@article {pmid41805199,
year = {2026},
author = {Marín-Sanz, M and Berlanga-Torres, JA and Guzmán-López, MH and Sánchez-León, S and Vallés, MP and Castillo, AM and Barro, F},
title = {Engineering ultra-low-gliadin wheat for celiac disease using an integrated RNAi, CRISPR, and doubled haploid strategy.},
journal = {Journal of experimental botany},
volume = {77},
number = {14},
pages = {4417-4434},
doi = {10.1093/jxb/erag131},
pmid = {41805199},
issn = {1460-2431},
support = {QUAL21_023 IAS//Junta de Andalucı́a/ ; //Conexión TRIGO, grant number 202490E049)./ ; },
mesh = {*Triticum/genetics/metabolism ; *Gliadin/genetics/metabolism ; *RNA Interference ; Haploidy ; *Celiac Disease/genetics ; *Gene Editing ; CRISPR-Cas Systems ; Glutens ; },
abstract = {The growing prevalence of gluten-related disorders in humans has driven the development of wheat varieties with reduced immunogenic gluten. This study aimed to integrate RNA interference (RNAi) and CRISPR genome editing within a doubled haploid (DH) platform to overcome challenges of gene redundancy and polyploidy in wheat gliadins. We generated DH lines from crosses between RNAi and CRISPR lines and elite wheat cultivars, enabling stable fixation of multiple genetic modifications in a single generation. Deep sequencing analysis of α-gliadin amplicons was conducted using a custom bioinformatics pipeline optimized for complex, repetitive gene families. Gluten protein profiles were evaluated using RP-HPLC and R5 monoclonal antibody. Several DH lines presented >70% reduction in immunogenic epitopes in α-gliadins, with lines outperforming both parents. Editing frequency was influenced by sgRNA efficiency and parental background. Silencing and editing combined led to nearly depleted gliadins in some lines, often with compensatory increases in other storage proteins linked to bread-making quality, such as high-molecular-weight glutenin subunits. Kernel and specific weight traits were largely maintained. This work demonstrates that combining RNAi and CRISPR in a DH platform enables efficient, heritable reduction of immunogenic gluten, providing a viable strategy for breeding wheat lines safer for individuals with gluten-related disorders.},
}

*Triticum/genetics/metabolism
*Gliadin/genetics/metabolism
*RNA Interference
Haploidy
*Celiac Disease/genetics
*Gene Editing
CRISPR-Cas Systems
Glutens

@article {pmid41928454,
year = {2026},
author = {Tietz, SM and Brenner, K and Moyo, T and Young, PR and Vivier, MA},
title = {Optimized protocol for efficient generation, confirmation, transformation, and CRISPR editing of grapevine hairy roots.},
journal = {Journal of experimental botany},
volume = {77},
number = {14},
pages = {4352-4374},
doi = {10.1093/jxb/erag165},
pmid = {41928454},
issn = {1460-2431},
support = {//South Africa Wine/ ; UID120460//National Research Foundation (NRF)/ ; },
mesh = {*Vitis/genetics/growth & development ; *Plant Roots/genetics/growth & development ; *Transformation, Genetic ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; },
abstract = {Hairy root cultures (HRCs) are powerful tools in plant biotechnology but show variable establishment efficiencies, limiting broader applications. Here, we present a standardized and optimized reference methodology for the routine generation, multiplication, and maintenance of HRCs across diverse grapevine genotypes. Our workflow evaluated three Rhizobium strains, seven grapevine cultivars (three Vitis vinifera cultivars; four Vitis rootstock hybrids), multiple explant types, infection protocols, co-cultivation times, growth media types, and anti-browning agents. The resulting protocol was effective for all grapevine genotypes and, with minor adjustments, also yielded HRCs from two other important South African plant species, namely Sutherlandia frutescens and Aspalathus linearis. Useful molecular tools were developed for transformation and selection of HRCs, including universal multiplex primers for confirmation of transformation, tested antibiotic resistance markers (kanamycin and hygromycin), and fluorescent reporters (DsRed and eyGFPuv), with DsRed found to be particularly versatile. To test the system, we overexpressed the VviMYBA1 transcription factor gene, leading to increased anthocyanin accumulation and red pigmentation in HRCs. Additionally, we achieved CRISPR/Cas9 editing of the VviPUB19 gene, the first report of CRISPR-edited grapevine HRCs. Gene editing combined with HRCs can facilitate rapid gene function studies, offering an efficient alternative or pre-screening system to whole-plant transformations, that could support advanced functional genomics and biotechnological applications in grapevine.},
}

*Vitis/genetics/growth & development
*Plant Roots/genetics/growth & development
*Transformation, Genetic
*Gene Editing/methods
*CRISPR-Cas Systems
Plants, Genetically Modified/genetics

@article {pmid42215861,
year = {2026},
author = {Liu, F and Zhang, X and Yang, Y},
title = {A comparison between CARLIN and DNA Typewriter in CRISPR-mediated lineage tracing.},
journal = {BMC bioinformatics},
volume = {27},
number = {1},
pages = {},
pmid = {42215861},
issn = {1471-2105},
support = {NCI U01-CA253553/CA/NCI NIH HHS/United States ; R01-CA251950/NH/NIH HHS/United States ; NCI U01-CA253553/CA/NCI NIH HHS/United States ; R01-CA251950/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *DNA/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Cell Lineage/genetics ; },
abstract = {BACKGROUND: CARLIN and DNA Typewriter are two major breakthroughs in CRISPR-based lineage tracing technology. It is essential to understand the potential and performance of these methods in lineage tracing, which provides important guidance on experimental design.

RESULTS: In this study, we systematically compare these two strategies using a unified stochastic simulation framework with known ground-truth lineages. By explicitly modeling CRISPR editing dynamics, barcode evolution, and cell division processes, the framework enables quantitative benchmarking of lineage reconstruction accuracy across diverse experimental parameter regimes. Both methods are evaluated using multiple accuracy metrics, including Robinson-Foulds accuracy and triplet accuracy, allowing a comprehensive assessment of lineage reconstruction performance under various editing probabilities, sampling depths, and lineage lengths.

CONCLUSIONS: DNA Typewriter consistently outperforms CARLIN in lineage reconstruction accuracy when sufficient numbers of recording targets are used, particularly in more cell divisions. Sequential and ordered recording in DNA Typewriter substantially reduces ambiguity in lineage inference compared to unordered CRISPR barcode editing. CARLIN's lineage-recording potential exhausts rapidly under continuous induction, limiting its effectiveness in long-term lineage tracing. Triplet accuracy provides a more permissive and informative metric than Robinson-Foulds accuracy, especially under partial sampling scenarios.},
}

*CRISPR-Cas Systems
*DNA/genetics
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Cell Lineage/genetics

@article {pmid42233593,
year = {2026},
author = {Zhao, W and Zheng, Z and Li, R and Xie, H and Yu, H and Zhang, Y and Wu, Y and Yang, Y and Zhang, Z and Gao, H and Li, Y and Zhang, K},
title = {In Situ Amplified Mutational mRNA Imaging Using a Spatially Confined CRISPR Nanoplatform.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {65},
number = {31},
pages = {e7088080},
pmid = {42233593},
issn = {1521-3773},
support = {22377110//National Natural Science Foundation of China/ ; 82402749//National Natural Science Foundation of China/ ; U23A20531//National Natural Science Foundation of China/ ; 22122409//National Natural Science Foundation of China/ ; 252300421073//Natural Science Foundation of Henan Province/ ; 252102311025//Science and Technology Department of Henan Province/ ; 2025SGAQZ-MS-03//State Key Laboratory of Metabolic Dysregulation & Prevention and Treatment of Esophageal Cancer/ ; 261111313300//Henan Provincial Key Research and Development Program/ ; },
mesh = {*RNA, Messenger/genetics/analysis ; Humans ; Mutation ; *CRISPR-Cas Systems/genetics ; },
abstract = {Highly sensitive spatial analysis of RNA mutations is essential for understanding cellular heterogeneity and disease mechanisms. Herein, we developed an integrated CRISPR/Cas13a-based nanoprobe system for rapid detection of RNA in tissue sections (Integrated CRISPR/Cas13a-based RNA Rapid Detection, InCasRD). Unlike conventional "always-on" probes that rely on accumulated probe hybridization, InCasRD leverages the trans-cleavage activity of Cas13a to achieve spatially confined signal amplification and a high signal-to-background ratio (SBR). Using InCasRD, we achieved imaging of multiple target RNAs in tumor cells within 0.5 h of incubation, including mRNA (survivin), microRNA (miR-21), and circular RNA (circ1785). Furthermore, the engineered InCasRD system enabled mapping of RNA mutations, such as the EGFR L858R and ovarian tumor domain (OTUD) single-nucleotide variant (SNV, 23439980 G>T), in tumor tissue sections, thereby facilitating clear tumor boundary delineation. Collectively, InCasRD is a powerful, one-step tool for in situ RNA analysis with potential for diagnosis and precision medicine.},
}

*RNA, Messenger/genetics/analysis
Humans
Mutation
*CRISPR-Cas Systems/genetics

@article {pmid42335403,
year = {2026},
author = {Luo, H and Gao, J and Huang, X and Fang, Y and Huang, T and Xia, Y and Yu, Z and Cao, C and Xiong, Z},
title = {Thermo-Responsive Living Microspheroids Enable a Regenerative Living Disk-Drive System for DNA Data Storage.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {42},
pages = {e73806},
pmid = {42335403},
issn = {1521-4095},
support = {53330200321//Tsinghua University/ ; },
mesh = {*DNA/chemistry/genetics ; *Temperature ; Plasmids/genetics ; *Information Storage and Retrieval/methods ; CRISPR-Cas Systems ; },
abstract = {DNA offers exceptional information density and long-term stability, yet its practical deployment is limited by destructive readout and the absence of a reusable, physically addressable architecture that connects nanoscale molecular information with macroscale device-level data organization. Here, we present a regenerative Living Disk-Drive system based on thermo-responsive engineered living memory microspheroids (ELMMs), in which data-encoded bacteria are encapsulated as discrete, file-level living storage units. Each ELMM contains a clonal bacterial population carrying both an information plasmid, which encodes 26 × 26 pixel icon payloads and one- to three-color intracellular fluorescent retrieval indices, and a help plasmid that enables CRISPR-Cas12a/λ-Red rewriting of the data sequence and retrieval tag. A lyophilized ELMM database forms the Living Disk, which is coupled to an Optical Retriever and desktop-scale Living Drive for closed-loop retrieval, regeneration, and database replenishment. Released bacteria regrow for downstream readout or rewriting, while a fraction is re-encapsulated into new ELMMs. The tested system retains retrieval, regrowth, and sequence recovery after four months of ambient dry storage and 13 lyophilization-rehydration cycles. Model-based performance estimates are reported only as theoretical architecture-level bounds. These results establish an experimentally bounded yet extensible architecture for physically manageable and regenerative DNA memory.},
}

*DNA/chemistry/genetics
*Temperature
Plasmids/genetics
*Information Storage and Retrieval/methods
CRISPR-Cas Systems

@article {pmid42372505,
year = {2026},
author = {Wang, R and Pan, Q and Huang, Y and Dai, W and Ping, Y and Jin, Q},
title = {Versatile hollow Ca[2+]-phenolic nanoparticles for intracellular delivery of diverse bioactive molecules and CRISPR-Cas9 genome editing.},
journal = {Biomaterials},
volume = {335},
number = {},
pages = {124407},
doi = {10.1016/j.biomaterials.2026.124407},
pmid = {42372505},
issn = {1878-5905},
mesh = {Humans ; *Nanoparticles/chemistry/ultrastructure ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Calcium/chemistry ; *Phenols/chemistry ; },
abstract = {The development of versatile nanoplatforms capable of universally encapsulating diverse bioactive molecules holds significant promise in biomedicine. In this study, size-tunable hollow Ca[2+]-tannic acid (TA) nanoparticles (HCT NPs) are synthesized as universally applicable drug nanocarriers by simply adding TA into amorphous calcium carbonate nanoparticles. The formation of HCT NPs is identified as a surface-protected self-etching process. A wide range of hydrophobic and hydrophilic small-molecule drugs, metal ions, and biomacromolecules including proteins and nucleic acids can be encapsulated in HCT NPs for efficient intracellular delivery. HCT NPs show rapid and efficient endosomal escape, which is crucial for maintaining the bioactivity of biomacromolecules. Remarkably, a wide array of cargo proteins, spanning different molecular weights and isoelectric points can be delivered into the cytosol by HCT NPs without compromising their bioactivities. The therapeutic potential of HCT NPs for intracellular cargo delivery is exemplified by cytosolic delivery of Cas9 plasmids and Cas9 ribonucleoprotein (RNP) for CRISPR-Cas9 genome editing both in vitro and in vivo. The facile and ultrafast synthesis, versatile cargo encapsulation capabilities, efficient cell uptake and endosomal escape, and excellent biocompatibility make HCT NPs a prominent candidate for intracellular delivery of diverse bioactive molecules, particularly in therapeutic applications such as genome editing.},
}

Humans
*Nanoparticles/chemistry/ultrastructure
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
*Calcium/chemistry
*Phenols/chemistry

@article {pmid42390232,
year = {2026},
author = {Duvenage, L and Chetty, A and Thomson, DD and Ballou, ER and Govender, NP and Rappleye, CA and Hoving, JC},
title = {Tools for genetic manipulation of the endemic fungal pathogen Emergomyces africanus and application of a fluorescent reporter strain in infection models.},
journal = {mSphere},
volume = {11},
number = {7},
pages = {e0018026},
pmid = {42390232},
issn = {2379-5042},
support = {209293/Z/17/Z/WT_/Wellcome Trust/United Kingdom ; 310933/Z/24/Z/WT_/Wellcome Trust/United Kingdom ; AI148561/NH/NIH HHS/United States ; },
mesh = {Animals ; Green Fluorescent Proteins/genetics ; *Genes, Reporter ; Mice ; Plasmids/genetics ; Macrophages/microbiology ; Disease Models, Animal ; *Mycoses/microbiology ; *Ascomycota/genetics/pathogenicity ; CRISPR-Cas Systems ; },
abstract = {UNLABELLED: Emergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa, which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterization of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of Histoplasma capsulatum were adapted for use in E. africanus. Agrobacterium-mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall α(1,3)-glucan by targeting the transcripts of the α-glucan synthase (AGS1). An episomal CRISPR/Cas9 system was evaluated for E. africanus, which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus.

IMPORTANCE: Emergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognized in recent years, and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (i) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (ii) methods for knocking down gene expression in E. africanus, and (iii) the permanent disruption of genes through CRISPR/Cas9 gene editing.},
}

Animals
Green Fluorescent Proteins/genetics
*Genes, Reporter
Mice
Plasmids/genetics
Macrophages/microbiology
Disease Models, Animal
*Mycoses/microbiology
*Ascomycota/genetics/pathogenicity
CRISPR-Cas Systems

@article {pmid42492264,
year = {2026},
author = {Shen, H and Nourmohammadi, S and Zhou, Y and Harata-Lee, Y and Qu, Z and Wang, W and Yool, AJ and Adelson, DL},
title = {Target pathway validation for Compound Kushen Injection active against cancer cells.},
journal = {Phytomedicine : international journal of phytotherapy and phytopharmacology},
volume = {159},
number = {},
pages = {158598},
doi = {10.1016/j.phymed.2026.158598},
pmid = {42492264},
issn = {1618-095X},
abstract = {BACKGROUND: Traditional Chinese medicines are based on complex mixtures of natural products and their multi-target mechanism of action. Therefore the discovery and validation of targets and mechanisms have always been challenging. In previous studies, using transcriptomic methods and Compound Kushen Injection (CKI) as a model drug, we identified multiple pathways and candidate target genes for validation, through which CKI exerts its pharmacological effects.

PURPOSE: This study aimed to demonstrate the involvement of multiple genetic targets in different pharmacological activities for natural products in Compound Kushen Injection.

METHODS: In this study, we selected eight key genes from four candidate pathways and used CRISPR/CAS technology to knock out these genes in four cell lines, validating their role in CKI activity.

RESULTS: Although the sensitivity of different cell lines to gene knockout varied, overall, it led to reductions in various cellular activities. After the addition of CKI, we observed that, except for the minor impact of CDKN1A gene knockout on the effect of CKI, knocking out the other genes significantly affected the pharmacological efficacy of CKI in different assays. Among them, knockout of MYD88 and NFkB genes enhanced the efficacy of CKI. At the same time, we found that the genes IL24 and CYP1B1 play a crucial role in CKI inhibition of tumour cell migration, and the CYP1A1 gene is critical for the cell cycle arrest induced by CKI.

CONCLUSIONS: These findings validate the results of our previous transcriptomic analysis and further demonstrate the complexity of pharmacological mechanisms of multi-target synergistic action of natural product mixtures.},
}

@article {pmid42492655,
year = {2026},
author = {Patel, S and Panchal, J and Patel, A and Chauhan, H and Sharma, K and Sabara, P and Vahora, S and Shrimali, M and Shekh, S and Thakor, A and Mohapatra, S and Hati, S},
title = {Unravelling the Resistome of Carbapenem-Resistant E. coli from Bovine Mastitis via Whole-Genome Sequencing.},
journal = {Veterinary journal (London, England : 1997)},
volume = {},
number = {},
pages = {106792},
doi = {10.1016/j.tvjl.2026.106792},
pmid = {42492655},
issn = {1532-2971},
abstract = {Carbapenem-resistant Escherichia coli (CREC) poses a growing threat to public health, particularly when emerging from animal reservoirs such as dairy cattle. This study aimed to characterize CREC isolates recovered from bovine mastitis cases in Gujarat, India, using a combination of phenotypic antibiotic susceptibility testing and whole-genome sequencing (WGS). Out of 130 confirmed E. coli isolates from 790 mastitic milk samples, 33 (25.38%) were resistant to imipenem. Of these, nine exhibited multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan-drug-resistant (PDR) phenotypes. WGS was performed on four representative isolates (SKN144, SKN685, SKN687, SKN926), revealing genome sizes ranging from 4.7 to 5.4Mb and GC content between 50.4% and 50.8%. Annotation identified numerous resistance determinants, including carbapenemase genes (blaNDM, blaOXA-48, blaTEM, blaCMY, blaCTX-M), aminoglycoside-modifying enzymes (APH, AAC), macrolide resistance genes (mphA, ermB), and multiple efflux pump systems (AcrAB-TolC, EmrAB, MdtEF-TolC). Functional genes associated with replication, repair, stress response, and mobile genetic elements (integrases, transposases, CRISPR-Cas) were also detected, indicating high genomic adaptability. Phenotypic testing revealed alarming resistance to key antimicrobials, including ampicillin (56.15%), amikacin (55.38%), ceftazidime (53.08%), and colistin (79.23%, including intermediate strains). Subsystem analysis highlighted metabolic versatility, defence mechanisms, and virulence-associated pathways. Phylogenetic analysis indicated that all isolates clustered within the same clade, suggesting possible clonal dissemination within the bovine population. The presence of CRISPR-Cas elements, integrases, and transposases suggests ongoing horizontal gene transfer and genome plasticity. These findings underscore the alarming prevalence of CREC in dairy environments and the urgent need for enhanced AMR surveillance, prudent antibiotic stewardship, and implementation of a One Health approach to prevent zoonotic transmission. This study contributes valuable genomic insights into livestock-associated CREC and highlights their close genomic parallels with high-risk human clinical clones.},
}

@article {pmid42493436,
year = {2026},
author = {Wang, Y and Song, H and DU, Y and Xu, T},
title = {[CRISPR-Cas9 activation screening identifies candidate chemokine regulators of ter-tiary lymphoid structure formation in bladder cancer].},
journal = {Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences},
volume = {58},
number = {4},
pages = {707-715},
pmid = {42493436},
issn = {1671-167X},
mesh = {Animals ; Mice ; *Urinary Bladder Neoplasms/genetics/pathology/immunology ; Humans ; *Chemokines/genetics/metabolism ; Cell Line, Tumor ; Mice, Inbred C57BL ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Chemokine CCL20/genetics ; },
abstract = {OBJECTIVE: To identify the cytokine genes influencing the formation of tertiary lymphoid structures (TLS) through CRISPR-Cas9 library screening, and to discover potential key regulatory molecules, providing new targets for enhancing the efficacy of bladder cancer immunotherapy.

METHODS: Based on a mouse whole-genome library, 44 chemokine-related genes were identified, and an single-guide RNA (sgRNA) library targeting these genes was designed and constructed, with three sgRNAs assigned to each gene. Using a lentiviral packaging system, the library plasmids were used to transfect HEK293T cells to generate a lentiviral library, which was then used to infect the mouse bladder cancer cell line MB49. Purinomycin selection was performed to obtain the MB49-mCherry cell line stably over-expressing chemokines. The cells were inoculated into the peritoneal cavity of C57BL/6 mice to establish a bladder cancer xenograft model, and tumor growth was monitored. Three weeks later, tumor tissue was excised, genomic DNA was extracted for high-throughput sequencing, and sgRNA enrichment was analyzed to screen for differentially expressed cytokine genes. Concurrently, immunohistochemical staining was performed to detect TLS markers CD20 and CD3, and the number, distribution, and maturity of TLS were assessed. The selected candidate genes were validated individually in vivo to further confirm their impact on TLS formation.

RESULTS: We successfully constructed a cytokine gene library containing 132 sgRNAs, covering 44 chemokine genes. Following lentiviral infection, we obtained the MB49-mCherry cell line, which stably expressed the library, and isolated dead Cas9-positive monoclonal cell lines via flow cytometry to ensure the homogeneity and reproducibility of subsequent experiments. Intratumoral tumor experiments in mice revealed that the number of TLS cells in the experimental group was significantly higher than in the control group, primarily distributed at the tumor margins. High-throughput sequencing results showed that, compared with the control group, in the experimental group, Cxcl16 sgDNA was significantly enriched, while Ccl20 and Cx3lc1 sgDNA levels decreased compared with baseline (P < 0.05). Further validation of the individual roles of each factor via intraperitoneal injection revealed that the number of TLSs in tumors decreased in the group treated with the CX3CL1 chemokine, suggesting that CX3CL1 might negatively regulate TLS formation. Immunohistochemical results showed that in the CX3CL1-treated group, the aggregation of CD20-positive B cells and CD3-positive T cells in the tumor tissue was reduced, and the TLS structure was incomplete.

CONCLUSION: Through CRISPR-Cas9 library screening combined with in vivo validation, this study successfully identified CX3CL1 as a potential negative regulator of TLS formation in bladder cancer. High CX3CL1 expression was associated with a reduction in TLS numbers, suggesting that it might exert an inhibitory role in the immune microenvironment of bladder cancer. This finding provides new clues and research directions for understanding the molecular mechanisms of TLS formation in bladder cancer. However, whether CX3CL1 can serve as an immunotherapeutic target remains to be further validated through clinical specimen analysis, multidimensional mechanistic investigation, and immunotherapy response correlation studies.},
}

Animals
Mice
*Urinary Bladder Neoplasms/genetics/pathology/immunology
Humans
*Chemokines/genetics/metabolism
Cell Line, Tumor
Mice, Inbred C57BL
*CRISPR-Cas Systems/genetics
HEK293 Cells
Chemokine CCL20/genetics

@article {pmid42494101,
year = {2026},
author = {Liu, K and Ren, C and Liu, X and Qi, X and Liu, Y and Ma, S and Zhang, S and Wong, XY and Wang, X and Hu, T and Hu, C},
title = {Shared CRISPR arrays underpin type I-A/I-B coexistence.},
journal = {Protein & cell},
volume = {},
number = {},
pages = {},
doi = {10.1093/procel/pwag050},
pmid = {42494101},
issn = {1674-8018},
abstract = {CRISPR-Cas systems provide adaptive immunity in prokaryotes, yet how multiple CRISPR-Cas subtypes coexist and coordinate within a single genome remains unclear. Comparative genomic analysis revealed that nearly one-third of type I-A CRISPR-Cas3 systems are adjacent to a type I-B system, often sharing a single CRISPR array. Using Thermococcus siculi RG-20 (Tsi) as a model, we show that purified TsiCas6a and TsiCas6b independently recognize and cleave the shared pre-crRNA, producing mature crRNAs with comparable efficiency. Plasmid interference assays further demonstrated that crRNAs produced by either Cas6a or Cas6b enzyme could guide both type I-A and type I-B interference complexes. This interchangeability shows that crRNAs generated by either Cas6a or Cas6b can be loaded into, and function with, both type I-A and type I-B interference complexes. Structural modelling revealed distinct but complementary recognition strategies for Cas6a and Cas6b, and mutational analysis of their RNA-binding residues impaired pre-crRNA cleavage and abolished interference activity. Together, these results uncover a shared-array logic in which Cas6a/Cas6b-compatible processing routes a single pre-crRNA to multiple type I effectors-providing a potential mechanism for subtype co-existence, a plausible explanation for array-less (or "orphan") interference modules, and an evolutionary bet-hedging strategy that prevents Acrs from shutting down immunity wholesale.},
}

@article {pmid42494113,
year = {2026},
author = {Ohtomo, M and Takarabe, S and Namiki, T and Kawata, Y and Kaneko, R and Ozawa, M and Yamada, Y and Mori, H and Kageyama, A and Kamoshita, M and Terakawa, J and Ito, J},
title = {Generation and Characterization of a Rdh1-iCre Line to Study Uterine Glandular Biology.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {4},
pages = {e70068},
pmid = {42494113},
issn = {1526-968X},
support = {JP21K09512//Japan Society for the Promotion of Science/ ; JP24K01950//Japan Society for the Promotion of Science/ ; JP25KJ2187//Japan Society for the Promotion of Science/ ; JP22H04922//Japan Society for the Promotion of Science/ ; JP25K22429//Japan Society for the Promotion of Science/ ; //Azabu University/ ; },
mesh = {Animals ; Female ; *Uterus/metabolism ; Mice ; *Alcohol Oxidoreductases/genetics/metabolism ; *Integrases/genetics/metabolism ; Pregnancy ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {The uterus is an essential organ for fetal development in most mammals. Uterine glands, highly conserved structures in the mammalian uterus, play critical roles in the establishment and maintenance of pregnancy and have been implicated in the pathogenesis of uterine diseases, including endometrial cancer and endometriosis. Previous studies have shown that Retinol dehydrogenase 1 (Rdh1) is specifically expressed in the glandular epithelium (GE) from the onset of gland formation through adulthood. In this study, to develop a GE-specific Cre driver line, we generated Rdh1-iCre mice by introducing an improved Cre recombinase (iCre) into the Rdh1 locus using the CRISPR/Cas9 system. To evaluate the utility of this model, Rdh1-iCre mice were crossed with ROSA26-H2B-mCherry reporter mice, and Cre-dependent reporter expression was analyzed. Robust mCherry fluorescence was observed throughout the uterine glands at 2 weeks after birth, coinciding with the active elongation and branching of the GE. These results demonstrate that the Rdh1-iCre mouse line is a valuable and highly efficient tool for investigating the physiological roles of uterine glands during development and pregnancy, as well as their contribution to the progression of GE-derived uterine diseases.},
}

Animals
Female
*Uterus/metabolism
Mice
*Alcohol Oxidoreductases/genetics/metabolism
*Integrases/genetics/metabolism
Pregnancy
Mice, Transgenic
CRISPR-Cas Systems

@article {pmid42494486,
year = {2026},
author = {Wu, W and Wang, R and Li, J and Zhang, Z and Yao, W and Zhang, N and Xu, W},
title = {Engineering climate-resilient horticultural crops: advances in transcriptional regulation, genome editing, and synthetic networks.},
journal = {Horticulture research},
volume = {13},
number = {8},
pages = {uhag119},
pmid = {42494486},
issn = {2662-6810},
abstract = {Abiotic stresses-particularly cold, drought, and salinity-pose significant threats to the productivity and sustainability of horticultural crops. Recent studies have revealed conserved and species-specific regulatory mechanisms that allow plants to adapt dynamically to these environmental constraints. This review synthesizes advances in understanding key transcription factor families-such as CBF/DREB, NAC, MYB, WRKY, and bHLH-that orchestrate stress-responsive gene networks and modulate physiological processes, including osmotic regulation, antioxidant defense, and ionic homeostasis. We also discuss the emerging roles of chromatin remodeling, DNA methylation, histone modifications, and noncoding RNAs in conferring transcriptional plasticity and stress memory. Beyond endogenous pathways, we evaluate transgenic strategies, CRISPR/Cas-based genome editing, and synthetic gene circuits for engineering abiotic stress tolerance. Particular attention is given to trade-offs between growth and defense, challenges in horticultural crop transformation, and gaps in field translation. We further examine the regulatory role of secondary metabolites-such as flavonoids and salicylic acid-as biochemical interfaces between signal transduction and adaptive responses. Finally, we propose a forward-looking roadmap integrating multi-omics, ideotype design, and precision breeding toward climate-resilient horticultural systems.},
}

@article {pmid42494498,
year = {2026},
author = {Banik, I and Coppé, JP},
title = {CRISPRing through time: How cutting-edge technology is revolutionizing life sciences and medicine.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {3},
pages = {103003},
pmid = {42494498},
issn = {2162-2531},
abstract = {Given the plethora of emerging technologies, none have truly captured the minds as CRISPR. From the groundbreaking research, the ultimate battle of the prizes and patents to a number of books, the science of CRISPR continues to be significant in the biomedical field. For many decades now, the emergence of synthetic biology as an intervention to correct diseases has become the foundation of biomedical research. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genetic editing has become a common place for routine investigation of scientific hypotheses in pre-clinical settings. More recently, CRISPR-based diagnostic testing kits for SARS-CoV-2 have showcased a translational output. Furthermore, a technological landmark was achieved when the Food and Drug Administration (FDA) approved the first CRISPR-based gene therapy (exa-cel) to edit erythroid specific enhancer region of BCL11A in hematopoietic stem cells, introduced in patients suffering from sickle cell anemia to achieve durable remission. In this review, we provide a snapshot into the most important milestones along the journey of CRISPR from its discovery in bacteria to its usage in precision medicine. The intervention of machine learning tools has now intertwined complex biology with high-throughput scalable outputs. Given the vast amount of information on CRISPR, we try to pin down key take-home messages for scientists as well as non-scientist readers. This review article attempts to understand why and how CRISPR remains significant and seamlessly integrates in the emerging era of new technologies.},
}

@article {pmid42496566,
year = {2026},
author = {Krishna, TPA and Harikrishnan, D and Veena, M and Maharajan, T and James, M and Udhayakumar, M and Arockiam Jeyasundar, PGS and David, SJ and Dineshkumar, R and Rajan, R and Rathinapriya, P},
title = {Next-Generation Metabolic Engineering of Capsaicinoids Biosynthesis in Chilli Pepper: Bridging Genomic Insights to Biotechnological Applications.},
journal = {Biotech (Basel (Switzerland))},
volume = {15},
number = {3},
pages = {},
pmid = {42496566},
issn = {2673-6284},
abstract = {Chilli peppers (Capsicum species) have been widely used around the world because of their economic value and distinctive sensory characteristics. They contain abundant functional metabolites, especially a group of vanillylamide compounds belonging to the family of capsaicinoids, which have been exploited for medicinal, nutritional, agricultural, and cosmetic uses. The demand for capsaicinoid molecules is increasing day by day due to their high economic value and wide range of applications. Therefore, increasing bioactive metabolites, especially capsaicinoids in chilli peppers, is a major priority in the current scenario. Multi-omics approaches such as genomics, transcriptomics, proteomics, and metabolomics have substantially contributed to understanding the complex regulatory networks governing capsaicinoid biosynthesis. Key structural genes, transcription factors, and signaling pathways involved in the phenylpropanoid and branched-chain fatty acid pathways have been identified, providing valuable targets for metabolic engineering in chilli pepper. Despite these advances, the integration of genetic modification approaches for the targeted enhancement of capsaicinoid production remains limited in chilli pepper. Recent developments in biotechnology, particularly CRISPR/Cas-mediated genome-editing, enable the precise genetic modification of metabolic pathways and regulatory networks in plants. Therefore, it can contribute to the precise modification of key genes involved in the capsaicinoid biosynthesis pathway, offering potential strategies to enhance the capsaicinoid content in chilli pepper. However, CRISPR/Cas-mediated genome editing in chilli pepper is still in its early stages. There are currently no reports available on the successful enhancement of capsaicinoid content in chilli peppers through CRISPR/Cas-mediated genome editing. To date, no comprehensive review has evaluated the CRISPR-Cas-mediated genome-editing approaches for capsaicinoid metabolic engineering in chilli pepper. This review critically evaluates the recent advances in CRISPR/Cas-mediated metabolic engineering in chilli peppers, with particular emphasis on regulatory genes involved in capsaicinoid biosynthesis. Furthermore, multi-omics approaches are expected to complement these strategies by enabling the identification of key regulatory genes, the optimization of genome-editing targets, and the prediction of metabolic outcomes for enhanced capsaicinoid production. Overall, this review provides insights into improving capsaicinoid accumulation in chilli peppers through advanced genome-editing technologies.},
}

@article {pmid42497250,
year = {2026},
author = {Zhou, Z and Yang, Y and Zhou, F and Liang, K and Gong, T and Zhou, X and Li, J and Luo, J and Li, J and Yang, J},
title = {Energy-assisted CRISPR cleavage and probiotic vesicle signaling platform: Microbiome reprogramming for homeostasis.},
journal = {Science advances},
volume = {12},
number = {30},
pages = {eaef1760},
pmid = {42497250},
issn = {2375-2548},
mesh = {*Probiotics ; *Homeostasis ; *Microbiota/genetics ; *Streptococcus mutans/genetics/metabolism ; Adenosine Triphosphate/metabolism ; *CRISPR-Cas Systems ; Quorum Sensing ; *Extracellular Vesicles/metabolism ; Humans ; Signal Transduction ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {Pathogenic infections drive microbial dysbiosis and persistent inflammation, posing therapeutic challenges due to difficulties in precise pathogen eradication and microbiome restoration. Although CRISPR-based therapeutics enable pathogen-specific antibacterial targeting, their effectiveness in treating pathogenic infections is constrained by difficulties in navigating complex microbial ecosystems, penetrating pathogenic barriers, sustaining energy-intensive intracellular cleavage, and, critically, restoring microbial balance after pathogen clearance. Here, we engineer a probiotic vesicle-synergized CRISPR platform by encapsulating gtfB-targeting CRISPR plasmids within hybrid extracellular vesicles from probiotics and pathogenic Streptococcus mutans. The pathogen-derived vesicle component enables targeted uptake by S. mutans, facilitating intracellular cleavage of the virulence gene gtfB. Vesicle-carried endogenous adenosine triphosphate (ATP) boosts CRISPR activity, amplifying targeted DNA cleavage for potent and selective pathogen elimination. Probiotic-derived vesicle components further remodel quorum-sensing networks and immunity, restoring microbial homeostasis. This probiotic vesicle-based strategy integrates ATP-enhanced CRISPR cleavage with microbiome and immune modulation, offering a next-generation therapeutic paradigm for microbiome-associated diseases.},
}

*Probiotics
*Homeostasis
*Microbiota/genetics
*Streptococcus mutans/genetics/metabolism
Adenosine Triphosphate/metabolism
*CRISPR-Cas Systems
Quorum Sensing
*Extracellular Vesicles/metabolism
Humans
Signal Transduction
*Clustered Regularly Interspaced Short Palindromic Repeats
Animals

@article {pmid42497869,
year = {2026},
author = {Terkelsen, T and Yumiceba, V and Kim, J and Melo, US and Axelgaard, E and Febbraro, F and Gunnarsson, AV and Balachandran, S and Dahl-Jessen, M and Christensen, R and Peters, BA and Asan, and Thelle, T and Nyegaard, M and Bak, RO and Denham, M and Spielmann, M and Jensen, UB},
title = {Position effect at the SOX3 locus by an interchromosomal insertion causes hereditary spastic paraplegia.},
journal = {American journal of human genetics},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ajhg.2026.07.001},
pmid = {42497869},
issn = {1537-6605},
abstract = {Pathogenic rewiring of the three-dimensional (3D) genome architecture is increasingly being identified as the cause of genetic diseases, but recognizing the cis-regulatory effects of structural variation remains a challenge. The Xq27.1 region contains a quasi-palindrome identified as a pleiotropic hotspot for disease-causing interchromosomal insertions. In a large Danish family affected by X-linked recessive complex spastic paraplegia, we identified the segregation of a 149-kb interchromosomal insertion at Xq27.1 originating from 4q24. To understand the disease mechanism, we generated induced pluripotent stem cells (iPSCs) from affected individuals. Using CRISPR perturbation and neural differentiation experiments combined with high-throughput chromatin conformation capture (Hi-C) and transcriptomic analyses, we identify a 3D regulatory rewiring of SOX3 and transcriptional dysregulation of SOX3 targets in iPSC-derived neurons. Consistent with regulatory partitioning of the SOX3 topologically associating domain (TAD) in affected individuals, our experiments show that upstream cis-regulatory elements have a reduced ability to activate SOX3 expression and that the observed dysregulation depends on CTCF-binding sites within the insertion. This work provides mechanistic evidence that a position effect at the SOX3 locus can cause hereditary spastic paraplegia.},
}

@article {pmid42500818,
year = {2026},
author = {Li, A and Zhang, X and Li, S and Wang, Y and Xu, C and Lv, C and Zhao, M and Liu, Y and Ding, M and Cao, C},
title = {ETTAS: a modular aptamer-recruited platform for programmable translational activation.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42500818},
issn = {1362-4962},
support = {2021YFA0911600//National Key Research and Development Program of China/ ; A2503021//Shenzhen Medical Research Fund/ ; A2303071//Shenzhen Medical Research Fund/ ; 82403183//National Natural Science Foundation of China/ ; 82303113//National Natural Science Foundation of China/ ; 82360603//National Natural Science Foundation of China/ ; 82560157//National Natural Science Foundation of China/ ; 82300871//National Natural Science Foundation of China/ ; 82474383//National Natural Science Foundation of China/ ; JCYJ20250604180714019//Shenzhen Science and Technology Program/ ; RCJC20221008092723011//Shenzhen Science and Technology Program/ ; JCYJ20240813140522029//Shenzhen Science and Technology Program/ ; RCBS20231211090747077//Shenzhen Science and Technology Outstanding Innovative Talent Training/ ; },
mesh = {Humans ; *Aptamers, Nucleotide/genetics/metabolism ; *Protein Biosynthesis/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; *CRISPR-Cas Systems ; PTEN Phosphohydrolase/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Cell Line, Tumor ; },
abstract = {Precise enhancement of endogenous protein synthesis offers a reversible therapeutic strategy without permanent genomic modification. However, existing Cas13-mediated translational activation systems are limited by modest potency and restricted modular expandability. Here, we developed the Enhanced Targeted Translational Activation System (ETTAS), a modular RNA-guided platform that combines dCas13a, the SINEB2 translational activation element, and an independently recruitable aptamer-mediated auxiliary module. Systematic ortholog screening identified dCas13a as the most effective scaffold for SINEB2-mediated translational activation, whereas direct tandem duplication of SINEB2 elements impaired rather than enhanced activity. To overcome this architectural limitation, we used aptamer-mediated recruitment to spatially separate target recognition from auxiliary activation. A binding-validated, non-interfering dCas13a-binding aptamer enabled construction of a dual-module system in which an aptamer-recruited SINEB2 element enhanced translation without altering target mRNA abundance or stability. Compared with the previously reported dCasRx-SINEB2 system, ETTAS produced stronger reporter activation, stronger endogenous induction of P53 and PTEN, and greater antiproliferative and pro-apoptotic effects in bladder cancer cells. Proteomic analyses showed selective target protein upregulation with limited global perturbation. In vivo, dual-AAV delivery of ETTAS activated endogenous P53 and suppressed tumor growth. ETTAS establishes a programmable framework for modular post-transcriptional upregulation of endogenous proteins.},
}

Humans
*Aptamers, Nucleotide/genetics/metabolism
*Protein Biosynthesis/genetics
Tumor Suppressor Protein p53/genetics/metabolism
*CRISPR-Cas Systems
PTEN Phosphohydrolase/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Animals
Cell Line, Tumor

@article {pmid42500819,
year = {2026},
author = {Wang, W and Chen, Y and Li, Z and Zhang, L and Gui, K and Wu, Y and Yin, N and Han, X and Zhang, Y and Lu, R and Zhang, Z and Wang, L and Xie, G},
title = {A structural accessibility principle for LbuCas13a activation by noncontiguous DNA.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42500819},
issn = {1362-4962},
support = {82372351//National Natural Science Foundation of China/ ; 82572673//National Natural Science Foundation of China/ ; CSTB2024NSCQ-MSX0521//Natural Science Foundation of Chongqing/ ; CSTB2024NSCQ-MSX1223//Natural Science Foundation of Chongqing/ ; 2024M763898//China Postdoctoral Science Foundation/ ; 2024CQBSHTB3005//Chongqing Postdoctoral Special Funding Project/ ; },
mesh = {*DNA/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry ; Models, Molecular ; Humans ; CRISPR-Cas Systems ; Molecular Dynamics Simulation ; },
abstract = {CRISPR-Cas13a is mainly known as an RNA-guided RNA endonuclease. Recent studies show that Leptotrichia buccalis Cas13a (LbuCas13a) can interact with DNA substrates too, without PAM or PFS constraints, but current understanding of DNA-mediated activation is largely based on continuous target strands. Here, we define a structural accessibility principle for LbuCas13a activation by noncontiguous DNA. We show that activation occurs only when overhang positioning creates an accessible protein-DNA interface. Outer overhangs near the crRNA repeat-adjacent side restore strong trans-cleavage activity by stabilizing key LbuCas13a-DNA contacts, whereas distal outer overhangs support only weak activation. In contrast, inner overhangs cause steric mismatch, destabilize the complex, and block formation of an active conformation. Molecular modeling and molecular dynamics simulations support this structure-dependent rule. Noncontiguous DNA also broadens the single-nucleotide discrimination window of LbuCas13a and enables accurate IDH1 R132H detection in glioma tissues. We further develop a one-step APE1-activated CRISPR-LbuCas13a reaction (ACROSS) for sensitive APE1 detection. Because activated LbuCas13a cleaves RNA reporters but not DNA-triggering products, ACROSS preserves the activating structure and supports stable signaling in vitro, in live cells, and in breast cancer serum samples.},
}

*DNA/chemistry/metabolism
*CRISPR-Associated Proteins/metabolism/chemistry
Models, Molecular
Humans
CRISPR-Cas Systems
Molecular Dynamics Simulation

@article {pmid42501080,
year = {2026},
author = {Gençoğlu, HS and Aydemir, E and Ayaz, F},
title = {Bacterial immune systems.},
journal = {Antonie van Leeuwenhoek},
volume = {119},
number = {8},
pages = {},
pmid = {42501080},
issn = {1572-9699},
mesh = {*Bacteria/immunology/virology/genetics ; Bacteriophages/immunology/physiology ; CRISPR-Cas Systems ; Immunity, Innate ; Adaptive Immunity ; },
abstract = {Bacterial immune systems encompass the multi-layered defense mechanisms that bacteria develop against bacteriophages and mobile genetic elements, such as plasmids. This review covers bacterial innate defense systems (surface defenses, superinfection exclusion, restriction-modification, abortive infection, and toxin-antitoxin systems), CRISPR-Cas-mediated adaptive immunity, and the escape strategies used by phages to overcome these defenses (genome modifications, anti-restriction proteins, and anti-CRISPR factors). Emerging evidence also highlights the role of outer membrane vesicles (OMVs) in anti-phage defense and their translational potential as vaccine and delivery platforms. In this context, a better understanding of bacterial defense systems contributes to the development of biotechnology and medical applications such as CRISPR technologies, diagnostic approaches, and phage therapy.},
}

*Bacteria/immunology/virology/genetics
Bacteriophages/immunology/physiology
CRISPR-Cas Systems
Immunity, Innate
Adaptive Immunity

@article {pmid42503339,
year = {2026},
author = {Yuan, YL and Dai, JL and Xiao, L and Jiang, JG},
title = {Advances in engineering microalgae for heterologous terpenoid synthesis: A review.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108993},
doi = {10.1016/j.biotechadv.2026.108993},
pmid = {42503339},
issn = {1873-1899},
abstract = {Terpenoids are a class of natural products widely distributed in living organisms, with isoprene as their fundamental structural unit. However, traditional plant extraction and chemical synthesis methods are often limited by low product purity, difficult separation, and complex synthetic steps, making it challenging to meet the demands of large-scale production. Conventional hosts such as Escherichia coli and Saccharomyces cerevisiae are utilized for terpenoid synthesis due to their advantages of short growth cycles and controllable cultivation conditions. Nevertheless, the complexity of terpenoid biosynthetic pathways poses significant challenges for these hosts in producing structurally complex terpenoids. In contrast, microalgae as photosynthetic microorganisms, possess well-developed endogenous terpenoid metabolic pathways, abundant precursor pools, and subcellular structures and regulatory mechanisms similar to those of plants, demonstrating significant advantages in the heterologous production of complex terpenoids. This review systematically summarizes recent advances in the production of heterologously synthesized terpenoids in eukaryotic microalgae, ranging from monoterpenes to triterpenes, and provides an in-depth analysis of key engineering strategies, including MEP/MVA pathway regulation, gene expression optimization, subcellular compartmentalization, and cultivation process intensification. In addition, the application potential of advanced tools such as CRISPR/Cas, microalgae-microorganism co-culture, and artificial intelligence is introduced. Finally, the major bottlenecks faced by microalgae as a sustainable green cell factory for terpenoid production are briefly analyzed, and future research directions are proposed.},
}

@article {pmid42503939,
year = {2026},
author = {Yang, XP and Gao, YQ and Huang, L and Zhao, LJ and Xiao, Y and Li, L and Zhang, TS},
title = {[Advances in research on the molecular mechanisms and gene therapy of hereditary hearing impairment].},
journal = {Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine]},
volume = {60},
number = {7},
pages = {1138-1149},
doi = {10.3760/cma.j.cn112150-20251230-01243},
pmid = {42503939},
issn = {0253-9624},
support = {82560219//National Natural Science Foundation of China/ ; 2025S129//Kunming Medical University 2025 Master's Degree Education Innovation Fund Project/ ; },
mesh = {Humans ; *Genetic Therapy ; Connexin 26 ; *Hearing Loss/genetics/therapy ; Animals ; Connexins ; },
abstract = {Hereditary hearing impairment represents a significant etiology of language and social dysfunction in both children and adults, primarily caused by genetic factors. To date, over 150 genes have been identified in association with this disorder. The pathogenic mechanisms involve multiple molecular levels, including abnormalities in hair cell cytoskeleton and stereociliary structure, dysfunction of intercellular gap junctions (e.g., GJB2, GJB6), dysregulation of ion channels and transporters (e.g., SLC26A4, KCNQ4), alterations in extracellular matrix composition, and disruption of intracellular signaling pathways. In recent years, research has expanded to investigate the role of the inner ear immune microenvironment in this condition, with emerging evidence suggesting that immune dysregulation may contribute to disease initiation and progression. Therapeutically, novel strategies such as adeno-associated virus (AAV)-based gene replacement therapy, CRISPR/Cas-mediated gene editing systems, and lipid nanoparticle (LNP)-delivered mRNA therapeutics have demonstrated partial restoration of auditory function in animal models of hereditary hearing impairment involving genes such as TMC1, OTOF, and GJB2, with some approaches having advanced to clinical trial stages. This article systematically summarizes recent advances in the molecular mechanisms, immune microenvironment involvement, and gene therapy strategies for hereditary hearing impairment, delineates the research trajectory from gene discovery and mechanistic elucidation to therapeutic development, and discusses future translational research directions and clinical challenges.},
}

Humans
*Genetic Therapy
Connexin 26
*Hearing Loss/genetics/therapy
Animals
Connexins

@article {pmid42507199,
year = {2026},
author = {Bhadra, M and Sachan, M and Nara, S},
title = {Recent advances in nanozyme assisted miRNA biosensing for disease diagnosis.},
journal = {Mikrochimica acta},
volume = {193},
number = {8},
pages = {},
pmid = {42507199},
issn = {1436-5073},
mesh = {*MicroRNAs/analysis ; *Biosensing Techniques/methods ; Humans ; *Nanostructures/chemistry ; Electrochemical Techniques/methods ; Colorimetry/methods ; },
abstract = {MiRNAs have emerged as key biomarkers for early disease detection and therapeutic monitoring; yet, their ultrasensitive and specific detection confronts challenges due to low expression levels in biological fluids and high sequence homology, therefore it demands innovative sensing strategies. Enzyme mimicking nanostructures or nanozymes, enable manifold signal amplification and ultrasensitive analyte detection. In recent years, nanozymes, mainly oxidases and peroxidases, have been used as signal amplifiers in miRNA sensors due to their detectable catalytic products at low concentrations. Considering the increasing use of nanozymes in miRNA detection, this dedicated review presents the latest advancements in the field of miRNA diagnostics and unravels the contributions of nanozymes in miRNA sensing. This work highlights two major approaches commonly used for miRNA detection in sensors; (i) assisted through nucleic acid amplification, and (ii) amplification-free approaches. It then comprehensively underpins nanozymes as signal amplifier in both aforementioned strategies through diverse sensing modalities such as colorimetry, electrochemical, SERS, and chemiluminescent. It further presents the enhancement of sensitivity through integration of nanozymes with latest technologies like machine learning based approaches, CRISPR-Cas, or towards designing point of care sensors. Lastly, the review explores the challenges in translating nanozyme-derived miRNA sensing platforms to clinical settings.},
}

*MicroRNAs/analysis
*Biosensing Techniques/methods
Humans
*Nanostructures/chemistry
Electrochemical Techniques/methods
Colorimetry/methods

@article {pmid42507484,
year = {2026},
author = {Liang, Y and Qi, X and Gao, S and Wang, Y and Kan, G and Valentovich, LN and Guo, J and An, Y},
title = {Multi-level precise regulation of gene transcription in the yeast Saccharomyces cerevisiae based on light-sensitive CRISPR/Cas systems.},
journal = {Nucleic acids research},
volume = {54},
number = {14},
pages = {},
pmid = {42507484},
issn = {1362-4962},
support = {32571457//National Natural Science Foundation of China/ ; JYTYB2024044//Scientific Research Projects of Liaoning Provincial Department of Education/ ; LJKZ0660//Scientific Research Projects of Liaoning Provincial Department of Education/ ; //Liaoning Provincial Academic Leadership Support Program/ ; //Liaoning Provincial Enterprise Sci-Tech Specialist Assignment Program/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems ; *Transcription, Genetic ; *Gene Expression Regulation, Fungal ; Luminescent Proteins/genetics/metabolism ; Light ; Optogenetics/methods ; Gene Regulatory Networks ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Red Fluorescent Protein ; },
abstract = {Regulation of gene transcription based on clustered regularly interspaced short palindromic repeats (CRISPR) is a powerful tool for constructing synthetic gene circuits in Saccharomyces cerevisiae. The current CRISPR-based regulatory approaches primarily focus on inhibiting the binding of dCas9 protein to single guide RNA (sgRNA) or blocking target site recognition. However, these regulation strategies are often at a single level, and their sensitivity still needs to be improved. In this study, the gene regulatory approaches at the translational and post-translational levels were integrated with optogenetic control patterns to attain very sensitive multi-level precision regulation of the dCas9 protein, thereby facilitating flexible regulation of transcription levels of target genes. This strategy was used to regulate the transcription levels of fluorescent proteins, resulting in up to 2.58-fold increase in the fluorescence intensity of mCherry compared to that without regulation. This CRISPR-based multi-level optogenetic system should be extremely helpful in understanding gene regulatory networks and in designing robust genetic circuits for synthetic biology.},
}

*Saccharomyces cerevisiae/genetics/metabolism
*CRISPR-Cas Systems
*Transcription, Genetic
*Gene Expression Regulation, Fungal
Luminescent Proteins/genetics/metabolism
Light
Optogenetics/methods
Gene Regulatory Networks
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Red Fluorescent Protein

@article {pmid42479319,
year = {2026},
author = {Eldemir, ME and Karaca, AN and Akçelik, N and Akçelik, M},
title = {CRISPR rewired: from adaptive immunity to a global virulence control network in Salmonella.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {8},
pages = {},
pmid = {42479319},
issn = {1573-0972},
mesh = {Virulence/genetics ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Bacterial ; *Salmonella enterica/genetics/pathogenicity/immunology ; *Adaptive Immunity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; Genomic Islands ; Biofilms/growth & development ; },
abstract = {The Type I-E CRISPR/Cas system in Salmonella enterica is increasingly hypothesized to function as a condition-dependent regulatory interface rather than exclusively as an adaptive immune module. Rooted in its evolutionary origins within mobile genetic elements such as casposons, this system reflects a functional transition toward influencing bacterial pathogenesis. A central hypothesis suggests that CRISPR components-specifically Cascade, Cas3, and Cas6-are integrated into core regulatory networks governing pathogenicity islands, biofilm formation, and oxidative stress adaptation. This regulatory control likely operates through a programmed deviation from the traditional immunity paradigm, where suboptimal PAM recognition or partial sequence complementarity allows Cascade to bind DNA without licensing Cas3 for lethal cleavage. Consequently, the machinery may facilitate transcriptional modulation through steric hindrance, acting as a natural CRISPR interference mechanism. Coordinated by global regulators like H-NS and LeuO in response to environmental cues such as pH fluctuations, this system effectively functions as a molecular rheostat. Collectively, these hypotheses offer a conceptual framework for novel translational strategies, including anti-CRISPR-based therapeutics and engineered evolutionary trap concepts.},
}

Virulence/genetics
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Bacterial
*Salmonella enterica/genetics/pathogenicity/immunology
*Adaptive Immunity
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics/metabolism
Genomic Islands
Biofilms/growth & development

@article {pmid42482458,
year = {2026},
author = {Hao, J and Xie, J and Ma, K and Li, X and Chen, X and Bao, G and Hu, J and Li, G},
title = {A novel acinetobacter phage reveals altered virulence traits in phage-resistant strains.},
journal = {Virulence},
volume = {},
number = {},
pages = {2707728},
doi = {10.1080/21505594.2026.2707728},
pmid = {42482458},
issn = {2150-5608},
abstract = {Phage therapy represents a promising alternative for combating bacterial infections. This study employed an A. baumannii isolate harboring the I-F CRISPR-Cas system as a host to isolate phage and evaluate its biological characteristics. Phage-resistant mutants were screened using a double-layer agar plate assay, and the underlying molecular mechanisms were identified through whole-genome sequencing, followed by validation via gene knockout. Transcriptome sequencing was subsequently applied to alterations in the global regulatory networks of these mutants. Our results demonstrate the successful isolation of a novel myovirus, stable at 40-50 ℃, which was successfully isolated and found to utilize the capsule as its adsorption receptor. Whole-genomic analysis confirmed its taxonomic distinction from currently published phages. Investigation into the primary resistance mechanism revealed that the capsule loss, due to an insertional mutation in the UDP-glucose 4-epimerase encoding gene galE. This conclusion was further validated through targeted gene knockout of galE. This defect concurrently attenuated bacterial virulence, as demonstrated by significantly reduced lethality in the Galleria mellonella infection model and enhanced susceptibility to serum killing, while concurrently enhancing the capacity for biofilm formation. Transcriptomic profiling indicated that the ΔgalE significantly upregulated multiple biofilm-associated genes and remodeled the transcriptomic-wide regulatory. Furthermore, the combination of carbenicillin or ceftazidime with the phage exhibited a synergistic effect in vitro, effectively inhibiting biofilm formation and suppressing the emergence of phage resistance. Overall, this work characterizes a novel phage and delineates the host's biological network changes triggered by phage resistance, offering valuable insights for developing phage-based antimicrobial strategies.},
}

@article {pmid42482984,
year = {2026},
author = {Ma, C and Zhang, J and Jiang, Y and Li, X},
title = {Hybridization chain reaction-assisted CRISPR/Cas12a strategy for rapid and visual detection of Haemophilus influenzae.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1844708},
pmid = {42482984},
issn = {2235-2988},
mesh = {*Haemophilus influenzae/genetics/isolation & purification ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Haemophilus Infections/diagnosis/microbiology ; Rapid Diagnostic Tests ; Colorimetry/methods ; *Nucleic Acid Hybridization/methods ; *Molecular Diagnostic Techniques/methods ; Benzidines ; },
abstract = {Haemophilus influenzae (H. influenzae) is a major pathogen causing community-acquired pneumonia in children, posing a serious threat to children's health. Rapid and convenient testing is needed for effective treatment. Traditional detection methods, such as bacterial culture and qPCR are cumbersome to operate, and require sophisticated instrumentation. Here, we developed a visual CRISPR/Cas detection platform that integrates the hybridization chain reaction (HCR) and horseradish peroxidase (HRP)-catalyzed 3,3',5,5'-Tetramethylbenzidine (TMB) colorimetric change, called Vi-CasHCP. The single-base recognition capability of CRISPR/Cas12a improves the specificity, while the efficiency of HCR shortens the detection time and improves the sensitivity. The peroxidase-like activity of HRP catalyzes the oxidation of TMB to oxTMB, resulting in a blue color change. Vi-CasHCP achieved a detection limit of 11.8 CFU/mL in 70 min, after Recombinase polymerase amplification. Clinical validation using 50 respiratory samples showed complete diagnostic agreement with qPCR, with 100% sensitivity, specificity, PPV, and NPV. Therefore, this platform offers rapid detection, requires no complex instruments, and has high sensitivity, providing fast and accurate diagnostic support for clinical practice, and is particularly suitable for resource-limited settings.},
}

*Haemophilus influenzae/genetics/isolation & purification
Humans
Sensitivity and Specificity
*CRISPR-Cas Systems
*Haemophilus Infections/diagnosis/microbiology
Rapid Diagnostic Tests
Colorimetry/methods
*Nucleic Acid Hybridization/methods
*Molecular Diagnostic Techniques/methods
Benzidines

@article {pmid42484920,
year = {2026},
author = {Dhall, RK and Rana, N and Kaur, G and Mandyal, SS},
title = {Genomics assisted breeding for mildew resistance in cucumber: from gene discovery to future innovations.},
journal = {Plant molecular biology},
volume = {116},
number = {4},
pages = {},
pmid = {42484920},
issn = {1573-5028},
support = {BT/Ag/CoE/PAU-GSKIG/2020-21//Ministry of Science and Technology, Department of Biotechnology, Government of India/ ; },
mesh = {*Disease Resistance/genetics ; *Cucumis sativus/genetics/microbiology ; *Plant Diseases/microbiology/genetics/immunology ; *Plant Breeding/methods ; *Genomics/methods ; Quantitative Trait Loci/genetics ; Genome, Plant ; Ascomycota ; Chromosome Mapping ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide ; },
abstract = {Cucumber is an economically important vegetable crop cultivated worldwide, but its productivity is severely affected by destructive foliar diseases particularly powdery mildew and downy mildew. These pathogens cause significant yield and quality losses and the continuous emergence of new races makes disease management increasingly challenging. Conventional approaches including cultural, biological and chemical control often provide limited and short-term effectiveness. Therefore, the development of host plant resistance remains the most sustainable and environmentally sound strategy for long-term disease control. Recent advances in cucumber genomics and molecular breeding have enabled the identification of resistance-associated loci through SNP genotyping, QTL mapping, genome-wide association studies and marker-assisted selection. Furthermore, multi-omics approaches such as transcriptomics, proteomics and metabolomics combined with innovative technologies like CRISPR/Cas-mediated genome editing, genomic selection and speed breeding are transforming resistance breeding. Therefore, by integrating advanced molecular tools with omics-driven insights, this review aims to accelerate genetic gains and facilitate the development of durable, broad-spectrum mildew-resistant cucumber cultivars for sustainable and resilient production systems.},
}

*Disease Resistance/genetics
*Cucumis sativus/genetics/microbiology
*Plant Diseases/microbiology/genetics/immunology
*Plant Breeding/methods
*Genomics/methods
Quantitative Trait Loci/genetics
Genome, Plant
Ascomycota
Chromosome Mapping
Genome-Wide Association Study
Polymorphism, Single Nucleotide

@article {pmid42485704,
year = {2026},
author = {Chen, J and Zheng, H and Guan, L and Kanaherarachchi, A and Munusamy, S and Kong, J and Zhou, S and Jahani, R and Guan, X},
title = {Chemical additives-enhanced CRISPR/Cas12a-based RNA detection.},
journal = {Biosensors & bioelectronics},
volume = {312},
number = {},
pages = {119048},
doi = {10.1016/j.bios.2026.119048},
pmid = {42485704},
issn = {1873-4235},
abstract = {The CRISPR/Cas12a system has revolutionized molecular diagnostics due to its RNA-guided trans-cleavage activity, enabling programmable and highly accurate nucleic acid detection. However, most Cas12a-based assays are optimized for DNA targets, while direct RNA detection constrained by limited sensitivity, typically at the nanomolar level. Existing strategies to improve the performance of RNA analysis often rely on additional DNA activators or complex auxiliary systems. Here, we report a simple yet effective chemical additive-based strategy that overcomes these limitations. This chemical additives-enhanced CRISPR/Cas12a-based RNA detection (CARD) enables femtomolar-level RNA detection using only a single crRNA, without the need for DNA activators, reverse transcription, or strand-displacement reactions. Notably, this approach can be adapted to single-stranded DNA, enabling ssDNA detection at attomolar levels. Collectively, CARD provides a straightforward, amplification-free, and highly sensitive diagnostic framework that might be readily extended to other CRISPR/Cas systems for ultrasensitive nucleic acid diagnostics.},
}

@article {pmid42486091,
year = {2026},
author = {Regmi, M and Ma, K and Bi, C and Zhang, X and Zhao, D and Yu, L and Yang, H and Wang, N and Yang, C},
title = {Base editing for precision therapeutics.},
journal = {Cell genomics},
volume = {},
number = {},
pages = {101298},
doi = {10.1016/j.xgen.2026.101298},
pmid = {42486091},
issn = {2666-979X},
abstract = {Base editing (BE), the precise installation of single-nucleotide changes in DNA or RNA without inducing double-strand breaks, holds substantial therapeutic promise for correcting single-nucleotide variants, which constitute more than half of the known pathogenic genetic variants. Recent advances have improved base editor specificity, efficiency, and delivery, enabling clinically oriented procedures. Clinically, BE has shown early success or strong translational promise in sickle cell disease, β-thalassemia, leukemia (via CAR T and epitope engineering), hypercholesterolemia (PCSK9 and ANGPTL3), alpha-1-antitrypsin deficiency, and glycogen storage disease type Ia. Key remaining challenges include bystander editing within the activity window, residual off-target DNA and RNA editing, delivery constraints (payload size, tissue targeting, and redosing limits), immunogenicity, and the need for durable long-term safety evidence across relevant cell types and disease contexts. Continued technological refinements, careful preclinical validation, and rigorous clinical assessment will be essential to fully realize BE's transformative potential in precision medicine.},
}

@article {pmid42486099,
year = {2026},
author = {Savulescu, J and Porsdam Mann, S and Gyngell, C and Schaefer, GO},
title = {Ethics of gene therapy.},
journal = {Med (New York, N.Y.)},
volume = {},
number = {},
pages = {101225},
doi = {10.1016/j.medj.2026.101225},
pmid = {42486099},
issn = {2666-6340},
abstract = {CRISPR-Cas systems, base editing, and prime editing have made precise genetic interventions possible, and several approved therapies now treat monogenic disorders that were previously untreatable. Heritable genome editing remains ethically contested. We argue that heritable interventions should not be treated as a single category subject to uniform prohibition. We distinguish three targets: catastrophic monogenic disorders, polygenic risk reduction, and non-disease trait enhancement. For catastrophic monogenic conditions in which preimplantation selection cannot yield unaffected embryos, heritable editing is permissible, and the duty of beneficence toward future persons may require it. When the alternative is certain severe suffering or early death, the expected benefits clearly outweigh the risks. For polygenic interventions, current scientific uncertainty makes clinical application premature: predictive validity remains insufficient and pleiotropic effects are poorly understood. For enhancement, the case is weaker still. Some of its benefits are positional; the risks of social stratification are significant; and the evidence base is absent. We conclude that governance frameworks should permit what the evidence supports under stringent safeguards and prohibit what it does not. The central ethical questions concern welfare, not appeals to nature or abstract notions of dignity. Where the evidence warrants it, failing to pursue heritable gene therapy responsibly may itself be an ethical failure. We outline a translational pathway for ethical germline gene editing.},
}

@article {pmid42486981,
year = {2026},
author = {Shu, X and Wang, R and Zhou, X and Cheng, F and Ma, J and Li, Z and Li, X and Wu, T and Wu, A and Xue, Q and Liu, C and Zhao, H and Cao, X and Wang, L and Zhang, S and Zhang, Y and Li, M},
title = {CRISPR-Cas regulates expression of embedded anti-phage defence systems.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {42486981},
issn = {1476-4687},
abstract = {Bacteria utilize diverse defence systems to protect against harmful foreign DNA such as bacteriophages[1,2], but how these systems coordinate with each other remains poorly understood. Here we uncover CRISIS (CRISPR-supervised immune system), a widespread regulatory paradigm whereby type I CRISPR-Cas loci embed and transcriptionally modulate diverse innate defences. Small non-canonical CRISPR RNA (crRNA)-like RNAs guide the I-C CRISPR-associated complex for antiviral defence (Cascade) effector complex to inhibit promoters of diverse immune cassettes-including composite multi-system clusters-enabling their basal expression for antiviral activity while mitigating fitness costs associated with hyperactivation, such as host growth impairment or exclusion of beneficial plasmids. When CRISPR-Cas is compromised by mutation or anti-CRISPR proteins, there is a burst in transcription of these embedded defence systems, leading to higher-level innate immunity at the expense of host fitness. Together, adaptive CRISPR-Cas systems orchestrate diverse innate immune systems into a layered defence network, comprising a prokaryotic 'immunity guard' strategy.},
}

@article {pmid42490001,
year = {2026},
author = {Feng, L and Fang, J and Xu, Y and Luo, L and Hong, B and Wang, J and Ma, Y},
title = {Functional Activity of the Lysis Protein E From Phage ID52: Dependence on SecB for Efficient Host Cell Lysis.},
journal = {Biotechnology journal},
volume = {21},
number = {7},
pages = {e70271},
pmid = {42490001},
issn = {1860-7314},
support = {XZ202601ZY0032//Science and Technology Projects of Xizang Autonomous Region, China/ ; Grant No. NERCGM-OF-20250301//Opening Foundation of National Engineering Research Center of Genetic Medicine, China/ ; 2022A1515010716//the Natural Science Foundation of Guangdong Province/ ; },
mesh = {*Bacteriophages/genetics/metabolism ; *Escherichia coli/virology/genetics/metabolism ; *Viral Proteins/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/genetics ; *Bacteriolysis ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; *Escherichia coli Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Single gene encoded phage lysis proteins offer a promising strategy for bacterial ghost production, yet their host-dependent regulatory mechanisms remain poorly understood. Here, we investigated the lysis protein E from phage ID52 (ID52-E), which exhibits stronger lytic activity than φX174 E. By screening ID52-E-resistant mutants, we identified a four-base insertion in secB as the genetic alteration associated with lysis resistance in BL21, and CRISPR-Cas9-mediated secB disruption confirmed that SecB is required for ID52-E-mediated lysis. Proteomic analysis revealed altered protein expression in resistant mutants despite preserved bacterial morphology. Co-immunoprecipitation and biolayer interferometry supported an apparent interaction between SecB and ID52-E, with an apparent KD of 3.541 × 10[-] [8] M under the tested 1:1 fitting model. Molecular docking, molecular dynamics simulations, mutagenesis, lysis assays, and binding measurements further implicated SecB Ala145 as a key interface residue. Together, these findings identify SecB as a host factor that facilitates ID52-E-mediated bacterial lysis and provide mechanistic insight for improving bacterial ghost production.},
}

*Bacteriophages/genetics/metabolism
*Escherichia coli/virology/genetics/metabolism
*Viral Proteins/metabolism/genetics/chemistry
*Bacterial Proteins/metabolism/genetics
*Bacteriolysis
Molecular Docking Simulation
Molecular Dynamics Simulation
*Escherichia coli Proteins/metabolism/genetics
CRISPR-Cas Systems

@article {pmid42490334,
year = {2026},
author = {Lv, Y and Kulsoom, and Jia, L and Wang, Z and Wang, F},
title = {Overcoming Liquid Biopsy Barriers: Nucleic Acid Biosensors Integrating DNA Nanotechnology and CRISPR-Cas System for Cancer Precision Theranostics.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c01673},
pmid = {42490334},
issn = {2379-3694},
abstract = {Liquid biopsy holds immense potential for the early detection of cancer, yet its clinical utility is hindered not by the lack of available tumor-associated biomarkers but by the inadequate sensitivity and clinical robustness of current molecular diagnostic tools. Nucleic acid-based biosensors have emerged as highly programmable platforms, enabling the detection of low-abundance cancer biomarkers such as microRNAs (miRNAs), circulating tumor DNA (ctDNA), and messenger RNAs (mRNAs) in complex biological fluids. Leveraging advances in DNA nanotechnology, CRISPR-Cas-mediated RNA sensing, and chemically engineered nucleic acid analogues, these biosensors achieve attomolar-level detection through nanoscale spatial confinement and enzyme-assisted signal amplification strategies. However, their clinical translation is hindered by biological sample variability, nonspecific amplification, probe degradation, and poor reproducibility. This review analyzes the core design principles of three major biosensor categories: functional DNA nanostructures, CRISPR-Cas-based sensing systems, and synthetic analogues (PNAs, SNAs). It elucidates their structural and enzymatic optimization mechanisms, distinguishes analytical from clinical sensitivity, and addresses key liquid biopsy challenges. Finally, it outlines promising strategies for clinical translation, including microfluidic integration, artificial intelligence-assisted data analysis, and theranostic nanostructures combining diagnosis with targeted therapy. This review provides a comprehensive theoretical and technical framework for the rational design of next-generation nucleic acid biosensors and offers critical insights to bridge the gap between nanoscale engineering innovation and clinical translation, ultimately advancing the development of minimally invasive and precise cancer theranostics in precision oncology.},
}

@article {pmid42491328,
year = {2026},
author = {Hussain, A and Mojgani, N and Siddiqui, MF and Khan, MI and Ali, SA},
title = {Exploring the emerging role of CRISPR-Cas systems in probiotic development.},
journal = {Engineering microbiology},
volume = {6},
number = {3},
pages = {100279},
pmid = {42491328},
issn = {2667-3703},
abstract = {Modification of the gut microbiota by beneficial microbes can enhance an organism's lifespan, giving rise to the concept of probiotics. Probiotics are live microorganisms that provide health benefits when taken in sufficient amounts. Owing to their outstanding health benefits, probiotics have experienced rapid expansion and gained interest for the development of new applications. The exploration of microbial applications via genetic modification is currently of great interest to researchers. Genetic engineering using the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has received considerable attention and has established applications. Owing to these enhanced properties, the CRISPR-Cas system is currently used in medicine, agriculture, food, and biotechnology. Considering the adaptive immune system in bacteria, this genetic tool is used to alter the microbial genome. Lactic acid bacteria (LAB) are widely recognized for their probiotic potential, and over 40% of LAB species contain the CRISPR-Cas system. The rising demand for probiotics and their expanding applications necessitate the enhancement of their existing characteristics. The CRISPR-Cas system, recognized for its precision, accuracy, and speed, has enabled researchers to modify the genomes of probiotics, thereby enhancing their beneficial attributes. This system can enhance probiotic properties through additive, subtractive, or modulatory mechanisms. Various approaches have been developed to improve probiotic functionalities using the CRISPR-Cas system, such as substituting slow promoters with efficient alternatives, eliminating undesirable components, boosting metabolism, and increasing tolerance levels. Furthermore, CRISPR-engineered probiotics have emerged as next-generation probiotics with enhanced properties and advanced applications across diverse fields, including the food, medicine, agriculture, and pharmaceutical sectors.},
}

@article {pmid42424384,
year = {2026},
author = {Källstig, E and Ruchti, E and Raman, M and Asadzadeh, J and Schneider, BL and McCabe, BD},
title = {Highly frequent undesired insertional mutagenesis during Drosophila genome editing.},
journal = {PLoS genetics},
volume = {22},
number = {7},
pages = {e1012192},
doi = {10.1371/journal.pgen.1012192},
pmid = {42424384},
issn = {1553-7404},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mutagenesis, Insertional/genetics ; Genome, Insect ; Recombinational DNA Repair/genetics ; Genetic Vectors/genetics ; },
abstract = {CRISPR/Cas9 based genome editing employing Homology Directed Repair (HDR) from template vector sequences is a widely used technique to enable precise insertions, deletions or modifications to genes. Here, we describe an undesired and highly frequent editing event when using conventional CRISPR/Cas9 plus HDR methods for Drosophila melanogaster germline genome editing. We find that the template vector employed for HDR repair unwantedly and commonly inserts into the genome. We observe this deviation from the desired edit at multiple genomic locations, with different HDR vectors and with multiple genome editing designs. To avoid these events, we have generated a novel HDR template vector that enables animals with these undesired insertions to be identified and excluded. Our results suggest that HDR based genome edited animals must be carefully screened for unwanted vector template genomic integration in order to avoid misleading interpretations of genome editing outcomes.},
}

Animals
*Drosophila melanogaster/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Mutagenesis, Insertional/genetics
Genome, Insect
Recombinational DNA Repair/genetics
Genetic Vectors/genetics

@article {pmid42475178,
year = {2026},
author = {Farhangmehr, S and Braunschweig, U and Wu, M and Nabeel-Shah, S and Brown, KR and Fine, JL and Moffat, J and Blencowe, BJ},
title = {A genome-wide functional analysis of conserved intronic regions reveals essential roles for speckle-associated retained introns.},
journal = {Cell reports},
volume = {45},
number = {7},
pages = {117696},
doi = {10.1016/j.celrep.2026.117696},
pmid = {42475178},
issn = {2211-1247},
abstract = {Hundreds of human introns harbor extended regions of high evolutionary conservation that have not been previously characterized. A survey of these sequences reveals that they are associated with intron retention and enriched in genes that function in RNA processing, chromatin remodeling and neuronal biology. Using a dual CRISPR-Cas editing approach, we targeted 2,600 of these regions for deletion and observed that a subset of these perturbations affects cell growth. Many of these "fitness" sequences affect intron retention and expression levels of their host genes. Deletions in nuclear speckle-associated retained introns in the FNBP4 and DDX5 genes further cause downstream effects on cell growth-related genes and intron retention, respectively. The intronic deletion in DDX5 additionally results in the accumulation of R-loops overlapping retained introns of speckle-proximal genes. Overall, the results highlight critical and multifaceted roles of highly conserved intronic sequences in the control of gene regulation, R-loop resolution, and cell growth.},
}

@article {pmid42477308,
year = {2026},
author = {Sikandar Zaman, M and Azeem, A and Nouman, A and Khalid, A and Ghafoor, S and Zia Ul Haq, M and Aslam, MT and El-Beltagi, HS},
title = {CRISPR-Cas systems for enhancing chilling tolerance in rice: recent advances and future prospects.},
journal = {Biologia futura},
volume = {},
number = {},
pages = {},
pmid = {42477308},
issn = {2676-8607},
abstract = {Rice (Oryza sativa L.) is an important staple crop in global food security and highly vulnerable to chilling stress, which greatly affects growth, development, and yield. The conventional breeding methods for enhancing chilling tolerance face numerous problems due to the polygenic nature of chilling tolerance and genetic complexities. The present review discusses the use of CRISPR-Cas genome editing technologies as an accurate and effective approach to increasing chilling tolerance in rice. We initially describe the physiological effects of chilling stress, such as membrane fluidity impairment, inhibition of photosynthesis, nutrient imbalance, and oxidative injury, and summarize major molecular pathways and genetic materials involved in chilling tolerance. The review then outlines the recent developments in CRISPR-Cas systems, including the modes of delivery (Agrobacterium-mediated transformation, protoplast transfection, and ribonucleoprotein techniques) and how they apply to rice genome editing. The precise examination of CRISPR-based functional genomics has shown that cold-responsive genes (OsMYB30, OsWRKY76, OsAnn3, OsPRP1, and OsKASI-2) are selectively manipulated, thus contributing to a clearer understanding of their functional roles in stress signaling, membrane stability, and antioxidant defense. Moreover, we also discuss recent CRISPR strategies, including multiplex editing, transcriptional reprogramming (CRISPRa/i), and omics-guided fine-tuning of gene networks. Synthesizing latest advancements, current review establishes a conceptual framework to overcome translational challenges in CRISPR-mediated improvement of complex traits in oilseed crops, through integrating the pivotal aspects of genotype-specific delivery, multi-gene network design, field validation, and the evolving regulatory landscape. The review concludes with a reflection of gaps in research and future opportunities, with a discussion on how integrated CRISPR technologies can be used to enhance the development of climate-resistant rice varieties.},
}

@article {pmid42477873,
year = {2026},
author = {Chen, Y and Gao, XH and Vichas, A and Wang, J and Golhar, R and Neuhaus, I},
title = {ALPINE: a scalable pipeline for comprehensive classification of gene-editing outcomes from long-read amplicon sequencing.},
journal = {Bioinformatics (Oxford, England)},
volume = {42},
number = {7},
pages = {},
pmid = {42477873},
issn = {1367-4811},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *High-Throughput Nucleotide Sequencing/methods ; *Software ; Humans ; *Sequence Analysis, DNA/methods ; DNA Repair ; },
abstract = {SUMMARY: CRISPR genome editing has enabled precise genetic modification for gene and cell therapies, but edits often produce heterogeneous on-target outcomes, including homology-directed repair (HDR) knock-ins, DNA repair template integrations, and structural variants. Existing tools are frequently limited to short reads or lack viral vector-specific integration categories needed for therapeutic development. Here, we present ALPINE (Amplicon Long-read Pipeline for INtegration Evaluation), a scalable and reproducible pipeline for classifying and quantifying gene-editing outcomes from long-read amplicon sequencing supporting both PacBio HiFi and Oxford Nanopore platforms. ALPINE classifies reads into 10+ categories, including DNA repair vector integration subtypes, and performs variant calling near the gene-edited site with batch, multi-sample reporting. Uniquely, ALPINE can distinguish between cells treated with multiple DNA repair vectors and identify distinct molecular features, such as inverted terminal repeats (ITRs), enabling comprehensive characterization of complex gene editing outcomes. Dual-target benchmarking on simulated datasets demonstrated high accuracy for transgene integration events. Independent validation on public crosslinked-HDR dataset confirmed ALPINE's integration detection capabilities, and application to edited T cell samples demonstrated comprehensive gene-editing outcome profiling.

AVAILABILITY: ALPINE is available under MIT license at https://github.com/Maggi-Chen/ALPINE and https://doi.org/10.5281/zenodo.20272510. All analysis scripts and visualization code used in this manuscript are available at https://github.com/Maggi-Chen/ALPINE-manuscript-analysis. Simulated datasets are deposited at Zenodo (https://doi.org/10.5281/zenodo.20260865). Public dataset PRJNA913199 is available through NCBI SRA.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*High-Throughput Nucleotide Sequencing/methods
*Software
Humans
*Sequence Analysis, DNA/methods
DNA Repair

@article {pmid42478499,
year = {2026},
author = {Du, Y and Shi, Y and Chen, D and Wang, S and Su, J and Liu, K and Zhang, H and Wang, F},
title = {Gene Editing-Driven Engineering of Microbial Systems and Metabolites for Precision Medicine and Sustainable Environmental Remediation.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e74410},
doi = {10.1002/smll.74410},
pmid = {42478499},
issn = {1613-6829},
support = {BJ-2023-118//National High Level Hospital Clinical Research Funding and Fundamental Research Funds for the Central Universities/ ; T2322025//National Natural Science Foundation of China/ ; 82272161//National Natural Science Foundation of China/ ; 22125701//National Natural Science Foundation of China/ ; 52372274//National Natural Science Foundation of China/ ; 22388101//National Natural Science Foundation of China/ ; 2024YFA0919300//National Key R&D Program of China/ ; 20240101175JC//Natural Science Foundation of Jilin Province, China/ ; XF012022C0200//Xiangfu Lab Research Project/ ; },
abstract = {The engineering of microorganisms is undergoing a fundamental paradigm shift, transitioning from the construction of static cell factories to the programming of dynamically responsive living materials. However, translating molecular interventions into robust macroscopic functions requires overcoming distinct microbial-specific barriers, including delivery bottlenecks and genetic stability. In this review, we establish a unified Edit-Reprogram-Functionalize conceptual framework that systematically delineates transient genetic regulation from permanent genomic engineering. We critically examine the evolutionary trajectories of five foundational technologies: plasmid engineering, CRISPR-Cas systems, base editors, prime editors, and enzyme engineering. Rather than analyzing these toolsets in isolation, we map their convergence into an integrated engineering continuum that drives the precise synthesis of two distinct output classes: engineered living microbial materials and robust microbial metabolite-derived materials. By evaluating representative breakthroughs-from ultrasound-actuated bacterial therapeutics to ultra-tough, biosynthesized protein composites-through the strict lens of host-dependent constraints, we reveal the mechanistic principles governing successful preclinical translation. Finally, we propose an actionable roadmap centered on systemic miniaturization, closed-loop control, and multi-scale integration, providing a definitive blueprint for the next generation of precision medicine, advanced biomanufacturing, and ecological remediation.},
}

@article {pmid42248880,
year = {2026},
author = {Wang, X and Jowsey, WJ and Cheung, CY and Dickerhof, N and Chapman, CL and Taka, JRH and Hampton, MB and Bashiri, G and Gardner, PP and Fineran, PC and Cook, GM and Jackson, SA and McNeil, MB},
title = {Genome scale CRISPRi reveals both shared and strain-specific vulnerabilities in genetically diverse drug-resistant strains of Mycobacterium tuberculosis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42248880},
issn = {2041-1723},
support = {20/459//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; 22/323//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; 22/156//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; 23/228//Manatu Hauora | Health Research Council of New Zealand (HRC)/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/metabolism/isolation & purification ; Bacterial Proteins/genetics/metabolism ; DNA-Directed RNA Polymerases/genetics ; Mutation ; *Genome, Bacterial ; Rifampin/pharmacology ; *Drug Resistance, Bacterial/genetics ; Antitubercular Agents/pharmacology ; Humans ; Genotype ; Amino Acyl-tRNA Synthetases/genetics/metabolism ; CRISPR-Cas Systems ; Microbial Sensitivity Tests ; Sulfur/metabolism ; Drug Resistance, Multiple, Bacterial/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {The global health burden caused by Mycobacterium tuberculosis is aggravated by the emergence and spread of drug resistance. Mutations that cause drug resistance can have collateral effects that increase the vulnerability of downstream pathways to inhibition. Here, using genome scale CRISPR interference we identified collateral effects associated with different drug-resistant genotypes of M. tuberculosis. We demonstrate that drug resistance generated shared vulnerabilities in several overlapping functional pathways. Most drug-resistant strains were more sensitive to tRNA synthetase knockdowns than the parental drug-sensitive strain, highlighting the potential of tRNA synthetases as high-value drug targets. Additionally, the rifampicin-resistant mutant RpoB(S450L) had increased sensitivity to the dysregulation of sulphur metabolism due to transcriptional dysregulation. This increased vulnerability did not translate to all rpoB genotypes but was linked to predicted effects on transcriptional dynamics. Amongst clinical isolates, non-synonymous mutations in sulphur metabolism genes have evolved in a geographic lineage specific manner to mitigate fitness costs associated with the collateral phenotypes of drug resistance. Combined, our findings highlight the power of functional genomics in pinpointing highly vulnerable drug targets across drug-resistant strains.},
}

*Mycobacterium tuberculosis/genetics/drug effects/metabolism/isolation & purification
Bacterial Proteins/genetics/metabolism
DNA-Directed RNA Polymerases/genetics
Mutation
*Genome, Bacterial
Rifampin/pharmacology
*Drug Resistance, Bacterial/genetics
Antitubercular Agents/pharmacology
Humans
Genotype
Amino Acyl-tRNA Synthetases/genetics/metabolism
CRISPR-Cas Systems
Microbial Sensitivity Tests
Sulfur/metabolism
Drug Resistance, Multiple, Bacterial/genetics
Gene Expression Regulation, Bacterial

@article {pmid42270637,
year = {2026},
author = {Seidel, S and Zwaans, A and Regalado, S and Choi, J and Shendure, J and Stadler, T},
title = {SciPhy: A Bayesian phylogenetic framework using sequential genetic lineage tracing data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42270637},
issn = {2041-1723},
mesh = {Bayes Theorem ; *Phylogeny ; Animals ; *Cell Lineage/genetics ; Mice ; *Software ; Computer Simulation ; CRISPR-Cas Systems ; },
abstract = {CRISPR-based lineage tracing offers a promising avenue to decipher single-cell lineage trees, especially in organisms not amenable to microscopy. Sequential genome editing records not only genetic edits but also the order in which they occur. To leverage this enriched information, we introduce SciPhy, a simulation and inference tool implemented in BEAST 2. SciPhy utilizes a Bayesian phylogenetic approach to jointly estimate time-scaled phylogenies and cell population parameters. After validation on simulated data, we use simulated and real data from a monoclonal cell culture to benchmark SciPhy against existing methods and find that it consistently reconstructs more accurate phylogenies. Compared to UPGMA, SciPhy additionally reports uncertainty and proliferation rates. Our second example applies SciPhy to murine gastruloids, demonstrating its ability to model time-varying population dynamics in early development. Together, these results establish a phylodynamic framework for the quantitative analysis of lineage tracing data. SciPhy's codebase is publicly available at https://github.com/azwaans/SciPhy .},
}

Bayes Theorem
*Phylogeny
Animals
*Cell Lineage/genetics
Mice
*Software
Computer Simulation
CRISPR-Cas Systems

@article {pmid42473674,
year = {2026},
author = {Ananda, H and Sahana, SR and Murthy, SR and Kunnath, AN and Prashant, A and Kaifi, JT and Kiran, PK and Suvilesh, KN},
title = {Genomic innovations in cancer prevention, diagnosis, prognosis and precision therapeutics.},
journal = {Frontiers in genetics},
volume = {17},
number = {},
pages = {1828450},
pmid = {42473674},
issn = {1664-8021},
abstract = {Cancer research has undergone a transformative change with the advent of high-throughput genomic technologies. Advances in next-generation sequencing accelerated the identification of somatic and germline alterations that drive tumorigenesis enabling the transition from traditional histology-based cancer classification to molecularly informed precision oncology. Large-scale sequencing initiatives and clinical genomic profiling facilitated the development of companion diagnostic assays and targeted therapies. Beyond targeted therapies, genomic innovations have also catalyzed the emergence of novel therapeutic strategies including immunogenomics-driven immunotherapies, RNA-based therapeutics, cancer vaccines and genome editing technologies based on CRISPR-Cas systems. This review summarizes the major technological developments in cancer genomics, including sequencing platforms, transcriptomic profiling, liquid biopsy, and functional genomic screening, and highlights the utility of these innovations in discovery of actionable biomarkers and next-generation therapeutic strategies. Collectively, these advances underscore the central role of genomic technologies in driving the evolution of precision oncology toward more personalized and effective cancer treatment strategies.},
}

@article {pmid42263439,
year = {2026},
author = {Guo, B and Li, Y and Shi, M and Zhang, J and Wu, Q and Wu, X and An, R and Wang, F},
title = {A triplex-readout CRISPR-Cas12a multimodal biosensing platform for point-of-care detection of avian influenza H5N1.},
journal = {Journal of hazardous materials},
volume = {514},
number = {},
pages = {142562},
doi = {10.1016/j.jhazmat.2026.142562},
pmid = {42263439},
issn = {1873-3336},
mesh = {*Influenza A Virus, H5N1 Subtype/isolation & purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Point-of-Care Systems ; Animals ; *Influenza in Birds/diagnosis/virology ; Rapid Diagnostic Tests ; RNA, Viral/analysis ; Humans ; },
abstract = {The highly pathogenic avian influenza A (H5N1) virus poses a significant zoonotic threat to public and animal health. Conventional detection methods often face limitations in complexity, time, and equipment requirements. In this study, we report a rapid and lightweight-equipment triplex diagnostic platform for H5N1 detection, integrating locked nucleic acid (LNA)-assisted target recognition, toehold-mediated strand displacement for signal amplification, and the high specificity of the CRISPR-Cas12a system. The developed assay achieved a detection limit of 3.7 × 10[2] copies/μL for H5N1 pseudovirus RNA within a 60-minute core detection process, and exhibited excellent specificity without cross-reactivity to other influenza subtypes or coronaviruses. Moreover, it successfully identified H5N1 in clinical swab samples, yielding consistent results across three independent readout formats: fluorescence, lateral flow strip, and a portable glucose meter. With advantages of low cost, rapid operation, and multimodal verification capability, this diagnostic system is well suited for point-of-care screening in resource-limited settings and represents a promising tool for frontline outbreak response.},
}

*Influenza A Virus, H5N1 Subtype/isolation & purification/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Point-of-Care Systems
Animals
*Influenza in Birds/diagnosis/virology
Rapid Diagnostic Tests
RNA, Viral/analysis
Humans

@article {pmid42320852,
year = {2026},
author = {Tadokoro, T and Liu, N and Olson, EN},
title = {Precision modification of heart failure signaling by CRISPR-Cas9 base editing.},
journal = {Journal of molecular and cellular cardiology},
volume = {217},
number = {},
pages = {89-93},
pmid = {42320852},
issn = {1095-8584},
mesh = {Humans ; *Heart Failure/genetics/therapy/metabolism ; *Signal Transduction/genetics ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; },
abstract = {Heart failure remains a leading cause of morbidity and mortality worldwide, and current therapies largely focus on symptom management and slowing disease progression rather than correcting the underlying molecular abnormalities. Recent advances in genome editing technologies have created new opportunities to treat heart failure. Among these approaches, CRISPR-Cas9 base editing has emerged as a particularly promising strategy because it enables precise nucleotide conversions without introducing double-strand DNA breaks and demonstrates relatively high efficiency in vivo. While correction of disease-causing mutations by CRISPR-Cas9 base editing represents an important application of genome editing, an alternative strategy is to directly modulate key signaling pathways that drive cardiac dysfunction. Protein kinase C alpha (PKCα) functions as a key regulator of cardiac contractility and pathological remodeling. Precision editing of phosphorylation sites that control PKCα stability or activation may therefore represent an effective strategy to suppress maladaptive kinase signaling in cardiomyocytes. This concept of "precision signaling modification" may provide a broadly applicable therapeutic approach for heart failure. Similar strategies may also be applicable to other signaling molecules, including Ca[2+]/calmodulin-dependent protein kinase II delta (CaMKIIδ), and illustrate the broader potential of signaling-focused genome editing approaches. Despite these advances, several challenges remain for clinical translation, including efficient delivery of genome editing components to the adult heart, long-term safety, and potential immune responses. Continued advances in delivery technologies and genome editing platforms may ultimately enable durable, potentially one-time therapeutic interventions for heart failure.},
}

Humans
*Heart Failure/genetics/therapy/metabolism
*Signal Transduction/genetics
*Gene Editing/methods
Animals
*CRISPR-Cas Systems/genetics

@article {pmid42470562,
year = {2026},
author = {Zhong, S and Leng, Y and Yang, S},
title = {Targeted Gene Editing in Wheat During Haploid Production via Wide Hybridization with Transgenic Maize Expressing Cas9 and Guide RNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3029},
number = {},
pages = {313-329},
pmid = {42470562},
issn = {1940-6029},
mesh = {*Triticum/genetics ; *Zea mays/genetics ; *Gene Editing/methods ; *Haploidy ; Plants, Genetically Modified/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Hybridization, Genetic ; Genome, Plant ; },
abstract = {The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system is an efficient and versatile genome engineering tool, which has been widely used for targeted mutagenesis and gene functional characterization in various organisms. This system is simple because it only requires a Cas9 enzyme serving as a nuclease and guide RNA (gRNA) containing a 20-nt sequence matching the target gene. Delivery of a vector expressing Cas9 and gRNA or the preassembled Cas9/gRNA complex as a ribonucleoprotein (RNP) into plant cells for gene targeting are usually via the biolistic- or Agrobacterium-mediated approach. However, most wheat genotypes suffer from low efficiency of callus induction and plant regeneration from explants receiving the vector or RNP delivered by the biolistic- or Agrobacterium-mediated transformation method, limiting the application of genome editing systems in many commercially grown wheat varieties. Here, we describe a stepwise protocol for targeted gene editing in wheat via wide hybridization with transgenic maize expressing Cas9 and gRNA. A binary vector expressing Cas9 and gRNA is constructed and used for Agrobacterium-mediated transformation to generate transgenic maize plants, which are used to pollinate emasculated spikes of wheat varieties. After fertilization, the maize chromosomes enter the transient hybrid zygote and the transgene (T-DNA) on a maize chromosome expresses the Cas9 enzyme and gRNA, which forms an RNP complex to edit the target gene in wheat genome. After several cell divisions, maize chromosomes in the hybrid zygote are eliminated, resulting in formation of haploid wheat embryos with the target gene edited, which can be rescued by embryo culture technique to produce haploid plants. Doubled haploid (DH) wheat plants with homozygous gene mutations are developed by chromosome doubling through colchicine treatment of the haploid plants. The wheat × maize hybridization combined with the CRISPR/Cas9 system provides a one-step approach for generating DH lines with the target gene edited from any wheat genotypes of interest.},
}

*Triticum/genetics
*Zea mays/genetics
*Gene Editing/methods
*Haploidy
Plants, Genetically Modified/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Hybridization, Genetic
Genome, Plant

@article {pmid42470563,
year = {2026},
author = {Shi, C and Zhang, X and Zhao, Z and Sun, MX},
title = {In Vivo Rice Haploid-Induction System by an Egg Cell-Specific Peptidase Knockout.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3029},
number = {},
pages = {331-344},
pmid = {42470563},
issn = {1940-6029},
mesh = {*Oryza/genetics ; *Haploidy ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; *Peptide Hydrolases/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; *Plant Proteins/genetics ; },
abstract = {Doubled haploid (DH) technology is a fast and convenient approach for crop breeding and genetic research. Currently, in vitro anther culture is the main method for rice haploid production. However, genotype dependence remains a major problem in the anther culture of most rice subspecies or cultivars for haploid induction. In this chapter, we describe a protocol for in vivo haploid induction in rice using egg cell-specific peptidase (ECS) knockout lines, including mutation of ECS using CRISPR-Cas9 system, selection of homozygous Osecs mutants, and identification of haploids in the offspring of Osecs by flow cytometry. The ECS mediated maternal in vivo haploid-induction system is a convenient, time-saving and labor-saving technique to produce rice DH lines.},
}

*Oryza/genetics
*Haploidy
*Gene Knockout Techniques/methods
CRISPR-Cas Systems
*Peptide Hydrolases/genetics
Plants, Genetically Modified/genetics
Plant Breeding/methods
*Plant Proteins/genetics

@article {pmid42472629,
year = {2026},
author = {Kimura, K and Tsukamoto, M and Shishida, K and Sugisaki, H and Katahira, J and Tanaka, M and Kuwamura, M and Kol, A and Okada, M and Iijima, M and Nakanishi, M and Sugiura, K and Hatoya, S},
title = {Red blood cell differentiation using canine-induced pluripotent stem cells.},
journal = {Stem cells translational medicine},
volume = {15},
number = {8},
pages = {},
pmid = {42472629},
issn = {2157-6580},
support = {JP18H02349//JSPS KAKENHI/ ; 22J14623//JSPS KAKENHI/ ; 22H02525//JSPS KAKENHI/ ; 23K23790//JSPS KAKENHI/ ; 26K01900//JSPS KAKENHI/ ; },
mesh = {Animals ; Dogs ; *Cell Differentiation ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Erythrocytes/cytology/metabolism ; Glycophorins/metabolism/genetics ; CRISPR-Cas Systems ; Green Fluorescent Proteins/metabolism ; },
abstract = {BACKGROUND: Red blood cell (RBC) transfusions are essential for treating various medical conditions, but global demand is difficult to meet due to a dwindling donor pool and compatibility issues. Pluripotent stem cells (PSCs) offer a promising alternative of blood dependent on volunteer donors for RBC production, and dogs serve as an excellent model for translational research due to their physiological and genetic similarities to humans.

METHODS: Canine induced pluripotent stem cells (ciPSCs) were differentiated toward hematopoietic and erythroid lineages. Differentiated cells were evaluated for hematopoietic marker expression, hemoglobinization, colony-forming capacity, enucleation, and hemoglobin gene expression. Glycophorin A (GYPA)-enhanced green fluorescent protein (EGFP) reporter ciPSC lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing to visualize GYPA expression during differentiation.

RESULTS: This study introduces a protocol for RBC differentiation using ciPSCs. We achieved generation of hemoglobinized RBCs, progressing through polychromatic and orthochromatic erythroblast-like stages. CiPSC-derived hematopoietic cells/RBCs were confirmed to have immature characteristics as determined by limited colony-forming capacities, low enucleation, and embryonic and fetal hemoglobin gene expression. Additionally, we created GYPA-EGFP reporter ciPSC lines using CRISPR-Cas9-mediated genome editing, enabling real-time visualization of GYPA expression. This innovation confirmed GYPA as a viable surface marker for ciPSC-derived RBCs.

CONCLUSION: Our findings mark an initial step toward establishing a canine PSC-based erythroid differentiation system, providing a foundation for future improvements and exploration of applications for canine PSC-derived RBCs.},
}

Animals
Dogs
*Cell Differentiation
*Induced Pluripotent Stem Cells/cytology/metabolism
*Erythrocytes/cytology/metabolism
Glycophorins/metabolism/genetics
CRISPR-Cas Systems
Green Fluorescent Proteins/metabolism

@article {pmid42464358,
year = {2026},
author = {Farooq, A and Rafique, A and Han, E and Park, SM and Kim, HS and Kim, MJ and Hussain, A and Sheeraz Ahmad, M and LaPointe, G},
title = {Interactions of antiphage defense systems in the ESKAPE pathogen plasmids.},
journal = {Mobile DNA},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13100-026-00410-2},
pmid = {42464358},
issn = {1759-8753},
support = {ALLRP 566176-2021//Natural Sciences and Engineering Research Council of Canada/ ; },
abstract = {BACKGROUND: The global rise of multidrug resistant (MDR) ESKAPE pathogens represents a serious threat to antimicrobial therapy. While phage therapy has re-emerged as a promising alternative, its effectiveness may be compromised by bacterial defense systems, particularly those encoded on plasmids. Comprehensive surveillance of the distribution, diversity, and mobilome context of plasmid-encoded defense systems in ESKAPE pathogens remains key to the design of effective phage therapies.

RESULTS: We analyzed 7,330 dereplicated plasmids from ESKAPE pathogens to characterize the prevalence, diversity, and co-occurrence of plasmid-encoded antiphage defense systems. Conjugative plasmids, especially from Enterobacter spp. and K. pneumoniae, harbored the highest prevalence and diversity of defense systems. Defense-positive plasmids showed larger sizes, higher GC content, and frequent co-occurrence of resistance genes, especially from β-lactam, aminoglycoside, and sulfonamide classes, along with transposable elements such as IS6, IS3, and Tn3. Random forest and correlation analyses confirmed TEs and ARGs as dominant predictors of defense system occurrence. Network analysis revealed structured and partially conserved interactions among defense genes, TEs, and ARGs. RM and CBASS systems were frequently linked to beta-lactam and aminoglycoside resistance genes, as well as TEs such as IS6 and IS3. Recurrent associations such as RM-IS6, RM-IS1380, CBASS-IS3 and RM-OXA suggest shared horizontal transfer mechanisms.

CONCLUSIONS: Plasmid-encoded antiphage defense systems in ESKAPE pathogens are widespread, structured, and linked to ARGs and mobile genetic elements. These findings highlight the contribution of plasmids to the dissemination of phage-resistance traits, underscore the importance of the mobilome in shaping phage-resistance landscapes in multidrug-resistant pathogens, and support the incorporation of plasmid defense profiling into phage therapy design.},
}

@article {pmid42465601,
year = {2026},
author = {Kumar, S and Singh, RM and Gadhave, KR},
title = {CRISPR-Cas systems for plant virus management: detection, surveillance, and host resistance.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1804262},
pmid = {42465601},
issn = {1664-462X},
abstract = {The CRISPR-Cas system has transformed genome manipulation by enabling precise and programmable modification of genetic material. Initially developed as a genome-editing tool, CRISPR technologies have expanded from fundamental research to applied use across plant, animal, and microbial systems due to their simplicity, accuracy, and versatility. In agriculture, CRISPR-Cas9 has progressed from crop improvement to host-directed strategies conferring resistance against a broad range of plant viruses. Concurrently, the discovery of additional Cas effector proteins, particularly Cas12a and Cas13a, has enabled highly sensitive nucleic acid-based diagnostic platforms supporting rapid, field-deployable pathogen detection. Here, we present a focused synthesis integrating CRISPR-mediated host resistance engineering with CRISPR-based diagnostic surveillance within a unified framework for plant virus management. Unlike previous reviews that treat these domains independently, we emphasize their convergence in enabling early detection, real-time surveillance, and targeted intervention across the disease cycle. Cas12a-based systems, currently the most widely implemented, have been coupled with isothermal amplification and visual readouts for rapid virus detection, whereas Cas13a-based platforms offer direct RNA targeting with potential for simplified workflows, although they remain less developed. We examine key design considerations, performance characteristics, and limitations of these platforms, including challenges related to sensitivity, multiplexing, and field deployment. Finally, we highlight future directions, including vector-based detection, multiplex diagnostics, and integration of CRISPR technologies into scalable surveillance systems. Collectively, this review positions CRISPR-based genome editing and diagnostics as complementary components of a next-generation strategy for plant virus detection, surveillance, and management.},
}

@article {pmid42467248,
year = {2026},
author = {Zhong, H and Ma, Q and Wei, J and Yang, A and Liu, Y and Zhen, D},
title = {CRISPR/cas-based biosensors for point-of-care testing: a comprehensive review of signal readout strategies.},
journal = {Archives of microbiology},
volume = {208},
number = {10},
pages = {},
pmid = {42467248},
issn = {1432-072X},
support = {202510555097//National College Students Innovation and Entrepreneurship Training Program/ ; 82503565//National Natural Science Foundation of China/ ; },
mesh = {*Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; Humans ; Colorimetry/methods ; Rapid Diagnostic Tests ; Point-of-Care Systems ; Electrochemical Techniques/methods ; },
abstract = {The CRISPR/Cas system has emerged as a transformative tool for nucleic acid detection, offering significant potential for point-of-care testing (POCT). However, translating CRISPR/Cas-based assays into practical POCT devices critically depends on the development of portable, sensitive, and user-friendly signal readout modalities. This review systematically compares four major readout modalities: fluorescence, electrochemical, colorimetric, and distance‑based readout, analyzing their mechanisms, analytical performance, and practical limitations. Key challenges, including sample preparation, amplification-free detection, multiplexing, and commercialization barriers, are critically assessed. Finally, future perspectives are proposed: integrating microfluidics with smartphone‑based readout, leveraging artificial intelligence and the Internet of Things for automated signal interpretation and cloud connectivity, and establishing regulatory pathways for clinical translation. This review aims to provide actionable insights for researchers developing next‑generation CRISPR diagnostics and to accelerate the transition from laboratory prototypes to deployable POCT devices.},
}

*Biosensing Techniques/methods/instrumentation
*CRISPR-Cas Systems
*Point-of-Care Testing
Humans
Colorimetry/methods
Rapid Diagnostic Tests
Point-of-Care Systems
Electrochemical Techniques/methods

@article {pmid42469036,
year = {2026},
author = {Portell-Montserrat, J and Höpfler, M},
title = {Nascent peptides emerge as regulators of mRNA stability.},
journal = {Trends in cell biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tcb.2026.06.010},
pmid = {42469036},
issn = {1879-3088},
abstract = {Mobile genetic elements and their hosts engage in continuous evolutionary conflict. Marino et al. recently uncovered an unusual anti-CRISPR mechanism: the phage protein AcrVA2 triggers translation-coupled mRNA degradation by recognizing nascent Cas12. The findings suggest that nascent peptides may signal an underappreciated layer of gene regulation across the kingdoms of life.},
}

@article {pmid42469498,
year = {2026},
author = {Khan, T and Abro, AA and Zulfiqar, U and Alotaibi, MS and Asadullaeva, D and Allaberdiev, R and Tang, X and Fan, G},
title = {CRISPR-enabled functional genomics for bolstering plant tolerance to abiotic and biotic stress; a comprehensive review.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42469498},
issn = {1438-7948},
mesh = {*Stress, Physiological/genetics ; *CRISPR-Cas Systems ; Drought Resistance ; *Gene Editing/methods ; *Genomics ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Climate change intensifies abiotic stresses including salinity, drought, and extreme temperatures alongside biotic threats such as pathogens and insect pests, collectively undermining global crop productivity and food security. Salinity and drought alone affect 20-50% of irrigated soils, with projections indicating that nearly half of global farmland could become saline by mid-century. Conventional breeding and earlier genome editing tools zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (ZFNs, TALENs) are constrained by genetic diversity limitations, technical complexity, and slow trait deployment. The CRISPR-Cas9 system has emerged as a transformative platform offering superior precision, efficiency, scalability, and affordability for crop improvement. This review systematically examines how CRISPR-Cas9 enables targeted engineering of stress tolerance in major crops (rice, wheat, maize, tomato, barley) through gene knockout and knock-in strategies. Key applications include editing transcription factors (ART1, DRO1, OsDST) for drought and salinity tolerance, modifying transporter genes (OsHMA2, OsNramp5) for heavy metal detoxification, and disrupting susceptibility genes (MLO, OsERF922, CsLOB1) for broad-spectrum disease and pest resistance. Beyond direct editing, we highlight emerging synergies with functional genomics, multi-omics integration, and high-throughput phenotyping to accelerate target discovery and validation. A central focus is placed on nanobiotechnology-enabled CRISPR delivery systems, including lipid nanoparticles (LNPs), exosomes, and engineered nanocarriers that overcome the plant cell wall barrier a major bottleneck in plant genetic transformation. These platforms enable efficient, genotype-independent delivery of ribonucleoprotein (RNP) complexes, facilitating DNA-free editing for sustainable crop protection. By integrating CRISPR-based precision with advances in nanodelivery and molecular breeding, this review outlines a road-map for developing climate-resilient, high-yielding, and nutritionally enhanced crops to safeguard global agricultural sustainability.},
}

*Stress, Physiological/genetics
*CRISPR-Cas Systems
Drought Resistance
*Gene Editing/methods
*Genomics
*Crops, Agricultural/genetics
Plants, Genetically Modified/genetics

@article {pmid42470537,
year = {2026},
author = {Sisay, T and Berhan, A and Mihrete, K and Hunie, E and Bizuye, A},
title = {Genomic regulation of the diphtheria toxin gene and Its implications for molecular diagnostics and surveillance in low-resource settings.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42470537},
issn = {1573-4978},
mesh = {*Diphtheria Toxin/genetics/metabolism ; *Corynebacterium diphtheriae/genetics/pathogenicity ; Humans ; *Diphtheria/diagnosis/genetics/microbiology ; Genomic Islands ; Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; Genome, Bacterial ; DNA-Binding Proteins ; },
abstract = {Corynebacterium diphtheriae remains a significant, though often underestimated, public health concern, particularly in low- and middle-income countries. The pathogenicity of the disease is primarily determined by diphtheria toxin (DT), which is produced by the tox gene, a bacteriophage-associated element, and is tightly regulated by the iron-dependent transcriptional repressor DtxR, encoded by the dtxR gene. Despite extensive investigation into the molecular biology of DT, its regulation within the broader genomic organization, as well as its implications for diagnostic methods and surveillance strategies, have not yet been fully elucidated. This review consolidates existing evidence regarding the genomic context and molecular regulation of the tox gene, encompassing chromosomal organization, variability in GC content, genomic islands, and mechanisms of horizontal gene transfer. Significant attention is focused on lysogenic conversion mediated by corynephages and regulatory pathways responsive to iron. We also evaluate both established and novel molecular diagnostic approaches, including PCR, real-time PCR, sequencing technologies, and isothermal amplification methods like loop-mediated isothermal amplification (LAMP). Recent genomic discoveries, including pan-genome variation, CRISPR-Cas mechanisms, and the emergence of non-toxigenic tox-bearing strains are analyzed in relation to diagnostic precision and epidemiological surveillance. Understanding the genomic regulation and evolutionary dynamics of toxin production is essential for improving diagnostic accuracy and strengthening surveillance systems, particularly in resource-limited settings where diphtheria is often underdiagnosed and underreported.},
}

*Diphtheria Toxin/genetics/metabolism
*Corynebacterium diphtheriae/genetics/pathogenicity
Humans
*Diphtheria/diagnosis/genetics/microbiology
Genomic Islands
Gene Expression Regulation, Bacterial
Bacterial Proteins/genetics/metabolism
Genome, Bacterial
DNA-Binding Proteins

@article {pmid41570009,
year = {2026},
author = {Tay, YL and Thomson, SB and Hnatova, S and Ng, S and Teo, SR and McCallum, R and Sim, B and Tarantini, L and Tai, FL and Bollati, V and Loh, M and Hayden, MR and Leavitt, BR and Pouladi, MA},
title = {Silencing of human HTT by targeted CRISPR/dCas9-mediated epigenetic editing.},
journal = {Journal of Huntington's disease},
volume = {15},
number = {3},
pages = {399-407},
doi = {10.1177/18796397251415368},
pmid = {41570009},
issn = {1879-6400},
mesh = {Humans ; *Huntingtin Protein/genetics ; *DNA Methylation/genetics ; *Gene Silencing ; *Huntington Disease/genetics/therapy ; DNA Methyltransferase 3A ; *Epigenesis, Genetic ; *CRISPR-Cas Systems ; Epigenome Editing ; DNA (Cytosine-5-)-Methyltransferases/genetics ; },
abstract = {BackgroundGene silencing is widely recognized as a promising therapeutic approach for dominant monogenic disorders. Current silencing strategies, many of which are transient, utilize RNA interference. Gene silencing may also be achieved through directed epigenetic editing using a CRISPR/dCas9 effector fused to DNA methyltransferase 3A (dCas9-DNMT3A). We used this system to direct DNA methylation to HTT, the causal gene underlying the autosomal dominant neurodegenerative disorder Huntington's disease, to assess the translational potential of this strategy for treating a genetic neurological disease.ObjectiveTo characterize the regulatory effect of targeted dCas9-DNMT3A-mediated DNA methylation at HTT.MethodsWe exploited DNA methylation profiles of high and low HTT-expressing tissues and targeted hypomethylated regions of HTT associated with high levels of HTT expression.ResultsDe novo DNA methylation of loci within defined upstream, promoter, intragenic and downstream regions of HTT resulted in robust, acute silencing of HTT. The best long-term silencing of HTT, which persisted up to 30 days, was observed when targeted DNA methylation was directed to the 5'UTR and promoter regions of HTT.ConclusionsHTT gene silencing may be achieved via targeted de novo DNA methylation within hypomethylated regulatory regions at the HTT locus. DNA methylation editing may be an attractive therapeutic approach for Huntington disease due to its potential for long-term silencing and reversibility.},
}

Humans
*Huntingtin Protein/genetics
*DNA Methylation/genetics
*Gene Silencing
*Huntington Disease/genetics/therapy
DNA Methyltransferase 3A
*Epigenesis, Genetic
*CRISPR-Cas Systems
Epigenome Editing
DNA (Cytosine-5-)-Methyltransferases/genetics

@article {pmid42045650,
year = {2026},
author = {Saha, A and Ocampo, RF and Wright, JT and Taylor, DW and Palermo, G},
title = {Molecular mechanisms and biotechnology applications of CRISPR-Cas12a.},
journal = {Nature reviews. Molecular cell biology},
volume = {27},
number = {8},
pages = {601-616},
pmid = {42045650},
issn = {1471-0080},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Biotechnology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Animals ; *Bacterial Proteins/genetics/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; },
abstract = {CRISPR-Cas12a is a versatile RNA-guided nuclease that has rapidly gained prominence for its dual functionality in genome editing and nucleic acid detection. In this Review, we discuss the structural, biochemical and mechanistic features of Cas12a that underpin its autonomous processing of the guide RNA and indiscriminate cleavage of single-stranded DNA, which enable Cas12a applications ranging from gene therapy to rapid diagnostics. We discuss key allosteric regulators and functional modules that orchestrate Cas12a activity, focusing on the core regulatory structural elements that control maturation of the guide RNA, target specificity, and both cis-cleavage and trans-cleavage activities, including the determinants of off-target cleavage. We provide a comparative analysis of Cas12a and the widely used Cas9, which further illuminates the distinctive attributes of Cas12a, and discuss recent advances in the characterization of its orthologues and in the development of engineered variants that expand its capabilities. Collectively, we present a comprehensive understanding of Cas12a and its increasing impact on biotechnology, therapeutics and molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Biotechnology/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Animals
*Bacterial Proteins/genetics/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/genetics/chemistry

@article {pmid42190664,
year = {2026},
author = {Zhang, H and Zhang, Z and Wang, P and Xu, T and Chen, X and Zhao, Y and Lin, S and Cai, W and Ren, P and Luo, C and Zhang, P and Wang, Y and Hou, S and Zhao, Y and Zeng, H and Liu, Z and Wang, C and Gao, Z and Feng, Y and Pan, D and Zeng, Z},
title = {Uncovering spatially resolved functional genomics with CRISPR screen sequencing.},
journal = {Cell},
volume = {189},
number = {15},
pages = {4594-4618.e48},
doi = {10.1016/j.cell.2026.04.049},
pmid = {42190664},
issn = {1097-4172},
mesh = {*Genomics/methods ; Animals ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Macrophages/metabolism/immunology ; Intercellular Adhesion Molecule-1/metabolism/genetics ; CD8-Positive T-Lymphocytes/metabolism/immunology ; Hyaluronan Receptors/metabolism ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Spatial omics has advanced our understanding of tissue-level biology, yet tools to systematically link gene functional perturbations to spatial phenotypes and signaling pathways remain limited. To address this, we developed spatial CRISPR screen sequencing (SPAC-seq), a high-throughput spatial CRISPR screen platform, and TARDIS (target prioritization toolkit for perturbation data in spatial omics), a statistical spatial perturbation analysis toolkit. Using SPAC-seq and TARDIS, we linked gene perturbations to spatial phenotypes and pathways, uncovering how Icam1 loss in tumor cells promotes metastasis via immune suppression and macrophage polarization. In CD8[+] T cells, we revealed Cd44's role in regulating spatial phenotypes by interacting with Spp1 on macrophages. We also demonstrated the model of the transcription factor-chemokine receptor axis coupling cell states with chemotaxis. SPAC-seq and TARDIS provide an effective framework to study spatially resolved functional genomics and pathways across diverse biological and disease contexts.},
}

*Genomics/methods
Animals
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Macrophages/metabolism/immunology
Intercellular Adhesion Molecule-1/metabolism/genetics
CD8-Positive T-Lymphocytes/metabolism/immunology
Hyaluronan Receptors/metabolism
Mice
CRISPR-Cas Systems
Cell Line, Tumor
High-Throughput Nucleotide Sequencing/methods

@article {pmid42192532,
year = {2026},
author = {Hwang, HY and Yi, H and Gwon, Y and Jeon, E and Kim, D},
title = {High-fidelity genome and prime editing enabled by the AI-designed openCRISPR-1.},
journal = {Genome medicine},
volume = {18},
number = {1},
pages = {},
pmid = {42192532},
issn = {1756-994X},
support = {RS-2025-00521074//National Research Foundation of Korea/ ; RS-2025-02214578, HR22C1363, RS-2024-02507183//Korea Health Industry Development Institute (KHIDI)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Genome, Human ; Induced Pluripotent Stem Cells/metabolism ; CRISPR-Associated Protein 9/genetics ; },
abstract = {BACKGROUND: RNA-guided nucleases such as CRISPR-Cas9 systems have revolutionized genome engineering by enabling programmable DNA modifications. Although structure-guided and evolution-derived high-fidelity Cas9 variants improve target specificity, they often compromise on-target activity or constrain guide RNA (gRNA) design.

METHODS: We performed head-to-head comparisons of OpenCRISPR-1 and Cas9 in human cells using amplicon sequencing, multiplex Digenome-seq, and off-target validation by targeted sequencing. Editing activity was assessed across 28 endogenous loci in HEK293T cells and further evaluated in human induced pluripotent stem cells (iPSCs) and MRC-5 fibroblasts. To test clinically relevant delivery, Cas9 and OpenCRISPR-1 ribonucleoproteins were delivered using engineered virus-like particles (eVLPs). We also generated OpenCRISPR-based prime editors, OpenCRISPR-PE2 and OpenCRISPR-PE7, and compared them with PE2max and PE7 using pegRNAs and engineered epegRNAs.

RESULTS: Here, we show that OpenCRISPR-1, an AI-designed, Cas9-like nuclease, retains Cas9-level editing efficiency across multiple genomic loci while significantly reducing off-target mutations. Using multiplex Digenome-seq and targeted deep sequencing, OpenCRISPR-1 exhibits up to a 553-fold reduction in off-target mutations compared to Cas9 and achieves off-target indices that match or surpass those of high-fidelity Cas9 variants. OpenCRISPR-1 also sustains robust editing across diverse gRNA formats (GX19, gX19, and gX20), highlighting its enhanced versatility. Furthermore, converting OpenCRISPR-1 into a prime editor yields comparable editing efficiencies while lowering the relative specificity ratio by up to 97%.

CONCLUSIONS: These findings establish generative AI-guided protein design as a powerful strategy to overcome the specificity-efficiency trade-off, expanding the genome editing toolkit for both research and therapeutic use, and ushering in a new era of rational protein design.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
*Genome, Human
Induced Pluripotent Stem Cells/metabolism
CRISPR-Associated Protein 9/genetics

@article {pmid42218140,
year = {2026},
author = {Di Bernardo, M and Kern, RS and Cepeda Diaz, AK and Mallar, A and Choi, SJ and Nutter-Upham, A and Lourido, S and Blainey, PC and Cheeseman, I},
title = {Brieflow: an integrated computational pipeline for high-throughput analysis of optical pooled screening data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42218140},
issn = {2041-1723},
support = {GM126930//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 000955563//National Science Foundation (NSF)/ ; Data Science Internship Program//Massachusetts Life Sciences Center (MLSC)/ ; UROP Program//Massachusetts Institute of Technology (MIT)/ ; R01HG009283//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01AI144369//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {*Computational Biology/methods ; *High-Throughput Screening Assays/methods ; CRISPR-Cas Systems ; Reproducibility of Results ; Humans ; *Genomics/methods ; Phenotype ; Software ; Large Language Models ; Pooled Testing ; },
abstract = {Optical pooled screening (OPS) has emerged as a powerful technique for functional genomics, enabling researchers to link genetic perturbations with complex cellular morphological phenotypes at scale. However, OPS data analysis presents challenges due to massive datasets, complex multi-modal integration requirements, and the absence of standardized frameworks. Here, we present Brieflow, a computational pipeline for end-to-end analysis of fixed-cell optical pooled screening data. We demonstrate Brieflow's capabilities through reanalysis of a CRISPR-Cas9 screen encompassing 5072 fitness-conferring genes, processing more than 70 million cells with multiple phenotypic markers. To accelerate biological interpretation, we additionally present MozzareLLM, a framework leveraging large language models to identify biological processes within phenotypic clusters and prioritize gene candidates for experimental validation. Our combined analysis recovers coherent biological modules missed by existing analytical approaches, including five core mitochondrial sub-programs absent from the original study. The modular design and open-source implementation of Brieflow facilitates the integration of new analytical components while ensuring computational reproducibility and improved performance for the use of high-content phenotypic screening in biological discovery.},
}

*Computational Biology/methods
*High-Throughput Screening Assays/methods
CRISPR-Cas Systems
Reproducibility of Results
Humans
*Genomics/methods
Phenotype
Software
Large Language Models
Pooled Testing

@article {pmid42230636,
year = {2026},
author = {Snetkova, V and Galan, C and Lopez, R and Rios, AR and Kudo, T and Dorighi, K and Warming, S and Haley, BJ},
title = {A tunable Cas12a platform for single-cell perturbation screening and CRISPRi.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42230636},
issn = {2041-1723},
mesh = {*Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; *Gene Editing/methods ; *Bacterial Proteins/genetics/metabolism ; Single-Cell Gene Expression Analysis ; Degrons ; HEK293 Cells ; Gene Knockout Techniques/methods ; Transcriptome ; Animals ; Endodeoxyribonucleases ; },
abstract = {Single-cell perturbation (Perturb-seq) screens have primarily relied on Cas9 for inducing loss-of-function phenotypes, whereas Cas12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas12a's guide RNA array (pre-crRNA) self-processing activity and the subsequent challenges associated with pre-crRNA sequence recovery during single-cell RNA sequencing library preparation. To overcome the self-processing constraint, we optimized pre-crRNA expression vectors and established a degron-based, enhanced Cas12a system for gene knock-out. As demonstrated across cell types, target genes, and with a minimized guide RNA library, this platform allows for accurate detection of pre-crRNAs and gene editing-induced effects on the transcriptome in single cells. Additionally, we show that HyperLbCas12a outperforms other existing variants for multiplexed gene suppression. While the rapid reversibility of this repressor highlights specific kinetic constraints for degron-based single-cell recording, the system provides a potent, modular tool for contexts requiring tunable, transient silencing. Together, this suite of technologies greatly expands the possibilities for future Perturb-seq efforts and broader application of Cas12a for genetic disruption at scale.},
}

*Single-Cell Analysis/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
Humans
*Gene Editing/methods
*Bacterial Proteins/genetics/metabolism
Single-Cell Gene Expression Analysis
Degrons
HEK293 Cells
Gene Knockout Techniques/methods
Transcriptome
Animals
Endodeoxyribonucleases

@article {pmid42236706,
year = {2026},
author = {Zhu, L and Nguyen, LT and Bell, AG and Krebel, T and Gillmann, KM and Cao, Q and Oatman, H and Hariri, J and Möglich, A and Myhrvold, C and Toettcher, JE},
title = {Multimodal control of Cas13d activity through domain insertion at an allosteric hotspot.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42236706},
issn = {2041-1723},
support = {R01GM144362//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Allosteric Regulation ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Protein Domains ; Blue Light ; HEK293 Cells ; Sirolimus/pharmacology/analogs & derivatives ; },
abstract = {CRISPR-Cas13d RNA nucleases are powerful tools for programmable RNA targeting. A light-controlled RNA nuclease could be transformative by enabling researchers to selectively knock down transcripts at desired positions in a cell or tissue or at timepoints of interest. Here, we develop a set of RfxCas13d tools that can be multimodally controlled by either light or small molecule addition. By screening an RfxCas13d library containing insertions of the AsLOV2 photoswitchable domain, we identify an OptoCas13d-off variant that induced target RNA cleavage in the dark and switched to an inactive state under blue light. We show that the same allosteric hotspot can be exploited to generate an OptoCas13d-on with an inverted light response and a ChemoCas13d that is activated by rapamycin analogs, enabling knockdown of endogenous mRNA and protein targets. Overall, our study shows that engineered allostery can produce stimulus-controlled Cas13d variants to modulate RNA with high spatial and temporal precision.},
}

*CRISPR-Cas Systems/genetics
Allosteric Regulation
Humans
*CRISPR-Associated Proteins/genetics/metabolism/chemistry
Protein Domains
Blue Light
HEK293 Cells
Sirolimus/pharmacology/analogs & derivatives

@article {pmid42236735,
year = {2026},
author = {Turocy, J and Jerabek, S and Hur, W and Kim, J and Xu, S and Zhao, Q and Xu, J and Robles, A and Liu, X and Treff, N and Marin, D and Hadjantonakis, AK and Egli, D},
title = {Asymmetric attrition and secondary chromosome destabilization after double-strand breaks in human embryonic development.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42236735},
issn = {2041-1723},
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *Embryonic Development/genetics ; *Chromosomal Instability/genetics ; Centromere/genetics/metabolism ; Chromatids/metabolism ; Telomere/genetics/metabolism ; DNA Repair ; Spindle Apparatus/metabolism ; CRISPR-Cas Systems ; },
abstract = {DNA repair in human embryos is poorly understood, and double-strand breaks (DSBs) can cause chromosome loss. We show that chromosomal alterations relative to an induced DSB are asymmetric: acentric arms show complementary gains and losses, while centric arms are biased toward losses. Centromeric to the cut site secondary breakage and attrition is extensive. In contrast, break sites at acentric arms are conserved with no secondary breakage. These differences reflect differential forces at the mitotic spindle. Telomeric arms detach from the pro-metaphase spindle while centric truncated chromosomes lag during anaphase, suggesting that the DSB impedes sister chromatid separation. Secondary breakage near the centromere concordant with extensive attrition at the DSB site indicates a DSB can destabilize a chromosome without end-joining of sister chromatids. These results highlight the risks of chromosomal-scale changes in CRISPR-Cas9 genome editing and show that a single DSB can destabilize a human embryo chromosome independent of fusion-breakage cycles.},
}

Humans
*DNA Breaks, Double-Stranded
*Embryonic Development/genetics
*Chromosomal Instability/genetics
Centromere/genetics/metabolism
Chromatids/metabolism
Telomere/genetics/metabolism
DNA Repair
Spindle Apparatus/metabolism
CRISPR-Cas Systems

@article {pmid42251067,
year = {2026},
author = {Gopal, N and Abay, T and Payne, C and Gomez, M and Rogers, MM and Fofanah, IU and Kallon, TPMS and Kamara, MS and Suk, HJ and Sandi, JD and Brock-Fisher, T and Stachler, E and Allan-Blitz, LT and Roach, DJ and Paye, MF and Wilkason, C and Grant, DS and Ozonoff, A and Sabeti, PC},
title = {Rapid development and field evaluation of a portable CRISPR-based assay for Mpox during the 2025 Sierra Leone outbreak.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42251067},
issn = {2041-1723},
mesh = {Sierra Leone/epidemiology ; Humans ; *Disease Outbreaks ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *CRISPR-Cas Systems/genetics ; *Hemorrhagic Fever, Ebola/epidemiology/diagnosis/virology ; *Ebolavirus/genetics/isolation & purification ; },
abstract = {The large 2025 Mpox clade IIb outbreak in Sierra Leone underscores the urgent need for portable, low-cost diagnostics in decentralized settings. While CRISPR-based assays offer high sensitivity and flexibility, their deployment during active outbreaks remains limited. Here we show the rapid development and field evaluation of Mpox SHINE, a CRISPR-Cas13 assay that integrates lyophilized reagents, ambient-temperature lysis, and automated fluorescence detection on the portable DxHub device. The assay achieves analytical sensitivity down to 10 copies/µL. Clinical validation in Sierra Leone, using 56 clinical specimens, confirms complete concordance with qPCR, demonstrating 100% sensitivity and 100% specificity. Crucially, Mpox SHINE also detects the virus directly from unextracted lesion swabs while maintaining 100% sensitivity and specificity. The mean time-to-result is fast, averaging 11.4 minutes for extracted samples and 27.9 minutes for unextracted samples. These findings demonstrate that CRISPR-based diagnostics translate quickly from genomic sequence to clinically validated, deployable tools within a single outbreak window.},
}

Sierra Leone/epidemiology
Humans
*Disease Outbreaks
Sensitivity and Specificity
Rapid Diagnostic Tests
*CRISPR-Cas Systems/genetics
*Hemorrhagic Fever, Ebola/epidemiology/diagnosis/virology
*Ebolavirus/genetics/isolation & purification

@article {pmid42269486,
year = {2026},
author = {Popsuj, S and Kalsang, T and Kim, K and Drummond, E and Manekar, P and Munagapati, P and Oleti, M and Sato, H and Vickery, I and Gigante, ED and Stolfi, A},
title = {Validated CRISPR/Cas9 guide RNAs targeting neurodevelopmental genes in the tunicate Ciona robusta.},
journal = {Differentiation; research in biological diversity},
volume = {150},
number = {},
pages = {100973},
doi = {10.1016/j.diff.2026.100973},
pmid = {42269486},
issn = {1432-0436},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Neurodevelopment/genetics ; *Ciona/genetics/growth & development ; Mutagenesis ; Transcription Factors/genetics ; Gene Expression Regulation, Developmental ; *Ciona intestinalis/genetics ; },
abstract = {The tunicate Ciona robusta provides a powerful and simplified model for dissecting the genetic control of developmental and cell biology. With a larval CNS composed of just over 200 neurons and sensory cells, it has also emerged as a model organism for neurobiology and the development of the nervous system. Although CRISPR/Cas9-mediated mutagenesis is now routinely used in Ciona as an important technique used to interrogate gene function in diverse biological processes, validated single-guide RNAs (sgRNAs) have yet to be validated for several key neural genes. Here, we report the design and experimental validation of 25 novel sgRNAs targeting eight conserved genes encoding conserved proteins involved in neurodevelopment and neural function, including six transcription factors (Cdx, Foxb, Sox1/2/3, Dmbx, Engrailed, and Mnx) and two neural effector genes (Tyrosinase and Slc18a3/VAChT). Candidate sgRNAs were selected and tested for mutagenesis efficiency using Illumina-based target site amplicon sequencing. All sgRNAs induced insertions or deletions at their target loci, with most genes yielding at least one sgRNA with mutagenesis efficacy exceeding 30%, with the exception of Dmbx, for which maximal efficacy reached 25%. We further compared measured mutagenesis rates to scores generated by different predictive algorithms, observing a modest but potentially improved correlation with predictions based on a newer algorithm. Based on these results, we recommend considering both scoring algorithms in combination, for improved predictive value for Ciona.},
}

Animals
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Neurodevelopment/genetics
*Ciona/genetics/growth & development
Mutagenesis
Transcription Factors/genetics
Gene Expression Regulation, Developmental
*Ciona intestinalis/genetics

@article {pmid42276496,
year = {2026},
author = {Wang, F and Zhang, M and Chen, S and Fu, S and Huai, M and Liu, M and Meng, G and Dong, C},
title = {CRISPR/Cas9-mediated disruption of Cmpks1 reveals its role as a key regulator of carotenoid biosynthesis and metabolic adaptation in Cordyceps militaris.},
journal = {International journal of biological macromolecules},
volume = {372},
number = {},
pages = {152987},
doi = {10.1016/j.ijbiomac.2026.152987},
pmid = {42276496},
issn = {1879-0003},
mesh = {*Cordyceps/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Carotenoids/metabolism ; Gene Expression Regulation, Fungal ; *Fungal Proteins/genetics/metabolism ; *Polyketide Synthases/genetics/metabolism ; Oxidative Stress ; *Adaptation, Physiological/genetics ; Light ; Mutation ; },
abstract = {Cordyceps militaris, a renowned edible mushroom, produces orange-yellow fruiting bodies (FBs), primarily due to carotenoid accumulation. However, genetic mechanisms and functional roles underlying carotenoid biosynthesis remain poorly understood. Here, we identified Cmpks1, a light-induced gene encoding a reducing type I polyketide synthase, as a key regulator of pigment biosynthesis. Transcription of Cmpks1 was CmWC-1-dependent and upregulated during FB development. CRISPR/Cas9-mediated loss-of-function mutants of Cmpks1 exhibited stable albino phenotypes but retained FB differentiation. In addition to abolishing carotenoid biosynthesis, the disruption of Cmpks1 increased sensitivity to high light and oxidative stress, indicating its role in redox homeostasis. Metabolomic profiling of the ΔCmpks1 mutant, including significantly reduced ergothioneine and elevated cordycepin, revealed extensive metabolic reprogramming, coupled with activation of compensatory survival mechanisms. These findings elucidate the genetic mechanisms governing pigment formation that influence the quality of Cordyceps products, offering new insights into the role of metabolites in fungal morphogenesis and stress adaptation.},
}

*Cordyceps/genetics/metabolism
*CRISPR-Cas Systems/genetics
Carotenoids/metabolism
Gene Expression Regulation, Fungal
*Fungal Proteins/genetics/metabolism
*Polyketide Synthases/genetics/metabolism
Oxidative Stress
*Adaptation, Physiological/genetics
Light
Mutation

@article {pmid42276945,
year = {2026},
author = {Meng, D and Zhang, Y and Zou, S and Wang, J and Tian, C and Gao, T and Liu, J and Yuan, X and Chen, S and Qiao, Y and Ma, N and Chang, H and Gao, X},
title = {Cas9-PALB2 fusion protein enhances CRISPR/Cas9 mediated gene knock-in efficiency.},
journal = {Journal of bioscience and bioengineering},
volume = {142},
number = {3},
pages = {187-195},
doi = {10.1016/j.jbiosc.2026.05.004},
pmid = {42276945},
issn = {1347-4421},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; HEK293 Cells ; *Fanconi Anemia Complementation Group N Protein/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Gene Editing/methods ; *Recombinant Fusion Proteins/genetics/metabolism ; Recombinational DNA Repair ; },
abstract = {Over the past decade, CRISPR-based technologies have revolutionized our capacity to manipulate genomes, thereby reshaping the landscape of functional genomics research. Among the CRISPR toolkit, CRISPR/Cas9-mediated homology-directed repair (HDR) enables precise genome editing with predefined mutations, rendering it an indispensable tool for gene functional analysis, disease model construction, and the development of gene therapy strategies. Nevertheless, despite the robust efficiency of CRISPR/Cas9 in mediating gene knockouts, HDR-dependent gene knock-in (KI) remains a major bottleneck due to its inherently low efficiency. Herein, we report that the co-expression of PALB2 with the CRISPR/Cas9 nuclease could trigger an enhanced HDR effect. Specifically, the fusion of Cas9 with PALB2 elevated KI efficiency by approximately 1.7-fold in human HEK293T cells. Furthermore, this Cas9-PALB2 fusion strategy exhibited cross-cell-type efficacy, demonstrating its broad applicability beyond a single cell line. Notably, the combined application of the Cas9-PALB2 fusion system and Nocodazole further boosted KI efficiency to a remarkable 25.5%. Collectively, these findings establish the Cas9-PALB2 fusion as a highly potent and versatile strategy to augment HDR-mediated KI efficiency, highlighting its substantial potential for widespread utilization in applications that demand high-fidelity genome editing.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
HEK293 Cells
*Fanconi Anemia Complementation Group N Protein/genetics/metabolism
*CRISPR-Associated Protein 9/genetics/metabolism
*Gene Editing/methods
*Recombinant Fusion Proteins/genetics/metabolism
Recombinational DNA Repair

@article {pmid42379534,
year = {2026},
author = {Lummerstorfer, M and Xue, Z and Zheng, D and König, L and Brunner, L and Lächelt, U},
title = {Lipid nanoparticles for Cas9 ribonucleoprotein delivery: design and evaluation of ionisable oligoamine-lipidoids.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {224},
number = {},
pages = {107596},
doi = {10.1016/j.ejps.2026.107596},
pmid = {42379534},
issn = {1879-0720},
mesh = {*Nanoparticles/chemistry/administration & dosage ; *Lipids/chemistry/administration & dosage ; *Ribonucleoproteins/administration & dosage/chemistry ; *CRISPR-Associated Protein 9/administration & dosage/chemistry ; Humans ; *Amines/chemistry/administration & dosage ; CRISPR-Cas Systems ; Gene Editing ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are the most advanced RNA delivery technology and are used with CRISPR-RNA in multiple clinical in vivo genome editing trials. By contrast, systemic delivery of Cas9 ribonucleoproteins (RNPs) - despite their high intrinsic efficiency - has lagged, largely due to a lack of mature delivery systems, and RNA‑optimised LNPs cannot readily be translated to RNPs. Differences arise from the pH-sensitive protein and cargo-specific optimal lipid compositions. With regard to the fundamental ionisable lipid component, comparatively less optimisation has been carried out for Cas9-RNPs than for RNA. In this work, C12-200, developed as a potent ionisable lipidoid for RNA-LNPs and also well-suited for Cas9-RNPs, served as the lead structure. Using an analogue synthesis strategy, 17 alternative C12-lipidoids were generated from different oligoamine precursors with structural differences, including the number of nitrogens (2 to ≈40), architecture (linear, branched or containing an N-heterocycle), and separating alkyl spacers (ethyl, propyl) between ionisable groups. Employing the different lipidoids in analogous LNP formulations enabled a systematic assessment at relevant stages of Cas9-RNP delivery and the identification of structure-activity relationships. Two C12-lipidoids with piperazine ring, ethyl spacers and three (C12-AEP) or four nitrogens (C12-BAEP) were identified as the most effective, exhibiting potencies comparable to or exceeding C12-200 in the in vitro knockout model. This study reports a systematic evaluation of ionisable oligoamine-lipidoids in Cas9-RNP-LNP formulations, highlights critical delivery bottlenecks, and provides recommendations for the design of potent candidates.},
}

*Nanoparticles/chemistry/administration & dosage
*Lipids/chemistry/administration & dosage
*Ribonucleoproteins/administration & dosage/chemistry
*CRISPR-Associated Protein 9/administration & dosage/chemistry
Humans
*Amines/chemistry/administration & dosage
CRISPR-Cas Systems
Gene Editing
Liposomes

@article {pmid42406843,
year = {2026},
author = {Morianou, I and Phillimore, L and Khatri, BS and Marston, L and Gribble, M and Burt, A and Bernardini, F and Hammond, AM and Nolan, T and Crisanti, A},
title = {Engineering resilient gene drives for sustainable malaria control by predicting, testing and overcoming target site resistance in Anopheles gambiae.},
journal = {PLoS biology},
volume = {24},
number = {7},
pages = {e3003879},
pmid = {42406843},
issn = {1545-7885},
mesh = {Animals ; *Anopheles/genetics ; *Gene Drive Technology/methods ; Female ; *Mosquito Control/methods ; *Malaria/prevention & control/transmission ; Mosquito Vectors/genetics ; *Insecticide Resistance/genetics ; Alleles ; CRISPR-Cas Systems ; },
abstract = {CRISPR-based gene drives are selfish genetic elements with the potential to spread through entire insect populations for sustainable vector control. Gene drives designed to disrupt the reproductive capacity of females can suppress laboratory populations of the malaria mosquito, Anopheles gambiae. However, any suppressive intervention will inevitably exert an evolutionary pressure for resistance, and the likelihood of resistance emerging at natural population scales remains poorly defined. Here, we present a pipeline to quantify the evolutionary space for resistance, enabling accelerated discovery, engineering, and testing of both natural and drive-induced variants that could reverse gene drive spread. We applied our approach to stress-test a best-in-class suppression gene drive that has evaded resistance in all laboratory-contained releases to date, known as Ag(QFS)1. We showed that previously undetected resistant alleles can arise at low frequency, including a novel type of partially resistant alleles that can perturb drive-invasion dynamics. Integrating experimentally derived resistance rates with population genetic modeling shows that single-target suppression drives are unlikely to be robust at natural mosquito population sizes, even at highly constrained loci. Here, we engineer and validate multiplexed gene drives in Anopheles gambiae, that target multiple conserved sites, actively removing resistant alleles. Our models predict that such gene drives could supress large natural mosquito populations in the field.},
}

Animals
*Anopheles/genetics
*Gene Drive Technology/methods
Female
*Mosquito Control/methods
*Malaria/prevention & control/transmission
Mosquito Vectors/genetics
*Insecticide Resistance/genetics
Alleles
CRISPR-Cas Systems

@article {pmid42454502,
year = {2026},
author = {Madhusudan, S and Eskici, N and Gomez-Sanchez, C and Pulli, K and Vaaralahti, K and Yellapragada, V and Conway, JRW and Wang, Y and Raivio, T},
title = {CRISPR activation of DLX5 drives neural progenitors to the GnRH cell fate.},
journal = {Journal of molecular endocrinology},
volume = {77},
number = {1},
pages = {},
doi = {10.1530/JME-26-0040},
pmid = {42454502},
issn = {1479-6813},
mesh = {*Gonadotropin-Releasing Hormone/metabolism ; Humans ; *Homeodomain Proteins/genetics/metabolism ; Cell Differentiation/genetics ; *Neural Stem Cells/metabolism/cytology ; Neurons/metabolism/cytology ; Animals ; Cell Movement/genetics ; *CRISPR-Cas Systems/genetics ; Fibroblast Growth Factor 8/metabolism/genetics ; Mice ; Hypothalamic-Pituitary-Gonadal Axis ; *Cell Lineage/genetics ; Transcription Factors ; },
abstract = {Gonadotropin-releasing hormone (GnRH) neurons regulate the hypothalamic-pituitary-gonadal (HPG) axis and are required for puberty onset and reproductive competence. However, the transcriptional regulators governing GnRH neuron specification and migration remain poorly defined. The homeodomain transcription factor DLX5 is expressed in fetal human GnRH neurons, its expression precedes that of GNRH1 in human pluripotent stem cell (hPSC)-derived GnRH neurons, and in mice, it serves as a guidance cue for GnRH neuron migration. We hypothesized that DLX5 may act as an upstream regulator of human GnRH neuron fate specification and migratory capacity. Using CRISPR activation, we upregulated DLX5 during FGF8b-directed differentiation of hPSCs to GnRH neurons via dual SMAD inhibition and Notch inhibition, as previously described. DLX5 activation increased neural progenitor motility (P < 0.001), upregulated FGF8 (P < 0.05), and induced GABAergic markers, including GAD1 and GAD2. Notably, DLX5 activation induced GNRH1 in the absence of exogenous FGF8b (P < 0.05), suggesting that in GnRH neurons, DLX5 regulates FGF8. When combined with exogenous FGF8b, DLX5 activation produced distinct neuronal patterning accompanied by upregulation of extracellular matrix genes, such as SPARC, which has been implicated in neurite outgrowth. Collectively, these data indicate that activation of DLX5 promotes GnRH neurogenesis from hPSCs, by driving GABAergic fate, inducing FGF8, and remodeling the extracellular matrix.},
}

*Gonadotropin-Releasing Hormone/metabolism
Humans
*Homeodomain Proteins/genetics/metabolism
Cell Differentiation/genetics
*Neural Stem Cells/metabolism/cytology
Neurons/metabolism/cytology
Animals
Cell Movement/genetics
*CRISPR-Cas Systems/genetics
Fibroblast Growth Factor 8/metabolism/genetics
Mice
Hypothalamic-Pituitary-Gonadal Axis
*Cell Lineage/genetics
Transcription Factors

@article {pmid42458955,
year = {2026},
author = {Kigaru, A and Ateka, EM and Pappu, HR and Nganga, EM and Murori, R and Toili, ME and Runo, S},
title = {Enhanced Rice Yellow Mottle Virus Resistance via CRISPR/Cas9-Targeted Mutagenesis of the Rice eIF(iso)4G Gene.},
journal = {Molecular plant pathology},
volume = {27},
number = {7},
pages = {e70312},
pmid = {42458955},
issn = {1364-3703},
support = {//African Union Commission/ ; },
mesh = {*Oryza/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/virology/genetics ; *Plant Viruses/physiology/pathogenicity ; *Mutagenesis/genetics ; *Eukaryotic Initiation Factor-4G/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Genes, Plant ; Mutation/genetics ; },
abstract = {Rice is a staple crop primarily recognised for its high content of carbohydrates and proteins. Rice yellow mottle disease (RYMD) is a destructive disease affecting rice fields in sub-Saharan Africa and is caused by the rice yellow mottle virus (RYMV). Development of virus-resistant genotypes is a highly recommended and effective approach to controlling RYMV. A genetic approach that exploits recessive mutations in susceptibility (S) genes may enhance resistance to the virus. Reports indicate that most rice genotypes grown in Kenya are vulnerable to RYMV infection. Genome editing has shown promise in enhancing agronomic traits in crops. We obtained enhanced resistance to RYMV in the Indica rice cv. IR2793-80-01 using the CRISPR-Cas9 system. The eIF(iso)4G susceptibility gene was targeted because natural mutations in this gene confer recessive resistance to RYMV. A Cas9-OseIF(iso)4G-gRNA-expressing vector targeting the eIF(iso)4G gene was introduced into rice calli via Agrobacterium-mediated transformation. Ten T2 homozygous mutant plant lines were assessed for their reaction to RYMV, and infection was significantly reduced. There were no significant differences in the agronomic characteristics between the T2 mutant lines and the wild-type plants. CRISPR/Cas9-mediated knockout alleles of the eIF(iso)4G gene conferred enhanced resistance to RYMV, which may be classified as partial. Findings underscore the need to embrace precise editing strategies, such as prime editing, to generate superior resistance alleles. Overall, the study provides an alternative resistance enhancement strategy that can create knockout resistance alleles that can be incorporated into breeding programmes for RYMV resistance.},
}

*Oryza/genetics/virology
*CRISPR-Cas Systems/genetics
*Disease Resistance/genetics
*Plant Diseases/virology/genetics
*Plant Viruses/physiology/pathogenicity
*Mutagenesis/genetics
*Eukaryotic Initiation Factor-4G/genetics/metabolism
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
*Genes, Plant
Mutation/genetics

@article {pmid42459963,
year = {2026},
author = {Dasgupta, R and Das, K},
title = {CasPINS: an integrated web-based platform for CRISPR/TALEN gRNA design, primer generation, and indel decomposition analysis.},
journal = {Bioinformatics advances},
volume = {6},
number = {1},
pages = {vbag189},
pmid = {42459963},
issn = {2635-0041},
abstract = {Genome editing researchers currently navigate multiple disconnected tools for guide RNA (gRNA) design, primer generation, and editing analysis-a fragmented workflow that introduces errors and limits reproducibility. CasPINS (Cas-Primer-Indel Suite) addresses this gap as an open-source, unified platform integrating the complete genome editing computational workflow into a single interactive web application accessible without programming expertise. The platform supports 90+ species, 14 CRISPR-Cas variants, TALEN design, and six editing modes. Primer design integrates with Ensembl and NCBI databases relative to cut sites, while indel quantification utilizes Non-Negative Least Squares (NNLS) decomposition of Sanger chromatograms with maximum signal extraction and R 2 -corrected conservative modes. Benchmarking demonstrates strong concordance with established tools, including a 68.8% recovery of CHOPCHOP gRNAs and 67.2% of CRISPOR gRNAs across five human benchmark genes, alongside an algorithmic agreement within 2.6 percentage points on gold-standard TIDE data. Ultimately, a step-count analysis shows that CasPINS significantly streamlines usability, reducing discrete user actions from 25 to 8 steps compared to the traditional sequential-tool pipeline.},
}

@article {pmid42460575,
year = {2026},
author = {Li, M and Huang, D and Xu, C and Fang, M and He, P and He, Y and Wang, X and Xu, Z},
title = {Thermally Unlocked One-Pot RPA-CRISPR Cas12b Assay Integrated with the Centrifugal Microfluidic Chip for Multiplex Detection of Porcine Viruses.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.6c02126},
pmid = {42460575},
issn = {1520-6882},
abstract = {Highly contagious porcine viruses, represented by African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), and pseudorabies virus (PRV), inflict severe economic losses on the swine industry and pose significant threats to global food security. Consequently, developing rapid, convenient, and efficient point-of-care testing (POCT) methods is essential for viral disease control. Although recombinase polymerase amplification (RPA) coupled with CRISPR/Cas systems demonstrates significant POCT potential, its practical application is currently restricted by operational complexity and limited throughput. Herein, a thermally unlocked one-pot RPA-CRISPR Cas12b assay integrated with the centrifugal microfluidic chip (TORCH) platform was developed in this paper. In this strategy, a thermal gating switch was utilized to physically isolate CRISPR reagents from the RPA during the initial phase, effectively addressing the inherent incompatibility in one-pot reactions. By employing the centrifugal microfluidic chip with a portable device, a highly integrated workflow enables fully automated processing ranging from sample lysis to multiplexed detection. Validated using pseudovirus-spiked porcine blood samples, TORCH successfully achieved multiplexed detection of ASFV, PRV, and PRRSV with the limits of detection as low as 0.5 copies/μL, while exhibiting exceptional resistance to interference and robust reagent stability. Overall, TORCH stands as a robust and user-friendly diagnostic solution, holding significant potential for early warning intervention and decentralized biosecurity control in resource-scarce environments.},
}

@article {pmid42462008,
year = {2026},
author = {Skopintsev, P and Esain-Garcia, I and DeTurk, EC and Yoon, PH and Zhou, Z and Weiss, T and Kamalu, M and Chamraj, A and Loi, KJ and Langeberg, CJ and Boger, RS and Nisonoff, H and Karp, HM and Chen, LX and Shi, H and Vohra, K and Banfield, JF and Cate, JHD and Jacobsen, SE and Doudna, JA},
title = {Structure and evolution-guided design of minimal RNA-guided nucleases.},
journal = {Science (New York, N.Y.)},
volume = {393},
number = {6808},
pages = {313-318},
doi = {10.1126/science.aed6123},
pmid = {42462008},
issn = {1095-9203},
mesh = {Humans ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Directed Molecular Evolution/methods ; DNA/chemistry ; *Gene Editing ; Models, Molecular ; Protein Conformation ; *Protein Engineering/methods ; RNA/chemistry ; *RNA, Guide, CRISPR-Cas Systems/chemistry ; *Bacterial Proteins/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; },
abstract = {The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.},
}

Humans
*CRISPR-Cas Systems
Cryoelectron Microscopy
*Directed Molecular Evolution/methods
DNA/chemistry
*Gene Editing
Models, Molecular
Protein Conformation
*Protein Engineering/methods
RNA/chemistry
*RNA, Guide, CRISPR-Cas Systems/chemistry
*Bacterial Proteins/chemistry/genetics
*Endodeoxyribonucleases/chemistry/genetics
*CRISPR-Associated Proteins/chemistry/genetics

@article {pmid42462085,
year = {2026},
author = {Effah, CY and Li, X and Zhang, Q and Ding, L and Drokow, EK and Zhang, X and Wu, Y},
title = {Integrating CRISPR/Cas Biosensors with Advanced Platforms: A Holistic Path Toward Preamplification-free, Multiplexed, and Continuous Molecular Monitoring.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00860},
pmid = {42462085},
issn = {2379-3694},
abstract = {The paradigm of molecular diagnostics has been transformed by the repurposing of CRISPR-Cas systems from being gene-editing tools to nucleic acid detection engines with remarkable specificity and programmability. Both the SHERLOCK and DETECTR platforms have shown high sensitivity and specificity; however, the requirement of a pre-amplification step to achieve clinically relevant detection limits adds another layer of complexity and cost and is also a potential source of contamination, precluding their use as true point-of-care (POC) tools. The next frontier for CRISPR diagnostics will be the design of biosensors that enable preamplification-free, multiplex, and continuous direct detection of targets. Achieving this goal will involve the very close integration of CRISPR biology with nano-biotechnology, microfluidics, orthogonal Cas enzyme systems, and artificial intelligence (AI). This review aims to provide a comprehensive overview of recent advancements and strategic thinking related to this integration. This review discusses how nanomaterials facilitate signal generation and transduction, how microfluidics automates, multiplexes, and miniaturizes "all-in-one" devices, and how orthogonal CRISPR systems can enable robust multiplexing. We will also probe into the emerging application of AI to accelerate guide RNA design and optimize the performance of CRISPR biosensors. Furthermore, the roles of orthogonality and nanomaterials in real-time, continuous molecular monitoring will be assessed. The review will finally discuss the transformative future applications of high-throughput biomarker discovery and theranostics potential through massively parallelized CRISPR sensing.},
}

@article {pmid42462700,
year = {2026},
author = {Gao, Y and Wang, B},
title = {Programmable cell killer: CRISPR-Cas12a2 eliminates cells via RNA identity.},
journal = {Molecular cell},
volume = {86},
number = {14},
pages = {2662-2664},
doi = {10.1016/j.molcel.2026.06.029},
pmid = {42462700},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; Humans ; *RNA/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; },
abstract = {In a recent issue of Nature, Scholz et al.[1] apply the RNA-triggered DNA shredding activity of CRISPR-Cas12a2 in eukaryotic cells to enable programmable elimination of yeast and human cells expressing target transcripts with single-nucleotide resolution specificity and non-detectable off-target activity.},
}

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

@article {pmid42462716,
year = {2026},
author = {Hong, A and Liu, M and Truta, A and Talaie, A and Smith, GR and Bondy-Denomy, J},
title = {Gabija restricts phage circularization and DNA replication.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2026.06.016},
pmid = {42462716},
issn = {1934-6069},
abstract = {Anti-bacteriophage systems such as restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable the identification of invaders. How Gabija, a highly prevalent nuclease-helicase antiphage system, limits phage replication while executing self- vs. non-self-discrimination remains unknown. Here, we show that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting a temperate lambda-like phage in Pseudomonas aeruginosa, Gabija prevents phage genome circularization and subsequent replication. DNA end-binding complexes, including a phage exonuclease and a single-stranded DNA (ssDNA)-annealing protein or GamMu dimers that prevent loading of the host repair complex RecBCD, are necessary and sufficient to license phage and plasmid sensitivity to Gabija. Mutant escape phages lacking these DNA end-binding proteins become protected from Gabija by RecBCD translocation activities. RecBCD activity on the bacterial genome, presumably whenever it is linearized, also prevents Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes the circularization and replication of linear DNA devoid of RecBCD as a mechanism to identify and antagonize foreign invaders.},
}

@article {pmid42463576,
year = {2026},
author = {Li, Q and Wang, H and He, Y and Hong, G and Bao, G},
title = {Gene editing of hematopoietic stem cells: applications and advances.},
journal = {International journal of hematology},
volume = {},
number = {},
pages = {},
pmid = {42463576},
issn = {1865-3774},
abstract = {Allogeneic hematopoietic stem cell transplantation remains the standard treatment for various hematologic genetic disorders resulting from single or multiple genes. However, this strategy is hindered by two main problems: failure to find a matching donor and the risk of graft-versus-host disease (GVHD) after transplantation. Recent advances in gene editing, particularly nucleases exemplified by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) and related derivative tools, have overcome the limitations posed by the poor specificity of traditional gene modification techniques. A robust groundwork has been established for developing efficient, precise, and diverse gene editing strategies, facilitating the clinical application of ex vivo modified autologous hematopoietic stem cells (HSCs). In contrast, autologous HSC transplantation does not have the previously mentioned problems associated with allogeneic transplantation. Consequently, gene editing involving ex vivo genetic modification of HSCs and subsequent reinfusion in a single patient has emerged, with related research progressing from investigation into fundamental mechanisms and proof-of-concept studies to clinical trials.},
}

@article {pmid42463755,
year = {2026},
author = {Nevard, K and Gonzalez, E and Harvey-Samuel, T and Sanders, C and Homem, RA},
title = {CRISPR-Cas9 mediated knockout of the white gene in the bluetongue virus vector, Culicoides sonorensis (biting midge).},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42463755},
issn = {2045-2322},
mesh = {Animals ; *Ceratopogonidae/genetics/virology ; *CRISPR-Cas Systems ; *Bluetongue virus ; Female ; *Insect Vectors/genetics/virology ; *Gene Knockout Techniques ; *Insect Proteins/genetics ; Gene Editing ; },
abstract = {Culicoides biting midges are small blood feeding insects responsible for the transmission of important arthropod-borne viruses (arboviruses) such as bluetongue virus (BTV), Schmallenberg virus (SBV) and epizootic hemorrhagic disease virus (EHDV), which cause major losses to livestock production worldwide. Culicoides sonorensis is the primary vector of BTV in North America and one of the few Culicoides species to be colonised and reared in artificial conditions. Gene editing technology has been used to explore virus-vector interactions in other vector groups, particularly within mosquitoes. Despite the availability of a reference genome since 2018, to date there have been no reports of gene editing in C. sonorensis. Here, we report the first instance of gene editing in C. sonorensis, achieved by intrathoracic injection of adult females with Cas9 and sgRNAs targeting the white gene. We generated heritable mutations in the white gene which produced both white eye and red eye phenotypes and went on to establish a homozygous knockout line carrying a single mutation. We observed gene editing efficiencies of up to 12.3%, making this an efficient protocol for genetic manipulation of Culicoides biting midges, opening the door to functional genomics studies and the development of control strategies in these important and understudied disease vectors.},
}

Animals
*Ceratopogonidae/genetics/virology
*CRISPR-Cas Systems
*Bluetongue virus
Female
*Insect Vectors/genetics/virology
*Gene Knockout Techniques
*Insect Proteins/genetics
Gene Editing

@article {pmid42463825,
year = {2026},
author = {Taha, BA and Addie, AJ and Haider, AJ and Ibnaouf, K and Arsad, N},
title = {Reversing cancer cell behavior using AI-guided CRISPR and quantum nanobiology: a systems-based approach to epigenetic reprogramming.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {42463825},
issn = {1476-5462},
abstract = {Treatment effectiveness is hindered by the phenotypic plasticity of cancer and the genetic complexity of tumors. However, CRISPR-Cas-based medicines face challenges with specificity, off-target effects, and tumor heterogeneity adaptability. This work investigates the possible combination of quantum biological processes, artificial intelligence, and nanomaterials to improve CRISPR gene editing and modulate or reverse selected malignant phenotypes. Quantum machine learning (QML) can be used to simulate quantum processes like electron tunneling in DNA repair and spin-dependent enzyme activity. To enable exact tumor phenotypic reversal, these models will be combined with optimization approaches powered by AI to direct CRISPR editing in oncogenic signaling networks. Graphene, gold nanoparticles, and lipid-based vectors are some of the nanomaterials that will be used as carriers to effectively and deliver CRISPR systems in a biocompatible manner to the cancer microenvironment. We hypothesize that selected homeostatic gene-expression states may be partially restored in experimental cancer models through the integration of quantum-informed AI, CRISPR gene alteration, and nanomaterial delivery. This integrated strategy could support future cancer therapies that move beyond tumor suppression toward controlled modulation of malignant cell states, although substantial preclinical and clinical validation remains necessary.},
}

@article {pmid42464282,
year = {2026},
author = {Zheng, W and Wang, M and Tu, Q and Bian, X and Zhang, Y and Wang, X},
title = {Establishment of SRLC: a multiplex genome editing technology for Saccharomyces cerevisiae and its application in metabolic engineering of malonyl-CoA pathway.},
journal = {Microbial cell factories},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12934-026-03068-w},
pmid = {42464282},
issn = {1475-2859},
support = {2023YFC3402003//National Key R&D Program of China/ ; },
abstract = {The development of advanced genome engineering tools is crucial for optimizing metabolic pathways in Saccharomyces cerevisiae and achieving efficient biomanufacturing. This study proposes an enhancing multiplex genome editing strategy in S. cerevisiae by employing Escherichia coli-derived single-stranded annealing proteins (SSAPs) combined with S. cerevisiae-derived homologous recombinases (Rad51 and Rad52). The strategy utilizes an SSAP-Rad-Linearized CRISPR (SRLC) platform, which supports efficient simultaneous editing of multiple genomic loci without constructing complex multi-gRNA expression vectors. Co-overexpressing Rad51/Rad52 and E. coli SSAP proteins significantly enhances homologous recombination (HR), allowing precise multi-locus genome editing mediated by short homologous arms. Furthermore, SRLC employs a linearized CRISPR-Cas system to stimulate homologous recombination and enable counter-selection in S. cerevisiae, thereby improving precise multiplex genome editing efficiency. We applied SRLC to engineer the malonyl-CoA metabolic pathway in S. cerevisiae. Through a single round of editing and screening, we constructed a chassis strain with 9 targets simultaneously modification and achieved a 9.6-fold increase in intracellular malonyl-CoA. Using this chassis, 3-hydroxypropionic acid production increased 4.5-fold relative to wild-type S. cerevisiae. This platform offers a robust and scalable tool for S. cerevisiae manipulation and a practical pathway-engineering strategy for building for malonyl-CoA-derived factories.},
}

@article {pmid42115602,
year = {2026},
author = {Larrosa-Godall, M and Shackleford, L and Edgington, MP and Leftwich, PT and Luk, JCY and Southworth, J and Rosell, S and Creasey, JT and Aked, JM and Nevard, K and Dodds, A and Mckee, M and Adedeji, E and Gonzalez, E and Ang, JXD and Anderson, MAE and Alphey, L},
title = {Integrating multiplexing into confineable gene drives effectively overrides resistance in Anopheles stephensi.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42115602},
issn = {2041-1723},
support = {INV-008549/GATES/Gates Foundation/United States ; BBS/E/I/00007033, BBS/E/I/00007038, BBS/E/I/00007039//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; INV-008549/GATES/Gates Foundation/United States ; },
mesh = {Animals ; *Anopheles/genetics ; CRISPR-Cas Systems/genetics ; Alleles ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Insecticide Resistance/genetics ; Malaria/transmission/prevention & control ; *Mosquito Control/methods ; },
abstract = {Anopheles stephensi is a major malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012 it has invaded several countries of eastern Africa, stimulating urgent efforts to develop more efficient strategies for vector control such as CRISPR/Cas9-based homing gene drives. Target site resistance due to end-joining repair is a significant challenge to the deployment of these systems. The use of multiple sgRNAs has the potential to solve this issue. Here we perform experimental crosses to assess the homing and cutting efficiency of both classical (e.g. four adjacent sgRNAs all in one construct) and additive (e.g. separate constructs each expressing a single sgRNA) multiplexing strategies targeting the cardinal locus, in the presence and absence of a resistance allele. We find resistance alleles at one sgRNA target site can be mitigated by the presence of the additional sgRNAs with either strategy, and do not significantly reduce the homing efficiency for either strategy, validating their effectiveness. Further modelling using parameters derived from the strains generated indicates that while both strategies can overcome resistance allele formation, the fitness of the drive-carrying alleles is a critical factor in determining the overall performance and persistence of a split drive.},
}

Animals
*Anopheles/genetics
CRISPR-Cas Systems/genetics
Alleles
*Gene Drive Technology/methods
*Mosquito Vectors/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Insecticide Resistance/genetics
Malaria/transmission/prevention & control
*Mosquito Control/methods

@article {pmid42154535,
year = {2026},
author = {Ziegler, M and Günter, C and Alecu, JE and Xue, X and Kim, HM and Saffari, A and Davies, AK and Sahin, M and Ebrahimi-Fakhari, D},
title = {CRISPR/Cas9 loss-of-function screen in a neuronal model of AP-4 deficiency identifies ATG9A trafficking modulators.},
journal = {JCI insight},
volume = {11},
number = {14},
pages = {},
doi = {10.1172/jci.insight.202204},
pmid = {42154535},
issn = {2379-3708},
mesh = {Humans ; *Membrane Proteins/metabolism/genetics ; *Vesicular Transport Proteins/metabolism/genetics ; CRISPR-Cas Systems ; *Autophagy-Related Proteins/metabolism/genetics ; *Neurons/metabolism ; Protein Transport/genetics ; *Adaptor Protein Complex 4/genetics/deficiency/metabolism ; trans-Golgi Network/metabolism ; *Spastic Paraplegia, Hereditary/genetics/metabolism ; Loss of Function Mutation ; },
abstract = {Biallelic loss-of-function variants in adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and a degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the "druggable genome," in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting that they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.},
}

Humans
*Membrane Proteins/metabolism/genetics
*Vesicular Transport Proteins/metabolism/genetics
CRISPR-Cas Systems
*Autophagy-Related Proteins/metabolism/genetics
*Neurons/metabolism
Protein Transport/genetics
*Adaptor Protein Complex 4/genetics/deficiency/metabolism
trans-Golgi Network/metabolism
*Spastic Paraplegia, Hereditary/genetics/metabolism
Loss of Function Mutation

@article {pmid42156946,
year = {2026},
author = {di Lillo, A and Tavella, S and Iannelli, F and Crisafulli, G and Gioia, U and Trastus, LA and Cabrini, M and d'Adda di Fagagna, F},
title = {Site-specific DNA double-strand break induces local transcription in cis and protein expression.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {42156946},
issn = {2399-3642},
support = {21762//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; 22458//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; 30471//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *Transcription, Genetic ; Humans ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; },
abstract = {The DNA damage response is a complex network of pathways that cells activate to safeguard genome integrity following DNA damage, including DNA double-strand breaks. We and others previously reported that RNA polymerase II, together with components of the preinitiation complex, is recruited to exposed DNA ends. This results in the assembly of a fully competent transcriptional apparatus and the synthesis of damage-induced long non-coding RNAs, which are necessary for full DNA damage response activation. Thus, DNA double-strand breaks could act as transcriptional promoters. Whether such DNA breaks, generated upstream of an open reading frame lacking a transcriptional promoter and followed by a polyadenylation signal, can induce the transcription of a coding RNA that is subsequently translated into a protein product remains unknown. Here, taking advantage of the CRISPR/Cas9 technology, we generate a sequence-specific double-strand break upstream of a promoter-less, and therefore silent, reporter gene in two distinct cellular systems. In both cell models, a DNA double-strand break is sufficient to trigger the expression of polyadenylated transcripts and a protein product. Collectively, our results demonstrate that DNA double-strand breaks can act as functional promoters capable of driving protein synthesis, revealing an additional mechanism through which DNA damage can regulate gene expression.},
}

*DNA Breaks, Double-Stranded
*Transcription, Genetic
Humans
Promoter Regions, Genetic
CRISPR-Cas Systems

@article {pmid42191711,
year = {2026},
author = {Feng, X and Ding, J and Liu, Y and Lopez Del Amo, V and Gantz, VM and Chen, XX and Champer, J and Liu, F},
title = {Self-limiting population suppression gene drive design in the West Nile vector mosquito, Culex quinquefasciatus.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42191711},
issn = {2041-1723},
support = {82372289//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82202559//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32270672//National Natural Science Foundation of China (National Science Foundation of China)/ ; MS25C140016//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; },
mesh = {Animals ; *Culex/genetics/virology ; Female ; *Mosquito Vectors/genetics/virology ; *Gene Drive Technology/methods ; West Nile virus ; Male ; CRISPR-Cas Systems ; *West Nile Fever/transmission/virology ; Mosquito Control/methods ; Alleles ; },
abstract = {Culex quinquefasciatus is a major vector of West Nile virus and other pathogens, yet genetic population suppression tools for this species remain limited. Here, we develop a self-limiting, CRISPR-based suppression gene drive system targeting doublesex, close to the male-determining locus, promoting male transmission. A recoded dsxM sequence converts females into sterile intersexes, preventing population-level spread. The drive achieves super-Mendelian inheritance (~ 71%) and generates resistance alleles that are fully or partially dominant female sterile. Single-release cage trials show extended but self-limiting population suppression. Population modeling of this RIDD (Release of Insects carrying a Dominant-sterile Drive) system further indicates that repeated releases can substantially reduce fertile female numbers at low release ratios and intrinsic growth rates, outperforming non-drive strategies under comparable conditions. Together, these results establish a self-limiting suppression gene drive platform for Culex, providing a confined and sustainable framework for vector population control.},
}

Animals
*Culex/genetics/virology
Female
*Mosquito Vectors/genetics/virology
*Gene Drive Technology/methods
West Nile virus
Male
CRISPR-Cas Systems
*West Nile Fever/transmission/virology
Mosquito Control/methods
Alleles

@article {pmid42192102,
year = {2026},
author = {Gong, Y and Shi, S and Li, L and Qian, Y and Lu, T and Zhang, Z and Jiang, L and Liu, G and Cui, M and Li, S and Li, Z and Lin, H},
title = {PIWIL3-piRNA pathway controls rabbit oogenesis and embryogenesis via broad regulation of the transcriptome and proteome.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42192102},
issn = {2041-1723},
mesh = {Animals ; Female ; *Oogenesis/genetics ; *Piwi-Interacting RNA/metabolism/genetics ; *Argonaute Proteins/metabolism/genetics ; *Embryonic Development/genetics ; Rabbits ; Oocytes/metabolism ; *Transcriptome/genetics ; *Proteome/metabolism/genetics ; *RNA, Small Interfering/metabolism/genetics ; Humans ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Fertility/genetics ; },
abstract = {Female infertility often arises from oogenic defects, yet the underlying molecular mechanisms remain elusive. The Piwi-piRNA pathway is crucial for gametogenesis, but its role in mammalian female fertility remains unclear, partly due to reliance on mouse models lacking PIWIL3. PIWIL3 exits in most other placental mammals and is highly expressed in human oocytes, but its function remains largely unexplored. Here, we show that rabbit PIWIL3 closely resembles its human counterpart and is the predominant PIWI protein in oocytes. Using CRISPR-Cas9 knockout, we demonstrate that PIWIL3 is essential for female fertility in rabbits, its loss leads to severe defects in oogenesis. Embryos lacking maternal PIWIL3 arrest by the 8-cell stage. Mechanistically, PIWIL3 binds ~18-nucleotide piRNAs, supports piRNA biogenesis, and regulates transcriptomic, proteomic, and transposable element dynamics during oocyte maturation and early embryogenesis. These findings establish PIWIL3 as an essential regulator of female fertility in non-rodent mammals, potentially including humans.},
}

Animals
Female
*Oogenesis/genetics
*Piwi-Interacting RNA/metabolism/genetics
*Argonaute Proteins/metabolism/genetics
*Embryonic Development/genetics
Rabbits
Oocytes/metabolism
*Transcriptome/genetics
*Proteome/metabolism/genetics
*RNA, Small Interfering/metabolism/genetics
Humans
Gene Expression Regulation, Developmental
CRISPR-Cas Systems
Fertility/genetics

@article {pmid42202890,
year = {2026},
author = {Li, L and Zhao, N and Ding, K and Zuo, L and Gan, Y and Liu, Y and Kong, L and Zhang, X and Zhao, Y},
title = {Development of a CRISPR/RspCas13d-based on-site rapid detection system for GII Norovirus.},
journal = {Journal of virological methods},
volume = {345},
number = {},
pages = {115421},
doi = {10.1016/j.jviromet.2026.115421},
pmid = {42202890},
issn = {1879-0984},
mesh = {*Norovirus/genetics/isolation & purification ; Humans ; Sensitivity and Specificity ; *Caliciviridae Infections/diagnosis/virology ; Feces/virology ; Rapid Diagnostic Tests ; *Gastroenteritis/virology/diagnosis ; RNA, Viral/genetics ; DNA Primers/genetics ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Noroviruses (NoVs) are major cause of acute viral gastroenteritis and a serious public health concern. Current detection methods are limited in rapidity, equipment requirements, or sensitivity. In this study, we developed a rapid, sensitive, and specific detection assay for GII NoV by combining RT-RAA, T7 transcription, and the RspCas13d system. The RspCas13d protein was expressed and purified. RT-RAA primers and crRNA were designed against the conserved region of GII Nov. The assay was optimized and evaluated for specificity, sensitivity, and clinical performance. Results showed that the RT-RAA-RspCas13d method exhibited high specificity without cross-reactivity to other common enteric viruses. The limit of detection was 5 copies/μL. In clinical fecal samples, the assay showed high consistency with RT-qPCR. This method is rapid, simple, sensitive, and specific, providing a reliable tool for the rapid on-site detection of GII NoV.},
}

*Norovirus/genetics/isolation & purification
Humans
Sensitivity and Specificity
*Caliciviridae Infections/diagnosis/virology
Feces/virology
Rapid Diagnostic Tests
*Gastroenteritis/virology/diagnosis
RNA, Viral/genetics
DNA Primers/genetics
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods

@article {pmid42270039,
year = {2026},
author = {Krishnamurthy, KA and Xiao, R and Rutten, MGS and Bos, T and Bleeker, A and Zhang, M and de Vries, HI and Koster, M and Huijkman, N and Smit, M and Kloosterhuis, N and Boer, T and Schomakers, B and van Weeghel, M and van de Sluis, B and Wolters, JC and Bakker, BM and Oosterveer, MH},
title = {Hepatocyte-specific Cas9-mediated editing of G6pc and Slc37a4 elicits comparable biochemical and regulatory responses between glycogen storage disease (GSD) type Ia and Ib mice.},
journal = {Molecular metabolism},
volume = {110},
number = {},
pages = {102393},
pmid = {42270039},
issn = {2212-8778},
mesh = {Animals ; Mice ; *Glycogen Storage Disease Type I/metabolism/genetics ; *Hepatocytes/metabolism ; Liver/metabolism/pathology ; *Glucose-6-Phosphatase/genetics/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems ; Male ; Female ; Humans ; Disease Models, Animal ; Proteomics ; *Antiporters/genetics/metabolism ; },
abstract = {BACKGROUND/OBJECTIVE: Glycogen storage disease type I (GSD I) is an autosomal recessive inborn error of carbohydrate metabolism. Patients with GSD type Ia and Ib exhibit overlapping and distinct symptoms and complications. Notably, GSD Ia patients show more severe hypertriglyceridemia and higher risk of hepatic tumors than GSD Ib patients.

METHODS: Given the liver's pivotal role in these processes, this study utilized hepatocyte-specific CRISPR/Cas9-mediated somatic gene editing to explore the pathophysiological and biochemical adaptations in hepatic GSD Ia and Ib side-by-side. Additionally, hepatic histology, transcriptomics, and proteomics analysis was performed.

RESULTS: Compared to controls, hepatic GSD Ia and Ib mice showed hepatomegaly, fasting hypoglycemia, hyperlactatemia, and increased uric acid in plasma, which was somewhat more pronounced in GSD Ia than Ib. Both GSD I subtypes showed similar reductions in hepatic acetyl-CoA precursor pool enrichment and increases in de novo biosynthesis of hepatic stearate and oleate. Interestingly, only GSD Ia mice showed mildly elevated plasma triglyceride and hepatic phosphate sugars. Metabolic changes were reflected at the transcriptomic and proteomic levels, with largely similar responses between GSD Ia and Ib livers. Moreover, altered mRNAs and protein levels related to nucleotide-binding oligomerization domain (NOD) signaling pathways, infection and inflammation, liver disease, and chemical carcinogenesis were somewhat more pronounced in hepatic GSD Ia than in GSD Ib mice.

CONCLUSIONS: Overall, the metabolic disturbance was more severe in hepatocyte-specific GSD Ia than in GSD Ib mice, consistent with the clinical phenotype in patients. The metabolic disorders and specific metabolites, genes, and proteins identified in this study provided new insights into the pathophysiological and biochemical phenotypes of GSD I subtypes in the liver.},
}

Animals
Mice
*Glycogen Storage Disease Type I/metabolism/genetics
*Hepatocytes/metabolism
Liver/metabolism/pathology
*Glucose-6-Phosphatase/genetics/metabolism
Gene Editing/methods
CRISPR-Cas Systems
Male
Female
Humans
Disease Models, Animal
Proteomics
*Antiporters/genetics/metabolism

@article {pmid42455481,
year = {2026},
author = {Marques, BS and Mendes, M and Alves, JL and Pais, A and Vitorino, C},
title = {Decoding and Overcoming Temozolomide Resistance Through CRISPR/Cas Technologies.},
journal = {Molecular diagnosis & therapy},
volume = {},
number = {},
pages = {},
pmid = {42455481},
issn = {1179-2000},
support = {2022.06174.PTDC//Fundação para a Ciência e a Tecnologia/ ; UID/PRR/00313/2025//Fundação para a Ciência e a Tecnologia/ ; UID/00313/2025//Fundação para a Ciência e a Tecnologia/ ; },
abstract = {Intrinsic and acquired resistance to temozolomide (TMZ), the standard chemotherapeutic agent for glioblastoma (GBM), is highly common and results in poor clinical outcomes. This review highlights the emerging dual role of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) technologies in addressing this challenge. First, it examines genome-wide CRISPR screens that revealed DNA damage repair networks, stress adaptations, stemness maintenance, and tumor heterogeneity as key drivers of resistance to TMZ. Second, it examines CRISPR/Cas-based strategies, including targeted gene disruption and epigenetic silencing of O[6]-methylguanine-DNA methyltransferase (MGMT), to restore TMZ sensitivity. Finally, it explores CRISPR/Cas-engineered brain tumor models. Alongside these approaches, CRISPR/Cas technologies highlight the value of decoding the multifactorial basis of TMZ resistance and guiding rational therapeutic strategies. Continued refinement of CRISPR/Cas tools may ultimately contribute to more effective treatments for GBM.},
}

@article {pmid42455488,
year = {2026},
author = {Wu, H and Jiang, F and Tian, S},
title = {Research progress on detection technologies for Mycoplasma pneumoniae.},
journal = {European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {},
number = {},
pages = {},
pmid = {42455488},
issn = {1435-4373},
abstract = {Mycoplasma pneumoniae (M. pneumoniae) is a primary pathogen responsible for community-acquired pneumonia (CAP), particularly prevalent among children and adolescents. The recent global resurgence of infection cases, coupled with the rapid dissemination of macrolide-resistant M. pneumoniae (MRMP), underscores the critical clinical need for rapid and precise diagnostic technologies. This article systematically reviews the evolutionary trajectory of detection technologies, covering the transition from traditional culture and serological testing to modern molecular diagnostic techniques. It critically analyzes emerging platforms, including isothermal amplification, CRISPR/Cas-based diagnostics, microfluidic chips, and biosensors. It explores their potential in facilitating point-of-care testing (POCT) and simultaneous antimicrobial resistance profiling. Despite continuous technological advancements, challenges remain regarding the differentiation between active infection and colonization, as well as the balancing of cost-effectiveness. The future of M. pneumoniae diagnostics lies in the deep integration of multidisciplinary biotechnologies with artificial intelligence, aiming to construct intelligent "sample-to-answer" solutions to optimize clinical antimicrobial stewardship.},
}

@article {pmid42455881,
year = {2026},
author = {Gao, X and Hao, J and Lu, S and Wang, S and Sun, Y and Ke, X and Gao, X and Su, Y and Sun, Y and Tian, Y and Yan, W and Wang, J and Zheng, Z and Hai, R and Zhang, Q and Wang, J and Hu, W and Wang, G},
title = {Genome-wide CRISPR screen reveals PEX11B as a host restriction factor against ORFV through membrane fluidity regulation.},
journal = {PLoS pathogens},
volume = {22},
number = {7},
pages = {e1013767},
pmid = {42455881},
issn = {1553-7374},
mesh = {Animals ; Sheep ; *Membrane Fluidity ; *Host-Pathogen Interactions ; *Parvoviridae Infections/genetics/virology/metabolism/veterinary ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Virus Replication ; Male ; Clustered Regularly Interspaced Short Palindromic Repeats ; Antigens, Differentiation ; },
abstract = {Host-pathogen interactions are shaped by cellular restriction factors that direct antiviral defenses. We built the first ovine genome-wide CRISPR knockout library in sheep testis (OA3.Ts) cells, targeting all protein-coding genes. Using this platform, we identified PEX11B, a peroxisomal membrane regulatory protein, as a strong restriction factor against orf virus (ORFV) infection. Removing PEX11B increased viral susceptibility and triggered severe cytopathic effects with membrane fusion and syncytia formation. Mechanistic studies showed that PEX11B knockout harmed peroxisomal integrity and disrupted lipid metabolism. This led to greater plasma membrane fluidity, creating a proviral environment that allowed more viral entry and replication. These results reveal a new antiviral function for PEX11B in blocking viral infection and underscore the importance of peroxisomal regulation in host-virus interactions.},
}

Animals
Sheep
*Membrane Fluidity
*Host-Pathogen Interactions
*Parvoviridae Infections/genetics/virology/metabolism/veterinary
*Membrane Proteins/genetics/metabolism
CRISPR-Cas Systems
Virus Replication
Male
Clustered Regularly Interspaced Short Palindromic Repeats
Antigens, Differentiation

@article {pmid42457405,
year = {2026},
author = {Hong, JF and Zou, QL and Xie, XY and Jiang, YP and Wang, SY and Ling, X and Zhou, C and Cai, X and Yang, YX and Chen, Y and Sun, W and Chen, B and Qiao, L},
title = {Engineered promoter system enables high-efficiency transgenic CRISPR editing in Malaria transmitting mosquito Anopheles sinensis.},
journal = {Zoological research},
volume = {47},
number = {4},
pages = {1045-1058},
doi = {10.24272/j.issn.2095-8137.2026.040},
pmid = {42457405},
issn = {2095-8137},
mesh = {Animals ; *Anopheles/genetics ; *Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems ; Animals, Genetically Modified ; Female ; *Gene Editing/methods ; Malaria/transmission ; *Mosquito Vectors/genetics ; },
abstract = {The binary CRISPR/Cas9 system deployed through crosses of transgenic lines facilitates efficient mutagenesis, but its application in non-model insects remains limited by the scarcity of validated species-specific regulatory elements. In the malaria vector Anopheles sinensis, we screened three germline-biased promoters (Asvasa2, Aszpg, Asnanos) for Cas9 expression, and found that Asvasa2 drove the highest editing efficiency with respect to target site mutagenesis. For gRNA transcription, comparative analysis identified AsU6-1 as the most active of four endogenous U6 promoters. Crossing stable transgenic lines harboring these components yielded F 1 progeny with complete germline editing penetrance at the Aswhite locus, a phenotype inherited in the F 2 generation. Quantitative sequencing of F 1 ovaries confirmed near-saturation (>99%) targeted mutagenesis using the optimal Asvasa2/ AsU6-1 combination, whereas alternative promoters showed markedly lower mutagenesis efficiency. Functional validation through knockout of Asdsx- F, a key sex differentiation regulator, efficiently induced complete female-to-male sexual reversal and sterility. This study provides a foundational genetic toolkit for genome engineering in this vector species, as well as an effective reference for binary transgenic manipulation in non-model insects.},
}

Animals
*Anopheles/genetics
*Promoter Regions, Genetic/genetics
*CRISPR-Cas Systems
Animals, Genetically Modified
Female
*Gene Editing/methods
Malaria/transmission
*Mosquito Vectors/genetics

@article {pmid41307817,
year = {2026},
author = {Zhang, F and Lu, Q and Qian, X and Xing, Y and Wang, W},
title = {Development of Knockout Cardiac Muscle Cell Lines Using Integrase-Deficient Lentivirus-Mediated CRISPR/Cas9 Gene Editing.},
journal = {Biochemical genetics},
volume = {64},
number = {4},
pages = {5394-5413},
pmid = {41307817},
issn = {1573-4927},
support = {82101314 to Y. X.; 81772559 to W. W//National Natural Science Foundation of China (NSFC) grants/ ; },
mesh = {*Lentivirus/genetics ; *CRISPR-Cas Systems ; *Myocytes, Cardiac/metabolism/cytology ; Animals ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Integrases/genetics/deficiency ; Cell Line ; Humans ; TRPM Cation Channels/genetics ; Rats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Vectors ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology is a highly efficient genome editing tool that can genetically disrupt genes and genetic elements, making it a timely, cost-effective, and powerful tool for studying gene function. The success of gene editing depends on the ability to introduce CRISPR components, including guide RNA (gRNA) and Cas9 nuclease, into the target cell, which is challenging in numerous difficult-to-transfect cell types, such as cardiomyocytes. Lentiviral vectors (LVs) are among the primary delivery methods for the CRISPR/Cas9 system as they can stably maintain robust expression in various dividing and non-dividing cells. However, stably integrated LVs consistently express CRISPR/Cas9 components at high levels, rendering them susceptible to off-target effects. New-generation integrase-deficient LV (IDLV) offers an attractive alternative approach for delivering CRISPR/Cas9 components. This study constructed transient receptor potential cation channel mucolipin subfamily member 1 gene knockout models in H9C2 cell lines using IDLVs. Strategies for gRNA design and screening, the IDLV packaging process, CRISPR delivery, and knockout validation are outlined. These protocols will assist researchers in the application of CRISPR technology to study gene function in mammalian cells.},
}

*Lentivirus/genetics
*CRISPR-Cas Systems
*Myocytes, Cardiac/metabolism/cytology
Animals
*Gene Editing/methods
*Gene Knockout Techniques/methods
*Integrases/genetics/deficiency
Cell Line
Humans
TRPM Cation Channels/genetics
Rats
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Vectors

@article {pmid42020342,
year = {2026},
author = {He, Z and Liu, M and Zhang, N and Yan, J and Li, F and Zhao, P and Guo, C},
title = {A CRISPR-Cas9 screen identifies LAPTM4A (lysosomal protein transmembrane 4 alpha) as a key host barrier against PRRSV infection.},
journal = {Autophagy},
volume = {22},
number = {8},
pages = {1882-1902},
doi = {10.1080/15548627.2026.2664607},
pmid = {42020342},
issn = {1554-8635},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Autophagy/genetics ; Lysosomes/metabolism ; *Porcine respiratory and reproductive syndrome virus/physiology ; Swine ; *Porcine Reproductive and Respiratory Syndrome/virology/genetics/metabolism ; Humans ; Host-Pathogen Interactions/genetics ; Virus Replication ; Signal Transduction ; Ubiquitination ; HEK293 Cells ; Sequestosome-1 Protein/metabolism ; },
abstract = {Porcine reproductive and respiratory syndrome virus (PRRSV) manipulates host intracellular processes, particularly macroautophagy/autophagy and lysosomal function, to facilitate its replication and spread. However, the precise host factors and molecular mechanisms by which PRRSV remodels the autophagy-lysosome axis remain poorly defined. Here, we performed a CRISPR-Cas9 knockout screen targeting 1,332 genes involved in protein degradation, metabolism, and vesicular trafficking, and identified LAPTM4A (lysosomal protein transmembrane 4 alpha) as a critical antiviral factor involved in the lysosomal pathway. A yeast two-hybrid screen identified LAPTM4A as an interactor of PRRSV GP5 (glycoprotein 5). Mechanistically, GP5 recruits the E3 ubiquitin ligase NEDD4 and the autophagy receptor SQSTM1/p62 to promote K63-linked polyubiquitination of LAPTM4A, leading to its autophagic degradation. This selective degradation activates the AMPK-ULK1-MAP1LC3/LC3 signaling cascade, initiating autophagy while facilitating MTOR-lysosome colocalization, thereby suppressing TFEB nuclear translocation and transcription of lysosome-related genes. The resulting incomplete autophagic flux enhances viral replication. Additionally, in terms of host defense, LAPTM4A maintains lysosomal homeostasis by restraining excessive autophagy through AMPK-ULK1-LC3 signaling and promoting TFEB-dependent lysosomal gene expression by impairing the binding of RPTOR/raptor to MTOR, thus providing broad antiviral protection against multiple RNA viruses. Collectively, our findings identify LAPTM4A as a central regulator of lysosome-autophagy homeostasis and reveal a viral strategy that dismantles this defense axis to facilitate infection.Abbreviations: ATG5: autophagy related 5; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; Baf A1: bafilomycin A1; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DMVT library: protein degradation, metabolism, and vesicular trafficking library; LAPTM4A: lysosomal protein transmembrane 4 alpha; MAGeCK: model-based analysis of genome-wide CRISPR-Cas9 knockout; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NC: negative control; PAMs: porcine alveolar macrophages; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PRRSV: porcine reproductive and respiratory syndrome virus; qRT-PCR: quantitative real-time PCR; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID50: 50% tissue culture infective dose; TFEB: transcription factor EB; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.},
}

Animals
*CRISPR-Cas Systems/genetics
Autophagy/genetics
Lysosomes/metabolism
*Porcine respiratory and reproductive syndrome virus/physiology
Swine
*Porcine Reproductive and Respiratory Syndrome/virology/genetics/metabolism
Humans
Host-Pathogen Interactions/genetics
Virus Replication
Signal Transduction
Ubiquitination
HEK293 Cells
Sequestosome-1 Protein/metabolism

@article {pmid42283463,
year = {2026},
author = {Sugiokto, FG and Liu, Y and Li, R},
title = {Proteomic screening identifies HNRNPA2B1 as an epigenetic repressor of Epstein-Barr virus reactivation.},
journal = {Journal of virology},
volume = {100},
number = {7},
pages = {e0061326},
doi = {10.1128/jvi.00613-26},
pmid = {42283463},
issn = {1098-5514},
mesh = {Humans ; *Herpesvirus 4, Human/physiology/genetics ; *Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism/genetics ; *Epigenesis, Genetic ; Proteomics/methods ; *Virus Activation/genetics ; Histone Demethylases/metabolism/genetics ; Promoter Regions, Genetic ; Gene Expression Regulation, Viral ; Trans-Activators/genetics/metabolism ; Virus Latency/genetics ; *Epstein-Barr Virus Infections/virology/genetics/metabolism ; Histones/metabolism ; Immediate-Early Proteins/genetics/metabolism ; Chromatin Immunoprecipitation ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; Cell Line, Tumor ; },
abstract = {Epstein-Barr virus (EBV) establishes lifelong persistent infection in over 90% of the world's population. The virus persists as an episome in the host cells during latency and periodically reactivates through transcriptional activation of the immediate-early (IE) genes. While epigenetic regulation is central to maintaining viral latency, the host factors that enforce repression at these promoters remain incompletely defined. Here, we employed a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation coupled with mass spectrometry (enChIP-MS) approach to identify proteins associated with the promoter of EBV IE gene ZTA. This approach revealed an enrichment of multiple heterogeneous nuclear ribonucleoproteins and identified HNRNPA2B1 as a potential regulator of EBV ZTA gene expression. Functional analyses across multiple EBV+ cancer cell models demonstrated that HNRNPA2B1 acts as a restriction factor for EBV lytic reactivation. Depletion of HNRNPA2B1 led to increased expression of IE and downstream lytic genes, enhanced RNA polymerase II recruitment to the ZTA and RTA promoters, and elevated the proportion of cells entering the lytic cycle. Conversely, enforced expression of HNRNPA2B1 suppressed EBV lytic reactivation. Mechanistically, HNRNPA2B1 enhances repressive viral chromatin states by facilitating recruitment of the histone demethylase LSD1 to EBV IE gene promoters, thereby limiting the activating histone H3 lysine 4 trimethylation. Together, these findings identify HNRNPA2B1 as a key epigenetic regulator of EBV latency and link RNA-binding proteins to epigenetic control of viral reactivation.IMPORTANCEThis study identifies HNRNPA2B1 as a previously unrecognized host factor that promotes Epstein-Barr virus (EBV) latency through direct regulation of viral chromatin at immediate-early gene promoters. By integrating locus-specific chromatin proteomics with functional and mechanistic analyses, our work reveals how an RNA-binding protein HNRNPA2B1 recruits a histone-modifying enzyme to control EBV reactivation. These findings provide new insights into host-virus interactions that control EBV latency and reactivation and highlight the role of RNA-binding proteins in chromatin regulation that may be broadly relevant to other latent DNA viruses.},
}

Humans
*Herpesvirus 4, Human/physiology/genetics
*Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism/genetics
*Epigenesis, Genetic
Proteomics/methods
*Virus Activation/genetics
Histone Demethylases/metabolism/genetics
Promoter Regions, Genetic
Gene Expression Regulation, Viral
Trans-Activators/genetics/metabolism
Virus Latency/genetics
*Epstein-Barr Virus Infections/virology/genetics/metabolism
Histones/metabolism
Immediate-Early Proteins/genetics/metabolism
Chromatin Immunoprecipitation
CRISPR-Cas Systems
Host-Pathogen Interactions
Cell Line, Tumor

@article {pmid42313514,
year = {2026},
author = {Xu, Y and Kou, S and Huang, X and Cui, D and Guo, Y and Sun, F},
title = {CRISPR-Based Programmable RNA-Responsive Protein Materials.},
journal = {ACS macro letters},
volume = {15},
number = {7},
pages = {1005-1012},
doi = {10.1021/acsmacrolett.6c00237},
pmid = {42313514},
issn = {2161-1653},
mesh = {Pseudomonas aeruginosa ; *CRISPR-Cas Systems ; Hydrogels/chemistry ; *RNA, Viral/analysis/genetics ; Biofilms ; },
abstract = {With the rapid expansion of RNA biology and associated biotechnologies, smart materials with programmable RNA responsiveness offer immense opportunities for biosensing, diagnostics, and therapeutics. Here, we present a programmable RNA-responsive protein material system leveraging CRISPR-Cas7-11, an RNA-guided protease complex. By immobilizing the protease complex and cleavable payload proteins onto protein scaffolds via SpyTag/SpyCatcher chemistry, we developed two platforms: (1) synthetic spider-silk fibers and (2) protein hydrogels. These materials enable sequence-specific RNA detection, triggering the controlled release of payloads such as GFP or the biofilm-degrading enzyme PslG. Applications demonstrated include viral RNA sensing and Pseudomonas aeruginosa detection with targeted biofilm degradation.},
}

Pseudomonas aeruginosa
*CRISPR-Cas Systems
Hydrogels/chemistry
*RNA, Viral/analysis/genetics
Biofilms

@article {pmid42424186,
year = {2026},
author = {Yang, Y and Pan, Q and Liu, M and Zeng, L and Zou, H and Liu, S and Fu, S and Xu, Y and Dai, Z and Fang, S and Pan, Y},
title = {Pan-Cancer Liquid Biopsy and Treatment Monitoring via a Split crRNA-Activated Label-Free CRISPR/Cas12a Platform for Ultrasensitive MicroRNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {28},
pages = {20968-20977},
doi = {10.1021/acs.analchem.6c02601},
pmid = {42424186},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *MicroRNAs/blood/genetics/analysis ; Liquid Biopsy/methods ; Limit of Detection ; *Neoplasms/diagnosis ; Biosensing Techniques/methods ; Cell Line, Tumor ; },
abstract = {Liquid biopsy based on circulating microRNAs (miRNAs) holds great promise for cancer diagnosis and treatment monitoring. However, the development of detection methods that are sensitive, specific, cost-effective, and compatible with diverse biofluids remains a challenge. Here, we report a sensitive and label-free detection platform, termed SCAN (Split crRNA-Activated CRISPR/Cas12a and Amplification Network), that integrates split CRISPR/Cas12a with catalytic hairpin assembly (CHA) for isothermal miRNA analysis. In this design, the target miRNA, serving as an alterable spacer RNA (sRNA), assembles with a conserved repeat RNA (rRNA) to reconstitute a functional full-length crRNA, activating the trans-cleavage activity of Cas12a. This cleaves a blocker probe and releases an initiator strand, which subsequently triggers a CHA cascade. The CHA reaction generates abundant G-quadruplex (G4) structures that bind specifically to N-methylmesoporphyrin IX (NMM), yielding a strong turn-on fluorescence signal. The optimized "signal-on" model achieved a detection limit of 2 fM for miR-21, offering approximately 5 orders of magnitude higher sensitivity than the basic split CRISPR/Cas12a system. The platform exhibited excellent specificity, capable of single-base mismatch discrimination, and could be readily adapted for detecting miR-128, miR-27a, and miR-155 through simple exchange of the double-stranded DNA activator. Importantly, by employing the label-free G4/NMM reporter, the cost of the signaling module was reduced by more than 45-fold compared to conventional dual-labeled probes. The SCAN platform reliably quantified miR-21 overexpression in colon cancer cell lines and robustly differentiated plasma samples from patients with multiple cancer types (colorectal, lung, cervical, breast, and thyroid cancers) from healthy individuals. Furthermore, it demonstrated utility in tracking treatment response through noninvasive urine analysis in prostate cancer and bladder cancer. This work establishes a sensitive, specific, low-cost, and versatile biosensing platform for miRNA-based liquid biopsy, holding strong potential for clinical diagnostic applications.},
}

Humans
*CRISPR-Cas Systems/genetics
*MicroRNAs/blood/genetics/analysis
Liquid Biopsy/methods
Limit of Detection
*Neoplasms/diagnosis
Biosensing Techniques/methods
Cell Line, Tumor

@article {pmid42424600,
year = {2026},
author = {Li, X and Gao, X and Gu, T and Li, Q and Wang, L and Deng, F and Guo, M and Huo, D and Hou, C},
title = {Tribos: A Modular Hairpin-Enhanced CRISPR/Cas12a Biosensor for Ultrasensitive Detection of HER2 Protein.},
journal = {Analytical chemistry},
volume = {98},
number = {28},
pages = {21055-21063},
doi = {10.1021/acs.analchem.6c03581},
pmid = {42424600},
issn = {1520-6882},
mesh = {Humans ; *Erb-b2 Receptor Tyrosine Kinases/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Female ; Breast Neoplasms/diagnosis ; Limit of Detection ; Aptamers, Nucleotide/chemistry/genetics ; Molecular Docking Simulation ; *Bacterial Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Inverted Repeat Sequences ; CRISPR-Associated Proteins ; },
abstract = {Accurate detection of human epidermal growth factor receptor 2 (HER2) is critical for early breast cancer screening and personalized therapy. This study constructed a target-triggered, hairpin-enhanced CRISPR/Cas12a biosensor named "Tribos" for ultrasensitive HER2 detection. The system integrates an aptamer hairpin switch (HAS), HAS-allosterically triggered rolling circle amplification (RCA), and a hairpin-enhanced CRISPR/Cas12a fluorescence reporter module. Taking advantage of Cas12a's high affinity for stem-loop structures, we designed a double-stem-loop reporter probe (DS-FQ) and validated its trans-cleavage enhancement mechanism via molecular docking. Under optimal conditions, Tribos exhibited a linear range from 10 fg/mL to 10 ng/mL, with a limit of detection as low as 1.08 fg/mL. In clinical validation with 29 breast cancer patients and 13 healthy controls, the sensor achieved a sensitivity of 82.76% and a specificity of 100%, which were highly consistent with clinical diagnoses and ELISA results, and it effectively distinguished different HER2 expression levels. The modular design of Tribos offers a new strategy for high-performance CRISPR diagnostics and lays a foundation for next-generation molecular diagnostic technologies based on nucleic acid conformational regulation.},
}

Humans
*Erb-b2 Receptor Tyrosine Kinases/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Female
Breast Neoplasms/diagnosis
Limit of Detection
Aptamers, Nucleotide/chemistry/genetics
Molecular Docking Simulation
*Bacterial Proteins/genetics
*Endodeoxyribonucleases/genetics/metabolism
Inverted Repeat Sequences
CRISPR-Associated Proteins

@article {pmid42451761,
year = {2026},
author = {Yilmaz, I and Yoğurtçu, BM and Aisida, S and Ezer, EB},
title = {Next-Generation Strategies to Encounter Antimicrobial Resistance (AMR): From Lariocidin to Gene Editing and Nanotechnology-Based Approaches.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {13},
pages = {},
pmid = {42451761},
issn = {1420-3049},
mesh = {*Gene Editing/methods ; *Anti-Bacterial Agents/pharmacology/chemistry ; *Nanotechnology/methods ; *Drug Resistance, Bacterial/drug effects/genetics ; Humans ; Biofilms/drug effects ; CRISPR-Cas Systems ; Nanoparticles/chemistry ; },
abstract = {The escalation of antimicrobial resistance (AMR) represents a serious global threat to public health, with AMR-associated mortality estimated to increase by 70% by 2050. As pathogens evolve through enzymatic inactivation, target modification, efflux-mediated clearance, biofilm formation, and broader genetic adaptation, conventional therapies are increasingly compromised, while the antibiotic development pipeline remains critically constrained by high discovery and development costs, weak commercial incentives, and the escalating complexity of resistance mechanisms. This review comprehensively synthesizes advanced pharmacological and biotechnological innovations designed to circumvent these entrenched resistance mechanisms. We highlight the development of novel therapeutic classes, particularly lariocidin, which disrupts bacterial protein synthesis via a previously unexploited ribosomal-binding site. Moreover, we critically evaluate molecular interventions, emphasizing CRISPR/Cas-based gene silencing and genome editing as precise tools to neutralize specific resistance determinants, such as the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA). Concurrently, we explore the integration of engineered nanoparticles to revitalize existing antimicrobials by overcoming biofilm barriers, improving drug solubility, and enabling targeted delivery. Collectively, mastering the evolving AMR landscape requires a multidimensional framework that seamlessly integrates these novel molecular targets with advanced rapid diagnostics and robust international governance.},
}

*Gene Editing/methods
*Anti-Bacterial Agents/pharmacology/chemistry
*Nanotechnology/methods
*Drug Resistance, Bacterial/drug effects/genetics
Humans
Biofilms/drug effects
CRISPR-Cas Systems
Nanoparticles/chemistry

@article {pmid42452230,
year = {2026},
author = {Song, S and Fan, X and Zhang, N and Lin, N and Wang, G},
title = {Molecular Crosstalk Between Flowering Time and Drought Adaptation in Cereal Crops.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {13},
pages = {},
pmid = {42452230},
issn = {2223-7747},
support = {32501990//National Natural Science Foundation of China/ ; 242300421572//Natural Science Foundation of Henan Province/ ; 242300421571//Natural Science Foundation of Henan Province/ ; },
abstract = {Increasingly frequent and severe drought events restrict global agricultural productivity. As sessile organisms, cereal crops have evolved phenotypic plasticity, drawing on drought escape (DE) and drought avoidance (DA) strategies to balance survival and reproduction. While the mechanisms governing photoperiodic flowering and drought responses are well characterized individually, their molecular intersection remains poorly understood. This review summarizes recent advances in the crosstalk between these two pathways. We highlight the divergent roles of core genetic hubs, such as florigen regulation, GIGANTEA (GI), DELLA proteins, and dual-function transcription factors (e.g., ZmCCT, Ghd7, Ppd-H1), and the breeding-selected alleles, including Green Revolution variants, that can partly uncouple stress tolerance from developmental penalties, though trade-offs often remain. Furthermore, we examine the internal networks driving this crosstalk, including circadian clock phase shifts, sugar and energy signaling through the trehalose-6-phosphate (T6P)-SNF1-related protein kinase 1 (SnRK1) module, and the antagonistic balance within phytohormone networks centered on abscisic acid (ABA). Finally, we propose that integrating epigenetic stress memory, systemic root-to-shoot signaling, and targeted CRISPR/Cas promoter engineering provides a useful conceptual framework for breeding climate-resilient, yield-stable crops.},
}

@article {pmid42452277,
year = {2026},
author = {Kim, HJ and Chae, J and Han, SJ and Kim, JH and Chung, YS and Karthik, S and Heo, JB},
title = {AI-Guided DNA-Free and Genotype-Independent Genome Editing for Soybean Improvement.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {13},
pages = {},
pmid = {42452277},
issn = {2223-7747},
abstract = {Soybean is a strategic crop for global protein and vegetable oil supply chains; however, genetic improvement remains constrained by genotype-dependent regeneration, variable transformation efficiency, and regulatory concerns regarding stable transgene integration. This review synthesizes emerging DNA-free and genotype-independent genome-editing frameworks for soybean, where genotype independence is defined as the ability to recover fertile, non-chimeric edited plants across elite germplasm. We critically examine the soybean genome-editing toolbox, including CRISPR-Cas9, Cas12a, multiplex editing systems, base editing, and prime editing, and discuss persistent bottlenecks associated with target selection, off-target assessment, editability, and plant recovery. Particular emphasis is placed on artificial intelligence (AI)-assisted approaches that integrate genomic, epigenomic, chromatin-accessibility, and multi-omics datasets to improve target prioritization, guide RNA design, off-target prediction, and locus- and genotype-specific editability assessment. We further evaluate DNA-free genome-editing technologies, including CRISPR-Cas ribonucleoproteins, transient RNA-based systems, and nanocarrier-mediated delivery platforms, highlighting their potential to generate non-integrative edits while reducing prolonged nuclease exposure. In addition, we discuss regeneration reprogramming strategies based on developmental regulators and morphogenic modules, including BBM-WUS, GRF-GIF, de novo meristem induction, and somatic embryogenesis, as enabling technologies for overcoming cultivar-dependent regeneration barriers. Importantly, this review proposes an integrated AI-to-field framework that connects target discovery, editability prediction, DNA-free editing, regeneration reprogramming, phenotypic validation, and breeding deployment into a unified soybean improvement pipeline. We further highlight emerging opportunities in multi-omics-guided target discovery, genotype-aware prediction models, regeneration-aware editing strategies, and closed-loop machine-learning systems that continuously improve editing decisions through experimental feedback. Collectively, these convergent innovations provide a practical foundation for accelerating the development of climate-resilient, nutritionally enhanced, and industry-ready soybean cultivars.},
}