@article {pmid41264695,
year = {2025},
author = {Rönspies, M and Khosravi, S and Helia, O and Valisi, A and Fajkus, J and Fojtová, M and Houben, A and Puchta, H},
title = {CRISPR-Cas-mediated heritable chromosome fusions in Arabidopsis.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6775},
pages = {843-848},
doi = {10.1126/science.adz8505},
pmid = {41264695},
issn = {1095-9203},
mesh = {*Arabidopsis/genetics ; *Chromosomes, Plant/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Genome, Plant ; Karyotype ; Meiosis ; Recombination, Genetic ; Gene Fusion ; },
abstract = {The genome of Arabidopsis thaliana consists of 10 chromosomes. By inducing CRISPR-Cas-mediated breaks at subcentromeric and subtelomeric sequences, we fused entire chromosome arms, obtaining two eight-chromosome lines. In one line, both arms of chromosome 3 were fused to chromosome 1. In another line, the arms were transferred to chromosomes 1 and 5. Both chromosome number-reduced lines were fertile. Phenotypic and transcriptional analyses revealed no differences compared with wild-type plants. After crossing with the wild type, the progeny showed reduced fertility. The meiotic recombination patterns of the transferred chromosome arms were substantially changed. Directed chromosome number changes in plants may enable new breeding strategies, redefining linkage groups and establishing genetic barriers. Moreover, our data indicate that plants are highly robust to engineered karyotype changes.},
}

*Arabidopsis/genetics
*Chromosomes, Plant/genetics
*CRISPR-Cas Systems
Gene Editing
*Genome, Plant
Karyotype
Meiosis
Recombination, Genetic
Gene Fusion

@article {pmid41263111,
year = {2025},
author = {Feng, X and Li, Y and Zheng, J and Chen, X and Yang, S and Chen, Y and Li, SC},
title = {MicrobialScope: an integrated genomic resource with rich annotations across bacteria, archaea, fungi, and viruses.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaf1234},
pmid = {41263111},
issn = {1362-4962},
support = {C2004-23Y//Young Collaborative Research/ ; JCYJ20220818101201004//Shenzhen Science and Technology Program/ ; 32300527//National Natural Science Foundation of China/ ; 32470695//National Natural Science Foundation of China/ ; 2022A1515110784//Guangdong Basic and Applied Basic Research Foundation/ ; 2023B0303040004//Key-Area Research and Development Program of Guangdong Province/ ; TC2024JC43//Basic Research Programs of Taicang, 2024/ ; //Shenzhen-Hong Kong Institute of Brain Science/ ; //SIAT-HKUST Joint Laboratory of Brain Science/ ; },
abstract = {Microorganisms, including bacteria, archaea, fungi, and viruses, are the most taxonomically diverse and ecologically dominant life forms on Earth, playing critical roles in ecosystems, human health, and industrial applications. While existing microbial databases such as BV-BRC and IMG archive both monoisolate and metagenome-assembled genomes (MAGs) across domains, challenges remain in standardized, multi-level annotations and interactive tools for all microbial groups. Here, we present MicrobialScope (https://microbial.deepomics.org/), a comprehensive microbial genomic platform that integrates large-scale genome collections, multilevel annotations, and interactive visualizations. MicrobialScope harbors 2 411 503 bacterial, 24 472 archaeal, 20 203 fungal, and 188 267 viral genomes derived from both monoisolate assemblies and MAGs. Integrating 15 state-of-the-art bioinformatics tools and 10 specialized databases, MicrobialScope provides extensive annotations encompassing basic genomic features, genomic element prediction (e.g., genes, tRNAs, tmRNAs, CRISPR-Cas and anti-CRISPR elements, secondary metabolite biosynthetic clusters, signal peptides, and transmembrane proteins), and functional and structural annotations. This includes 1 072 114 935 proteins with diverse annotations, 24 640 186 tRNAs and tmRNAs, 140 888 CRISPR-Cas systems, 173 256 anti-CRISPR elements, 105 121 secondary metabolite biosynthetic clusters, 13 235 096 signal peptides, and 50 811 729 transmembrane proteins. In addition, MicrobialScope offers unrestricted access to all data resources, interactive visualization tools, and built-in online analytical modules for intuitive exploration and comparative analysis. With its extensive genome collection, comprehensive annotations, and user-friendly interface, MicrobialScope serves as a scalable platform to advance genome research across diverse microbial domains.},
}

@article {pmid41262458,
year = {2025},
author = {Gao, Z and Gao, Y and Wang, S and Li, X and Cao, W and Deng, W and Yao, L and Wei, X and Zhang, Z and Wang, S and Zhang, Y and Li, M and Xie, Y},
title = {Application progress and biosafety challenges of gene editing and synthetic biotechnology in diagnosis, treatment and prevention of infectious diseases.},
journal = {Biosafety and health},
volume = {7},
number = {5},
pages = {312-322},
pmid = {41262458},
issn = {2590-0536},
abstract = {Global infectious disease prevention faces escalating challenges due to the continual emergence of novel pathogens and rapid viral mutations. Synthetic biology has revolutionized this field by enabling precise diagnostics, innovative vaccine platforms, and targeted therapeutics, yet it simultaneously raises concerns regarding dual-use potential, biosafety, and ethical governance. This systematic review (2015-2025, PubMed, Web of Science, Scopus) focuses on CRISPR-based diagnostics, synthetic vaccines, and engineered probiotics. CRISPR/Cas systems such as DETECTR (Cas12a) and SHERLOCK (Cas13a) demonstrate high sensitivity and rapid pathogen detection (e.g., SARS-CoV-2, Ebola), but their misuse could enhance pathogen virulence or enable bioweapon development. mRNA and viral vector vaccines offer flexible and rapid responses to emerging infections but encounter limitations in molecular stability, delivery system toxicity, and ecological safety. Engineered probiotics, designed as "living therapeutics," can detect pathogens and modulate immune responses, yet pose potential risks of horizontal gene transfer and host-specific variability. Overall, while synthetic biology provides transformative tools for infectious disease control, it necessitates robust global regulatory frameworks, standardized biosafety practices, and ethical oversight to ensure responsible and sustainable application.},
}

@article {pmid41262327,
year = {2025},
author = {Jia, Y and Horvath, K and Rananaware, SR and Jain, PK and Sampath, J},
title = {Exploring the temperature stability of CRISPR-Cas12b using molecular dynamics simulations.},
journal = {Molecular systems design & engineering},
volume = {},
number = {},
pages = {},
pmid = {41262327},
issn = {2058-9689},
abstract = {The thermal stability of CRISPR-Cas nucleases is a critical factor for their successful application in 'one-pot' diagnostic assays that utilize high-temperature isothermal amplification. To understand the atomistic mechanism of stabilization in a previously engineered variant of the thermostable BrCas12b protein, we performed all-atom molecular dynamics (MD) simulations on the wild-type and mutant forms of apo BrCas12b. High-temperature simulations reveal a small structural change along with greater flexibility in the PAM-interacting domain of the mutant BrCas12b, with marginal structural and flexibility changes in the other mutated domains. Comparative essential dynamics analysis between the wild-type and mutant BrCas12b at both ambient and elevated temperatures provides insights into the stabilizing effects of the mutations. Our findings offer comprehensive insights into the important protein motions induced by these mutations. These results provide insights into thermal stability mechanisms in BrCas12b that may inform the future design of CRISPR-based tools.},
}

@article {pmid41262251,
year = {2025},
author = {Stelcer, E and Wozniak, A and Magner, D and Zeyland, J},
title = {Genetically modified pigs with α1,3-galactosyltransferase knockout and beyond: a comprehensive review of xenotransplantation strategies.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1663246},
pmid = {41262251},
issn = {1664-3224},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Galactosyltransferases/genetics/deficiency ; *Animals, Genetically Modified ; Swine ; *Graft Rejection/immunology/genetics/prevention & control ; Gene Knockout Techniques ; Humans ; Heterografts/immunology ; },
abstract = {Xenotransplantation holds promise to eliminate the shortage of organs intended for humans in need. Pigs constitute the most suitable organ xenograft donor due to the fact that their organ anatomy physiological metabolism and immune system resemble those of humans. However, swine organs rapidly cause hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR) after transplantation. HAR and AHXR are caused by the presence of xenoreactive natural immunoglobulins directed toward a galactose alpha1-3-galactose (alpha-Gal) epitope on porcine vascular endothelium. In order to suppress both types of rejection, pigs with alpha1,3-galactosyltransferase gene knockout (GT-KO) and other genetic modifications (like simultaneous expression of the human complementary regulatory proteins) are intensively investigated. This review highlights the usefulness of GT-KO pig - derived organs such as kidney, heart, corneal, and lung in xenotransplantation. To obtain transgenic pigs researchers can use several techniques based on pronuclear and cytoplasmic microinjection, somatic cell nuclear transfer (SCNT), viral transduction of DNA and DNA transposable element -based technology, site specific nucleases and modifications of the CRISPR/Cas bacterial immune system. Some additional strategies like targeted immunosuppression or tolerance induction of B and T cells will be essential for sustained survival of xenografts. Although xenotransplantation with the use of pigs is a very rapidly evolving field, more research is needed to create perfectly compatible with the human immune system organs.},
}

Animals
*Transplantation, Heterologous/methods
*Galactosyltransferases/genetics/deficiency
*Animals, Genetically Modified
Swine
*Graft Rejection/immunology/genetics/prevention & control
Gene Knockout Techniques
Humans
Heterografts/immunology

@article {pmid41261856,
year = {2025},
author = {Xiao, Y and Zhao, R and Bao, Y and Lu, B and Jiang, Y and Tang, Y and Li, B},
title = {Cas12a-assisted split crRNA complex for analysis and detection of diverse entities.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
pmid = {41261856},
issn = {1362-4962},
support = {SKL202402017//The Science and Technology Development Plan Project of Jilin Province/ ; SKL202302030//The Science and Technology Development Plan Project of Jilin Province/ ; 20240101005JJ//Jilin Province Science Fund for Distinguished Young Scholars/ ; 23GZZ03//Major Project of Changchun State Key Laboratory/ ; 20230203193SF//Key R&D Program of Jilin Province/ ; 22525405//National Nature Science Foundation of China/ ; 22504138//National Nature Science Foundation of China/ ; 22474135//National Nature Science Foundation of China/ ; 22374142//National Nature Science Foundation of China/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Cas Systems ; MicroRNAs/genetics/analysis ; DNA, Single-Stranded/genetics ; DNA/genetics ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/methods ; },
abstract = {The Cas12a-crRNA system possesses inherent sequence-specific recognition of double-stranded DNA/single-stranded DNA (dsDNA/ssDNA) coupled with trans-cleavage activity toward ssDNA, making it a powerful tool for nucleic acid diagnostics. However, its application beyond nucleic acid targets remains challenging, limiting its potential as a universal detection platform. In this study, we systematically explore the key parameters governing the activation of a Cas12a-split crRNA system and established a comprehensive set of design guidelines. Building on these findings, we developed CASCADE (Cas12a-Assisted Split crRNA Complex for Analysis and Detection of Diverse Entities), an adaptable detection platform that extends Cas12a's application beyond nucleic acids. Using microRNA as a model, we validated the system's sensitivity, specificity, and mismatch discrimination capability. Additionally, we successfully demonstrated its capability for non-nucleic acid target detection by detecting tobramycin, kanamycin, biotin, and tetracycline repressor protein, confirming its sensitivity and specificity. Finally, by integrating a lateral flow assay (LFA), we enhanced the portability of CASCADE, enabling user-friendly, on-site detection. This work expands the application scope of the Cas12a system and offers a promising strategy for point-of-care diagnostics or environmental monitoring.},
}

*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*CRISPR-Cas Systems
MicroRNAs/genetics/analysis
DNA, Single-Stranded/genetics
DNA/genetics
*Bacterial Proteins/genetics/metabolism
*Biosensing Techniques/methods

@article {pmid41261174,
year = {2025},
author = {Seker-Polat, F and Rogozinska, M and Ban, Y and Abdula, F and Buyukcelebi, K and Xie, P and Fan, J and Abbaszadeh, N and Kingham, Y and Paylakhi, SZ and Zhang, B and Adli, M},
title = {Druggable genome CRISPR screening identifies the KEAP1/NRF2 axis as a mediator of PD-L1 expression.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1610},
pmid = {41261174},
issn = {2399-3642},
support = {R01CA267544//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; U54CA268084//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*B7-H1 Antigen/genetics/metabolism ; Humans ; *Kelch-Like ECH-Associated Protein 1/metabolism/genetics ; *NF-E2-Related Factor 2/metabolism/genetics ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Animals ; Female ; Mice ; CRISPR-Cas Systems ; Pancreatic Neoplasms/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Ovarian Neoplasms/genetics ; },
abstract = {Cancer cells rapidly induce PD-L1 expression in response to inflammatory cytokines such as IFNγ from cytotoxic T cells. Increased surface PD-L1 is a primary mechanism of cancer cells evading cytotoxic T-cell-mediated immune clearance. Identifying how cancer cells increase PD-L1 expression may yield clinically relevant immune checkpoint regulators. However, the key regulators and molecular mechanisms mediating rapid PD-L1 induction are yet to be understood entirely. To identify targetable mechanisms controlling cytokine-induced PD-L1 expression, we performed functional CRISPR gene KO screening with a custom-designed sgRNA library that targets "druggable" genes. We performed the screening in 6 different cancer lines: 3 ovarian (OVCAR4, CaOV3, and SKOV3) and three pancreatic cancer (MiaPaca2, ASPC1 and KP4) cell lines. The screening recovered the known regulators of PD-L1 expression and uncovered several novel regulators of PD-L1 that control its expression in all cell lines or in a cancer-type-specific fashion. For example, while genetic or pharmacological depletion of CSNK1A1 results in reduced PD-L1 expression in ovarian cancer cells, CDK1 depletion modulates PD-L1 in pancreatic cancer cell lines. Significantly, we discovered that KEAP1 depletion or pharmacological inhibition diminishes PD-L1 in all cell lines tested (n = 6). Mechanistically, KEAP1 depletion-mediated reduced PD-L1 is due to transcriptional repression of the PD-L1 gene by NRF2 activation. As such, depletion of NRF2 restores PD-L1 expression, while its overexpression leads to diminished PD-L1 expression. Supporting this, pharmacological NRF2 activation resulted in significant antitumor immunity with increased cytotoxic effector T cell infiltration and reduced exhausted T cells, resulting in smaller xenografted tumors. These findings establish the KEAP1/NRF2 axis as a novel and potentially druggable mechanism of IFNγ-meditated PD-L1 expression in cancer cells.},
}

*B7-H1 Antigen/genetics/metabolism
Humans
*Kelch-Like ECH-Associated Protein 1/metabolism/genetics
*NF-E2-Related Factor 2/metabolism/genetics
Cell Line, Tumor
*Gene Expression Regulation, Neoplastic
Animals
Female
Mice
CRISPR-Cas Systems
Pancreatic Neoplasms/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Signal Transduction
Ovarian Neoplasms/genetics

@article {pmid41260544,
year = {2025},
author = {Gupta, A and Beg, MA and Badhwar, S and Srivastava, S and Srivastava, R and Puri, N and Saxena, A and Abdin, MZ and Selvapandiyan, A},
title = {Nucleoside diphosphate kinase (LdNDK2): A metacyclogenesis-regulating kinase essential for Leishmania parasite survival within eukaryotic host cells.},
journal = {Microbial pathogenesis},
volume = {210},
number = {},
pages = {108192},
doi = {10.1016/j.micpath.2025.108192},
pmid = {41260544},
issn = {1096-1208},
abstract = {Nucleoside diphosphate kinase (NDK) transfers phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs) via a ping-pong mechanism, benefiting both prokaryotes and eukaryotes. In Leishmania donovani, we identified a putative NDK2 (LdNDK2), hypothesized to play a crucial role in nucleotide metabolism and cellular energy regulation. To investigate its function and enzymatic properties, we cloned, expressed, and purified recombinant LdNDK2, confirming enzymatic activity via the ADP-Glo assay. The secondary structure and thermal stability were analyzed using circular dichroism spectroscopy, while intrinsic tryptophan fluorescence assays revealed that ATP's gamma phosphate is first transferred to rLdNDK2, forming an intermediate phospho-enzyme complex. We further evaluated the role of rLdNDK2 in ATP-mediated cytolysis of LPS-activated THP-1 cells by measuring lactate dehydrogenase release. Using the CRISPR-Cas9 method, we fluorescently tagged LdNDK2, localizing it to distinct regions of the endomembrane system. In vitro growth studies of LdNDK2 deleted (LdNDK2[-/-]) procyclic promastigotes, through CRISPR-Cas9, revealed that these mutants exhibit a longer flagellum, a reduced cell body, an altered cell division cycle, and increased growth compared to wild-type parasites. LdNDK2[-/-] parasites were arrested at the metacyclic stage, contrasting with continuous differentiation in parental counterparts. Morphological alterations and growth defects were confirmed by re-expressing LdNDK2 in knockouts. Infection with LdNDK2[-/-] parasites significantly reduced the viability and recovery of mast cells and THP-1 macrophages and flow cytometry indicated increased apoptosis in host cells, likely due to parasites inability to utilize NDK enzyme for preventing cytolysis. Our findings highlight LdNDK2's crucial role in metacyclogenesis and immune evasion, underscoring its therapeutic potential for leishmaniasis.},
}

@article {pmid41205603,
year = {2025},
author = {Regan, SB and Medhi, D and Xu, Y and White, TB and Jiang, YZ and Kim, JE and Wang, SC and Deng, Q and Jia, S and Baasan, D and Connelly, JP and Chang, TC and Pruett-Miller, SM and Jasin, M},
title = {Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4119-4137.e10},
doi = {10.1016/j.molcel.2025.10.015},
pmid = {41205603},
issn = {1097-4164},
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *CRISPR-Cas Systems ; Animals ; *Loss of Heterozygosity ; Mice ; DNA End-Joining Repair ; Gene Editing/methods ; Flow Cytometry ; CRISPR-Associated Protein 9/genetics/metabolism ; Epithelial Cells/metabolism ; },
abstract = {Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), a common feature of tumor genomes. To interrogate this risk, we developed a flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of mouse embryonic and human epithelial cells following a DSB. Inhibition of both non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) massively increases LOH, although the dependence on individual pathways differs in the two cell types. Multiple mechanisms lead to LOH, including chromosome truncations with de novo telomere addition and whole chromosome loss. LOH spans megabases distal from the DSB but also frequently tens of megabases centromere-proximal, which can arise from breakage-fusion-bridge events. Unlike DSBs, Cas9 nicks and adenine base editing did not noticeably impact LOH. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end-joining inhibition.},
}

*DNA Breaks, Double-Stranded
Humans
*CRISPR-Cas Systems
Animals
*Loss of Heterozygosity
Mice
DNA End-Joining Repair
Gene Editing/methods
Flow Cytometry
CRISPR-Associated Protein 9/genetics/metabolism
Epithelial Cells/metabolism

@article {pmid41194386,
year = {2025},
author = {Sanchez-Quirante, T and Kužmová, E and Riopedre-Fernandez, M and Golojuch, S and Vopálenský, P and Raindlová, V and El-Sagheer, AH and Brown, T and Hocek, M},
title = {Enzymatic Synthesis of Modified RNA Containing 5-Methyl- or 5-Ethylpyrimidines or Substituted 7-Deazapurines and Influence of the Modifications on Stability, Translation, and CRISPR-Cas9 Cleavage.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2755-2767},
doi = {10.1021/acschembio.5c00692},
pmid = {41194386},
issn = {1554-8937},
mesh = {*CRISPR-Cas Systems ; *Pyrimidines/chemistry/metabolism ; *RNA/chemistry/metabolism ; Humans ; *Purines/chemistry/metabolism ; Protein Biosynthesis ; DNA-Directed RNA Polymerases/metabolism ; Viral Proteins ; },
abstract = {A set of modified 5-methyl- and 5-ethylpyrimidine (uracil and cytosine) and 7-methyl-, 7-ethyl-, and 7-unsubstituted 7-deazapurine (deazaadenine and deazaguanine) ribonucleoside triphosphates was synthesized and used for enzymatic synthesis of base-modified RNA using in vitro transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding Renilla luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in in vitro and in cellulo translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the in vitro translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, in cellulo experiments showed a significant enhancement of translation with mRNA containing 7-deazaguanine and moderate enhancement with 5-methyl- or 5-ethylcytosine. Most of the modifications had a minimal effect on the efficiency of the gene cleavage in CRISPR-Cas9 except for 7-alkyl-7-deazaadenines that completely inhibited the cleavage. The results are important for further design of potential base-modified RNA therapeutics.},
}

*CRISPR-Cas Systems
*Pyrimidines/chemistry/metabolism
*RNA/chemistry/metabolism
Humans
*Purines/chemistry/metabolism
Protein Biosynthesis
DNA-Directed RNA Polymerases/metabolism
Viral Proteins

@article {pmid41131871,
year = {2025},
author = {Campbell, KB and Ouye, RB and Wong, BL and Jiang, A and Okada, K and McKenney, RJ and Fisher, AJ and Beal, PA},
title = {Control of ADAR2 Dimerization and RNA Editing Efficiency by Site-Specific 2'-Fluoro Modification of Guide RNAs.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2637-2648},
doi = {10.1021/acschembio.5c00493},
pmid = {41131871},
issn = {1554-8937},
mesh = {*Adenosine Deaminase/metabolism/chemistry/genetics ; *RNA Editing ; *RNA-Binding Proteins/metabolism/chemistry/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; RNA, Double-Stranded/metabolism/chemistry ; Protein Multimerization ; Binding Sites ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) are an important class of RNA editing enzymes that catalyze the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Since inosine is typically read as guanosine (G) during translation, ADARs can produce A to G transitions in dsRNA. Site-directed RNA editing (SDRE) is a promising therapeutic tool wherein guide RNAs can be used to direct endogenous human ADARs to reverse disease-causing mutations in specific RNA transcripts. Guide RNA (gRNA) modifications at locations that contact the ADAR active site are often used to improve editing efficiency. However, little is known about rate-enhancing chemical modifications in the gRNA at the dsRNA binding domain (dsRBD)-RNA interface. Analysis of published crystal structures of ADAR2 bound to dsRNA suggested positions at this interface would be sensitive to gRNA modification. In this work, gRNAs bearing 2'-modifications in the dsRBD binding site were synthesized and subsequently tested to determine their effects on the editing rate of therapeutically relevant ADAR targets. We found that replacing a single 2'-OH at specific positions on the gRNA with a 2'-F substantially increased the rate of in vitro ADAR2-catalyzed adenosine deamination for two different sequences, whereas 2'-OMe at these positions was inhibitory. This effect was also validated in cellulo. The rate of ADAR1-catalyzed deamination is not stimulated by these 2'-F modifications. A crystal structure of an ADAR2 fragment bound to duplex RNA bearing a single 2'-F at guide position +13 suggested a favorable interaction between the side chain of N241 of the auxiliary ADAR2 monomer and the 2'-F modification. Furthermore, electrophoretic mobility shift assays and mass photometry indicate 2'-F at position +13 facilitates ADAR2 dimerization on the RNA substrate. This work advances our understanding of the RNA features that define superior ADAR substrates and inform the design of gRNAs for therapeutic RNA editing.},
}

*Adenosine Deaminase/metabolism/chemistry/genetics
*RNA Editing
*RNA-Binding Proteins/metabolism/chemistry/genetics
Humans
*RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics
RNA, Double-Stranded/metabolism/chemistry
Protein Multimerization
Binding Sites

@article {pmid41099664,
year = {2025},
author = {Spavieri, JM and Inacio, TG and Seguchi, G and de Souza, BC and Pereira, GAG and de Mello, F},
title = {Impact of CRISPRi-Mediated Titration of GPD Genes on the Fermentative Performance of S. cerevisiae.},
journal = {ACS synthetic biology},
volume = {14},
number = {11},
pages = {4412-4423},
doi = {10.1021/acssynbio.5c00316},
pmid = {41099664},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Fermentation/genetics ; Ethanol/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; Glycerol/metabolism ; *CRISPR-Cas Systems/genetics ; *Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Glycerol is one of the main byproducts in ethanol fermentation due to its importance in redox balance and response to osmotic stress in Saccharomyces cerevisiae. Since its production diverts carbon from alcohol production, traditional gene-editing methods have been applied to the glycerol synthesis pathway. However, such approaches generate undesirable phenotypes for industrial applications. In the present study, we employed the CRISPR-dCas9 system to moderately downregulate the expression of GPD1 and GPD2, the two main genes involved in this metabolism. GPD2 gene expression downregulation and a graded reduction in glycerol production after repression of four different target sites in each paralogue were achieved. Employment of the CRISPRi approach for GPD gene modulation resulted in higher specific ethanol productivity (SEP) than that of single knockout cells. Targeted modulation in a region -140 basepairs upstream of the transcription start site (TSS) of GPD1 resulted in a 3% increase in ethanol production compared to the wild type and gpd Δ strains. Such regulation, combined with GPD2 deletion, revealed the higher SEP among all tested strains. Furthermore, a GPD1-modulated strain maintained tolerance to high osmolarity in very high-gravity (VHG) fermentation while maintaining its ethanol production levels above those observed in the control strain.},
}

*Saccharomyces cerevisiae/genetics/metabolism
Fermentation/genetics
Ethanol/metabolism
*Saccharomyces cerevisiae Proteins/genetics/metabolism
Glycerol/metabolism
*CRISPR-Cas Systems/genetics
*Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism
Gene Editing/methods

@article {pmid41072420,
year = {2025},
author = {Zhao, G and Liu, Y and Zhang, G and Wang, J and Zou, Z and Wang, Y and Xu, S and Han, D and Xu, Z and Chen, Z and Yang, M and Zeng, Z and Wu, Y and Zhou, X and Huang, J},
title = {Engineered hypercompact Fanzor-ωRNA system with enhanced genome editing activity.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4138-4151.e4},
doi = {10.1016/j.molcel.2025.09.031},
pmid = {41072420},
issn = {1097-4164},
mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; Animals ; Dependovirus/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genome, Human ; CRISPR-Cas Systems ; Mice ; },
abstract = {As programmable RNA-guided DNA nucleases found in eukaryotes, Fanzors have shown promise in genome editing with their compact size. A wide variety of RuvC-containing Fanzor nucleases have been identified in various eukaryotes and their viruses. However, low editing efficiency limits the application of Fanzor in mammalian genome editing. In this study, we introduce SpuFz1 V4, an engineered RNA-guided DNA endonuclease with vigorous editing activity in the human genome. Furthermore, we demonstrate the substantial potential of the Fanzor system as a base editor. SpuFz1 V4 is currently the most active RNA-guided DNA nuclease of eukaryotic origin, belonging to the Fanzor1 family. Due to its compact size, SpuFz1 V4 can be efficiently delivered via a single adeno-associated virus (AAV) into the retina, achieving robust in vivo genome editing, which has the potential to be applied in both basic research and disease treatment applications.},
}

Humans
*Gene Editing/methods
HEK293 Cells
Animals
Dependovirus/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Genome, Human
CRISPR-Cas Systems
Mice

@article {pmid41017740,
year = {2025},
author = {Cao, X and Zeng, Z and Cao, X and He, Y and Wang, L and Li, D and Zhang, X},
title = {Generation and Phenotypic Analysis of the IL-10RAR104W/R104W Mouse Model.},
journal = {Inflammatory bowel diseases},
volume = {31},
number = {11},
pages = {3149-3159},
doi = {10.1093/ibd/izaf099},
pmid = {41017740},
issn = {1536-4844},
support = {32025023//National Natural Science Foundation of China/ ; 22YF1437700//Shanghai Science and Technology Program/ ; },
mesh = {Animals ; Mice ; *Disease Models, Animal ; Phenotype ; *Inflammatory Bowel Diseases/genetics/pathology ; *Interleukin-10 Receptor alpha Subunit/genetics ; *Colitis/pathology/genetics ; *Point Mutation ; Humans ; Macrophages ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; },
abstract = {BACKGROUND: Very-early-onset inflammatory bowel disease (VEO-IBD) is a form of IBD that manifests in infants and young children, with a significant proportion of them carrying interleukin 10 receptor alpha (IL-10RA) mutations. Despite the increasing incidence rate, the pathogenesis of VEO-IBD remains elusive, and treatment options are limited. The utilization of a humanized mouse model holds promise for further investigation into VEO-IBD. Previous study has revealed that VEO-IBD patients had a homozygous C > T mutation at IL-10RA position 301, which can be pathogenic.

METHODS: We generated the corresponding point mutation mouse model via CRISPR/Cas9 technology. Subsequently, we performed various experiments to assess the colitis phenotype in mice and conducted a preliminary exploration of the model's utility.

RESULTS: The mouse model progressively developed spontaneous colitis between 6 and 12 weeks. Hematoxylin and eosin (H&E) staining revealed abnormal colonic structure and massive local immune cell infiltration. The mouse model has abnormal levels of inflammatory cytokines in the colonic tissue, with an expansion of F4/80+ macrophages, CD4+ T cells, and B220+ B cells. Among the macrophages, the level of tissue-resident macrophages associated with anti-inflammation was reduced in IL-10RAR104W/R104W mice, while the level of immature macrophages associated with pro-inflammation was increased. Furthermore, we found that bone marrow transplantation can alter the composition of intestinal macrophage populations and treat intestinal inflammation in mutant mice. Finally, the result of subcutaneous tumor-bearing experiments indicated a faster tumor growth rate in the mutant mice.

CONCLUSIONS: In summary, we have successfully constructed a humanized mouse model with a stable spontaneous colitis phenotype, which is a valuable model for the therapeutic exploration of VEO-IBD.},
}

Animals
Mice
*Disease Models, Animal
Phenotype
*Inflammatory Bowel Diseases/genetics/pathology
*Interleukin-10 Receptor alpha Subunit/genetics
*Colitis/pathology/genetics
*Point Mutation
Humans
Macrophages
CRISPR-Cas Systems
Mice, Inbred C57BL

@article {pmid40954215,
year = {2025},
author = {Romero-Moya, D and Torralba-Sales, E and Calvo, C and Marin-Bejar, O and Magallon-Mosella, M and Distefano, M and Pera, J and Castaño, J and De Giorgio, F and Gonzalez, J and Iglesias, A and Berenguer-Balaguer, C and Schilling, M and Plass, M and Pasquali, L and Català, A and Molina, O and Wlodarski, MW and Bigas, A and Giorgetti, A},
title = {CRISPR-engineered human GATA2 deficiency model uncovers mitotic dysfunction and premature aging in HSPCs, impairing hematopoietic fitness.},
journal = {Leukemia},
volume = {39},
number = {12},
pages = {3015-3025},
pmid = {40954215},
issn = {1476-5551},
support = {PID2020-15591RB-100//Ministry of Economy and Competitiveness | Agencia Estatal de Investigación (Spanish Agencia Estatal de Investigación)/ ; FORT23/00032//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; AC23_2/00040//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; AC23_2/00014//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; KOG-202109-01162//European Hematology Association (EHA)/ ; 101029927//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (H2020 Excellent Science - Marie Skłodowska-Curie Actions)/ ; LCF-PR-HR24-00150//"la Caixa" Foundation (Caixa Foundation)/ ; PID2023-151556OB-I00//"la Caixa" Foundation (Caixa Foundation)/ ; PID2022-142966OB-I00//Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism/pathology ; *GATA2 Transcription Factor/genetics/deficiency ; *CRISPR-Cas Systems ; *Mitosis/genetics ; *GATA2 Deficiency/genetics/pathology ; Animals ; Mutation ; Mice ; Cell Proliferation ; *Cellular Senescence/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {GATA2 deficiency is a monogenic transcriptopathy disorder characterized by bone marrow failure (BMF), immunodeficiency, and a high risk of developing myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Although informative mouse models have been developed, the mechanisms by which GATA2 haploinsufficiency drives disease initiation in humans remain incompletely understood. To address this, we developed a novel humanized model using CRISPR/Cas9 technology to knock-in GATA2-R398W variant in primary cord blood CD34[+] cells. Additionally, we introduced specific mutations in SETBP1 and ASXL1 to model distinct premalignant stages of GATA2 deficiency. Through clonal competition and serial transplantation assays, we demonstrated that human CD34[+] cells harboring the GATA2 mutation exhibit significantly reduced fitness in vivo when compete with wild-type cells. Notably, this fitness disadvantage persists even when GATA2 mutations are combined with oncogenic SETBP1 and ASXL1 drivers, underscoring the dominant, deleterious effect of GATA2 deficiency on hematopoietic stem cell function. Functional in vitro analyses revealed that GATA2-R398W mutation impairs cell proliferation, disrupts cell cycle progression, and induces mitotic defects, which may contribute to hematopoietic stem/progenitor cell loss and impaired self-renewal. Transcriptomic profiles of GATA2-mutant cells revealed that these functional defects are associated with reduced HSC self-renewal capacity and upregulation of the pre-aging phenotype. Our work highlights the feasibility of generating a human GATA2 deficiency model suitable for studying the biological consequences of various GATA2 variants and the generation of a platform to test potential phenotype-rescuing therapeutics.},
}

Humans
*Hematopoietic Stem Cells/metabolism/pathology
*GATA2 Transcription Factor/genetics/deficiency
*CRISPR-Cas Systems
*Mitosis/genetics
*GATA2 Deficiency/genetics/pathology
Animals
Mutation
Mice
Cell Proliferation
*Cellular Senescence/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40753279,
year = {2025},
author = {Tan, GZH and Maurya, KS and Krishnamoorthi, S and Boonyaves, K and Urano, D},
title = {Regulatory helix deletion in glutamate decarboxylase reduces GABA and enhances Agrobacterium-mediated transient expression in lettuce.},
journal = {Journal of plant research},
volume = {138},
number = {6},
pages = {1033-1044},
pmid = {40753279},
issn = {1618-0860},
support = {A19E4a0101//Agency for Science, Technology and Research/ ; },
mesh = {*gamma-Aminobutyric Acid/metabolism ; *Glutamate Decarboxylase/genetics/metabolism ; *Lactuca/genetics/metabolism/enzymology ; *Agrobacterium/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; *Plant Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Gamma-aminobutyric acid (GABA) is a metabolite involved in plant growth and stress responses, with its synthesis regulated by glutamate decarboxylase (GAD). Plant GAD enzymes have an autoinhibitory α-helix at the C-terminus, which calmodulin (CaM) binding typically relieves. Eliminating this C-terminal motif usually increases GABA levels in crops. In this case study, we generated a CRISPR/Cas9-edited lettuce line with a 14-amino acid deletion in the C-terminal helix of LsGAD2, the isozyme primarily expressed in most tissues. This targeted truncation removes CaM-binding residues while retaining the key Lys cluster (Lys489, Lys490, Lys491) responsible for autoinhibition, resulting in a significant reduction in GABA content without affecting growth. The LsGAD1/2-ΔC line showed a transcriptomic profile resembling stress responses in the wildtype under unstressed conditions. Reduced GABA levels appeared to upregulate genes involved in stress perception, signalling, and defense-related metabolic and hormonal changes, potentially mediated by WRKY-family transcription factors. Likely due to lower GABA levels and altered defense responses, LsGAD1/2-ΔC plants showed increased Agrobacterium-mediated transient expression of β-glucuronidase. Overall, our study suggests that targeted genetic manipulation of the C-terminal helix of GAD enzymes can reduce GABA levels while enhancing transformation efficiency in lettuce, thus presenting a means for engineering for such purposes.},
}

*gamma-Aminobutyric Acid/metabolism
*Glutamate Decarboxylase/genetics/metabolism
*Lactuca/genetics/metabolism/enzymology
*Agrobacterium/genetics
Gene Expression Regulation, Plant
Plants, Genetically Modified
*Plant Proteins/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41259334,
year = {2025},
author = {Siroosi, M and Ghasemi, F and Jabalameli, F and Emaneini, M and Salehi, M and Beigverdi, R and Amoozegar, MA},
title = {Investigating the impact of type I-E CRISPR-Cas systems and acrEI10 on multidrug-resistance in clinical isolates of Klebsiella pneumoniae.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0335756},
doi = {10.1371/journal.pone.0335756},
pmid = {41259334},
issn = {1932-6203},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; Humans ; *Bacterial Proteins/genetics ; Anti-Bacterial Agents/pharmacology ; Klebsiella Infections/microbiology/drug therapy ; Microbial Sensitivity Tests ; beta-Lactamases/genetics ; },
abstract = {Klebsiella pneumoniae is a pathogen related to nosocomial infections with a high rate of antibiotic resistance. The aim of this study was to understand the impact of the presence of CRISPR-Cas systems and an anti-CRISPR gene on multidrug-resistance in K. pneumoniae isolates. The study analyzed 100 clinical K. pneumoniae isolates obtained from a hospital setting. The investigation involved determining antibiotic resistance profiles, including ESBL production, identifying specific carbapenemase and aminoglycoside resistance genes, detecting the presence of CRISPR-Cas systems, identifying the anti-CRISPR gene acrEI10, and sequencing CRISPR arrays. Correlation analysis between resistance genes and CRISPR-Cas systems was also performed. All isolates in this study were determined to be multidrug-resistant (MDR), with resistance rates exceeding 70% for the majority of antibiotics tested. The most prevalent carbapenemase genes were blaOXA-48 and blaNDM, while aminoglycoside resistance was primarily mediated by aac(6´)-Ia and ant(2")-Ia. Only 7% of the isolates harbored CRISPR-Cas systems and the gene acrEI10, which encodes an anti-CRISPR protein, was detected in one of the CRISPR-Cas positive isolates. Sequencing of the CRISPR array from this isolate showed similarities between the spacers and sequences found in plasmids and K. pneumoniae chromosome. No strong correlation was identified between the antibiotic resistance genes and CRISPR-Cas systems. Findings from this study suggest a complex interplay between these factors in MDR isolates of K. pneumoniae and show that further investigations are needed to have a better understanding of the mechanisms related to the coexistence of these elements and their impact on dissemination of antibiotic resistance genes.},
}

*Klebsiella pneumoniae/genetics/drug effects/isolation & purification
*CRISPR-Cas Systems/genetics
*Drug Resistance, Multiple, Bacterial/genetics
Humans
*Bacterial Proteins/genetics
Anti-Bacterial Agents/pharmacology
Klebsiella Infections/microbiology/drug therapy
Microbial Sensitivity Tests
beta-Lactamases/genetics

@article {pmid41257956,
year = {2025},
author = {Feng, C and Wang, Y and Liu, C and Dong, F and Guo, M and Liu, F and Li, Y and Zhang, L},
title = {Programmable no-nonspecific genetic analytical system via dual-circle-based rolling circle amplification with an efficient CRISPR/Cas12a biosensing strategy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40723},
pmid = {41257956},
issn = {2045-2322},
support = {CSTB2024NSCQ-MSX0785//Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission/ ; 2023YQB061//Young Ph.D. Talent Incubation Program of the Second Affiliated Hospital of Army Medical University/ ; CQYC20220303658//Chongqing Science and Technology Innovation Leading Talent Support Program/ ; 2024ZDXM012//Chongqing Municipal Science and Health Joint Medical Research Project Key Project/ ; 82472384//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; DNA, Single-Stranded/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; DNA, Circular/genetics ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive and specific detection of DNA is highly important for early cancer screening and diagnosis. Nucleic acid amplification technology is the most commonly used method for oncogene detection, but nonspecific amplification may occur. We designed a nicking endonuclease (NEase)-mediated exponential rolling circle amplification (RCA) that avoids nonspecific amplification for the CRISPR/Cas12a preamplification process. The purpose was to construct a NEase-assisted target recycling (NATR)-triggered no-nonspecific exponential RCA (NER) reaction integrated with a CRISPR/Cas12a (NATR-NER/Cas12a) system, enabling ultrasensitive and high-fidelity target detection. Innovatively, two circular single-stranded DNAs (ssDNAs) with NEase recognition sites were designed as the preprimer and template for RCA. In the presence of the target, the endonuclease Nt.BstNBI cleaves the circular preprimers into linear fragments, triggering the NER reaction. This generates many short ssDNA fragments, which are recognized by CRISPR/Cas12a and generates a fluorescence signal. The proposed strategy exhibited a wide linear range (10 fM-1 nM), a low detection limit (0.77 fM), and specifically recognized single mismatched DNA. In serum samples, this method exhibited good agreement with real-time quantitative polymerase chain reaction (qPCR) results at lower cost. The developed NATR-NER/Cas12a system provides a promising tool for the early screening and clinical diagnosis of cancer in resource-limited areas.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Humans
DNA, Single-Stranded/genetics
*Endodeoxyribonucleases/metabolism/genetics
DNA, Circular/genetics
Limit of Detection
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41256794,
year = {2025},
author = {Zhou, X and Ye, C and Xie, M and Wei, Y and Zhao, Y and Liu, X and Ma, J and Qing, J and Chen, Z},
title = {Advances in the application of CRISPR technology in pathogen detection: amplification-based and amplification-free strategies.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1645699},
pmid = {41256794},
issn = {2235-2988},
mesh = {Humans ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics/isolation & purification ; *Communicable Diseases/diagnosis ; },
abstract = {CRISPR technology, with its high specificity and programmability, has become an important tool for the detection of human pathogens. The timely and accurate detection of pathogens is crucial for public health. In recent years, significant progress has been made in the application of CRISPR technology for pathogen detection. However, several challenges remain, including detection sensitivity, specificity, and operational convenience. This review summarizes the latest advances in CRISPR technology for pathogen detection, with a focus on the principles and performance comparisons of amplification-based CRISPR (such as those combined with isothermal amplification techniques like RPA and LAMP) and amplification-free CRISPR (such as cascade CRISPR, sensor technologies, and digital droplet CRISPR). It also discusses their applications in pathogen detection. In addition, the article analyzes the advantages and limitations of CRISPR detection technology and looks forward to future development trends, providing a theoretical basis for the optimization of rapid diagnostic techniques for pathogens.},
}

Humans
*Molecular Diagnostic Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Sensitivity and Specificity
*Clustered Regularly Interspaced Short Palindromic Repeats
*Bacteria/genetics/isolation & purification
*Communicable Diseases/diagnosis

@article {pmid41255389,
year = {2025},
author = {Pizzoccheri, R and Falchi, FA and Alloni, A and Caldarulo, M and Camboni, T and Zambelli, F and Pavesi, G and Visentin, C and Camilloni, C and Sertic, S and Briani, F},
title = {Pathological PNPase variants with altered RNA binding and degradation activity affect the phenotype of bacterial and human cell models.},
journal = {NAR molecular medicine},
volume = {2},
number = {1},
pages = {ugae028},
pmid = {41255389},
issn = {2976-856X},
abstract = {Human PNPase (hPNPase) is an essential RNA exonuclease located in mitochondria, where it contributes to RNA import from the cytoplasm, degradation of mitochondrial RNA and R-loop homeostasis. Biallelic mutations in the hPNPase PNPT1 gene cause different genetic diseases, ranging from hereditary hearing loss to Leigh syndrome. In this work, we used an Escherichia coli model to test the effects of four pathological PNPT1 mutations associated with diseases of different severity. Moreover, we generated a new human cell model by introducing PNPT1 mutations into 293T cells via CRISPR-Cas editing. Notably, the bacterial cells expressing the different mutant alleles exhibited similar phenotypes consistent with hPNPase loss of function. In contrast, the human cell model responded differently to the two mutations tested, with responses correlating with the severity of the respective pathologies. We interpreted the data derived from both models in the light of the in vitro RNA binding and degradation activity of the wild-type and mutated hPNPase variants. We found that all pathogenic mutations tested caused defects in protein assembly and affected the degradation and RNA binding efficiency to varying degrees. However, the severity of the conditions caused by different mutations did not correlate with the catalytic activity of the mutant proteins.},
}

@article {pmid41254867,
year = {2025},
author = {Podkowik, M and Tillman, A and Takats, C and Carion, H and Putzel, G and McWilliams, J and See, B and Wang, G and Munoz-Gomez, S and Otto, C and Drlica, K and Marraffini, L and Pironti, A and Hochman, S and Kerantzas, C and Shopsin, B},
title = {CRISPR-Cas-associated SCCmec variants in methicillin-resistant Staphylococcus aureus evade rapid diagnostic detection.},
journal = {The Journal of infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1093/infdis/jiaf575},
pmid = {41254867},
issn = {1537-6613},
abstract = {Rapid molecular assays guiding treatment of methicillin-resistant Staphylococcus aureus (MRSA) detect SCCmec (Xpert) or the SCCmec-orfX junction (BCID2). Sequence variation in this region can disrupt primer binding, yielding false-negative results. Investigation of a missed bloodstream infection linked escape to a CRISPR-Cas-associated SCCmec variant, leading to identification of 64 variants from 45 patients-2% of 2,432 screened. Misdiagnosis was restricted to clonal complex 5, a hospital-associated lineage; 11 of 40 SCCmec/junctions evaded detection by BCID2 or Xpert. Variants had mecA instability and circulated in healthcare settings. Our findings reveal a unique escape mechanism and underscore a threat to diagnostic accuracy.},
}

@article {pmid41254256,
year = {2025},
author = {Wang, Q and Wang, Z and Liu, H and Lv, Y and Zhou, C and Li, C and Fan, H and Ouyang, X and Tao, L and Pei, X and Xie, T},
title = {PAM-readID is a rapid, simple, and accurate PAM determination method for CRISPR-Cas enzymes in mammalian cells.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1601},
pmid = {41254256},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; HEK293 Cells ; *CRISPR-Associated Protein 9/metabolism/genetics ; Animals ; DNA Breaks, Double-Stranded ; },
abstract = {One CRISPR-Cas enzyme's recognized protospacer adjacent motif (PAM) profile always shows intrinsic differences between assays with different working environments, such as in vitro, in bacterial cells, or in mammalian cells. The developed methods in mammalian cells are technically complex and not readily amenable to be broadly adopted, highlighting the urgent need for a well-established PAM-determining method in mammalian cells. In this study, we construct a rapid, simple, and accurate method for determining the PAM recognition profile of CRISPR-Cas nucleases in mammalian cells. The developed method is termed PAM-readID, PAM REcognition-profile-determining Achieved by Double-stranded oligodeoxynucleotides Integration in DNA double-stranded breaks. Using PAM-readID, the PAM recognition profiles of SaCas9, SaHyCas9, Nme1Cas9, SpCas9, SpG, SpRY, and AsCas12a in mammalian cells are well produced. An accurate PAM preference for SpCas9 can be identified by analysis with extremely low sequence depth (500 reads). PAM-readID can also define a PAM recognition profile of Cas9 based on Sanger sequencing with a significantly lower cost of time and price than that of high-throughput sequencing. We present an easy-to-use method for comprehensively revealing functional PAM of CRISPR-Cas nucleases in mammalian cells, which can contribute towards accelerating the advancement of exploiting novel genome editing nucleases.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
HEK293 Cells
*CRISPR-Associated Protein 9/metabolism/genetics
Animals
DNA Breaks, Double-Stranded

@article {pmid41253931,
year = {2025},
author = {Ni, J and Gong, J and Ran, Y and Bai, R and Huang, P and Zheng, Z and Zhou, M and Lan, F and Gu, W and Liu, X},
title = {Optimization of gene knockout approaches and sgRNA selection in hPSCs with inducible Cas9 expression.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40461},
pmid = {41253931},
issn = {2045-2322},
support = {JCYJ20220531091615034//Shenzhen Fundamental Research Program/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line ; INDEL Mutation ; },
abstract = {CRISPR/Cas9 has been extensively used for gene knockout, enabling functional studies of genetic loss-of-function in human pluripotent stem cells (hPSCs). However, commonly used Cas9 systems usually exhibit limited and variable efficiencies, and identifying single-guide RNAs (sgRNAs) with high cleavage activity-while avoiding ineffective ones-remains a major challenge. To address these issues, we generated a doxycycline-inducible spCas9-expressing hPSCs (hPSCs-iCas9) line and developed it into an optimized gene knockout system through systematically refining critical parameters. Through this optimization, the system achieved stable INDELs (Insertions and Deletions) efficiencies of 82-93% for single-gene knockouts, over 80% for double-genes knockouts, and up to 37.5% homozygous knockout efficiency for large DNA fragment deletions. Moreover, using this optimized system, we precisely evaluated three widely used gRNA scoring algorithms and integrated Western blotting to rapidly identify the ineffective sgRNA. As a result, among the tested algorithms, Benchling provided the most accurate predictions. Notably, we identified an ineffective sgRNA targeting exon 2 of ACE2, where the edited cell pool exhibited 80% INDELs but retained ACE2 protein expression. Together, these findings provide a robust framework for improving gene knockout efficiency in hPSCs and offer practical guidance for reliable sgRNA selection in gene editing experiments.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
Gene Editing/methods
*Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Associated Protein 9/genetics/metabolism
Cell Line
INDEL Mutation

@article {pmid41252463,
year = {2025},
author = {Fehrenbach, A and Mitrofanov, A and Alkhnbashi, OS and Backofen, R and Baumdicker, F},
title = {An evolutionary approach to predict the orientation of CRISPR arrays.},
journal = {PLoS computational biology},
volume = {21},
number = {11},
pages = {e1013706},
doi = {10.1371/journal.pcbi.1013706},
pmid = {41252463},
issn = {1553-7358},
abstract = {CRISPR-Cas is a defense system of bacteria and archaea against phages. Parts of the foreign DNA, called spacers, are incorporated into the CRISPR array which constitutes the immune memory. The orientation of CRISPR arrays is crucial for analyzing and understanding the functionality of CRISPR systems and their targets. Several methods have been developed to identify the orientation of a CRISPR array. To predict the orientation, different methods use different features such as the repeat sequences between the spacers, the location of the leader sequence, the Cas genes, or PAMs. However, those features are often not sufficient to predict the orientation with certainty, or different methods disagree. Remarkably, almost all CRISPR systems have been found to insert spacers in a polarized manner at the leader end of the array. We introduce CRISPR-evOr, a method that leverages the resulting patterns to predict the acquisition orientation for (a group of) CRISPR arrays by reconstructing and comparing the likelihood of their evolutionary history with respect to both possible acquisition orientations. The new method is independent of Cas type, leader existence and location, and transcription orientation. CRISPR-evOr is thus particularly useful for arrays that other CRISPR orientation tools cannot predict confidently and to verify or resolve conflicting predictions from existing tools. CRISPR-evOr currently confidently predicts the orientation of 28.3% of the arrays in the considered subset of CRISPRCasdb, which other tools like CRISPRDirection and CRISPRstrand cannot reliably orient. As our tool leverages evolutionary information we expect this percentage to grow in the future when more closely related arrays will be available. Additionally, CRISPR-evOr provides confident decisions for rare subtypes of CRISPR arrays, where knowledge about repeats and leaders and their orientation is limited.},
}

@article {pmid41252186,
year = {2025},
author = {Calhoun, CCS and Capps, MES and Muya, K and Gannaway, WC and Martina, V and Conklin, CL and Klein, MC and Webster, JM and Torija-Olson, EG and Thyme, SB},
title = {Removal of developmentally regulated microexons has a minimal impact on larval zebrafish brain morphology and function.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {41252186},
issn = {2050-084X},
mesh = {Animals ; *Zebrafish/genetics/growth & development ; *Brain/anatomy & histology/growth & development/physiology ; Larva/growth & development/genetics ; *Exons/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; *Alternative Splicing ; Phenotype ; },
abstract = {Microexon splicing is a vertebrate-conserved process through which small, often in-frame, exons are differentially included during brain development and across neuron types. Although the protein sequences encoded by these exons are highly conserved and can mediate interactions, the neurobiological functions of only a small number have been characterized. To establish a more generalized understanding of their roles in brain development, we used CRISPR/Cas9 to remove 45 microexons in zebrafish and assessed larval brain activity, morphology, and behavior. Most mutants had minimal or no phenotypes at this developmental stage. Among previously studied microexons, we uncovered baseline and stimulus-driven phenotypes for two microexons (meA and meB) in ptprd and reduced activity in the telencephalon in the tenm3 B0 isoform. Although mild neural phenotypes were discovered for several microexons that have not been previously characterized, including in ppp6r3, sptan1, dop1a, rapgef2, dctn4, vti1a, and meaf6. This study establishes a general approach for investigating conserved alternative splicing events and prioritizes microexons for downstream analysis.},
}

Animals
*Zebrafish/genetics/growth & development
*Brain/anatomy & histology/growth & development/physiology
Larva/growth & development/genetics
*Exons/genetics
*Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems
*Gene Expression Regulation, Developmental
*Alternative Splicing
Phenotype

@article {pmid41232532,
year = {2025},
author = {Yang, Y},
title = {What we can learn from the first personalized CRISPR-treated baby to tackle genetic brain disorders.},
journal = {Neuron},
volume = {113},
number = {22},
pages = {3697-3702},
doi = {10.1016/j.neuron.2025.10.023},
pmid = {41232532},
issn = {1097-4199},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; *Brain Diseases/genetics/therapy ; *Precision Medicine/methods ; Infant ; },
abstract = {The landmark report on personalized CRISPR genome editing to treat an infant (baby KJ) with a life-threatening liver disease sparked widespread attention,[1] ushering in a new era of precision genetic intervention. This piece discusses the key challenges and opportunities in translating this milestone into treatments for genetic brain disorders.},
}

Humans
*Gene Editing/methods
*Genetic Therapy/methods
*CRISPR-Cas Systems/genetics
*Brain Diseases/genetics/therapy
*Precision Medicine/methods
Infant

@article {pmid41222482,
year = {2025},
author = {Chen, W and He, Y and Yuan, J and Yang, F},
title = {Catalytic hairpin assembly cascade-initiated proximity with self-priming amplification for CRISPR-enhanced ultrasensitive detection of coronary heart disease-associated microRNAs.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {45},
pages = {9254-9261},
doi = {10.1039/d5ay01456e},
pmid = {41222482},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Coronary Disease/genetics/diagnosis ; *Biosensing Techniques/methods ; },
abstract = {Accurate detection of specific microRNAs (miRNAs) is essential for the early diagnosis of coronary heart disease. Emerging technologies, including functional nuclease-mediated target amplification and DNA nanotechnology, offer substantial potential for precise miRNA identification in clinical diagnostics. This study presents a highly sensitive and specific biosensing platform that integrates catalytic hairpin assembly (CHA) cascade-initiated proximity based self-priming amplification and CRISPR/Cas12a-mediated signal generation for miRNA quantification. Target miRNA initiates the CHA cascade, yielding a toehold-bearing CHA product. This toehold subsequently enables "Variable primer" extension, transcribing double-stranded DNA (dsDNA). The resultant dsDNA activates CRISPR/Cas12a, triggering collateral cleavage and signal amplification. Leveraging this dual-amplification strategy (CHA and CRISPR/Cas12a), the assay achieves a sub-femtomolar detection limit (0.36 fM). Dual-sequence verification, including CHA and CRISPR/Cas12a recognition, ensures exceptional specificity. Validation using spiked serum samples confirmed precise miRNA quantification. Collectively, this biosensor demonstrates significant promise for clinical molecular diagnostics.},
}

*MicroRNAs/genetics/analysis/blood
Humans
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
*Coronary Disease/genetics/diagnosis
*Biosensing Techniques/methods

@article {pmid41217062,
year = {2025},
author = {Vieyra, F and Pindi, C and Lisi, GP and Morzan, UN and Palermo, G},
title = {Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER and EQR variants.},
journal = {The journal of physical chemistry. B},
volume = {129},
number = {46},
pages = {11949-11958},
doi = {10.1021/acs.jpcb.5c06153},
pmid = {41217062},
issn = {1520-5207},
mesh = {Molecular Dynamics Simulation ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; Gene Editing ; },
abstract = {Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target noncanonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135 V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.},
}

Molecular Dynamics Simulation
*CRISPR-Cas Systems
DNA/chemistry/metabolism/genetics
*CRISPR-Associated Protein 9/chemistry/metabolism/genetics
Gene Editing

@article {pmid41183414,
year = {2026},
author = {Luo, Q and Zheng, C and Huang, Y and Liang, L and Gong, Y and Zhang, J and Tang, Q and Zhang, K and Liao, X},
title = {Polydopamine-stabilized CsPbBr3 enables toxicity-reduced ECL detection of MMP-2 via CRISPR/Cas12a trans-cleavage.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118193},
doi = {10.1016/j.bios.2025.118193},
pmid = {41183414},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Indoles/chemistry ; *Polymers/chemistry ; *Matrix Metalloproteinase 2/isolation & purification/chemistry/blood ; Gold/chemistry ; Electrochemical Techniques/methods ; CRISPR-Cas Systems ; Humans ; Limit of Detection ; Luminescent Measurements/methods ; Lead/chemistry ; Titanium/chemistry ; },
abstract = {Lead-halide perovskites offer bright electrochemiluminescence (ECL) but suffer from aqueous instability and Pb[2+] safety concerns. We construct a CsPbBr3@PDA-Au nanointerface that couples polydopamine (PDA) passivation with Au-assisted charge transfer, integrated into a peptide-to-CRISPR/Cas12a amplification scheme for ultrasensitive detection of matrix metalloproteinase-2 (MMP-2). PDA conformally coats CsPbBr3, suppressing trap-mediated quenching and mitigating Pb[2+] release, while providing catechol/amine anchors for a tetrahedral DNA scaffold bearing a ferrocene-labeled hairpin. Upon MMP-2 cleavage of a GPLG↓VRG peptide-DNA chimera, the released strand activates Cas12a trans-cleavage, removing the proximal quencher and switching the interface from "off" to "on." Under unified processing (baseline subtraction and renormalization), the platform achieves a 5.6 aM detection limit with high selectivity over non-target proteases and excellent inter-device reproducibility (triplicate error bars reported). TEM/HRTEM visualize uniform nanocubes; XPS/FTIR evidence PDA catechol/quinone/amine coordination; XRD confirms phase-pure CsPbBr3; and corrected CV/EIS analyses verify accelerated interfacial kinetics after Au decoration. The sensor retains ∼98 % intra-day and ≥91 % 7-day signal, supporting operational robustness. By integrating interfacial stabilization, catalytic electron transfer, and molecular amplification, this modular design advances eco-conscious, perovskite-based ECL diagnostics for low-abundance protease monitoring and broader clinical bioanalysis.},
}

*Biosensing Techniques/methods
*Indoles/chemistry
*Polymers/chemistry
*Matrix Metalloproteinase 2/isolation & purification/chemistry/blood
Gold/chemistry
Electrochemical Techniques/methods
CRISPR-Cas Systems
Humans
Limit of Detection
Luminescent Measurements/methods
Lead/chemistry
Titanium/chemistry

@article {pmid41183412,
year = {2026},
author = {Liu, Y and Li, H and Wang, J and Liu, J and Zhou, X},
title = {Dual-acting CRISPR/Cas12a system enhanced hydrogel fluorescent aptasensor for one-pot detection of tetracycline in water.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118181},
doi = {10.1016/j.bios.2025.118181},
pmid = {41183412},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Tetracycline/analysis/isolation & purification ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; Hydrogels/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis/isolation & purification ; *Anti-Bacterial Agents/analysis ; Water/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid yet ultrasensitive and accurate detection of tetracycline (TC) in aquatic environments subjected to composite pollution remains challenging. CRISPR-mediated biosensors have been extensively studied to achieve ultrasensitive detection. However, these biosensors have always been coupled with a time-consuming nucleic acid amplification process to improve the sensitivity, which may cause erratic signal due to the sophisticated biochemical reaction cascades, hence leading to inaccurate results. To address this issue, we proposed a novel amplification-free fluorescent aptasensor based on polyacrylamide-deoxyribonucleic acid hydrogel integrated with a dual-acting CRISPR/Cas12a system for ultrasensitive and rapid detection of TC in water. With this well-designed dual-acting CRISPR/Cas12a system, a single aptamer-target specific molecular binding event can synchronously initiate pairing between two distinct CRISPR RNA and their target nucleic acid modified on the hydrogel, enabling dual activation of Cas12a. The aptasensor instantly emitted quantifiable strong fluorescence due to the efficient cleavage of reporter probes by the twofold activated Cas12a, demonstrating a TC detection limit of 0.035 μg/L, with approximately 10.6-fold and 5.6-fold sensitivity improvement over the two corresponding single-crRNA systems. The entire detection process can be accomplished in one pot within 10 min. The one-step hydrogel aptasensor shows superior resistance to matrix interference over the conventional solution-phase system, achieving satisfactory recovery percentages (92 %-105 %) for TC in different water matrices. This study offers a new perspective on CRISPR/Cas12a biosensor design and advances the environmental antibiotic monitoring field.},
}

*Biosensing Techniques/methods
*Tetracycline/analysis/isolation & purification
*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems/genetics
Hydrogels/chemistry
Limit of Detection
*Water Pollutants, Chemical/analysis/isolation & purification
*Anti-Bacterial Agents/analysis
Water/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41175692,
year = {2026},
author = {Hao, X and Qin, C and He, C and Yuan, Q and Yang, P and Wen, Y and Wu, Y and Cui, R and Zhang, Y and Chen, W and Liu, Y and Ramadan, S and Ying, Y and Li, D and Xu, L},
title = {In situ detection of bacteria from skin interstitial fluid via CRISPR microneedles: An amplification-free platform for point-of-care diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118123},
doi = {10.1016/j.bios.2025.118123},
pmid = {41175692},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Staphylococcus aureus/isolation & purification/genetics ; Humans ; *Skin/microbiology ; *Extracellular Fluid/microbiology ; CRISPR-Cas Systems ; Gold/chemistry ; Graphite/chemistry ; Limit of Detection ; *DNA, Bacterial/genetics/isolation & purification ; Wearable Electronic Devices ; Needles ; Point-of-Care Systems ; *Staphylococcal Infections/microbiology/diagnosis ; },
abstract = {We report a CRISPR/Cas12a-functionalized microneedle (MN) biosensor for in situ, amplification-free detection of pathogenic bacteria directly from skin interstitial fluid. The platform integrates conductive poly(styrene)/gold/graphene oxide MNs with a minimized electrochemical signal transducer, enabling real-time and highly specific sensing of bacterial DNA through CRISPR-mediated cleavage of ferrocene-labeled ssDNA reporters. Using Staphylococcus aureus as a model target, this strategy achieves direct detection limits of 0.69 pM in ex situ assays and 6.3 pM in situ, without the need for target amplification. The sensor also detects bacterial loads as low as 4.27 × 10[5] CFU/mL in interstitial fluid, which is below typical clinical thresholds. Engineered for biocompatibility and minimal invasiveness, the MNs effectively access interstitial fluid, capture targets and produce quantifiable electrochemical signals within 1 h. Paired with a portable reader and smartphone interface, this wearable system offers a user-friendly tool for point-of-care diagnostics and personalized infectious disease monitoring.},
}

*Biosensing Techniques/instrumentation
*Staphylococcus aureus/isolation & purification/genetics
Humans
*Skin/microbiology
*Extracellular Fluid/microbiology
CRISPR-Cas Systems
Gold/chemistry
Graphite/chemistry
Limit of Detection
*DNA, Bacterial/genetics/isolation & purification
Wearable Electronic Devices
Needles
Point-of-Care Systems
*Staphylococcal Infections/microbiology/diagnosis

@article {pmid41161233,
year = {2026},
author = {Shan, J and Wang, W and Sheng, Y and Hong, B and Luo, L and Liu, X and Ma, Y and Wang, J},
title = {Ultra-fast one-pot isothermal detection of respiratory virus: ADNA-initiated CRISPR/Cas12a-mediated RCA cycle.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118141},
doi = {10.1016/j.bios.2025.118141},
pmid = {41161233},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *SARS-CoV-2/isolation & purification/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; *Molecular Diagnostic Techniques/methods ; *COVID-19/diagnosis/virology ; Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Respiratory virus detection is a prominent area of molecular diagnostics, yet current clinical diagnostic methods lack ultra-rapid and highly sensitive detection capabilities. Here, we reported an ultra-rapid, one-pot isothermal assay called "ADNA-initiated CRISPR-Cas12a-mediated RCA cycle" (ACRE). ACRE was developed through computational studying, engineered design of nucleic acid, and enzyme kinetics analysis, combining rolling circle amplification (RCA) with Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for respiratory virus detection. The assay involved an engineered assistant DNA (ADNA) initiation reaction, followed by leveraging the cis-cleavage activity of Cas12a coupled with an engineered Padlock, which converted the linear RCA into the RCA cycle. When the RCA cycle is continuously in operation, the trans-cleavage activity of Cas12a facilitates both signal output and amplification. The limit of detection (LOD) for three respiratory viruses (SARS-CoV-2, Inf A, and Inf B) was as low as several hundred attomoles (751 aM, 3.7 fM, and 863 aM), with single-nucleotide specificity. Remarkably, the current assay can detect targets with concentrations above 10 pM within 2.5 min, without the reverse transcription step or specialized instrumentation. Given its exceptional speed, sensitivity, and specificity, ACRE could serve as a robust assay for detecting respiratory virus, enabling molecular diagnostics in clinical settings.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*SARS-CoV-2/isolation & purification/genetics
*Biosensing Techniques/methods
Humans
Limit of Detection
*Molecular Diagnostic Techniques/methods
*COVID-19/diagnosis/virology
Endodeoxyribonucleases/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41138445,
year = {2026},
author = {Hu, M and Liu, F and Zhang, J and Yang, L and Yan, Y and Wang, Y and Liu, M and An, H and Zhu, L and Ai, Y and Jiang, X},
title = {Integrated CRISPR/Cas12a-activated liposomal SERS amplification in microfluidic chips for ultrasensitive ampicillin detection.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118131},
doi = {10.1016/j.bios.2025.118131},
pmid = {41138445},
issn = {1873-4235},
mesh = {*Ampicillin/analysis/isolation & purification ; Liposomes/chemistry ; CRISPR-Cas Systems/genetics ; *Spectrum Analysis, Raman/methods ; *Biosensing Techniques ; Limit of Detection ; *Anti-Bacterial Agents/analysis ; Animals ; Gold/chemistry ; Lab-On-A-Chip Devices ; Milk/chemistry ; Silver/chemistry ; Humans ; Metal Nanoparticles/chemistry ; },
abstract = {The misuse of antibiotics has created a vicious cycle: from excessive use in livestock breeding to elevated residue levels in food products, ultimately leading to heightened antibiotic resistance in humans, a phenomenon with significant public health consequences. Food-based antibiotic testing is crucial for preventing antibiotic misuse and protecting human health. However, traditional analytical methods exhibit high equipment dependency, restricting on-site detection in hills, plateaus, and other regions with inconvenient transportation. This study developed an integrated CRISPR/Cas12a-activated liposomal Surface-enhanced Raman scattering (SERS) amplification system within a microfluidic chip for ultrasensitive ampicillin detection. The method achieves primary signal amplification by converting ampicillin concentration into a DNA signal through catalytic hairpin assembly (CHA), followed by secondary signal amplification using an Au@Ag substrate coupled with SERS technology for 4-MPBA@liposome marker detection. The platform demonstrated a linear detection range of 1 fM-1 nM with a limit of detection as low as 740 aM. Notably, the method exhibited excellent stability, selectivity, and sensitivity, successfully detecting ampicillin residues in environmental water and milk samples. It provides a technological approach for environmental monitoring and food safety, demonstrating significant practical application value.},
}

*Ampicillin/analysis/isolation & purification
Liposomes/chemistry
CRISPR-Cas Systems/genetics
*Spectrum Analysis, Raman/methods
*Biosensing Techniques
Limit of Detection
*Anti-Bacterial Agents/analysis
Animals
Gold/chemistry
Lab-On-A-Chip Devices
Milk/chemistry
Silver/chemistry
Humans
Metal Nanoparticles/chemistry

@article {pmid41108536,
year = {2025},
author = {Siddiqui, S and Siddiqui, H and Riguene, E and Nomikos, M},
title = {Zebrafish: A Versatile and Powerful Model for Biomedical Research.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {12},
pages = {e70080},
pmid = {41108536},
issn = {1521-1878},
support = {//NPRP-Blue Skies Research Award/ ; NPRP-BSRA1-0504-210082//Qatar Research Development and Innovation Council (QRDI), Doha, Qatar/ ; },
mesh = {*Zebrafish/genetics ; Animals ; Humans ; *Biomedical Research/methods ; *Disease Models, Animal ; Precision Medicine/methods ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Zebrafish (Danio rerio) have become a versatile model in precision medicine, bridging fundamental biology with translational applications. Their optical transparency, rapid development, and high genetic conservation with humans enable real-time imaging and cost-efficient high-throughput screening. Advances in CRISPR/Cas9, prime editing, and morpholino approaches have expanded their utility for modeling diverse human diseases. In addition to well-established roles in cardiovascular, neurological, metabolic, oncological, and infectious disease research, emerging applications include non-invasive larval urine assays, functional validation of rare human variants, host-microbiome interactions, and automated behavioral profiling for neuropsychiatric conditions. Limitations such as species-specific lipid metabolism and limited antibody availability remain, yet recent integration of single-cell transcriptomics, computational modeling, and machine learning is enhancing translational relevance. Collectively, these innovations position zebrafish as a scalable and powerful platform for disease modeling and personalized therapeutic strategies, underscoring their growing impact in the evolving landscape of precision medicine.},
}

*Zebrafish/genetics
Animals
Humans
*Biomedical Research/methods
*Disease Models, Animal
Precision Medicine/methods
CRISPR-Cas Systems
Gene Editing

@article {pmid41101316,
year = {2025},
author = {Chitboonthavisuk, C and Martin, C and Huss, P and Peters, JM and Anantharaman, K and Raman, S},
title = {Systematic genome-wide mapping of host determinants of bacteriophage infectivity.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101427},
doi = {10.1016/j.cels.2025.101427},
pmid = {41101316},
issn = {2405-4720},
mesh = {*Escherichia coli/virology/genetics ; *Bacteriophages/genetics/pathogenicity ; *Host-Pathogen Interactions/genetics ; CRISPR-Cas Systems/genetics ; Genome, Viral/genetics ; Bacteriophage T7/genetics/pathogenicity ; Host Microbial Interactions/genetics ; },
abstract = {Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed phage-host analysis using genome-wide CRISPR interference and phage packaging ("PHAGEPACK"), a pooled assay that systematically and comprehensively measures each host gene's impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways that affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates that phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers insights into phage-host interactions, phage evolution, and bacterial resistance.},
}

*Escherichia coli/virology/genetics
*Bacteriophages/genetics/pathogenicity
*Host-Pathogen Interactions/genetics
CRISPR-Cas Systems/genetics
Genome, Viral/genetics
Bacteriophage T7/genetics/pathogenicity
Host Microbial Interactions/genetics

@article {pmid41101079,
year = {2026},
author = {Ma, Q and Rong, Z and Shen, J and Yang, M and Han, Y},
title = {Integrated one-pot RPA-CRISPR/Cas13a platform enables ultrasensitive and field-deployable JAK2 V617F detection for myeloproliferative neoplasm diagnosis.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {268},
number = {},
pages = {117197},
doi = {10.1016/j.jpba.2025.117197},
pmid = {41101079},
issn = {1873-264X},
mesh = {Humans ; *Janus Kinase 2/genetics ; *CRISPR-Cas Systems/genetics ; *Myeloproliferative Disorders/genetics/diagnosis ; Mutation ; Sensitivity and Specificity ; Recombinases ; Real-Time Polymerase Chain Reaction/methods ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies driven by mutations like JAK2 V617F, a constitutively activating variant diagnostic for polycythemia vera and subsets of essential thrombocythemia and primary myelofibrosis. Sanger sequencing is the gold standard for detecting JAK2 mutations, but it faces limitations in terms of cost, sensitivity, and portability. CRISPR diagnostics generally requires preamplification, which increases the risk of contamination. There remains an unmet need for field-deployable, rapid MPNs diagnostics in resource-limited settings. We developed ONE-CASPR, a one-pot RPA-CRISPR/Cas13a system enabling ultrasensitive (0.1 % mutant allele frequency), rapid (30 min), portable JAK2 V617F detection. The platform synergizes recombinase polymerase amplification (RPA) with Cas13a trans-cleavage activity in a single-tube reaction at 37 °C, executed via a miniaturized wireless analysis device (CPod) to eliminate cross-contamination while preserving accuracy. Clinical validation across 36 patient samples demonstrated 100 % concordance with real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) in both sensitivity and specificity. ONE-CASPR provides a rapid, sensitive, field-deployable, and user-friendly solution for MPNs point-of-care diagnosis in resource-limited settings, with broad molecular screening application potential.},
}

Humans
*Janus Kinase 2/genetics
*CRISPR-Cas Systems/genetics
*Myeloproliferative Disorders/genetics/diagnosis
Mutation
Sensitivity and Specificity
Recombinases
Real-Time Polymerase Chain Reaction/methods
Nucleic Acid Amplification Techniques/methods

@article {pmid41047505,
year = {2025},
author = {Rajalekshmi, S and Sathyan, KM},
title = {Direct Protein Degradation: Emerging Tools to Probe Biological Complexity in Mammalian Systems.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {12},
pages = {e70075},
doi = {10.1002/bies.70075},
pmid = {41047505},
issn = {1521-1878},
support = {//School of Medicine, Southern Illinois University/ ; },
mesh = {Animals ; *Proteolysis ; Humans ; Gene Editing/methods ; Mammals/metabolism ; *Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Conditional degron approaches for acute and reversible protein depletion have become standard tools for studying gene function in cells and model organisms. Traditional gene perturbation methods have advanced gene function studies but are limited by slow kinetics, potential irreversibility, and lethality when targeting essential genes. To overcome these limitations, tag-based and antibody-based direct protein degradation technologies have been developed. These direct protein degradation systems utilize endogenous protein degradation pathways to achieve rapid and reversible protein depletion. When combined with genome editing, these systems provide precise temporal-and in some cases, spatial-control over endogenous protein expression. In this review, we will discuss the current status of tag-based and antibody-based direct protein degron technologies. We aim to provide a comprehensive guide for selecting these tools, highlighting their context-dependent applications and potential improvements to enhance efficiency and reliability.},
}

Animals
*Proteolysis
Humans
Gene Editing/methods
Mammals/metabolism
*Proteins/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41045937,
year = {2025},
author = {Koo, BM and Todor, H and Sun, J and van Gestel, J and Hawkins, JS and Hearne, CC and Banta, AB and Huang, KC and Peters, JM and Gross, CA},
title = {Comprehensive genetic interaction analysis of the Bacillus subtilis envelope using double-CRISPRi.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101406},
doi = {10.1016/j.cels.2025.101406},
pmid = {41045937},
issn = {2405-4720},
mesh = {*Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Regulatory Networks/genetics ; *Cell Wall/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Peptidoglycan ; CRISPR-Cas Systems/genetics ; },
abstract = {Understanding bacterial gene function remains a major challenge. Double-mutant genetic interaction analysis addresses this challenge by uncovering the functional partners of targeted genes, enabling association of genes of unknown function with known pathways and unraveling of connections among well-studied pathways, but such approaches are difficult to implement at the genome scale. Here, we use double-CRISPR interference (CRISPRi) to systematically quantify genetic interactions at scale for the Bacillus subtilis cell envelope, including essential genes. We discover >1,000 genetic interactions, some known and others novel. Our analysis pipeline and experimental follow-ups reveal the shared and distinct roles of paralogous genes such as mreB and mbl in peptidoglycan and teichoic acid synthesis and identify additional genes involved in the well-studied process of cell division. Overall, our study provides valuable insights into gene function and demonstrates the utility of double-CRISPRi for high-throughput dissection of bacterial gene networks, providing a blueprint for future studies in diverse species. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Bacillus subtilis/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Gene Regulatory Networks/genetics
*Cell Wall/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Peptidoglycan
CRISPR-Cas Systems/genetics

@article {pmid41045936,
year = {2025},
author = {Dénéréaz, J and Eray, E and Jana, B and de Bakker, V and Todor, H and van Opijnen, T and Liu, X and Veening, JW},
title = {Dual CRISPRi-seq for genome-wide genetic interaction studies identifies key genes involved in the pneumococcal cell cycle.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101408},
doi = {10.1016/j.cels.2025.101408},
pmid = {41045936},
issn = {2405-4720},
mesh = {*Streptococcus pneumoniae/genetics ; *Cell Cycle/genetics ; *CRISPR-Cas Systems/genetics ; Genome, Bacterial/genetics ; Computational Biology/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Regulatory Networks/genetics ; Genome-Wide Association Study/methods ; },
abstract = {Uncovering genotype-phenotype relationships is hampered by genetic redundancy. For example, most genes in Streptococcus pneumoniae are non-essential under laboratory conditions. A powerful approach to unravel genetic redundancy is by identifying gene-gene interactions. We developed a broadly applicable dual CRISPRi-seq method and analysis pipeline to probe genetic interactions (GIs) genome-wide. A library of 869 dual single-guide RNAs (sgRNAs) targeting high-confidence operons was created, covering over 70% of the genetic elements in the pneumococcal genome. Testing these 378,015 unique combinations, 4,026 significant GIs were identified. Besides known GIs, we found previously unknown positive and negative interactions involving genes in fundamental cellular processes such as division and chromosome segregation. The presented methods and bioinformatic approaches can serve as a roadmap for genome-wide gene interaction studies in other organisms. All interactions are available for exploration via the Pneumococcal Genetic Interaction Network (PneumoGIN), which can serve as a starting point for new biological discoveries. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Streptococcus pneumoniae/genetics
*Cell Cycle/genetics
*CRISPR-Cas Systems/genetics
Genome, Bacterial/genetics
Computational Biology/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Regulatory Networks/genetics
Genome-Wide Association Study/methods

@article {pmid40991130,
year = {2025},
author = {Kapoor, SA and Choudhary, P and Kasana, RC},
title = {Exploring CRISPR/Cas9-Mediated Gene Editing Advances in Conventional and Non-conventional Yeast Species.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {11},
pages = {7083-7122},
pmid = {40991130},
issn = {1559-0291},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Yeasts/genetics ; *Saccharomyces cerevisiae/genetics ; },
abstract = {In recent years, using modern technologies, researchers have harnessed the potential of yeast species for various industrial uses, such as the bioproduction of biopharmaceuticals, food additives, industrial biocatalysts, and biofuels. To improve the efficiency and potential of yeast species for industrial uses, genetic modification is carried out. Various genome engineering techniques, including Cre-loxP, homing endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), have been employed by different research groups for the genetic manipulation of yeast species. Among different genome engineering techniques, CRISPR/Cas9 has become popular because of its precise editing at targeted loci with increased efficiency. The ease of use, effectiveness, and adaptability of CRISPR/Cas9 make multiplexing possible for simultaneously targeting multiple genes, which was earlier very challenging through traditional methods. Moreover, the ability to perform marker-free editing is the significant advantage offered by CRISPR/Cas9. This review focuses on the applications of the CRISPR/Cas9 system in both conventional and non-conventional yeast species. Further, we discussed the advancements of CRISPR/Cas9, including the regulation of gene transcription-activation/repression and other genome engineering aspects. Additionally, innovations in CRISPR/Cas9, such as cloning-free CRISPR/Cas9 assembly, CRISPR-targeted in vivo editing (ACtive), CRISPR/Cas9-induced gene conversion, and selective ploidy ablation (CRI-SPA) are also discussed for enhancing the potential applications of CRISPR/Cas9 in diverse yeast species.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Yeasts/genetics
*Saccharomyces cerevisiae/genetics

@article {pmid41249581,
year = {2025},
author = {Turgeman-Grott, I and Golan, N and Neri, U and Naki, D and Altman-Price, N and Eizenshtein, K and Choudhary, DK and Levy, R and Navok, S and Cohen, L and Shalev, Y and Singla, H and Reshef, L and Gophna, U},
title = {A previously undescribed archaeal virus suppresses host immunity.},
journal = {EMBO reports},
volume = {},
number = {},
pages = {},
pmid = {41249581},
issn = {1469-3178},
support = {787514//EC | European Research Council (ERC)/ ; 1599/24//Israel Science Foundation (ISF)/ ; },
abstract = {Extremophilic archaea can have chronic viral infections that are well-tolerated by the hosts and may potentially protect against more lethal infections. Here we show that a natural Haloferax strain (48N), is chronically infected by a lemon-shaped virus. This viral infection is not cleared spontaneously, despite the multiple defense systems of the host. Curing 48N of its virus led to radical changes in the gene expression profile of 48N and a dramatic improvement in its growth rate. Remarkably, the cured 48N is the fastest-growing haloarchaeon reported to date, with a generation time of ~107 min at 45 °C, and faster than any known Haloferax species at this temperature. The virus subverts host defenses by reducing its transcription, including the CRISPR spacer acquisition machinery. Nonetheless, even in the virus-cured background, spacer acquisition is very low, indicating that another genetic element is disrupting CRISPR activity. Our results suggest that the slow growth of some halophilic archaea could be due to the effects of proviruses within their genomes that consume resources and alter the gene expression of their hosts.},
}

@article {pmid41249545,
year = {2025},
author = {Warkad, S and Kumar, A and Gampa, M and Goswami, S and T, V and Kumar, S and Dalal, M and Mishra, D and Jha, GK and C, V and Kumar, RR},
title = {Small RNAs big impact: a review on microRNA-mediated tolerance in wheat under terminal heat.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {245},
pmid = {41249545},
issn = {1438-7948},
support = {CABin//Indian Council of Agricultural Research/ ; NICRA//Indian Council of Agricultural Research/ ; },
mesh = {*Triticum/genetics/physiology ; *MicroRNAs/genetics/metabolism ; Gene Expression Regulation, Plant ; *Thermotolerance/genetics ; *Heat-Shock Response/genetics ; *RNA, Plant/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {MicroRNAs (miRNAs) are key regulators of gene expression in plant responses to abiotic stresses, including heat stress. High temperatures during the critical developmental stages of wheat (Triticum aestivum L.) drastically limit growth and production. Recent research has found that specific miRNAs regulate molecular complexes and physiological responses by targeting transcription factors, heat shock proteins, and signaling components, thereby modulating heat stress tolerance pathways. This review highlights current knowledge about heat-responsive miRNAs in wheat, including their validated targets and functional involvement in thermotolerance. In addition, we summarized the potential CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats /CRISPR associated protein 9) genome editing tool for precise alteration of miRNA genes or their targets, degradome profiling, the secondary structure of miRNA, and how interplay of miRNAs with HSFs and HSPs in target gene regulation to improve heat resilience. A comprehensive understanding of miRNA-regulated networks presents novel possibilities for developing climate-resilient wheat varieties, thereby ensuring food security in the face of global warming.},
}

*Triticum/genetics/physiology
*MicroRNAs/genetics/metabolism
Gene Expression Regulation, Plant
*Thermotolerance/genetics
*Heat-Shock Response/genetics
*RNA, Plant/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41249169,
year = {2025},
author = {Ramadoss, GN and Namaganda, SJ and Kumar, MM and Hamilton, JR and Sharma, R and Chow, KG and Workley, LA and Macklin, BL and Sun, M and Ha, AS and Liu, JC and Fellmann, C and Watry, HL and Dierks, PH and Bose, RS and Jin, J and Perez, BS and Sandoval Espinoza, CR and Matia, MP and Lu, SH and Judge, LM and Shy, BR and Nussenzweig, A and Adamson, B and Murthy, N and Doudna, JA and Kampmann, M and Conklin, BR},
title = {Characterizing and controlling CRISPR repair outcomes in nondividing human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9883},
pmid = {41249169},
issn = {2041-1723},
mesh = {Humans ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *DNA Repair/genetics ; *CRISPR-Cas Systems/genetics ; *Neurons/metabolism/cytology ; Myocytes, Cardiac/metabolism ; DNA Damage ; T-Lymphocytes/metabolism ; },
abstract = {Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.},
}

Humans
Induced Pluripotent Stem Cells/metabolism/cytology
*Gene Editing/methods
*DNA Repair/genetics
*CRISPR-Cas Systems/genetics
*Neurons/metabolism/cytology
Myocytes, Cardiac/metabolism
DNA Damage
T-Lymphocytes/metabolism

@article {pmid41248011,
year = {2025},
author = {Dereki, I and Chondrou, V and Lagoumintzis, G and Sgourou, A},
title = {Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {224},
pages = {},
doi = {10.3791/69328},
pmid = {41248011},
issn = {1940-087X},
mesh = {Humans ; *DNA Methylation/genetics ; K562 Cells ; DNA Methyltransferase 3A ; *DNA (Cytosine-5-)-Methyltransferases/genetics/biosynthesis ; Plasmids/genetics ; *Histone Deacetylase 1/genetics/metabolism ; Genetic Vectors/genetics ; Recombinant Fusion Proteins/genetics/biosynthesis ; *Epigenesis, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; },
abstract = {Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns presents significant challenges due to the scarcity of versatile tools capable of inducing site-specific and long-term epigenetic modifications for modulating gene expression. The study aimed to develop and validate an innovative episomal-based system to facilitate stable DNA methylation at a targeted gene locus, potentially useful for both basic epigenetic research and therapeutic applications. To achieve this, the K562 cell line was co-transfected with two distinct episomal vectors. Both vector types were engineered to express guide RNAs (gRNAs) targeting a 367 bp unmethylated region within the CpG island 326, located upstream of the ZBTB7A gene. Each vector encoded a deactivated form of endonuclease Cas9 (dead or dCas9) fused to either the catalytic domain of DNA methyltransferase DNMT3A (dCas-DNMT3A-CD) or the full-length histone deacetylase HDAC1 (dCas-HDAC1). The dCas sequence included two Nuclear Localization Signals (NLS) to ensure the protein's nuclear import. This dual system expression cassette promotes a chromatin state potentially conducive to long-term epigenetic silencing, promising robust and durable epigenetic results. This intervening approach to the host epigenome via utilization of self-replicating episomal vectors offers several advantages: maintenance and expression of vectors at low copy numbers throughout multiple cell divisions without integration into the host genome, hence minimizing off-target effects and preserving genome integrity. We report the precise and significant increase of DNA methylation at the targeted ZBTB7A CpG island 326. The findings validate that the engineered episomal CRISPR/dCas systems can elicit durable, site-specific DNA methylation. Therefore, this system is a valuable research tool for evaluating the functional effects of targeted methylation changes and a promising platform for developing future epigenetic treatments.},
}

Humans
*DNA Methylation/genetics
K562 Cells
DNA Methyltransferase 3A
*DNA (Cytosine-5-)-Methyltransferases/genetics/biosynthesis
Plasmids/genetics
*Histone Deacetylase 1/genetics/metabolism
Genetic Vectors/genetics
Recombinant Fusion Proteins/genetics/biosynthesis
*Epigenesis, Genetic
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems

@article {pmid41243026,
year = {2025},
author = {Menelih, A and Girma, A and Assamere, A},
title = {Mechanisms and applications of epigenome editing in plants: current status, challenges and future perspectives.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {243},
pmid = {41243026},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *Epigenome ; CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Plants/genetics ; DNA Methylation ; *Genome, Plant ; Epigenome Editing ; },
abstract = {Epigenome editing has become a leading-edge technology of programmable, heritable and reversible control of gene expression in plants without changing the DNA sequence. CRISPR/dCas9 systems along with transcription activator-like effectors (TALEs) and zinc finger systems have made it possible to manipulate DNA methylation, histone modifications, and RNA epigenetic marks in a precise and locus-specific fashion. These tools have been used on major regulatory genes of flowering time, stress adjustment, and yield maximization in model and crop plants. This review synthesizes the current status of plant epigenome editing advances and highlights mechanistic innovations including SunTag, CRISPRoff/on and RNA m6A editing. It also emphasizes new paradigm shifts in chromatin reprogramming, including transcription-resistive chromatin states, locus-specific H3K27me3 demethylation, and nanobody-mediated chromatin targeting. Furthermore, it considers the consequences of these shifts in the context of trait stability and epigenetic inheritance. Moreover, the relative evaluation of dCas9-, TALE-, and ZFP-based platforms indicated that there are still enduring problems in the performance of delivery, off-target effects, and transgenerational stability. The review concludes with a conceptual framework connecting epigenome editing to climate-smart crop improvement and outlines future research priorities focused on combinatorial multi-omics integration and the development of environmentally responsive editing platforms.},
}

*Gene Editing/methods
*Epigenome
CRISPR-Cas Systems
*Epigenesis, Genetic
*Plants/genetics
DNA Methylation
*Genome, Plant
Epigenome Editing

@article {pmid41242874,
year = {2025},
author = {Akter, T and Baishnab, SM and Damtie, M and Dumpati, S and Eshete, TB and Irfan, S and Kumar, M and Sara, M and Urmi, UL and Willcox, M},
title = {A review of technological advances in the management of bacterial, fungal, viral, and Acanthamoeba keratitis.},
journal = {Clinical & experimental optometry},
volume = {},
number = {},
pages = {1-11},
doi = {10.1080/08164622.2025.2579169},
pmid = {41242874},
issn = {1444-0938},
abstract = {Microbial keratitis is a major cause of monocular blindness worldwide. Trauma and contact lens wear are major risk factors. Bacteria are a major cause, but fungi, viruses, and Acanthamoeba can also cause the disease. Therapeutic options are dwindling with the rise of antimicrobial resistance in all of these microbes, but especially bacteria. Infection with resistant strains results in worse clinical outcomes. New antimicrobial therapies are being developed but most have yet to be either used to treat ocular infections or become commercially available. For bacterial keratitis, antimicrobial peptides (AMPs) are a potential new option, as are bacteriophages and even bacteria that eat other bacteria or maggot secretions. For fungal keratitis, ocular natamycin, with oral ketoconazole or voriconazole treatments, offer improved outcomes for severe cases. For viral keratitis, AMPs again offer hope as new therapeutics, as do RNA interference and CRISPR-Cas systems. The recent release of a commercially available polyhexamethylene biguanide eye drop is a step forward, but new therapies that can kill both Acanthamoeba trophozoites and cysts are still needed. Beyond antibiotics, photoactivated chromophores for keratitis-corneal collagen cross-linking and photodynamic therapy have been successfully used. Therapies for large ulcers or scarring include corneal gluing, tissue scaffolds such as amniotic membrane or conjunctival flaps, and tissue replacement through penetrating or lamellar keratoplasty.},
}

@article {pmid41242543,
year = {2025},
author = {Hillary, VE and Rajan, V and Ceasar, SA},
title = {CRISPR's impact on cancer: From fundamental models to clinical solutions.},
journal = {Life sciences},
volume = {},
number = {},
pages = {124087},
doi = {10.1016/j.lfs.2025.124087},
pmid = {41242543},
issn = {1879-0631},
abstract = {CRISPR is a programmable genome editing method that has transformed genome engineering and advanced cancer research. Various engineering iterations of CRISPR, including base and prime editing, facilitate more accurate and adaptable genomic manipulations, opening new frontiers in understanding and combating cancer. This review emphasizes CRISPR's significant influence in cancer biology, from its ability to accurately recreate somatic mutations in cellular and animal models to its application in developing precise diagnostic tools. We also examine advancements in cancer therapies, particularly through the ex vivo engineering of robust chimeric antigen receptor (CAR) T cells that perform effectively in restrictive tumor microenvironments. Lastly, we consider the existing limitations of CRISPR-Cas systems and outline future directions that could further enhance the application of this powerful technology against cancer.},
}

@article {pmid41213415,
year = {2026},
author = {Xu, YY and Zhou, SM and Wang, LY and Zhang, R and Li, K and Qian, ZY and Xiao, L},
title = {Methods for detecting off-target effects of CRISPR/Cas9.},
journal = {Biotechnology advances},
volume = {86},
number = {},
pages = {108750},
doi = {10.1016/j.biotechadv.2025.108750},
pmid = {41213415},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; },
abstract = {The CRISPR/Cas9 system has emerged as a revolutionary tool for gene editing, widely used in the biomedical field due to its simplicity, efficiency, and cost-effectiveness. However, evidence suggests that CRISPR/Cas9 can induce off-target effects, leading to unintended mutations that may compromise the precision of gene modifications. Consequently, predicting，detecting and evaluating these off-target effects is crucial for optimizing the accuracy and reliability of CRISPR/Cas9 system. This paper provides an overview of the various methodologies and strategies, used or to be used for identifying off-target effects in CRISPR/Cas9-based genome editing, offering insights to improve the precision and safety of CRISPR applications in research and therapeutics.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
Animals

@article {pmid41208769,
year = {2025},
author = {Luo, D and Wang, N and Major-Monfried, H and Ralls, J and Rha, S and Maitland, SA and Ponnienselvan, K and Yamada, M and Bauer, DE and Wolfe, SA and Kentsis, A},
title = {Modular Platform for Therapeutic Drug Delivery Using Trifunctional Bio-Orthogonal Macromolecular Conjugates.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {11},
pages = {2472-2486},
doi = {10.1021/acs.bioconjchem.5c00440},
pmid = {41208769},
issn = {1520-4812},
mesh = {Humans ; *Cell-Penetrating Peptides/chemistry ; *Drug Delivery Systems/methods ; *Macromolecular Substances/chemistry ; Gene Editing ; Cell Line, Tumor ; Peptidomimetics/chemistry/pharmacology ; Ligands ; CRISPR-Cas Systems ; },
abstract = {Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling the modulation of protein-protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargo to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent antileukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables the customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications.},
}

Humans
*Cell-Penetrating Peptides/chemistry
*Drug Delivery Systems/methods
*Macromolecular Substances/chemistry
Gene Editing
Cell Line, Tumor
Peptidomimetics/chemistry/pharmacology
Ligands
CRISPR-Cas Systems

@article {pmid41186998,
year = {2025},
author = {Wang, W and Liu, H and Yang, Z and Wang, L and Li, W and He, H and Chen, S and Xu, M and Lu, Y},
title = {Enhancement of Spinosyn Production by Integrating a Static and Dynamic CRISPRi-Mediated Metabolic Switch in Saccharopolyspora spinosa.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {46},
pages = {29717-29728},
doi = {10.1021/acs.jafc.5c08276},
pmid = {41186998},
issn = {1520-5118},
mesh = {*Saccharopolyspora/genetics/metabolism/growth & development ; *Macrolides/metabolism ; *Bacterial Proteins/metabolism/genetics ; Metabolic Engineering/methods ; CRISPR-Cas Systems ; Multigene Family ; Gene Expression Regulation, Bacterial ; *Insecticides/metabolism ; },
abstract = {Spinosyns, polyketide-derived macrolide insecticides produced by Saccharopolyspora spinosa, have broad market prospects. However, their large-scale production is still challenged by the low titers of industrial strains. Here, we developed an integrated static and dynamic CRISPRi strategy to redirect metabolic flux toward spinosyn overproduction without affecting cell growth. Initially, static CRISPRi-mediated repression of four genes (gltA1, fabH3, fabH4, and glgC) in three key nodes of primary metabolism boosted spinosyn titers by 19-55% without growth impairment. Simultaneous repression of these genes led to a further increase of spinosyn production. However, static repression of two other genes (pyc or gltA2) severely hindered bacterial growth and reduced spinosyn biosynthesis. To address this, two growth phase-dependent promoters from the spinosyn biosynthetic gene cluster (BGC) were characterized and employed for dynamic repression of pyc and gltA2, resulting in increased spinosyn titers by 44-68% while maintaining robust growth. Finally, combined static and dynamic repression of these six targets simultaneously achieved over a 2-fold increase of spinosyn production. Collectively, integrated static and dynamic CRISPRi-based reprogramming of primary metabolic pathways in S. spinosa achieved a balance between growth and secondary metabolism, thereby leading to spinosyn overproduction. This strategy has broad potential applicability in other actinomycetes for natural product overproduction.},
}

*Saccharopolyspora/genetics/metabolism/growth & development
*Macrolides/metabolism
*Bacterial Proteins/metabolism/genetics
Metabolic Engineering/methods
CRISPR-Cas Systems
Multigene Family
Gene Expression Regulation, Bacterial
*Insecticides/metabolism

@article {pmid41170688,
year = {2025},
author = {Wang, S and Guan, X and Sun, S},
title = {A CRISPR/Cas12a-based aptasensor enhanced by functionalized AuNPs for sensitive full-range C-reactive protein detection.},
journal = {Journal of materials chemistry. B},
volume = {13},
number = {45},
pages = {14749-14760},
doi = {10.1039/d5tb01727k},
pmid = {41170688},
issn = {2050-7518},
mesh = {*C-Reactive Protein/analysis ; *Gold/chemistry ; *Aptamers, Nucleotide/chemistry/metabolism ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Particle Size ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {C-reactive protein (CRP) is a well-established biomarker whose plasma levels increase significantly during inflammatory and infectious episodes. It plays a vital role in the diagnosis of bacterial infections and autoimmune diseases, and cardiovascular risk assessment. However, conventional detection techniques often struggle to balance high sensitivity with a broad dynamic range. Here, we developed and validated a novel fluorescence-based aptasensor named AuCA (AuNP-enhanced CRISPR Aptasensor), which integrates nucleic acid aptamers, magnetic beads (MBs), gold nanoparticles (AuNPs), and the CRISPR/Cas12a system for robust CRP quantification. In this system, MBs functionalized with aptamer 1 selectively captured the target protein, while AuNPs co-modified with aptamer 2 and a Cas12a activation sequence (Trigger) enabled efficient signal transduction. Upon target binding, the Trigger activated the Cas12a/crRNA complex, initiating trans-cleavage of fluorescent reporters and producing a markedly amplified signal. The optimized AuCA platform achieved a low detection limit of 60 ng mL[-1] and a quantifiable range of 0.1-150 μg mL[-1]. It exhibited excellent specificity and resistance to biological interference, ensuring reliable measurements even in complex sample matrices. When applied to clinical human plasma, AuCA demonstrated results that are in strong concordance with results from a commercial immunoturbidimetric assay. AuCA allowed the simultaneous detection of both standard and hypersensitive CRP (hsCRP), supporting comprehensive full-range CRP (frCRP) analysis with strong potential for clinical applications.},
}

*C-Reactive Protein/analysis
*Gold/chemistry
*Aptamers, Nucleotide/chemistry/metabolism
Humans
*Metal Nanoparticles/chemistry
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
Particle Size
*Endodeoxyribonucleases/metabolism
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41151122,
year = {2025},
author = {Cea Salazar, VI and Boender, AJ and Seelke, AMH and Gaard, L and Mederos, SL and Rogers, S and Gutierrez, XZ and Bales, KL and Young, LJ and Trainor, BC},
title = {CRISPR-mediated knockdown of oxytocin receptor in extended amygdala reduces stress-induced social avoidance in female California mice.},
journal = {Hormones and behavior},
volume = {176},
number = {},
pages = {105845},
doi = {10.1016/j.yhbeh.2025.105845},
pmid = {41151122},
issn = {1095-6867},
mesh = {Animals ; *Receptors, Oxytocin/genetics/metabolism ; Female ; *Stress, Psychological/genetics/metabolism ; Peromyscus ; Mice ; *Amygdala/metabolism ; *Social Behavior ; Gene Knockdown Techniques ; Nucleus Accumbens/metabolism ; CRISPR-Cas Systems ; Septal Nuclei/metabolism ; *Avoidance Learning/physiology ; Behavior, Animal/physiology ; },
abstract = {Oxytocin receptors (OTRs) within the extended amygdala and nucleus accumbens (NAc) have been implicated in modulating social behaviors, particularly following stress. The effects of OTR could be mediated by modulating the activity of pre-synaptic axon terminals or via receptors in post-synaptic neurons or glia. Using a viral-mediated CRISPR/Cas9 gene editing system in female California mice (Peromyscus californicus), we selectively knocked down OTR in the anteromedial bed nucleus of the stria terminalis (BNST) or NAc to examine their roles modulating social approach and vigilance behaviors. Knockdown of OTR in the BNST attenuated stress-induced decreases of social approach and had less robust effects on vigilance when interacting with a target mouse behind a wire barrier. In this large arena, where mice could control their proximity to a target mouse, BNST OTR knockdown also increased investigation of a non-social stimulus (empty cage). Behavioral effects of BNST OTR knockdown were weaker in the small arena where focal mice physically interacted with target mice. Interestingly, OTR knockdown in the NAc, reduced stress-induced social vigilance without affecting social approach. These effects could mediate altered encoding of socially aversive experiences, as knockdown manipulations were performed before stress exposure. Together, these results highlight effects of local OTR on social behavior that are region-specific.},
}

Animals
*Receptors, Oxytocin/genetics/metabolism
Female
*Stress, Psychological/genetics/metabolism
Peromyscus
Mice
*Amygdala/metabolism
*Social Behavior
Gene Knockdown Techniques
Nucleus Accumbens/metabolism
CRISPR-Cas Systems
Septal Nuclei/metabolism
*Avoidance Learning/physiology
Behavior, Animal/physiology

@article {pmid41123407,
year = {2025},
author = {Xu, Z and Huang, Y and Dong, Y and An, X and Tong, Y and Li, M},
title = {Engineering broad-spectrum phage-resistant Escherichia coli via adaptive and programmable defense strategies.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {11},
pages = {e0159625},
doi = {10.1128/aem.01596-25},
pmid = {41123407},
issn = {1098-5336},
support = {2022FY101100//Science & Technology Fundamental Resources Investigation Program/ ; 202423m10050004//Anhui Province Scientific and Technological Research Project/ ; 22322908D//S&T Program of Hebei/ ; 82341119//National Natural Science Foundation of China/ ; XK2025-05//Interdisciplinary Research Center of Beijing University of Chemical Technology/ ; },
mesh = {*Escherichia coli/virology/genetics ; *Coliphages/genetics/physiology/isolation & purification ; CRISPR-Cas Systems ; Genome, Viral ; Fermentation ; },
abstract = {UNLABELLED: Phage contamination, which impacts product quality and production efficiency, remains a major challenge in industrial fermentation. Although bacteria have evolved various defense systems to combat phage infection, these systems often suffer from narrow host specificity and limited efficacy. In this study, we isolated and characterized a novel lytic Escherichia coli phage, TR2, from a contaminated fermentation substrate. Its strong environmental stability, short latency period, and high lytic activity render it a significant threat to fermentation processes. Genomic sequencing revealed that phage TR2 has a linear, double-stranded DNA genome of 45,171 bp with a G+C content of 44% and 74 coding sequences. On the basis of the physiological characteristics and genomic features of this phage, we developed two strategies to generate phage-resistant E. coli strains: (i) selection of spontaneous mutations in bacterial surface receptors to prevent phage adsorption and infection and (ii) integration of an exogenous CRISPR/Cas9 system to confer sequence-specific immunity. Spontaneous mutation provides broad-spectrum resistance but at the cost of fitness and evolutionary stability, whereas CRISPR/Cas9 ensures long-term, programmable immunity with minimal growth defects. Importantly, both strategies successfully protected bacterial cultures from phage infection without compromising recombinant protein production, highlighting their potential for industrial application. Our findings provide a practical approach for mitigating phage contamination in industrial fermentation processes. This study also highlights the advantages and limitations of spontaneous mutations and natural phage defense systems, offering valuable insights for the design of more effective phage-resistant microbial platforms.

IMPORTANCE: Phage contamination is a significant challenge in industrial fermentation and severely impacts product quality and production efficiency. We systematically compared spontaneous mutation and CRISPR/Cas9-mediated immunity as two strategies for engineering phage-resistant E. coli strains. Both approaches effectively protected bacterial cultures from phage infection without compromising recombinant protein production, underscoring their potential for industrial applications. Notably, spontaneous mutation conferred broad-spectrum resistance but was associated with fitness costs and limited evolutionary stability. In contrast, CRISPR/Cas9-based immunity offered long-term, programmable protection with minimal growth impairment. By delineating the trade-offs between these two strategies, our work provides a framework for selecting tailored phage resistance solutions suited to diverse biomanufacturing scenarios.},
}

*Escherichia coli/virology/genetics
*Coliphages/genetics/physiology/isolation & purification
CRISPR-Cas Systems
Genome, Viral
Fermentation

@article {pmid40962686,
year = {2025},
author = {Chen, H and Fan, S and Chen, K and Wang, F and Lu, M and Wu, Y and Lu, H and Li, J},
title = {CRISPR-edited iPSCs reveal BSN gene mutations induce neuronal hyperexcitability via astrocyte lipid accumulation.},
journal = {Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics},
volume = {22},
number = {6},
pages = {e00740},
doi = {10.1016/j.neurot.2025.e00740},
pmid = {40962686},
issn = {1878-7479},
mesh = {*Astrocytes/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; Humans ; *Neurons/metabolism/physiology ; *Mutation/genetics ; *Lipid Metabolism/genetics/physiology ; Gene Editing/methods ; CRISPR-Cas Systems ; *Nerve Tissue Proteins/genetics ; Cells, Cultured ; Epilepsy/genetics ; },
abstract = {Mutations in the BSN gene, encoding the presynaptic protein Bassoon, are implicated in epilepsy, but their impact on astrocytes remains unclear. Using CRISPR/Cas9, we introduced patient-derived BSN mutations (p.M1903V and c.5672insCG) into human induced pluripotent stem cells (iPSCs) and differentiated them into astrocytes. We found that mutant astrocytes exhibited significant lipid accumulation, evidenced by elevated free cholesterol, reduced arginase activity, and increased lipid droplets. Proteomic analysis revealed upregulation of lipid metabolism regulators, such as APOE and FASN. Electrophysiological recordings showed impaired Kir4.1 potassium channel function, depolarized resting membrane potential, and increased capacitance in mutant astrocytes following kainic acid stimulation. Co-culture experiments with neurons demonstrated that BSN-mutant astrocytes led to reduced neurite outgrowth, elevated neuronal apoptosis, increased pro-inflammatory cytokines (IL-1β, TNF-α), and neuronal hyperexcitability. These findings demonstrate that BSN mutations disrupt astrocyte lipid homeostasis and impair neurosupportive functions, thereby driving neuronal hyperexcitability. This study establishes astrocytes as critical mediators of epilepsy pathogenesis in BSN-related disorders and highlights lipid metabolism as a potential therapeutic target.},
}

*Astrocytes/metabolism
*Induced Pluripotent Stem Cells/metabolism
Humans
*Neurons/metabolism/physiology
*Mutation/genetics
*Lipid Metabolism/genetics/physiology
Gene Editing/methods
CRISPR-Cas Systems
*Nerve Tissue Proteins/genetics
Cells, Cultured
Epilepsy/genetics

@article {pmid41242302,
year = {2025},
author = {Ishihara, K and Kitagawa, S and Adachi, N and Akutsu, M and Senda, T and Inanaga, H and Numata, T},
title = {Cryo-EM structure of Archaeoglobus fulgidus type III-B CRISPR-Cas effector and intermediate crRNA processing during effector assembly.},
journal = {Biochemical and biophysical research communications},
volume = {792},
number = {},
pages = {152978},
doi = {10.1016/j.bbrc.2025.152978},
pmid = {41242302},
issn = {1090-2104},
abstract = {Type III CRISPR-Cas effectors recognize target RNAs complementary to the crRNA guide, activating diverse downstream antiviral responses. In contrast to type III-A systems, the architecture of the type III-B effector (Cmr), comprising six proteins (Cmr1-Cmr6) and a crRNA, remains incompletely defined. Moreover, although maturation of the 3' region of type III crRNA has been attributed to polynucleotide phosphorylase (PNPase), an alternative maturation pathway has been suggested but remains to be elucidated. Here we determined the cryo-EM structure of the Cmr1-lacking Archaeoglobus fulgidus Cmr (AfCmrΔ1) bound to a target analog at 3.4 Å resolution. The complex forms a continuous basic channel that accommodates a crRNA-target heteroduplex. Comparative interface analysis explains why the previously reported cross-species Cmr assembly retains activity, revealing interface flexibility that enables compatible Cmr3-Cmr4 and Cmr2-Cmr5 interactions. Furthermore, we show the cooperative, site-specific processing of an intermediate crRNA that requires both AfCmrΔ1 and AfCmr1 and proceeds without divalent cations. In addition to identifying the cleavage site within the intermediate crRNA, mutational analysis of AfCmr1 reveals residues critical for the reaction. These findings suggest an alternative pathway for crRNA maturation during type III effector assembly that complements PNPase-mediated trimming of the intermediate crRNA, thereby expanding the mechanistic landscape of type III CRISPR-Cas systems.},
}

@article {pmid41242144,
year = {2025},
author = {Yu, ES and Jang, H and Kwon, JM and Jeong, H and Park, J and Kang, BH and Rho, D and Son, S and Kang, T and Jeong, KH},
title = {Nanoplasmonic real-time RT-RPA and CRISPR/Cas12a detection for rapid point-of-care molecular diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {294},
number = {},
pages = {118216},
doi = {10.1016/j.bios.2025.118216},
pmid = {41242144},
issn = {1873-4235},
abstract = {The rapid and precise detection of nucleic acids is crucial for effective disease diagnosis and management at the point-of-care (POC) level. Here we report a palm-sized plasmonic photothermal platform for real-time on-chip recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection. An ultrathin photothermal nanoplasmonic cavity (PNC) of Au nanoislands (AuNIs) and an aluminum reflector delivers uniform and efficient photothermal heating under white LED illumination. The configuration drives isothermal amplification and CRISPR-mediated cleavage in a single microchamber while a fluorescence microlens array (FMLA) camera records real-time emission. The compact platform detects the SARS-CoV-2 E gene in 25 min at 25.7 copies per cartridge and achieves 100 % concordance with RT-qPCR across 42 clinical samples. This all-in-one platform can offer a robust and cost-effective solution for molecular diagnostics, facilitating scalable and real-time testing of infectious diseases in decentralized POC settings.},
}

@article {pmid41241907,
year = {2026},
author = {Treichel, AJ and Bazzini, AA and Tornini, VA},
title = {Functional Testing of Microproteins in a Vertebrate Model of Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2992},
number = {},
pages = {183-201},
pmid = {41241907},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; CRISPR-Cas Systems ; *Zebrafish Proteins/genetics/metabolism ; Ribosomes/metabolism/genetics ; Gene Expression Regulation, Developmental ; Embryonic Development/genetics ; Micropeptides ; },
abstract = {While thousands of putative microproteins have been identified through ribosome profiling, reporter assays, and mass spectrometry-based methods, their functional testing has remained challenging. Advances in genome sequencing and CRISPR/Cas technologies enable the prioritization and testing of candidate microprotein functions for roles in development, for example, in the maternal-to-zygotic transition or in neurodevelopment. Here, we describe the functional testing of microproteins in vivo using a vertebrate model of early development, Danio rerio (zebrafish).},
}

Animals
*Zebrafish/genetics/embryology/metabolism
CRISPR-Cas Systems
*Zebrafish Proteins/genetics/metabolism
Ribosomes/metabolism/genetics
Gene Expression Regulation, Developmental
Embryonic Development/genetics
Micropeptides

@article {pmid41241835,
year = {2025},
author = {Zhang, J and Li, L and Zhu, Y and Qian, K and Xu, Q and Qin, Y and Wu, L},
title = {Amplification-Free CRISPR-Cas System Integrated Centrifugal Digital Microfluidic Platform Developed for Multiplexed Respiratory Pathogen Nucleic Acid Analysis.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04524},
pmid = {41241835},
issn = {1520-6882},
abstract = {In response to the urgent demand for highly sensitive and rapid multiplex detection technologies in the prevention and control of respiratory infectious diseases, this study presents the development of an integrated CRISPR-Cas9/Cas13a detection platform based on a centrifugal digital microfluidic chip. It aims to overcome the reliance of traditional real-time fluorescence quantitative PCR on specialized equipment and trained personnel. Additionally, it addresses the issue of false positives commonly associated with existing isothermal amplification technologies, while also meeting the requirement for preamplification in sensitive CRISPR-based detection methods. In this study, Methicillin-resistant Staphylococcus aureus (MRSA) and influenza A virus subtype H1N1 were selected as model pathogens. The off-chip CRISPR-Cas9/Cas13a dual nucleic acid detection system was initially developed and optimized to enable highly specific detection of MRSA-mecA DNA at a concentration of 173 pM and H1N1-HA RNA at a concentration of 117 pM. Subsequently, the optimal centrifugal digital chip structure was designed and screened to achieve a droplet filling rate of 99.6%. The optimized CRISPR system was finally integrated into the digital chip, resulting in significantly improved sensitivity, reaching 0.7 copies/μL for MRSA DNA and 1.2 copies/μL for H1N1 RNA within a 20 min reaction time at 37 °C. Furthermore, both the negative and positive detection rates achieved 100% accuracy across all 20 simulated clinical samples. The platform integrates centrifugal digital droplet segmentation technology with the CRISPR-Cas system in an innovative manner, enabling subcopy sensitivity detection without the need for nucleic acid preamplification. Therefore, this convenient, cost-effective, and contamination-resistant method provides a reliable solution for the rapid detection of respiratory pathogens in resource-constrained scenarios.},
}

@article {pmid41241617,
year = {2025},
author = {Kalogeropoulos, K and van Beljouw, SPB and Feldmann, D and van den Berg, DF and Brouns, SJJ},
title = {Proteases in bacteriophage defense systems and their potential in bioengineering.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibs.2025.10.006},
pmid = {41241617},
issn = {0968-0004},
abstract = {Novel phage defense systems featuring diverse enzymatic activities are continually being discovered. Among these, defense systems employing proteolytic enzymes have been identified, revealing a previously unrecognized enzymatic activity in phage defense. These protease-associated defense systems represent an untapped reservoir for new biotechnological tools and may serve as a springboard for the development of proteome editors. This review outlines recent advancements in the discovery and characterization of protease-containing defense systems, proposes methods for further exploration and investigation of protease activity, and considers the prospect of protease defense systems for modulating protein processing and cell fate.},
}

@article {pmid41220317,
year = {2025},
author = {Rayad, N and Chowdhury, EA and Meno-Tetang, GML},
title = {The Impact of QSP Modeling on the Design and Optimization of Gene Therapy Approaches.},
journal = {CPT: pharmacometrics & systems pharmacology},
volume = {14},
number = {11},
pages = {1760-1764},
doi = {10.1002/psp4.70131},
pmid = {41220317},
issn = {2163-8306},
mesh = {*Genetic Therapy/methods ; Humans ; Gene Editing/methods ; Dependovirus/genetics ; Genetic Vectors ; Animals ; RNA, Messenger/administration & dosage/genetics ; Nanoparticles/administration & dosage ; CRISPR-Cas Systems ; *Models, Biological ; Lipids/chemistry ; Tissue Distribution ; Liposomes ; },
abstract = {Quantitative Systems Pharmacology (QSP) is increasingly utilized to support the design and translation of gene therapies. This perspective outlines the application of QSP modeling across three domains of gene therapy: mRNA-based therapeutics, adeno-associated virus (AAV) vectors, and genome editing systems. We highlight opportunities for dose optimization, biomarker interpretation, and mechanistic understanding, while addressing current limitations in model generalizability, data sparsity, and translational relevance. Examples include QSP platforms for lipid nanoparticle (LNP)-delivered mRNA, physiologically based pharmacokinetics (PBPK)-informed AAV biodistribution models, and CRISPR-Cas9-based editing systems. These case studies demonstrate QSP's value in de-risking development and personalizing therapies for rare and complex diseases.},
}

*Genetic Therapy/methods
Humans
Gene Editing/methods
Dependovirus/genetics
Genetic Vectors
Animals
RNA, Messenger/administration & dosage/genetics
Nanoparticles/administration & dosage
CRISPR-Cas Systems
*Models, Biological
Lipids/chemistry
Tissue Distribution
Liposomes

@article {pmid41191401,
year = {2025},
author = {Guo, Y and Xiong, Y and Tong, W and Huang, X and Xiong, Y},
title = {CRISPR/Cas12a-Based Dynamic Light Scattering Assay for Nucleic Acid Amplification-Free and Ultrasensitive Detection of Listeria monocytogenes.},
journal = {Analytical chemistry},
volume = {97},
number = {45},
pages = {25232-25244},
doi = {10.1021/acs.analchem.5c04740},
pmid = {41191401},
issn = {1520-6882},
mesh = {*Listeria monocytogenes/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Dynamic Light Scattering/methods ; Limit of Detection ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques/methods ; Particle Size ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive signal transduction systems are essential for developing rapid, robust, and sensitive nucleic acid tests using the amplification-free CRISPR-Cas12a system. In this study, we introduce a novel platform termed CRISPR-DART (dynamic light scattering Assisted Rapid Test), which combines CRISPR-Cas12a with a dynamic light scattering (DLS) signal readout. We systematically evaluated the effects of nanoparticle size (20, 60, and 100 nm) and morphology (spherical, cubic, and flower-like) on DLS sensing performance. Results demonstrate that larger and more structurally complex nanoparticles significantly enhance scattering intensity, allowing stable DLS signals at lower concentrations and improving sensitivity for detecting low-abundance nucleic acid targets. By utilizing the high specificity and trans-cleavage activity of CRISPR-Cas12a, target-triggered cleavage of single-stranded DNA linkers modulates nanoparticle aggregation, enabling quantitative nucleic acid detection. Notably, CRISPR-DART based on 100 nm gold nanoflowers with large size and complex morphology achieves a detection limit (LOD) of 32 aM, an improvement of 5 orders of magnitude over conventional CRISPR-Cas12a assays using fluorophore-quencher as signal output. Furthermore, the amplification-free CRISPR-DART platform also achieves a LOD of 92 CFU/mL for Listeria monocytogenes in food samples, and after a brief preincubation step, successfully detects 1 CFU in 25 g of food sample. In summary, the CRISPR-DART platform provides a straightforward, highly sensitive, and specific tool for rapid on-site diagnostics and food safety monitoring.},
}

*Listeria monocytogenes/isolation & purification/genetics
*CRISPR-Cas Systems
*Dynamic Light Scattering/methods
Limit of Detection
Gold/chemistry
Metal Nanoparticles/chemistry
Nucleic Acid Amplification Techniques
*Biosensing Techniques/methods
Particle Size
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41052028,
year = {2025},
author = {Khan, S and Chen, L and Chou, CL and Khundmiri, SJ and Knepper, MA},
title = {Inducible Avp knockout mouse line.},
journal = {American journal of physiology. Renal physiology},
volume = {329},
number = {6},
pages = {F784-F795},
doi = {10.1152/ajprenal.00340.2025},
pmid = {41052028},
issn = {1522-1466},
support = {ZIA-HL001285//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; ZIA-HL006129//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {Animals ; Mice, Knockout ; *Arginine Vasopressin/genetics/deficiency/metabolism ; Aquaporin 2/metabolism/genetics ; *Tamoxifen/pharmacology ; Mice ; Male ; *Kidney/metabolism/pathology ; Female ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; },
abstract = {Arginine vasopressin (AVP) is a peptide hormone synthesized in the hypothalamus and secreted by the posterior pituitary. Previous studies toward understanding AVP physiology relied heavily on Brattleboro rats, which have a spontaneous mutation in the Avp gene and lack circulating AVP. However, these rats are difficult to breed due to high neonatal death and behavioral issues, causing commercial breeders to stop production. To address this, we developed a mouse line with tamoxifen-inducible deletion of Avp. We used CRISPR/Cas9 to insert loxP sites into the Avp gene. These mice were then bred with mice expressing a tamoxifen-inducible Cre recombinase. The resulting conditional knockout mice (Avp[flx/flx]Cre[+]) are viable, fertile, and healthy before induction. Administration of tamoxifen in 8-12-wk-old mice successfully deleted Avp, as confirmed by Sanger sequencing. This deletion caused a significant decrease in urine osmolality, a hallmark of AVP deficiency. The kidney structure remained normal, with no signs of medullary atrophy. In addition, these mice exhibited a substantially decreased expression of the aquaporin 2 water channel (AQP2), which is involved in water reabsorption in the kidney inner medulla. We illustrate the use of this model by using RNA-seq to profile the consequences of Avp deletion on gene expression in the kidney. The curated RNA-seq data can be browsed, searched, or downloaded at https://esbl.nhlbi.nih.gov/Databases/AVP-KO/. In conclusion, we successfully created an inducible Avp knockout mouse line that has been made available to the research community. This model will be valuable for studying water balance regulation, polycystic kidney disease, and the neural, vascular, and metabolic functions of vasopressin.NEW & NOTEWORTHY We developed an inducible Avp knockout mouse line that will be shared with the research community and is likely to be useful for further study of the regulation of water balance and polycystic kidney disease, as well as neural, vascular, and metabolic roles of vasopressin.},
}

Animals
Mice, Knockout
*Arginine Vasopressin/genetics/deficiency/metabolism
Aquaporin 2/metabolism/genetics
*Tamoxifen/pharmacology
Mice
Male
*Kidney/metabolism/pathology
Female
Mice, Inbred C57BL
CRISPR-Cas Systems

@article {pmid41241097,
year = {2025},
author = {Hołubowicz, R and Gao, F and Du, SW and Menezes, CR and Zhang, J and Hołubowicz, MW and Chen, PZ and Armbrust, N and Geilenkeuser, J and Liu, DR and Jeffery Truong, DJ and Westmeyer, GG and Palczewska, G and Palczewski, K},
title = {Scalable purification enables high-quality virus-like particles for therapeutic translation.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110946},
doi = {10.1016/j.jbc.2025.110946},
pmid = {41241097},
issn = {1083-351X},
abstract = {Emerging molecular therapies introduce enzymatic activity into cells by delivering genes, transcripts, or proteins. Owing to their robust cell-entry capacity, virus-like particles (VLPs) represent a technology of choice in genome editing, where low doses of heterologous proteins and nucleic acids are essential. However, clinical translation of VLP vectors is hindered by inadequate purification methods. Current approaches, relying primarily on ultracentrifugation, suffer from inconsistent product quality and poor scalability. Here, we report the development of a broadly applicable purification strategy that improves the purity and therapeutic efficacy of genome-editing VLPs. Considering the characteristic properties of murine leukemia virus (MLV)-derived engineered VLPs (eVLPs) and HIV-derived engineered nucleocytosolic vehicles for loading of programmable editors (ENVLPEs+), we developed a workflow that involves single- and multi-modal chromatographic steps, effectively removing host cell proteins and cell-culture contaminants while improving VLP integrity and biological activity. Our purified VLPs displayed superior protein composition, consistency, and enhanced functional delivery compared to VLPs partially purified by conventional ultracentrifugation methods. Mass spectrometric analysis revealed a substantial decrease in contaminants, with VLP-specific proteins comprising >90% of the final product. In vivo studies confirmed improved therapeutic outcomes when chromatographically purified VLPs were used. Our scalable purification platform addresses critical manufacturing bottlenecks and constitutes a starting point for further development of VLP therapeutics, enabling robust production of pure VLPs for diverse applications such as genome editing, vaccine development, and other uses that require intracellular protein delivery.},
}

@article {pmid41240805,
year = {2025},
author = {Ghoreshi, ZA and Ali-Hassanzadeh, M and Mashayekhi-Sardoo, H and Askarpour, H and Arefinia, N},
title = {The identification of tuberculosis using CRISPR technique: A systematic review and meta-analysis.},
journal = {Diagnostic microbiology and infectious disease},
volume = {114},
number = {2},
pages = {117183},
doi = {10.1016/j.diagmicrobio.2025.117183},
pmid = {41240805},
issn = {1879-0070},
abstract = {BACKGROUND: Rapid and precise detection of Mycobacterium tuberculosis (MTB) is essential for effective management and control of tuberculosis. The diagnostic challenge is particularly acute for extrapulmonary TB, which accounts for approximately 15-20% of cases and often presents with paucibacillary samples. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for pathogenic diagnosis across diverse sample types owing to its specificity and adaptability. This systematic review and meta-analysis aimed to appraise the diagnostic accuracy of CRISPR-based techniques in identifying MTB.

METHODS: A comprehensive search was conducted in Medline, Scopus, Embase, and ISI Web of Science to retrieve relevant studies, adhering to PRISMA guidelines. Quality was assessed using the Joanna Briggs comprehensive checklist. Data synthesis and analyses, including subgroup analyses, were performed with Meta-Disc 1.4, examining variables like CRISPR variants, gene targets, pre-amplification techniques, and signal readout methods.

RESULTS: From 341 identified studies, 13 met the inclusion criteria, encompassing 1,572 MTB strains. The pooled sensitivity and specificity of CRISPR-based techniques were 0.91 (95%CI: 0.89-0.92) and 0.97 (95%CI: 0.95-0.98), respectively. The pooled diagnostic odds ratio was 498.67 (95%CI: 255.1-974.7), with an AUC of 0.99 in the SROC curve, denoting excellent diagnostic accuracy. Subgroup analysis by sample type revealed that sputum samples achieved sensitivity and specificity of 0.92 (95%CI: 0.89-0.94) and 0.97 (95%CI: 0.94-0.99), while extrapulmonary samples (BALF, pus, CSF) showed comparable performance with sensitivity of 0.89 (95%CI: 0.84-0.93) and specificity of 0.98 (95%CI: 0.94-0.99).

CONCLUSION: CRISPR-based methods exhibit substantial diagnostic sensitivity and specificity for detecting MTB across both pulmonary and extrapulmonary samples, with notable variances across different CRISPR variants and methodological approaches.},
}

@article {pmid41240616,
year = {2025},
author = {Zong, W and Xie, S and Chu, H and Han, S and Zhang, X},
title = {Beyond traditional antibacterial agents: Novel approaches to combat resistant pathogens.},
journal = {European journal of medicinal chemistry},
volume = {302},
number = {Pt 2},
pages = {118362},
doi = {10.1016/j.ejmech.2025.118362},
pmid = {41240616},
issn = {1768-3254},
abstract = {As bacterial infections caused by antibiotic-resistant strains become increasingly prevalent, traditional antibacterial therapies face mounting challenges. These resilient pathogens not only complicate the treatment of common infections but also undermine the efficacy of therapies for major diseases. This growing threat underscores the urgent need for innovative therapeutic strategies. In recent years, the combinatorial use of antibacterial agents has emerged as a promising approach to enhance efficacy and combat resistant bacteria. This review first provides an overview of antibacterial classifications and their mechanisms of action against bacterial infections. It then explores two combined treatment strategies: antibacterial-antibacterial combinations and antibacterial-non-antibacterial pairings, alongside a drug delivery technology: antibacterial-loaded liposomes. Furthermore, we highlight emerging frontiers in antimicrobial strategies, including CRISPR-Cas technologies, AI-driven discovery platforms, nanomaterials beyond liposomes, microbiota-based therapies, and immunotherapeutic approaches. Finally, we offer a forward-looking perspective on the challenges and opportunities shaping the future of antibacterial development in the biomedical field.},
}

@article {pmid41178406,
year = {2025},
author = {Pei, C and Yan, B and Wang, Y and Chen, T and Du, K and Ma, L and Wang, J},
title = {TdT/Cas12a cascade amplification biosensor for sensitive ALP activity detection.},
journal = {The Analyst},
volume = {150},
number = {23},
pages = {5330-5337},
doi = {10.1039/d5an00938c},
pmid = {41178406},
issn = {1364-5528},
mesh = {Humans ; *Biosensing Techniques/methods ; *Alkaline Phosphatase/metabolism/analysis ; HeLa Cells ; CRISPR-Cas Systems ; Limit of Detection ; *DNA Nucleotidylexotransferase/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {This study presents a novel biosensor based on TdT and CRISPR-Cas12a, which integrates the catalytic activity of terminal deoxynucleotidyl transferase (TdT) with the trans-cleavage property of CRISPR-Cas12a to achieve ultra-sensitive biomolecular detection. The biosensor exhibited a broad linear detection range from 0 to 0.2 U L[-1] and a remarkably low detection limit of 1.7 × 10[-3] U L[-1], demonstrating high specificity and sensitivity. In practical validation, the biosensor successfully quantified alkaline phosphatase (ALP) activity in both cervical cancer cells and HeLa cell lysates, even at a dilution factor of up to 10[6]-fold. Its sensitivity allowed precise detection at the single-cell level. This technology offers a robust, simple, and cost-effective platform for cancer diagnosis, treatment monitoring, and enzyme inhibitor screening, while maintaining excellent detection performance in complex biological samples. This breakthrough establishes a foundation for serological tumor screening and early disease diagnosis, while also opening new avenues for enhanced cancer management and clinical translation, indicating significant potential in translational medicine.},
}

Humans
*Biosensing Techniques/methods
*Alkaline Phosphatase/metabolism/analysis
HeLa Cells
CRISPR-Cas Systems
Limit of Detection
*DNA Nucleotidylexotransferase/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics

@article {pmid41171167,
year = {2025},
author = {Tharmatt, A and Guha, S and Kumeria, T and Yadav, S and Mittal, A and Chitkara, D},
title = {Lipopolymeric Nanoplex-Mediated CRISPR/Cas9 Delivery for VEGF-A Knockdown in Psoriatic Angiogenesis.},
journal = {ACS applied bio materials},
volume = {8},
number = {11},
pages = {10137-10157},
doi = {10.1021/acsabm.5c01478},
pmid = {41171167},
issn = {2576-6422},
mesh = {*CRISPR-Cas Systems ; *Psoriasis/drug therapy/metabolism/genetics/pathology ; Humans ; *Vascular Endothelial Growth Factor A/genetics/metabolism/antagonists & inhibitors ; Animals ; *Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Particle Size ; *Neovascularization, Pathologic/drug therapy/genetics ; Mice ; Materials Testing ; *Polymers/chemistry ; Gene Knockdown Techniques ; *Nanoparticles/chemistry ; Angiogenesis ; },
abstract = {Psoriasis is a chronic, incurable inflammatory skin disease characterized by immune cell infiltration, aberrant keratinocyte differentiation, and enhanced angiogenesis. Overexpression of the vascular endothelial growth factor-A (VEGF-A) gene promotes angiogenesis and is essential for endothelial cell migration, adhesion, and proliferation. Therefore, downregulating VEGF-A represents a promising therapeutic strategy for angiogenesis-related disorders. We investigated the application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein complexes (sgRNA/eGFP-Cas9 RNPs) targeting VEGF-A in psoriasis. To enable efficient delivery in vitro and in vivo, we developed lipopolymeric nanoplexes (NPXs) encapsulating sgRNA/eGFP-Cas9 RNPs. These NPXs exhibited a particle size of 142.2 nm (polydispersity index: 0.144), a zeta potential of +4.27 mV, and achieved >70% transfection efficiency in HaCaT (human immortalized keratinocyte) cells. Ex vivo skin permeation studies demonstrated 66% of permeation after 24 h. The optimized NPX formulation was incorporated into a Carbopol-based gel, which displayed non-Newtonian, shear-thinning behavior with variable thixotropy and achieved 48% of skin permeation after 24 h. In vivo efficacy assessment in an imiquimod-induced psoriasis model in Swiss albino mice showed significantly improved Psoriasis Area and Severity Index (PASI) scores, reduced epidermal damage, and suppressed keratinocyte proliferation compared to naked RNPs and blank gel controls. Gene editing analysis revealed an indel frequency of 40.7% by T7 endonuclease I assay and 14% by Sanger sequencing. Enhanced cellular uptake, efficient skin permeation and retention, and improved therapeutic efficacy collectively highlight the potential of NPX-mediated CRISPR/Cas9 delivery as a noninvasive strategy for psoriasis treatment.},
}

*CRISPR-Cas Systems
*Psoriasis/drug therapy/metabolism/genetics/pathology
Humans
*Vascular Endothelial Growth Factor A/genetics/metabolism/antagonists & inhibitors
Animals
*Biocompatible Materials/chemistry/pharmacology/chemical synthesis
Particle Size
*Neovascularization, Pathologic/drug therapy/genetics
Mice
Materials Testing
*Polymers/chemistry
Gene Knockdown Techniques
*Nanoparticles/chemistry
Angiogenesis

@article {pmid40982615,
year = {2025},
author = {Kanwal, F and Aslam, A and Torriero, AAJ},
title = {Microalgae-based biodiesel: integrating AI, CRISPR and nanotechnology for sustainable biofuel development.},
journal = {Emerging topics in life sciences},
volume = {8},
number = {3},
pages = {131-143},
pmid = {40982615},
issn = {2397-8554},
mesh = {*Microalgae/metabolism/genetics ; *Biofuels ; *Nanotechnology/methods ; *Artificial Intelligence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; },
abstract = {Microalgae are a promising feedstock for biodiesel due to their rapid growth, high lipid content and ability to use non-arable land and wastewater. This review synthesises recent advances in artificial intelligence (AI)-driven strain optimisation, engineering, nanotechnology-assisted processing, and life cycle and technoeconomic insights to evaluate pathways for industrialisation. Over the past decade (2015-2024), genetic engineering and, more recently, AI-guided strain selection have improved lipid productivity by up to 40%. Cultivation advances, including hybrid photobioreactor-open pond systems and precision pH/CO2 control, have enhanced biomass yields while reducing costs. Innovation in lipid extraction, such as supercritical CO2 and microwave-assisted methods, now achieves >90% yields with lower toxicity, while magnetic nanoparticle-assisted harvesting and electroflocculation have reduced energy inputs by 20-30%. Life cycle analyses (net energy ratio ~2.5) and integration of high-value co-products (e.g. pigments and proteins) underscore the need to align biological innovations with techno-economic feasibility. This review uniquely integrates advances in AI, CRISPR and nanotechnology with life cycle and techno-economic perspectives, providing a comprehensive framework that links laboratory-scale innovation to industrial feasibility and positions microalgal biodiesel as a viable contributor to global decarbonisation strategies.},
}

*Microalgae/metabolism/genetics
*Biofuels
*Nanotechnology/methods
*Artificial Intelligence
*Clustered Regularly Interspaced Short Palindromic Repeats
*CRISPR-Cas Systems

@article {pmid40447760,
year = {2025},
author = {Hebert, JD and Xu, H and Tang, YJ and Ruiz, PA and Detrick, CR and Wang, J and Hughes, NW and Donosa, O and Siah, VP and Andrejka, L and Karmakar, S and Aboiralor, I and Tang, R and Sotillo, R and Sage, J and Cong, L and Petrov, DA and Winslow, MM},
title = {Efficient and multiplexed somatic genome editing with Cas12a mice.},
journal = {Nature biomedical engineering},
volume = {9},
number = {11},
pages = {1982-1997},
pmid = {40447760},
issn = {2157-846X},
support = {T34FT8013//Tobacco-Related Disease Research Program (TRDRP)/ ; K00 CA234962/CA/NCI NIH HHS/United States ; DGE-2146755//National Science Foundation (NSF)/ ; R01 CA231253/CA/NCI NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; R35-CA231997//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 CA234349/CA/NCI NIH HHS/United States ; R01-GM141627//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01-CA231253//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; K00CA234962//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; PF-21-112-01-MM//American Cancer Society (American Cancer Society, Inc.)/ ; P01 CA244114/CA/NCI NIH HHS/United States ; R35-HG011316//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; P30-CA124435//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01-CA234349//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01 CA230025/CA/NCI NIH HHS/United States ; R01 GM141627/GM/NIGMS NIH HHS/United States ; R35 CA231997/CA/NCI NIH HHS/United States ; P30 CA124435/CA/NCI NIH HHS/United States ; P01-CA244114//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Mice, Transgenic ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Acidaminococcus/genetics/enzymology ; *Bacterial Proteins/genetics ; Genome ; *Endodeoxyribonucleases/genetics ; Genotype ; },
abstract = {Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations. However, existing models have a limited ability to create multiple targeted edits, hindering our understanding of complex genetic interactions. Here we generate transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a), which robustly generates compound genotypes, including diverse cancers driven by inactivation of trios of tumour suppressor genes or an oncogenic translocation. We integrate these modular CRISPR RNA (crRNA) arrays with clonal barcoding to quantify the size and number of tumours with each array, as well as the impact of varying the guide number and position within a four-guide array. Finally, we generate tumours with inactivation of all combinations of nine tumour suppressor genes and find that the fitness of triple-knockout genotypes is largely explainable by one- and two-gene effects. These Cas12a alleles will enable further rapid creation of disease models and high-throughput investigation of coincident genomic alterations in vivo.},
}

Animals
*Gene Editing/methods
Mice
Mice, Transgenic
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Acidaminococcus/genetics/enzymology
*Bacterial Proteins/genetics
Genome
*Endodeoxyribonucleases/genetics
Genotype

@article {pmid41240306,
year = {2026},
author = {Harada, A},
title = {Golgi Units as Modules in the Ribbon.},
journal = {Sub-cellular biochemistry},
volume = {110},
number = {},
pages = {35-42},
pmid = {41240306},
issn = {0306-0225},
mesh = {*Golgi Apparatus/metabolism/ultrastructure/genetics ; Glycosylation ; Humans ; Glycosaminoglycans/metabolism/biosynthesis ; CRISPR-Cas Systems ; HeLa Cells ; Animals ; },
abstract = {Using super-resolution microscopies, SCLIM and STORM, and CRISPR/Cas9 knockin technology, we show that the Golgi complex is assembled by a number of small "Golgi units" that are 1-3 μm in diameter. Each Golgi unit can be considered a functional and morphological module because it contains all sets of glycosylation enzymes that occupy small domains named "zones." Golgi units change shape dynamically. They attach, detach, fuse, and separate from each other. The zones of glycosylation enzymes rapidly move near the rim of the unit. However, the behaviors of N- and O-linked glycosylation enzyme zones differ from those of glycosaminoglycan (GAG)-synthesizing enzyme zones in localization and dynamics. Since depletion of giantin dissociates the Golgi units, it is responsible for their attachment. Giantin depletion also prevents the movement of GAG-synthesizing enzymes between units, leading to incomplete GAG synthesis. Here, we present the model of the Golgi and its glycosylation enzymes that can explain a number of characteristics of the glycosylation processes.},
}

*Golgi Apparatus/metabolism/ultrastructure/genetics
Glycosylation
Humans
Glycosaminoglycans/metabolism/biosynthesis
CRISPR-Cas Systems
HeLa Cells
Animals

@article {pmid41240134,
year = {2025},
author = {Gong, J and Soleimani Samarkhazan, H and Siavashi, M and Servatian, N and Pirsavabi, F},
title = {CRISPR-Cas9 in leukemia immunotherapy: precision engineering of CAR-T cells and tumor-microenvironment modulation.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {90},
pmid = {41240134},
issn = {1573-4978},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Leukemia/therapy/immunology/genetics ; Gene Editing/methods ; Receptors, Chimeric Antigen/genetics/immunology ; *Tumor Microenvironment/immunology/genetics ; *Immunotherapy, Adoptive/methods ; Precision Medicine/methods ; *Immunotherapy/methods ; T-Lymphocytes/immunology ; Animals ; },
abstract = {Together with base editors, prime editors, and alternative nuclease platforms, CRISPR-Cas9 technology has transformed the field of genetic engineering by providing unprecedented precision in genome editing and creating new opportunities for therapeutic interventions. This technology comprises a versatile genome-editing toolkit for achieving a variety of therapeutic objectives. In the context of leukemia, a group of life-threatening hematologic malignancies, CRISPR-Cas9 has emerged as a transformative tool for immunotherapy. By enabling targeted modifications of immune cells, such as T cells, this technology enhances their ability to recognize and eradicate leukemic cells. CRISPR-Cas9 facilitates the disruption of immune checkpoint inhibitors, the insertion of chimeric antigen receptors (CARs), and the correction of genetic mutations that drive leukemia progression. These advancements have led to the development of more potent and personalized immunotherapies, such as CAR-T cell therapies, with improved efficacy and reduced off-target effects. Moreover, CRISPR-Cas9 allows researchers to model leukemia in vitro, providing deeper insights into disease mechanisms and accelerating the discovery of novel therapeutic targets. Despite challenges such as delivery efficiency and potential immunogenicity, the integration of CRISPR-Cas9 into leukemia immunotherapy represents a paradigm shift, offering hope for durable remissions and potentially curative outcomes. As clinical trials progress, this technology promises to redefine the standard of care for leukemia patients, ushering in a new era of precision medicine. This narrative review explores the revolutionary applications of CRISPR-Cas9 in redefining therapeutic strategies for leukemia.},
}

Humans
*CRISPR-Cas Systems/genetics
*Leukemia/therapy/immunology/genetics
Gene Editing/methods
Receptors, Chimeric Antigen/genetics/immunology
*Tumor Microenvironment/immunology/genetics
*Immunotherapy, Adoptive/methods
Precision Medicine/methods
*Immunotherapy/methods
T-Lymphocytes/immunology
Animals

@article {pmid41240131,
year = {2025},
author = {Behera, L and Samal, KC and Mishra, A and Sahoo, JP and Dash, M and Mishra, A},
title = {CRISPR-driven innovations in rice (Oryza sativa L.) breeding: precision development of male sterile lines.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {89},
pmid = {41240131},
issn = {1573-4978},
mesh = {*Oryza/genetics ; *Plant Infertility/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; Pollen/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Male sterility is a vital trait exploited in hybrid seed production to boost crop yield and improve quality. In rice, different male sterility systems have been developed, significantly advancing the production of high-yielding hybrids. The three main types of male sterility in rice are cytoplasmic male sterility (CMS), photoperiod-sensitive genic male sterility (PGMS), and genic male sterility (GMS). Among these, CMS is the most widely used, arising from interactions between mitochondrial (cytoplasmic) and nuclear genes, resulting in pollen dysfunction. PGMS, on the other hand, is influenced by environmental cues such as day length and temperature, while GMS is attributed to mutations in specific nuclear genes affecting anther or pollen development. A thorough understanding of the genetic and molecular mechanisms underlying these systems is essential for efficient hybrid rice breeding. CMS lines are typically crossed with maintainer and restorer lines carrying fertility-restoring genes to produce fertile F1 hybrids. Recent advancements in molecular biology, genomics, and genome editing technologies have accelerated the development of novel male-sterile and fertility-restoring lines, thereby enhancing the precision and scalability of hybrid breeding programs. These innovations are not only expanding the genetic base of hybrid rice but also making the production process more sustainable. As global food demand rises alongside climate uncertainties, the strategic use of male sterility in rice breeding holds immense potential for improving agricultural productivity.},
}

*Oryza/genetics
*Plant Infertility/genetics
*Plant Breeding/methods
Gene Editing/methods
Pollen/genetics
*CRISPR-Cas Systems/genetics

@article {pmid41239469,
year = {2025},
author = {Ma, J and Zhang, J and Guo, X},
title = {Harnessing CRISPR-Cas9 for Lactobacillus improvement in silage production: current knowledge and future perspectives.},
journal = {Journal of animal science and biotechnology},
volume = {16},
number = {1},
pages = {150},
pmid = {41239469},
issn = {1674-9782},
support = {U20A2002//National Natural Science Foundation of China/ ; },
abstract = {High-quality silage is the cornerstone to sustainable livestock development and animal food production. As the core fermentation bacteria of silage, Lactobacillus directly regulates silage fermentation by producing lactic acid, enzymes, and other bioactive molecules. However, traditional screening methods for functional strains are labor-intensive and time-consuming. Recent advances in synthetic biology, particularly the development of CRISPR-Cas genome editing technology, offer a revolutionary approach to designing Lactobacillus strains with customized traits. This review systematically reviewed the importance of silage in sustainable agricultural development and the limitations of current silage preparation and promotion. It also discussed the application of strain engineering approaches in optimizing the phenotypic performance of Lactobacillus for better silage. Building on this, we reviewed the research progress of CRISPR-Cas9 gene editing in Lactobacillus and discussed how to leverage its high efficiency and precision to optimize the strain's traits for improved silage quality and functionality. CRISPR-Cas9 toolkits are expected to achieve directed evolution of strain performance, ultimately yielding next-generation silage microbial inoculants with multiple functions, adaptability to multiple substrates, and eco-friendly characteristics. The use of such innovative biotechnologies would facilitate resource-efficient utilization, promote animal performance and health for sustainable development in livestock production.},
}

@article {pmid41239088,
year = {2025},
author = {Xu, H and Chen, H and Li, Y and Wang, Y and Zeng, H and Xu, Z and Chen, T and Kong, D and Huang, W and Cheng, H and Zhou, H and Jiang, X and Feng, J},
title = {CRISPR/Cas12a-SERS biosensor based on sea urchin-like AuNPs for the detection of β-thalassemia mutant gene CD31.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {810},
pmid = {41239088},
issn = {1436-5073},
mesh = {*Biosensing Techniques/methods ; *beta-Thalassemia/genetics/diagnosis ; *CRISPR-Cas Systems ; Animals ; *Spectrum Analysis, Raman/methods ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; Mutation ; Sea Urchins/chemistry ; Limit of Detection ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; DNA, Single-Stranded/chemistry/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Sulfhydryl Compounds/chemistry ; Bacterial Proteins ; Benzoates ; },
abstract = {Beta-thalassemia is a single-gene recessive disorder caused by mutations in the HBB gene, and approximately 1.5% of the global population are carriers of β-thalassemia. It is therefore vital to establish a rapid and sensitive method to detect the mutant genes of β-thalassemia. In this study, a CRISPR/Cas12a-mediated amplification-free surface-enhanced Raman spectroscopy (SERS) biosensor was developed. This biosensor uses sea urchin-shaped gold nanoparticles (SUGNPs) as the SERS enhancement substrate and 4-mercaptobenzoic acid (4-MBA) as the Raman reporter. It couples the SUGNPs/4-MBA with magnetic beads through single-strand DNA (ssDNA) to form an SERS probe with magnetic responsiveness. The presence of the β-thalassemia target mutation gene CD31 activates the cleavage activity of Cas12a, leading to non-specific cleavage of single-stranded DNA (ssDNA) on the probe. This results in a significant reduction in SERS intensity. This signal change enables quantitative detection of the target gene, thereby significantly enhancing the sensitivity of nucleic acid testing. We employed magnetic separation technology to enrich target nucleic acids in serum while eliminating matrix interference, enabling specific recognition and quantitative detection of the mutated CD31 gene. This method exhibits excellent linearity over a concentration range 0.1 fM to 10 pM, with a detection limit of 0.1 fM and a detection time of only 40 min. Compared to traditional qPCR and other CRISPR methods, this approach is simple, rapid, and offers advantages such as high sensitivity, high specificity, and cost-effectiveness. By simply replacing the crRNA, it can detect multiple β-thalassemia and other disease genes, demonstrating broad clinical application potential.},
}

*Biosensing Techniques/methods
*beta-Thalassemia/genetics/diagnosis
*CRISPR-Cas Systems
Animals
*Spectrum Analysis, Raman/methods
*Gold/chemistry
*Metal Nanoparticles/chemistry
Mutation
Sea Urchins/chemistry
Limit of Detection
Humans
*Endodeoxyribonucleases/genetics/metabolism
DNA, Single-Stranded/chemistry/genetics
*CRISPR-Associated Proteins/metabolism/genetics
Sulfhydryl Compounds/chemistry
Bacterial Proteins
Benzoates

@article {pmid41236708,
year = {2025},
author = {Zahedi, S},
title = {CRISPR-Based Functional Genomics in Pluripotent Stem Cells.},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {41236708},
issn = {2629-3277},
abstract = {The integration of CRISPR-based functional genomics with pluripotent stem cell (PSC) technologies has been recognized as a transformative approach for investigating gene function, modeling human disease, and advancing regenerative medicine. The aim of this review is to provide a comprehensive evaluation of recent developments in CRISPR-Cas platforms, including gene knockouts, base and prime editing, and CRISPR activation or interference (CRISPRa/i), as applied to PSC systems. Studies employing human PSCs, including embryonic stem cells and induced pluripotent stem cells, have been examined to summarize methodologies for genome-wide screening, lineage tracing, and therapeutic engineering. Advances in editing efficiency, delivery strategies, and genomic safety have been reported, while limitations persist in the form of off-target modifications, epigenetic variability, and cell-type-specific responses. Notable applications include the generation of immune-evasive PSC lines, the development of organoid models for physiological and pathological studies, and the implementation of phenotypic screening for disease-relevant traits. Collectively, these technological and methodological advancements have established functional genomics of PSC-CRISPRSPR as a versatile and powerful framework for elucidating fundamental aspects of human biology, dissecting complex traits, and accelerating the translation of discoveries from experimental research to clinical implementation.},
}

@article {pmid41130353,
year = {2026},
author = {Lee, SY and Kim, HS and Kim, YG and Shin, S and Kweon, S and Lee, JJ and Lee, GM},
title = {Genome-wide CRISPR screening identifies genes in recombinant human embryonic kidney 293 cells for increased ammonia resistance.},
journal = {Metabolic engineering},
volume = {93},
number = {},
pages = {184-193},
doi = {10.1016/j.ymben.2025.10.008},
pmid = {41130353},
issn = {1096-7184},
mesh = {Humans ; *Ammonia/pharmacology/metabolism ; HEK293 Cells ; *CRISPR-Cas Systems ; *Antibodies, Monoclonal/genetics/biosynthesis ; Gene Knockout Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome, Human ; },
abstract = {Ammonia, a byproduct of glutamine metabolism, inhibits cell growth and reduces product yield and quality in mammalian cell culture. To identify novel genes associated with ammonia resistance, a genome-wide CRISPR knockout screening was conducted in monoclonal antibody (mAb)-producing human embryonic kidney 293 (HEK-mAb) cells using a virus-free, recombinase-mediated cassette exchange-based gRNA interrogation method. The knockout cell library was subcultured for five consecutive passages under 20 mM NH4Cl, enriching cells with a sgRNA that conferred a proliferation advantage under high-ammonia conditions. Next-generation sequencing analysis of the enriched population identified three target genes -WNT3, TSPAN1, and CYHR1-among 19,114 genes. Knockout of these genes in HEK-mAb cells resulted in a 1.33- to 1.56-fold increase in maximum viable cell concentration and a 1.28- to 1.58-fold increase in maximum mAb concentration under 20 mM NH4Cl. Notably, WNT3 knockout maintained N-glycan galactosylation proportions of mAb despite ammonia stress. These findings highlight the effectiveness of genome-wide CRISPR knockout screening in identifying novel gene targets for ammonia-resistant HEK293 cell, offering a promising strategy for improving mAb production.},
}

Humans
*Ammonia/pharmacology/metabolism
HEK293 Cells
*CRISPR-Cas Systems
*Antibodies, Monoclonal/genetics/biosynthesis
Gene Knockout Techniques
*Clustered Regularly Interspaced Short Palindromic Repeats
*Genome, Human

@article {pmid41110569,
year = {2025},
author = {Cui, Z and Liang, W and Li, J and Bai, Z},
title = {CRISPR/Cas9 gene editing strategy for cancer therapy: non-viral nanocarrier-mediated delivery of plasmids, RNA and ribonucleoprotein complexes.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 1},
pages = {148389},
doi = {10.1016/j.ijbiomac.2025.148389},
pmid = {41110569},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Ribonucleoproteins/genetics ; *Neoplasms/therapy/genetics ; *Plasmids/genetics ; Animals ; *RNA/genetics ; *Gene Transfer Techniques ; Genetic Therapy/methods ; Nanoparticles/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing systems are among the most prevalent tools in modern genetic research and have been extensively studied in the context of cancer therapy. CRISPR/Cas9 systems can be categorized into three main delivery forms: plasmid DNA (pDNA) containing both Cas9 and guide RNA (sgRNA), messenger RNA (mRNA) of Cas9 along with sgRNA, and ribonucleoprotein (RNP) complexes consisting of Cas9 protein bound to sgRNA. This article will review the various CRISPR/Cas9 systems, focusing on the non-viral especially nanocarrier delivery strategies and common protocols used for their delivery and the challenges encountered during this process. This review will offer guidance on the potential applications of CRISPR/Cas9 gene editing technology for the treatment of malignant tumors in the future.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
*Ribonucleoproteins/genetics
*Neoplasms/therapy/genetics
*Plasmids/genetics
Animals
*RNA/genetics
*Gene Transfer Techniques
Genetic Therapy/methods
Nanoparticles/chemistry
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41093194,
year = {2025},
author = {Yang, G and Rao, Q and Dong, W and Yin, X and Shen, R and Wang, R and Deng, X and Peng, X and Tao, Y and Li, S and Wang, X and Tang, Y and Du, D},
title = {Rapid and sensitive one-pot CRISPR-Cas12a detection platform for common fungal species in ocular infections using multiplex crRNAs.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 1},
pages = {148279},
doi = {10.1016/j.ijbiomac.2025.148279},
pmid = {41093194},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Fungi/genetics/isolation & purification ; *Eye Infections, Fungal/microbiology/diagnosis/genetics ; DNA, Fungal/genetics ; },
abstract = {Corneal fungal infection is a major cause of visual impairment and can cause blindness if not diagnosed promptly. Traditional diagnostic methods are often time-consuming and require specialized expertise, UNDERSCORING the urgent need for rapid, sensitive, and user-friendly diagnostic tools. In this study, we utilized multiplex crRNAs in the Cas12a system to develop a highly sensitive and specific platform for the molecular detection of common fungal species causing corneal infections in a single reaction. We enhanced detection sensitivity through recombinase-aided amplification (RAA) and designed a streamlined tube with insert one-pot protocol to minimize aerosol contamination risk. Remarkably, our platform achieved a limit of detection of just 4 copies of fungal genomic DNA per reaction in only 32 min. It allows for direct detection from simulated tear samples, providing a potential for convenient and non-invasive testing option. Furthermore, our platform effectively tests clinical samples such as aqueous humor (AH) and vitreous humor (VH), demonstrating superior sensitivity compared to traditional PCR-based methods. These results highlight the potential of our platform as an on-site diagnostic solution for both clinical and non-clinical settings, contributing to the preservation of visual function.},
}

Humans
*CRISPR-Cas Systems/genetics
*Fungi/genetics/isolation & purification
*Eye Infections, Fungal/microbiology/diagnosis/genetics
DNA, Fungal/genetics

@article {pmid41043753,
year = {2025},
author = {Yang, Q and Zhao, H and Zhai, Y and Wang, Q and Zhu, Z and Rui, C and Yuan, H and Cui, L},
title = {Ryanodine receptor I4734M mutation confers diamide resistance but reduces invasiveness in Spodoptera frugiperda.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 2},
pages = {148059},
doi = {10.1016/j.ijbiomac.2025.148059},
pmid = {41043753},
issn = {1879-0003},
mesh = {Animals ; *Spodoptera/genetics/drug effects ; *Ryanodine Receptor Calcium Release Channel/genetics ; *Mutation ; *Diamide/pharmacology ; *Insecticide Resistance/genetics ; ortho-Aminobenzoates/pharmacology ; Insecticides/pharmacology ; CRISPR-Cas Systems ; Gene Editing ; Pyrazoles ; },
abstract = {Although Spodoptera frugiperda populations with the ryanodine receptor (RyR) I4734M mutation occurred in their native regions, this mutation has not been detected in the Eastern Hemisphere. In order to clarify the functional role of this mutation, the first HDR-mediated RyR[I4734M] mutation was introduced into S. frugiperda using CRISPR/Cas9 technology and a homozygous SfRyR[I4734M] strain was successfully established. Meanwhile, a novel, non-invasive genotyping method based on insect fecal DNA to efficiently identify genome-edited individuals was developed. Compared with the wild type, the genome-edited SfRyR[I4734M] strain showing high-level resistance to chlorantraniliprole (396.7-fold) and tetraniliprole (149.1-fold), and moderate resistance to cyantraniliprole (32.3-fold) and flubendiamide (29.5-fold). Reciprocal crossing experiments indicated that resistance to chlorantraniliprole was inherited in an autosomally incompletely recessive mode. Furthermore, the SfRyR[I4734M] substitution adversely reduced the fitness and flight ability of S. frugiperda. The mutant strain exhibited significantly decreased fecundity and severely impaired flight distance and velocity. These findings provided in vivo genetic validation of SfRyR[I4734M] mutation in diamide resistance, demonstrating this mutation conferred subtle differences on the binding affinities of four diamides. Moreover, our results firstly demonstrated that SfRyR[I4734M] mutation could affect muscle function, thereby reducing the flight ability of S. frugiperda.},
}

Animals
*Spodoptera/genetics/drug effects
*Ryanodine Receptor Calcium Release Channel/genetics
*Mutation
*Diamide/pharmacology
*Insecticide Resistance/genetics
ortho-Aminobenzoates/pharmacology
Insecticides/pharmacology
CRISPR-Cas Systems
Gene Editing
Pyrazoles

@article {pmid41236489,
year = {2025},
author = {Wang, Y and Kong, Y and Zhang, Y and He, Y and Wang, W and Lu, Y and Meng, J and Yuan, H},
title = {Long-Term Attenuation of Vascular Hyperpermeability in a Hereditary Angioedema Mouse Model by Adenine Base Editing.},
journal = {Allergy},
volume = {},
number = {},
pages = {},
doi = {10.1111/all.70152},
pmid = {41236489},
issn = {1398-9995},
support = {//the National Natural Science Foundation of China/ ; //West China Hospital, Sichuan University/ ; },
abstract = {BACKGROUND: Hereditary angioedema (HAE) is a rare and potentially life-threatening disorder caused by dysregulated kallikrein-kinin signaling and bradykinin-induced vascular hyperpermeability. Current therapies targeting this pathway are effective but require lifelong administration, underscoring the need for durable and potentially curative interventions. Adenine base editors (ABEs), engineered from CRISPR/Cas systems, enable precise single-nucleotide modifications with minimal genomic disruption, offering a promising strategy for long-term gene silencing.

METHODS: NG-ABE8e was delivered via AAV8 or lipid nanoparticles (LNP) to disrupt the exon 2 splice donor site of Klkb1 in Serping1[+/-] mice. Editing outcomes were quantified by high-throughput sequencing; serum kallikrein levels were measured by ELISA; and vascular permeability was evaluated using Evans blue dye extravasation, fluorescent tracer leakage, and VE-cadherin immunostaining. Safety evaluations included off-target analysis, histopathology, serum biochemistry, activated partial thromboplastin time (aPTT), and systemic hemodynamic stability.

RESULTS: AAV8-NG-ABE8e induced > 60% A•T-to-G•C conversion at the target site, promoting exon 2 skipping and reducing Klkb1 mRNA and serum kallikrein levels by > 85%, an effect sustained for at least 1 year. AAV8-NG-ABE8e also reversed bradykinin-driven vascular hyperpermeability and attenuated inflammatory gene signatures. Prolongation of aPTT was observed only when circulating kallikrein levels declined below 2 μg/mL. Similarly, LNP-mediated delivery of NG-ABE8e mRNA resulted in > 90% reductions in Klkb1 expression and serum kallikrein levels. No evidence of significant long-term toxicity was detected.

CONCLUSION: These findings demonstrate that, in a murine model, NG-ABE8e-mediated disruption of Klkb1 enables durable suppression of serum kallikrein and vascular stabilization, suggesting its potential as a promising single-intervention strategy for the treatment of HAE.},
}

@article {pmid41236435,
year = {2025},
author = {Chen, Y and Wang, Y and Liu, L and Yu, X and Zhang, Y and Xi, M and Xu, J and Yang, H and Xie, C and Wang, D},
title = {Targeting BnNAC038 improves drought tolerance with low yield penalty in Brassica napus.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {3},
pages = {e70571},
doi = {10.1111/tpj.70571},
pmid = {41236435},
issn = {1365-313X},
support = {241111112400//Key Research and Development Project of Henan Province/ ; 2023-YBNY-023//Key Research and Development Project of Shaanxi Province/ ; 32070324//National Natural Science Foundation of China/ ; 32301811//National Natural Science Foundation of China/ ; 2024YFD1200400//the National Key Research and Development Project from the Ministry of Agriculture and Rural Affairs of China/ ; },
mesh = {*Brassica napus/genetics/physiology/growth & development ; Droughts ; *Plant Proteins/genetics/metabolism/physiology ; Gene Expression Regulation, Plant ; Photosynthesis/genetics ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Drought stress severely limits crop productivity in Brassica napus, yet strategies to enhance drought tolerance without compromising yield remain elusive. Here, we identify BnNAC038 as a negative regulator of drought responses in Brassica napus. CRISPR/Cas9-generated bnnac038 mutants exhibited improved drought survival, reduced water loss, and enhanced stomatal closure under drought conditions compared to wild-type (WT) plants. RNA-sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-seq) analyses revealed that BnNAC038 directly represses drought-responsive genes, including BnSnRK2.6 (a key ABA signaling kinase), and genes involved in photosynthesis (BnPPC2) and gluconeogenesis (BnPGK). Field trials demonstrated that bnnac038 plants exhibit enhanced photosynthesis, accumulate more sucrose and glucose under drought, and exhibit increased biomass and seed yield compared to WT. Genetic interaction studies further showed that BnSnRK2.6 acts downstream of BnNAC038 to mediate drought tolerance. Our results indicate that targeted editing of BnNAC038 enhances drought tolerance while minimizing yield loss, providing a new strategy for developing drought-resilient Brassica napus varieties with minimal yield penalty.},
}

*Brassica napus/genetics/physiology/growth & development
Droughts
*Plant Proteins/genetics/metabolism/physiology
Gene Expression Regulation, Plant
Photosynthesis/genetics
CRISPR-Cas Systems
Drought Resistance

@article {pmid41236144,
year = {2025},
author = {Apsley, EJ and Riepsaame, J and Cheng, YC and Cowley, SA and Becker, EBE},
title = {Cerebellar organoids model cell type-specific FOXP2 expression during human cerebellar development.},
journal = {Disease models & mechanisms},
volume = {18},
number = {11},
pages = {},
doi = {10.1242/dmm.052290},
pmid = {41236144},
issn = {1754-8411},
support = {BB/M011224/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //UK Research and Innovation/ ; },
mesh = {Humans ; *Forkhead Transcription Factors/metabolism/genetics ; *Organoids/metabolism ; *Cerebellum/metabolism/growth & development/embryology/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; Purkinje Cells/metabolism ; Gene Expression Regulation, Developmental ; Cell Differentiation/genetics ; *Models, Biological ; CRISPR-Cas Systems/genetics ; },
abstract = {Human cerebellar development is unique and cannot be fully replicated in animal models. Although human stem cell-derived cerebellar organoid models are increasingly being applied to model cerebellar diseases, their potential to provide insight into normal human cerebellar development remains underexplored. Here, we used CRISPR-based gene editing in cerebellar organoids as an approach for modelling specific features of early human cerebellar development. Forkhead box protein P2 (FOXP2) is a transcription factor associated with speech and language development that is highly expressed in the developing brain. However, little attention has been directed to the study of FOXP2 in the early developing cerebellum. We generated a fluorescent FOXP2 reporter line in human induced pluripotent stem cells to enable the characterisation of FOXP2-expressing cells during cerebellar organoid differentiation. Through transcriptomic profiling of FOXP2 reporter cerebellar organoids and cross-referencing with existing cerebellar datasets, we describe the expression and identify potential downstream targets of FOXP2 in the early developing human cerebellum. Our results highlight expression of FOXP2 in early human Purkinje cells and cerebellar nuclei neurons, and the vulnerability of these cell populations to neurodevelopmental disorders.},
}

Humans
*Forkhead Transcription Factors/metabolism/genetics
*Organoids/metabolism
*Cerebellum/metabolism/growth & development/embryology/cytology
Induced Pluripotent Stem Cells/metabolism/cytology
Purkinje Cells/metabolism
Gene Expression Regulation, Developmental
Cell Differentiation/genetics
*Models, Biological
CRISPR-Cas Systems/genetics

@article {pmid41234307,
year = {2025},
author = {Tuncel, A and Kim, HU and Kim, MC},
title = {Editorial: Engineering future crops through genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1720325},
pmid = {41234307},
issn = {1664-462X},
}

@article {pmid41233858,
year = {2025},
author = {Mayer, J and Pack, M and Montenarh, M and Götz, C},
title = {Gene expression changes in pancreatic α-cell lines following knock-out Of either CK2α or CK2α'.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {69},
pmid = {41233858},
issn = {0717-6287},
mesh = {*Casein Kinase II/genetics/metabolism ; *Glucagon-Secreting Cells/metabolism ; Animals ; Mice ; Cell Line ; Gene Knockout Techniques ; *Gene Expression/genetics ; },
abstract = {BACKGROUND: Protein kinase CK2 is known to exist as a tetramer of two catalytic α- or α'- subunits and two non-catalytic β-subunits, or as multimers of this tetramer. Moreover, CK2α (CSNK2A1) and CK2α' (CSNK2A2) are also active in the absence of CK2β (CSNK2B). Very little is known about specific functions of the individual subunits of protein kinase CK2.

RESULTS: In order to study the effects of CK2α and CK2α' on gene expression, we used the Mus musculus pancreatic α-cell line αTC1 and two derivatives with either CK2α (KO1 cells) or CK2α' (KO2 cells) expression knocked-out by CRISPR/Cas technology. We found numerous genes deregulated in both KO1 and KO2 cells compared to the parental cells. Applying stringent thresholds, 266 genes were found down-regulated and 153 genes up-regulated in KO1 cells, 233 genes were found down-regulated and 84 genes up-regulated in KO2 cells. Dozens of genes were found deregulated in a similar fashion in both KO1 and KO2 cells. We found altered expression of genes involved in the differentiation of pancreatic cells, including Hox genes, and in the regulation of glucagon synthesis or secretion. Moreover, many of the deregulated genes play an important role in developmental processes and in neuronal cell biology.

CONCLUSION: Our findings reveal individual and shared functions of the CK2α and CK2α' catalytic subunits, in particular regarding their involvement in regulating gene expression.},
}

*Casein Kinase II/genetics/metabolism
*Glucagon-Secreting Cells/metabolism
Animals
Mice
Cell Line
Gene Knockout Techniques
*Gene Expression/genetics

@article {pmid41232012,
year = {2025},
author = {Maire, A and Bikard, D},
title = {Programmable DNA insertion in native gut bacteria.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6774},
pages = {676-677},
doi = {10.1126/science.aec3823},
pmid = {41232012},
issn = {1095-9203},
mesh = {Animals ; *Gastrointestinal Microbiome/genetics ; Mice ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Bacteria/genetics ; *Mutagenesis, Insertional ; DNA, Bacterial/genetics ; },
abstract = {A gene-editing approach enables modification of bacteria within the mouse gut.},
}

Animals
*Gastrointestinal Microbiome/genetics
Mice
*Gene Editing/methods
CRISPR-Cas Systems
*Bacteria/genetics
*Mutagenesis, Insertional
DNA, Bacterial/genetics

@article {pmid41231980,
year = {2025},
author = {Gelsinger, DR and Ronda, C and Ma, J and Kar, OB and Edwards, M and Huang, Y and Mavros, CF and Sun, Y and Perdue, T and Vo, PL and Ivanov, II and Sternberg, SH and Wang, HH},
title = {Metagenomic editing of commensal bacteria in vivo using CRISPR-associated transposases.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6774},
pages = {eadx7604},
doi = {10.1126/science.adx7604},
pmid = {41231980},
issn = {1095-9203},
mesh = {Animals ; Mice ; *Gastrointestinal Microbiome/genetics ; *Gene Editing/methods ; *Bacteroides/genetics/growth & development ; Humans ; Metagenomics/methods ; *CRISPR-Cas Systems ; Symbiosis ; Mice, Inbred C57BL ; Metagenome ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Although metagenomic sequencing has revealed a rich microbial biodiversity in the mammalian gut, methods to genetically alter specific species in the microbiome are highly limited. Here, we introduce Metagenomic Editing (MetaEdit) as a platform technology for microbiome engineering that uses optimized CRISPR-associated transposases delivered by a broadly conjugative vector to directly modify diverse native commensal bacteria from mice and humans with new pathways at single-nucleotide genomic resolution. Using MetaEdit, we achieved in vivo genetic capture of native murine Bacteroides by integrating a metabolic payload that enables tunable growth control in the mammalian gut with dietary inulin. We further show in vivo editing of segmented filamentous bacteria, an immunomodulatory small-intestinal microbial species recalcitrant to cultivation. Collectively, this work provides a paradigm to precisely manipulate individual bacteria in native communities across gigabases of their metagenomic repertoire.},
}

Animals
Mice
*Gastrointestinal Microbiome/genetics
*Gene Editing/methods
*Bacteroides/genetics/growth & development
Humans
Metagenomics/methods
*CRISPR-Cas Systems
Symbiosis
Mice, Inbred C57BL
Metagenome
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41231855,
year = {2025},
author = {Liu, S and Bai, J and Zhan, B and Yao, J and Zhang, J and Yi, J and Dong, M and Li, Q and Shen, Y and Chen, Y and Zhao, Y},
title = {Development of an RNA aptamer-assisted CRISPR/Cas9 system for efficiently generating and isolating Cas9-free mutants in plant.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011931},
pmid = {41231855},
issn = {1553-7404},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Aptamers, Nucleotide/genetics ; Plants, Genetically Modified/genetics ; Mutation ; *Arabidopsis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 gene-editing system is a powerful tool in plant genetic engineering; however, screening for Cas9-free edited plants remains complex and time-consuming. To address this limitation, we developed an RNA aptamer-assisted CRISPR/Cas9 system, termed 3WJ-4 × Bro/Cas9. In this system, the engineered RNA aptamer 3WJ-4 × Bro functions as a transcriptional reporter, serving as an alternative to traditional fluorescent proteins and thus avoiding their potential interference with Cas9 activity. Compared to the conventional GFP/Cas9 system, 3WJ-4 × Bro/Cas9 showed more efficient transformation and higher accuracy in fluorescence-based selection of positive T1 transformants, without significantly affecting plant growth. Furthermore, 3WJ-4 × Bro/Cas9 achieved a 78.6% increase in the T1 mutation rate compared to GFP/Cas9, with the homozygous mutation rate reaching 1.78%. In addition, 3WJ-4 × Bro/Cas9 enabled fluorescence-based visual screening in the T2 generation for rapid identification of Cas9-free mutants, improving sorting efficiency by 30.2% over the GFP-based method. Moreover, 3WJ-4 × Bro/Cas9 enabled more efficient generation of homozygous double-target mutants compared to GFP/Cas9. These results demonstrate that the 3WJ-4 × Bro/Cas9 system provides a non-transgenic, efficient, and broadly applicable strategy for plant genome editing and selection.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Aptamers, Nucleotide/genetics
Plants, Genetically Modified/genetics
Mutation
*Arabidopsis/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40958664,
year = {2025},
author = {Yang, L and Zhao, Z and Lei, R and Zhang, Y and Wu, P},
title = {A one-pot RPA/CRISPR-bio-dCas9 lateral flow assay for rapid and on-site detection of Monilinia fructicola in stone and pome fruits.},
journal = {Pest management science},
volume = {81},
number = {12},
pages = {8564-8574},
doi = {10.1002/ps.70159},
pmid = {40958664},
issn = {1526-4998},
support = {//the National Key Research and Development Program of China (2021YFC2600400)/ ; //the Basic Scientific Research Foundation of the Chinese Academy of Quality and Inspection & Testing (2024JK051)/ ; },
mesh = {*Plant Diseases/microbiology ; *Fruit/microbiology ; *Ascomycota/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Brown rot, a devastating fungal disease affecting stone and pome fruits, leads to substantial economic losses worldwide, impacting production, post-harvest storage, and transportation. Monilinia fructicola, the primary causal agent of brown rot, is especially challenging to manage because of its ability to establish latent infections in fruit, making early detection and control extremely difficult, enabling the disease to spread unnoticed. Therefore, it is essential to develop fast and accurate detection technologies.

RESULTS: In this study, a one-pot detection method for M. fructicola, integrating CRISPR-bio-dCas9, recombinase polymerase amplification (RPA), and a lateral flow assay (LFA) was developed. This method could detect the M. fruticola genome in less than 30 min from sample collection to result, with a detection limit of 4 copies/μL of M. fructicola. The RPA/CRISPR-bio-dCas9 LFA method simplified M. fructicola detection by eliminating the need for probes, additional reporters, or specialized equipment, thereby reducing costs and complexity. Furthermore, the streamlined single-tube workflow minimized cross-contamination risks, enabling non-expert workers to perform efficient pathogen screening.

CONCLUSIONS: The RPA/CRISPR-bio-dCas9 LFA enabled advanced brown rot management by addressing the limitations of traditional detection (slow speed, high cost, complexity). With high sensitivity and a short detection time using an equipment-free design, it enabled practical on-site detection of M. fructicola, allowing timely interventions to reduce pre- and post-harvest losses in fruit production. © 2025 Society of Chemical Industry.},
}

*Plant Diseases/microbiology
*Fruit/microbiology
*Ascomycota/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems

@article {pmid40810834,
year = {2025},
author = {Xu, M and Chu, J and Li, M and Ren, X and Chen, X and Li, X and Cheng, H and Wang, C and Yang, F},
title = {A Streamlined One-Step Bioprocess for Isomaltulose Production in Bacillus subtilis Through Multicopy Genomic Integration of Sucrose Isomerase Gene.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {10},
pages = {6820-6833},
pmid = {40810834},
issn = {1559-0291},
support = {GXTZYKF202406//Opening Project of Guangxi Key Laboratory of Green Processing of Sugar Resources/ ; 2024-MSLH-037//Liaoning Provincial Science and Technology Program Joint Program/ ; 202410152013//Dalian Polytechnic University College Student Innovation and Entrepreneurship Training Program Project/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism/enzymology ; *Isomaltose/analogs & derivatives/biosynthesis ; *Glucosyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Isomaltulose, a sucrose isomer with a low glycemic index and non-cariogenic properties, is extensively used in the food industry. The industrial production of this functional sugar relies on enzymatic biotransformation using sucrose isomerase (SIase). However, conventional bioprocesses involve expressing and isolating the SIase enzyme, followed by using the purified SIase to convert sucrose into isomaltulose, resulting in a multi-step and high-cost process that hindered the broader applications of isomaltulose. In this study, we reported a streamlined one-step bioprocess that integrates extracellular SIase secretion and direct isomaltulose biosynthesis in the culture medium of an engineered B. subtilis strain. Using CRISPR/Cas9 technology, we engineered B. subtilis to integrate multiple SIase expression cassettes into the genome while concurrently replacing genes within the sacP-sacA-ywdA and sacB-levB-yveA operons, which are crucial for sucrose hydrolysis in B. subtilis. This strategy synergistically increased the genomic copy number of SIase gene while limited sucrose consumption by native pathways, thereby maximizing substrate availability for SIase-mediated catalysis. The resulting engineered strain, containing four copies of the SIase expression cassettes, achieved an extracellular SIase activity of 8.2 U/mL in shake flasks. When cultured in a medium containing 200 g/L sucrose, this strain produced a maximum isomaltulose titer of 162.1 g/L with a yield of 0.81 g/g and a productivity of 13.5 g/L/h. These findings demonstrate an integrated bioprocess that eliminates costly enzyme isolation procedure and reduces fermentation complexity, presenting a commercially feasible strategy for sustainable isomaltulose production.},
}

*Bacillus subtilis/genetics/metabolism/enzymology
*Isomaltose/analogs & derivatives/biosynthesis
*Glucosyltransferases/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40759871,
year = {2025},
author = {Luo, X and Su, B and Lai, P and Li, M and Deng, MR and Zhu, H},
title = {Development of an NADPH Regeneration System for L-threonine Production in Escherichia coli.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {10},
pages = {6575-6591},
pmid = {40759871},
issn = {1559-0291},
support = {2021YFC2100900//National Key Research and Development Program of China/ ; 2021JC06N628//Guangdong Special Support Program/ ; 2022GDASZH-2022010101//GDAS' Project of Science and Technology Development/ ; },
mesh = {*NADP/metabolism ; *Escherichia coli/metabolism/genetics ; *Threonine/biosynthesis ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {NADPH is essential for the biosynthesis of L-threonine, and a deficiency in its supply significantly constrains L-threonine production. To address the challenge of inadequate NADPH availability that adversely affects L-threonine synthesis, we developed an NADPH regeneration system aimed at enhancing the NADPH supply and subsequently improving L-threonine production. Through overexpression of the zwf and gnd genes, which are involved in NADPH generation within the pentose phosphate pathway (PPP), the NADPH/NADP[+] ratio in the strain was elevated 4.1-fold compared with the control strain, resulting in a 2.0-fold increase in L-threonine production. Subsequently, integration of the asd and thrA1034 genes, which are linked to NADPH consumption, enhanced L-threonine production by 3.6-fold. Moreover, the application of promoter engineering facilitated a 7.1-fold increase in L-threonine production compared with the control strain. Finally, we employed the CRISPR-Cas12f1 system to delete the pgi gene to further examine its impact on L-threonine production. The results indicated an increase in the NADPH/NADP[+] ratio and a subsequent enhancement in L-threonine production following deletion of the pgi gene. Consequently, the NADPH regeneration system developed in this study demonstrates potential to effectively improve L-threonine production and may serve as a novel strategy for L-threonine synthesis.},
}

*NADP/metabolism
*Escherichia coli/metabolism/genetics
*Threonine/biosynthesis
Escherichia coli Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40751408,
year = {2025},
author = {Bao, Z and Chang, X and Cheng, L and Lin, W and Xu, W and Shi, J},
title = {A highly specific and ultrasensitive approach to detect Hylurgus ligniperda based on RPA-CRISPR-LbaCas12a-LFD system.},
journal = {Pest management science},
volume = {81},
number = {12},
pages = {7874-7884},
doi = {10.1002/ps.70099},
pmid = {40751408},
issn = {1526-4998},
support = {2023YFE0116200//National Key Research and Development Program of China/ ; 2023YFC2605200//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Weevils/genetics/classification ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; China ; Recombinases ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Hylurgus ligniperda is an invasive bark beetle that poses a serious threat to global coniferous forests and the timber trade. Its broad host range, high reproductive potential, and strong environmental adaptability enable it to establish and spread rapidly in newly invaded regions. In October 2020, H. ligniperda was first reported in Shandong Province, China. Developing a rapid, sensitive, and accurate field detection method is critical for early interception and effective management.

RESULTS: We developed a detection method for H. ligniperda based on recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12a, with results monitored via fluorescence signals and lateral flow dipstick (LFD). The mitochondrial COI gene was selected as the target sequence, and key parameters-including incubation time, temperature, and concentrations of Cas12a protein and CRISPR RNA (crRNA)-were optimized. The RPA-CRISPR-LbaCas12a-LFD assay exhibited high specificity and sensitivity, successfully distinguishing H. ligniperda from five closely related species, and detecting target DNA at concentrations as low as 1 copy per μL. Finally, The field applicability of the detection system was validated using samples from global geographic populations.

CONCLUSION: This study establishes a portable, rapid, and sensitive visual detection system for H. ligniperda based on RPA-CRISPR-LbaCas12a-LFD, suitable for both laboratory and field applications. The method enables field detection without the need for specialized equipment, offering a robust tool for invasive pest surveillance, port quarantine, and early warning. © 2025 Society of Chemical Industry.},
}

Animals
*CRISPR-Cas Systems
*Weevils/genetics/classification
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
China
Recombinases
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41231321,
year = {2025},
author = {Kumari, S and Keshari, AK and Singh, SK and Pandey, S and Singh, A},
title = {CRISPR-Based genome editing in pulses: current approaches, challenges, and future prospects.},
journal = {Plant molecular biology},
volume = {115},
number = {6},
pages = {126},
pmid = {41231321},
issn = {1573-5028},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *Fabaceae/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; },
abstract = {Legumes are the second most important food crop after cereals for the world population. It is a significant protein source for developing countries and integral to global food security. However, various agroecological constraints and biotic and abiotic factors often compromise the production of pulses. Legumes are long-term neglected crops worldwide and follow traditional breeding, leading to a time-consuming, labor-intensive, less economically feasible program associated with linkage drag. Recent sequencing attempts in the twenty-first century, with the development of an enormous repertoire of genetic and genomic resources, allowed scientists to accelerate the improvement of legumes with modern genome editing tools. One such promising tool is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which has revolutionized and transformed the landscape of genetic engineering. The emergence of CRISPR/Cas systems has redefined precision breeding, offering unprecedented control over genome manipulation in legume crops. It has tremendous potential for crop improvement and can precisely make changes at genomic locations with incredible accuracy. Therefore, identifying the desired genes and their precise manipulation has enormous implications for legume crop improvement. This review will give an overview of the genome editing tools available for crop improvement and the efficiency of different transformation methods in legume crops. It will also discuss the current status of genome editing in legume crops, including challenges and future perspectives.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Genome, Plant/genetics
*Crops, Agricultural/genetics
*Fabaceae/genetics
Plant Breeding/methods
Plants, Genetically Modified/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Engineering/methods

@article {pmid41230228,
year = {2025},
author = {Park, S and Mani, V and Ha, K and Kim, JA and Lee, S},
title = {Plant synthetic biology: from knowledge to biomolecules.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1562216},
pmid = {41230228},
issn = {1664-462X},
abstract = {Plant synthetic biology is rapidly emerging as an innovative approach to solving complex problems in human health and agriculture. Although conventional metabolic engineering primarily focuses on microbial systems for large-scale biomolecules production, these platforms often face limitations in expressing plant-derived enzymes and synthesizing structurally complex molecules. In contrast, recent advances in plant synthetic biology have integrated multidisciplinary tools, from molecular biology and biochemistry to synthetic circuit design and computational modeling, to engineer plant systems with enhanced traits. These include improved yield, nutritional quality, environmental resilience, and synthesis of pharmaceutically relevant functional biomolecules. This review focuses on the fundamental technologies that have enabled such advances, which include DNA synthesis, programmable gene circuits, and CRISPR/Cas-based genome editing. We discussed recent applications of reprogramming plant metabolic pathways and existing obstacles, such as transformation efficiency, regulatory bottlenecks, and pathway instability. This review provides key case studies and a forward-looking perspective on the evolution of plant synthetic biology as a robust foundation for sustainable biomanufacturing and production of functional biomolecules.},
}

@article {pmid41229344,
year = {2025},
author = {Sures, K and Esser, SP and Bornemann, TLV and Moore, CJ and Soares, AR and Plewka, J and Figueroa-Gonzalez, PA and Ruff, SE and Moraru, C and Probst, AJ},
title = {Acquisition of Spacers from Foreign Prokaryotic Genomes by CRISPR-Cas Systems in Natural Environments.},
journal = {Genome biology and evolution},
volume = {17},
number = {11},
pages = {},
pmid = {41229344},
issn = {1759-6653},
mesh = {*CRISPR-Cas Systems ; *Genome, Archaeal ; *Archaea/genetics ; Genome, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics ; Metagenome ; Evolution, Molecular ; *DNA, Intergenic/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems of bacteria and archaea provide immunities against mobile genetic elements, like viruses. In addition, protospacer analyses revealed a very specific acquisition of CRISPR spacers derived from genomes of related species or from closely interacting episymbiont genomes as recently shown for subsurface archaea. However, the origin of most of the spacers that can be found in CRISPR-Cas systems from natural environments has not been deciphered. Here, by analyzing CRISPR-Cas systems of metagenome-assembled genomes (MAGs) from two subsurface environments spanning more than 1 Tb of sequencing data, we show that a substantial proportion of CRISPR spacers are acquired from DNA of other prokaryotes inhabiting the same environment. As such, we found that the number of respective spacers can be up to three times higher than the number of self-targeting spacers. Statistical analyses demonstrated that the acquisition of CRISPR spacers from other prokaryotic genomes is partly explained by the relative abundance of the MAG containing the protospacer, as well as by other factors, such as the total number of CRISPR arrays present in a MAG with the respective spacers. Further, we found that spacer acquisition from foreign prokaryotic DNA occurs in almost all types of CRISPR-Cas systems, but shows preferences for subtypes of CRISPR-Cas systems that differ across the investigated ecosystems. Taken together, our results shed new light on the diversity of CRISPR spacers in natural microbial communities and provide an explanation for some of the many unmatched spacers in public databases.},
}

*CRISPR-Cas Systems
*Genome, Archaeal
*Archaea/genetics
Genome, Bacterial
*Clustered Regularly Interspaced Short Palindromic Repeats
*Bacteria/genetics
Metagenome
Evolution, Molecular
*DNA, Intergenic/genetics

@article {pmid41229123,
year = {2025},
author = {Ma, Y and Wei, W and Yang, Z and Zhou, Y and Dong, T and Wang, T and Xia, X and Ma, Y and Zhou, M and Gao, Y and Yu, B and Wang, C and Ruan, L and Hong, K and Gu, C},
title = {Exosomes as Nonviral Carrier for Targeted Delivery of CRISPR/Cas12a for Therapeutic HIV-1Proviral DNA Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.11.012},
pmid = {41229123},
issn = {1525-0024},
abstract = {Current strategies to treat HIV infection including traditional cART and immunotherapy can effectively suppress viral replication but unable to eliminate the latent viral reservoir, particularly within circulating immune cells. Although genome editing by CRISPR-Cas provides a promising cure for HIV-1, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we developed an exosome-mediated targeted CRISPR-Cas12a delivery system (EMT-Cas12a), an engineered exosome system enabling targeted delivery of mRNA of Cas12a and crRNAs to CD4+ T cells. The EMT-Cas12a system uniquely optimizes cell-specific targeting, CRISPR-Cas12a expression, crRNAs maturation, nuclear entry efficiency, accuracy cleavage with major Delins and achieving dramatically HIV suppression in both cellular and humanized mouse models. Compared to single-crRNA approaches, the multiple crRNA arrays strategy demonstrates enhanced antiviral efficacy in HIV -infected mouse model, ex vivo expanded PBMCs from HIV+ subjects and especially in vitro cell line without detectable HIV DNA. Critically, the system exhibits no detectable off-target effects and restores CD4+ T cell counts in vivo and ex vivo PBMCs, indicating its dual therapeutic potential for viral clearance and immune reconstitution. Altogether, in vitro and in vivo excision of HIV-1 proviral DNA can be achieved via EMT-Cas12a delivery, which could advance efforts toward human clinical trials.},
}

@article {pmid41139833,
year = {2025},
author = {Neo, DM and Ben-Zion, I and Bagnall, J and Solomon, MY and Bond, AN and Gath, E and Zhang, S and Shoresh, N and Gomez, J and Hung, DT},
title = {A Multiplexed, Target-Based Phenotypic Screening Platform Using CRISPR Interference in Mycobacterium abscessus.},
journal = {ACS infectious diseases},
volume = {11},
number = {11},
pages = {3263-3275},
doi = {10.1021/acsinfecdis.5c00623},
pmid = {41139833},
issn = {2373-8227},
mesh = {*Mycobacterium abscessus/genetics/drug effects ; Microbial Sensitivity Tests ; Mycobacterium Infections, Nontuberculous/microbiology/drug therapy ; *Anti-Bacterial Agents/pharmacology ; Drug Discovery/methods ; Bacterial Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Phenotype ; Humans ; },
abstract = {The rise of difficult-to-treat Mycobacterium abscessus infections presents a growing clinical challenge due to the immense arsenal of intrinsic, inducible and acquired antibiotic resistance mechanisms that render many existing antibiotics ineffective against this pathogen. Moreover, the limited success in discovery of novel compounds that inhibit novel pathways underscores the need for innovative drug discovery strategies. Here, we report a strategic advancement in PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), which is an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of bacterial mutants, each depleted of a different essential protein target, to identify whole-cell active compounds with high sensitivity. Applying this modified strategy to M. abscessus, in contrast to previously described versions of PROSPECT which utilized protein degradation or promoter replacement strategies for generating engineered hypomorphic strains, here we leveraged CRISPR interference (CRISPRi) to more efficiently generate mutants each depleted of a different essential gene involved in cell wall synthesis or located at the bacterial surface. We applied this platform to perform a pooled PROSPECT pilot screen of a library of 782 compounds using CRISPRi guides as mutant barcodes. We identified a range of active hits, including compounds targeting InhA, a well-known mycobacterial target but under-explored in the M. abscessus space. The unexpected susceptibility to isoniazid, traditionally considered to be ineffective in M. abscessus, suggested a complex interplay of several intrinsic resistance mechanisms. While further complementary efforts will be needed to change the landscape of therapeutic options for M. abscessus, we propose that PROSPECT with CRISPRi engineering provides an increasingly accessible, high-throughput target-based phenotypic screening platform and thus represents an important step toward accelerating early stage drug discovery.},
}

*Mycobacterium abscessus/genetics/drug effects
Microbial Sensitivity Tests
Mycobacterium Infections, Nontuberculous/microbiology/drug therapy
*Anti-Bacterial Agents/pharmacology
Drug Discovery/methods
Bacterial Proteins/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
*CRISPR-Cas Systems
Phenotype
Humans

@article {pmid41086891,
year = {2025},
author = {Sun, J and Huang, Y and Bai, J and Wang, J and Langford, PR and Zhang, Y and Li, G},
title = {A CRISPR/Cas12a-based DNAzyme chemiluminescence platform for rapid detection of all Streptococcus suis and individually S. suis serotypes 7 and 9.},
journal = {International journal of biological macromolecules},
volume = {330},
number = {Pt 4},
pages = {148280},
doi = {10.1016/j.ijbiomac.2025.148280},
pmid = {41086891},
issn = {1879-0003},
mesh = {*Streptococcus suis/genetics/isolation & purification/classification ; *DNA, Catalytic/metabolism/genetics ; Serogroup ; *CRISPR-Cas Systems/genetics ; *Luminescent Measurements/methods ; Swine ; Animals ; Biosensing Techniques/methods ; Luminescence ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Streptococcus suis is a zoonotic pathogen that can infect pigs and humans with causing meningitis, sepsis, endocarditis, and arthritis. S. suis serotypes 7 and 9 cause substantial economic losses to the swine industry and pose a major threat to public health, thus, accurate and rapid detection is important for the prevention and control of epidemic disease. In this study, we developed a platform, combining recombinase polymerase amplification (RPA) with a CRISPR/Cas12a detection system to rapidly detect all S. suis serotypes and individually differentiates serotypes 7 and 9. This was achieved by targeting recN of S. suis and serotype-specific cpsH genes of serotypes 7 and 9, respectively. Both fluorescence and G4 DNAzyme chemiluminescence visualization biosensing methods had high specificity and sensitivity, no cross-reaction was found with common pig pathogens, closely related Streptococcus spp., or other S. suis serotypes. Compared to traditional identification techniques, these two methods are rapid and convenient. Notably, the G4 DNAzyme chemiluminescence method provides a clear, direct visual interpretation of results without the need for specialized equipment, which is particularly advantageous for point-of-care testing. Thus, this platform has the potential to significantly enhance diagnostic capabilities and ultimately benefit both animal and public health.},
}

*Streptococcus suis/genetics/isolation & purification/classification
*DNA, Catalytic/metabolism/genetics
Serogroup
*CRISPR-Cas Systems/genetics
*Luminescent Measurements/methods
Swine
Animals
Biosensing Techniques/methods
Luminescence
*Endodeoxyribonucleases/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40865049,
year = {2025},
author = {Shang, Y and Li, S and Xu, T and Sun, Y and Li, F},
title = {An Optimized Platform Overcomes Excessive Tumor Immune Rejection Induced by the CRISPR/Cas9 Lentiviral System.},
journal = {Cancer research},
volume = {85},
number = {22},
pages = {4315-4328},
doi = {10.1158/0008-5472.CAN-25-0809},
pmid = {40865049},
issn = {1538-7445},
support = {2022YFA1103900//National Natural Science Foundation of China (NSFC)/ ; 82372794//National Natural Science Foundation of China (NSFC)/ ; 82172744//National Natural Science Foundation of China (NSFC)/ ; GZB20240544//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 2024M752432//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 24ZR1469100//Natural Science Foundation of Shanghai Municipality ()/ ; //Shanghai Municipal Education Commission ()/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; *Lentivirus/genetics ; Mice ; Humans ; Gene Knockout Techniques/methods ; Integrases/genetics ; *Neoplasms/immunology/genetics/therapy ; Genetic Vectors/genetics ; Adenoviridae/genetics ; Cell Line, Tumor ; Female ; },
abstract = {UNLABELLED: The clustered regularly interspaced short palindromic repeats/Cas9 lentiviral system has emerged as a powerful tool for gene knockout in cancer immunology research. However, the persistent expression of exogenous elements, such as Cas9 and resistance markers, often triggers excessive tumor immune rejection. This can lead to prolonged experimental timelines, increased data variability, biased outcomes, and even experimental failures. To address this challenge, several strategies have been investigated that offer partial solutions, but they have failed so far to comprehensively resolve the issue. In response, we developed the v2-Blast-lox2272 (VL)-adenovirus expressing Cre recombinase (AdCre) system, a strategy that enables efficient excision of exogenous expression elements following gene knockout. The VL-AdCre system effectively reduced tumor immune rejection in allograft models, streamlined experimental workflows, and improved the reliability of research outcomes. Overall, this optimized clustered regularly interspaced short palindromic repeats/Cas9 lentiviral gene knockout system offers a robust and practical solution for studying gene functions in vivo and advancing immunotherapeutic strategies.

SIGNIFICANCE: The VL-AdCre system enables effective removal of exogenous components following gene knockout, thus avoiding tumor immune rejection and enhancing the accuracy and reproducibility of in vivo cancer immunology research.},
}

*CRISPR-Cas Systems
Animals
*Lentivirus/genetics
Mice
Humans
Gene Knockout Techniques/methods
Integrases/genetics
*Neoplasms/immunology/genetics/therapy
Genetic Vectors/genetics
Adenoviridae/genetics
Cell Line, Tumor
Female

@article {pmid41226739,
year = {2025},
author = {Arana, ÁJ and Veiga-Rua, S and Cora, D and Gónzalez-Gómez, MA and Seijas, A and Carballeda, M and Polo, D and Cuesta, A and Piñeiro, Y and Rivas, J and Novo, M and Al-Soufi, W and Martínez, P and Sánchez, L and Robledo, D},
title = {Comparative Analysis of CRISPR/Cas9 Delivery Methods in Marine Teleost Cell Lines.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110703},
pmid = {41226739},
issn = {1422-0067},
support = {CRISPR-NANOXEN//Universidade de Santiago de Compostela/ ; Optimizing CRISPR/Cas9 genome editing to improve disease resistance in aquaculture//Fundación Caixa Rural Galega Tomás Notario Vacas/ ; investment line nº 1 and component number 17, which includes the Complementary RTDI Plan for Marine Science//Spain's Recovery and Resilience Plan/ ; No 101076432 (FishTRIM)//European Union ERC Starting Grant programme 2022/ ; (BBS/E/20002172, BBS/E/D/30002275, BBS/E/D/10002070 and BBS/E/RL/230002A)//Oportunius programme of the Axencia Galega the Innovación (GAIN, Xunta de Galicia)/ ; RYC2023-044793-I//Ministerio de Ciencia, Innovación y Universidades/ ; 10.13039/501100011033//Agencia Estatal de Investigación/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Line ; Electroporation/methods ; *Fishes/genetics ; Transfection/methods ; *Gene Transfer Techniques ; },
abstract = {Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies-electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)-for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific genomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate editing (~25%) in DLB-1 but yielded only minimal editing in SaB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluorescence correlation spectroscopy suggested that nuclear localization and Cas9 aggregation may influence editing success, highlighting the importance of intracellular trafficking in CRISPR/Cas9 delivery. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquaculture.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Cell Line
Electroporation/methods
*Fishes/genetics
Transfection/methods
*Gene Transfer Techniques

@article {pmid41226719,
year = {2025},
author = {Amiri, S and Adibzadeh, S and Khazaei Monfared, Y and Kaboli, S and Arashkia, A and Barkhordari, F and Mahmoudian, M and Kheirandish, MH and Trotta, F and Davami, F},
title = {Efficient Delivery of CRISPR-Cas9 RNP Complexes with Cyclodextrin-Based Nanosponges for Enhanced Genome Editing: TILD-CRISPR Integration.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110682},
pmid = {41226719},
issn = {1422-0067},
support = {BD-66002183//Pasteur Institute of Iran/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Cyclodextrins/chemistry ; CHO Cells ; Cricetulus ; *Ribonucleoproteins/genetics ; Green Fluorescent Proteins/genetics ; *Nanostructures/chemistry ; },
abstract = {The CRISPR-Cas9 system has transformed biomedical research by enabling precise genetic modifications. However, efficient delivery of CRISPR components remains a major hurdle for therapeutic applications. To address this, we employed a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) system to deliver an integrating GFP gene using the TILD-CRISPR method, which couples donor DNA linearization with RNP complexes. The physicochemical properties, loading efficiency, and cellular uptake of RNP with Ppoly were studied. After transfection, antibiotic selection and single-cell cloning were performed. Junction PCR was then performed on the isolated clones, and we compared the knock-in efficiency of Ppoly with that of the commercial CRISPRMAX™ reagent (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The results demonstrate the encapsulation efficiency of over 90% for RNP and Ppoly, and cell viability remaining above 80%, reflecting the minimal toxicity of this approach. These attributes facilitated successful GFP gene integration using the TILD-CRISPR with RNP delivered via cyclodextrin-based nanosponges. The present method achieved a remarkable 50% integration efficiency in CHO-K1 cells, significantly outperforming the 14% observed with CRISPRMAX™ while maintaining lower cytotoxicity. This study highlights a promising platform for precise and efficient genome editing, with strong potential for therapeutic and regenerative medicine applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
*Cyclodextrins/chemistry
CHO Cells
Cricetulus
*Ribonucleoproteins/genetics
Green Fluorescent Proteins/genetics
*Nanostructures/chemistry