal desmoplasia, a histologic finding we describe as associated with reduced anti-PD1 response in human HNSCCs. COL17A1, a collagen highly and specifically expressed in HNSCC, mediates in part Uchl5-mediated immune evasion. Our findings suggest an unappreciated role for UCHL5 in promoting EMT in HNSCC and highlight ECM modulation as a strategy to improve immunotherapy responses.},
}

Animals
Humans
*Ubiquitin Thiolesterase/genetics/metabolism/immunology
Mice
*Head and Neck Neoplasms/genetics/immunology/therapy/pathology
*Immunotherapy/methods
*Squamous Cell Carcinoma of Head and Neck/genetics/immunology/therapy/pathology
Epithelial-Mesenchymal Transition/genetics/immunology
Cell Line, Tumor
CD8-Positive T-Lymphocytes/immunology
CRISPR-Cas Systems
Extracellular Matrix/metabolism
Immune Checkpoint Inhibitors/pharmacology/therapeutic use
Tumor Escape/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Expression Regulation, Neoplastic
Female

@article {pmid41022133,
year = {2025},
author = {Jin, X and Ouyang, C and Sun, T and Li, C and Gu, J and An, B and Wang, Z},
title = {A CRISPR/Cas9 mutant resource for OsSm RNA-binding genes in rice.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70475},
doi = {10.1111/tpj.70475},
pmid = {41022133},
issn = {1365-313X},
support = {2021CX02N173//Guangdong Pearl River Talents Program/ ; 2025B03J0025//Guangzhou Science and Technology Plan Project/ ; 2024KJ31//the Provincial Rural Revitalization Strategy Special Project of Guangdong in 2024/ ; ZDYF2024XDNY179//Hainan Province Science and Technology Special Fund/ ; 32100252//National Natural Science Foundation of China/ ; 32100294//National Natural Science Foundation of China/ ; 32171292//National Natural Science Foundation of China/ ; 211207157080997//Zhanjiang plan for navigation/ ; QT2024-017//Young Talent Support Project of Guangzhou Association for Science and Technology/ ; 2023A1515010428//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {*Oryza/genetics/growth & development/metabolism ; *CRISPR-Cas Systems/genetics ; *Plant Proteins/genetics/metabolism ; Mutation ; Gene Expression Regulation, Plant ; *RNA-Binding Proteins/genetics/metabolism ; Genes, Plant ; RNA Splicing ; },
abstract = {Pre-mRNA, produced by eukaryotic DNA transcription, undergoes splicing by the spliceosome, which removes introns and joins exons to form mRNA. The spliceosome is a large and highly dynamic molecular machine. Its core components include five small nuclear ribonucleoproteins (snRNPs) and the various spliceosome-related proteins. The conserved Smith (Sm) complex and the Sm-like proteins (LSm) serve as primary components of the snRNPs. Sm proteins are involved in processes such as pre-mRNA splicing and mRNA degradation, which can regulate gene expression, thereby influencing plant growth, development, and stress responses. While 25 Sm proteins have been identified in rice, their specific roles in regulating rice growth and development remain unclear. In this study, we employed the CRISPR/Cas9 system to edit 15 OsSm genes, and 13 mutants were obtained, with mutation rates ranging from 20.83 to 83.87%. In comparison to the wild type (WT), the mutants exhibited dwarfism, reduced tiller numbers, lower seed-setting rates or sterility, and increased susceptibility to diseases. One Sm mutant, ossmf-2, exhibited dwarfism, delayed flowering, and small grains. Through transcriptome analysis, three target genes, OsMRG702, OsRGG2, and OsLA1, were identified. Mutations of the OsSmF protein may lead to the abnormal splicing of these genes and finally lead to the inhibition of growth and development. Our study first edited the OsSm genes and generated a mutant library in rice. Most of the mutants exhibited abnormal growth and development, underscoring the essential roles of OsSm proteins in rice physiology. Furthermore, this work addresses a critical gap in the functional characterization of Sm proteins in rice. The resulting mutant collection offers valuable germplasm resources and lays a theoretical foundation for elucidating the molecular regulatory networks involving spliceosomal components and their target genes in the control of crop growth, development, and reproduction.},
}

*Oryza/genetics/growth & development/metabolism
*CRISPR-Cas Systems/genetics
*Plant Proteins/genetics/metabolism
Mutation
Gene Expression Regulation, Plant
*RNA-Binding Proteins/genetics/metabolism
Genes, Plant
RNA Splicing

@article {pmid41021290,
year = {2025},
author = {Johnson, KA and Cooper, C and Philippe, C and Catchpole, RJ and Mitchell, S and Terns, MP},
title = {A Phage Variable Region Encodes Anti-CRISPR Proteins Inhibiting All Streptococcus thermophilus CRISPR Immune Systems.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1177/25731599251369720},
pmid = {41021290},
issn = {2573-1602},
abstract = {Bacteria and archaea utilize CRISPR-Cas systems to defend against invading mobile genetic elements (MGEs) such as phages and plasmids. In turn, MGEs have evolved anti-CRISPR (Acr) proteins to counteract these defenses. While several type II-A Acrs have been identified in Streptococcus thermophilus (Sth) phages, a more comprehensive understanding of Acr diversity in Sth phages has yet to be explored. Guided by the genomic context of known Acrs, we systematically screened uncharacterized phage proteins and identified several novel Acrs that inhibit type I-E, type II-A or type III-A Sth CRISPR-Cas systems. These acr genes are clustered within a variable phage genomic region, indicating a hotspot for anti-defense activity. We also identified neighboring proteins with predicted enzymatic or structural domains that may modulate phage-host interactions through Acr-independent mechanisms. Together, our findings expand the known repertoire of Sth Acrs and highlight the phage variable region as a key reservoir of immune-modulating factors.},
}

@article {pmid41021273,
year = {2025},
author = {Sgodda, M and Gebel, E and Dignas, L and Alfken, S and Eggenschwiler, R and Stalke, A and Dröge, C and Pfister, ED and Baumann, U and Luedde, T and Esposito, I and Keitel, V and Cantz, T},
title = {iPSC-based hepatic organoids reveal a heterozygous MYO5B variant as driver of intrahepatic cholestasis.},
journal = {Hepatology communications},
volume = {9},
number = {10},
pages = {},
pmid = {41021273},
issn = {2471-254X},
mesh = {Humans ; *Cholestasis, Intrahepatic/genetics/metabolism ; *Organoids/metabolism ; ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics/metabolism ; *Induced Pluripotent Stem Cells ; Heterozygote ; *Myosin Type V/genetics ; *Myosin Heavy Chains/genetics ; ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism ; Liver/metabolism ; Multidrug Resistance-Associated Protein 2 ; },
abstract = {BACKGROUND: Hereditary intrahepatic cholestasis is caused by variants of various genes involved in enterohepatic bile circulation, metabolization, and conjugation. Originally classified into 3 groups, the number of contributing genes is still increasing, underlining the need for a deeper understanding of the molecular interaction during intrahepatic cholestasis.

METHODS: In the present study, we investigate the interplay of heterozygous variants in 3 cholestasis-associated genes (ABCB11, ABCB4, and MYO5B) by exploiting iPSC-based hepatic organoids from a patient suffering from recurrent intrahepatic cholestasis.

RESULTS: Functional characterization of MRP2-mediated cholyl-lysyl-fluorescein (CLF) and BSEP-mediated Tauro-nor-THCA-24-DBD transport demonstrated a marked reduction of transport in MYO5B-deficient organoids, in comparison to unaffected control organoids. Moreover, iPSC-based organoids derived from the patient carrying 3 heterozygous variants in ABCB11, ABCB4, and MYO5B also exhibited absence of BSEP-mediated Tauro-nor-THCA-24-DBD transport, but functional MRP2-mediated CLF-transport. Interestingly, CRISPR/Cas9-mediated correction of the mutated ABCB11 allele could not restore the impaired BSEP function, suggesting the heterozygous MYO5B variant as the main driver of the transport deficiency. In fact, CRISPR/Cas-mediated correction of the MYO5B variant finally resulted in a restoration of the BSEP-mediated Tauro-nor-THCA-24-DBD transport.

CONCLUSIONS: iPSC-based organoids serve as an authentic model for functional assessment of the hepatobiliary transport with fluorescent substrates. This allows the characterization of variants of uncertain significance and other variants in cholestasis-associated genes and revealed that a heterozygous MYO5B variant increases the susceptibility to defective hepatobiliary BSEP-mediated transport.},
}

Humans
*Cholestasis, Intrahepatic/genetics/metabolism
*Organoids/metabolism
ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics/metabolism
*Induced Pluripotent Stem Cells
Heterozygote
*Myosin Type V/genetics
*Myosin Heavy Chains/genetics
ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism
Liver/metabolism
Multidrug Resistance-Associated Protein 2

@article {pmid41021067,
year = {2025},
author = {Zou, S and Chen, W and Cao, Y and Liu, X and Wang, J and Wang, Y and Zhou, S},
title = {Lethal endotoxin (ccdB) based counterselection improved the efficiency of sequential gene editing in Escherichia coli.},
journal = {Biotechnology letters},
volume = {47},
number = {5},
pages = {118},
pmid = {41021067},
issn = {1573-6776},
support = {Guike AA24206048//Science and Technology Major Project of Guangxi/ ; AA24206050//Science and Technology Major Project of Guangxi/ ; 4301/00960//Hubei University of Technology High-Level Talent Research Startup Fund Program/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; Plasmids/genetics ; CRISPR-Cas Systems ; *Endotoxins/genetics ; *Escherichia coli Proteins/genetics ; },
abstract = {The CRISPR/Cas9-based technology has been used for sequential gene editing in E. coli. The plasmids carrying the sgRNA and/or Cas9 genes need to be cured after each round of editing. Curing of these plasmids, particularly the sgRNA plasmid, limits the efficiency of sequential gene editing. In this study, a lethal endotoxin (ccdB) based counterselection was established for improving the overall efficiency of sequential gene editing in E. coli. This approach was validated for sequential editing (deletion) of cstA and ppsA genes in HBUT-P2 strain (W derivative). The experimental results showed that the transformation efficiency of sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20) reached 10[8]-10[9] cfu/μg-DNA, resulting in a 100% and 93.75% recombination rate for cstA and ppsA gene, respectively. Upon completion of cstA gene editing, the sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (cstA)) was effectively cured through ccdB based counterselection at 42 °C, with a 43.75% efficiency. At the end of sequential editing of ppsA gene, both Cas9 (25A) and sgRNA (pTargetF-tcr-PL-ccdB-N20 (ppsA)) plasmids were cured simultaneously through the sacB and ccdB based counterselections by incubating the cells on LB-sucrose (5%) plate at 42 °C, achieving a curing rate of 100% for Cas9 plasmid (25A), 37.5% for sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (ppsA)), and 37.5% for both Cas9 and sgRNA plasmids. Moreover, this approach was further validated through efficient site-specific insertion of the csc operon into the slmA gene in DH5α (K12 derivative) and S322 (C derivative) strains. These results demonstrated that the endotoxin (ccdB) based counterselection improved the transformation efficiency of sgRNA plasmid, the recombination rate of the editing target gene, the curing rate of sgRNA plasmid, and the overall efficiency of sequential gene editing.},
}

*Gene Editing/methods
*Escherichia coli/genetics
Plasmids/genetics
CRISPR-Cas Systems
*Endotoxins/genetics
*Escherichia coli Proteins/genetics