@article {pmid39898483,
year = {2025},
author = {Guerra-Resendez, RS and Lydon, SL and Ma, AJ and Bedford, GC and Reed, DR and Kim, S and Terán, ER and Nishiguchi, T and Escobar, M and DiNardo, AR and Hilton, IB},
title = {Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00569},
pmid = {39898483},
issn = {2161-5063},
abstract = {Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.},
}

@article {pmid39895071,
year = {2025},
author = {Choi, YJ and Eom, H and Nandre, R and Kim, M and Oh, YL and Kim, S and Ro, HS},
title = {Simultaneous gene editing of both nuclei in a dikaryotic strain of Ganoderma lucidum using Cas9-gRNA ribonucleoprotein.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {1},
pages = {e.2409006},
doi = {10.71150/jm.2409006},
pmid = {39895071},
issn = {1976-3794},
support = {2023R1A 2C1007213//National Research Foundation of Korea/ ; //Rural Development Administration/ ; RS-2024-00322425//New Breeding Technologies Development Program/ ; },
mesh = {*Gene Editing/methods ; *Cell Nucleus/metabolism/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Reishi/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; Orotidine-5'-Phosphate Decarboxylase/genetics/metabolism ; Protoplasts/metabolism ; },
abstract = {The presence of multiple nuclei in a common cytoplasm poses a significant challenge to genetic modification in mushrooms. Here, we demonstrate successful gene editing in both nuclei of a dikaryotic strain of Ganoderma lucidum using the Cas9-gRNA ribonucleoprotein complex (RNP). The RNP targeting the pyrG gene was introduced into dikaryotic protoplasts of G. lucidum, resulting in the isolation of 31 mycelial colonies resistant to 5-fluoroorotic acid (5-FOA). Twenty-six of these isolates were confirmed as dikaryotic strains by the presence of two distinct A mating type markers, denoted as A1 and A2. All dikaryons exhibited clamp connections on their mycelial hyphae, while the remaining 5 transformants were monokaryotic. Subsequent sequence analysis of PCR amplicons targeting pyrG revealed that two dikaryons harbored disrupted pyrG in both nuclei (pyrG-/pyrG-), while 10 and 14 displayed pyrG+/pyrG- (A1/A2) and pyrG-/pyrG+ (A1/A2) configurations, respectively. The disruption was achieved through non-homologous end joining repair, involving deletion or insertion of DNA fragments at the site of the double-strand break induced by RNP. Importantly, the nuclei were stable throughout 10 serial transfers over a period of 6 months. These findings highlight the capability of RNP to target genes across multiple nuclei within the same cytoplasm.},
}

*Gene Editing/methods
*Cell Nucleus/metabolism/genetics
*Ribonucleoproteins/genetics/metabolism
*Reishi/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Fungal Proteins/genetics/metabolism
CRISPR-Associated Protein 9/genetics/metabolism
Orotidine-5'-Phosphate Decarboxylase/genetics/metabolism
Protoplasts/metabolism

@article {pmid39894896,
year = {2025},
author = {Banday, S and Mishra, AK and Rashid, R and Ye, T and Ali, A and Li, J and Yustein, JT and Kelliher, MA and Zhu, LJ and Deibler, SK and Malonia, SK and Green, MR},
title = {The O-glycosyltransferase C1GALT1 promotes EWSR1::FLI1 expression and is a therapeutic target for Ewing sarcoma.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1267},
pmid = {39894896},
issn = {2041-1723},
mesh = {Humans ; Animals ; *Galactosyltransferases/genetics/metabolism ; *RNA-Binding Protein EWS/genetics/metabolism ; Cell Line, Tumor ; *Sarcoma, Ewing/genetics/metabolism/pathology/drug therapy ; Mice ; *Oncogene Proteins, Fusion/genetics/metabolism ; *Gene Expression Regulation, Neoplastic ; Proto-Oncogene Protein c-fli-1/metabolism/genetics ; Bone Neoplasms/genetics/pathology/metabolism/drug therapy ; Signal Transduction/drug effects ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems/genetics ; Hedgehog Proteins/metabolism/genetics ; Female ; },
abstract = {Ewing sarcoma (ES) is an aggressive bone cancer driven by the oncogenic fusion-protein EWSR1::FLI1, which is not present in normal cells and is therefore an attractive therapeutic target. However, as a transcription factor, EWSR1::FLI1 is considered undruggable. Factors that promote EWSR1::FLI1 expression, and thus whose inhibition would reduce EWSR1::FLI1 protein levels and function, are potential drug targets. Here, using genome-scale CRISPR/Cas9 knockout screening, we identify C1GALT1, a galactosyltransferase required for the biosynthesis of many O-glycoproteins, as a factor that promotes EWSR1::FLI1 expression. We show that C1GALT1 acts by O-glycosylating the pivotal Hedgehog (Hh) signaling component Smoothened (SMO), thereby stabilizing SMO and stimulating the Hh pathway, which we find directly activates EWSR1::FLI1 transcription. Itraconazole, an FDA-approved anti-fungal agent that is known to inhibit C1GALT1, reduces EWSR1::FLI1 levels in ES cell lines and suppresses growth of ES xenografts in mice. Our study reveals a therapeutically targetable mechanism that promotes EWSR1::FLI1 expression and ES tumor growth.},
}

Humans
Animals
*Galactosyltransferases/genetics/metabolism
*RNA-Binding Protein EWS/genetics/metabolism
Cell Line, Tumor
*Sarcoma, Ewing/genetics/metabolism/pathology/drug therapy
Mice
*Oncogene Proteins, Fusion/genetics/metabolism
*Gene Expression Regulation, Neoplastic
Proto-Oncogene Protein c-fli-1/metabolism/genetics
Bone Neoplasms/genetics/pathology/metabolism/drug therapy
Signal Transduction/drug effects
Xenograft Model Antitumor Assays
CRISPR-Cas Systems/genetics
Hedgehog Proteins/metabolism/genetics
Female

@article {pmid39894193,
year = {2025},
author = {Saini, A and Dilbaghi, N and Yadav, N},
title = {CRISPR integrated biosensors: A new paradigm for cancer detection.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {569},
number = {},
pages = {120179},
doi = {10.1016/j.cca.2025.120179},
pmid = {39894193},
issn = {1873-3492},
abstract = {Cancer remains one of the leading causes of morbidity and mortality globally, necessitating need for advancements of technologies for early therapeutics. Conventional detection methodologies often lag behind in terms of sensitivity, specificity, and cost-effectiveness, leading to delayed diagnosis and inadequate treatment. The need of advanced diagnostic techniques has considerably increased and led to the development of biosensors. Biosensing technologies offer several advantages over conventional methods hence, overcome limitations and improve diagnostic accuracy. Biosensors, particularly CRISPR-Cas based biosensors have emerged as a revolutionary technology for oncology diagnostics due to their high precision and adaptability. CRISPR-based biosensors provide remarkable precision, sensitivity, multiplexing capabilities, specificity, and rapidness for developing a cost-effective and portable point of care diagnostic device for cancer detection. In this review, we have discussed cancer pathogenicity, assessed the traditional detection techniques, and explored the advancements and advantages of biosensors, particularly CRISPR-based biosensors, in the detection of some major cancer types, namely lung, liver, colorectal, prostate, and cervical cancers. CRISPR-based biosensors represent a significant potential in cancer diagnostics, offering precise, cost-effective, and rapid detection of cancer biomarkers. The integration of CRISPR technology with biosensors holds substantial promise for enhancing early detection and improving patient outcomes in cancer diagnostics.},
}

@article {pmid39841710,
year = {2025},
author = {Park, BJ and Heo, ST and Kim, M and Yoo, JR and Bae, EJ and Kang, SY and Park, S and Han, KR and Lee, KH and Lee, JM and Lee, H and Song, YJ},
title = {A CRISPR-Cas12a-based universal rapid scrub typhus diagnostic method targeting 16S rRNA of Orientia tsutsugamushi.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {1},
pages = {e0012826},
doi = {10.1371/journal.pntd.0012826},
pmid = {39841710},
issn = {1935-2735},
mesh = {*Scrub Typhus/diagnosis/microbiology ; *Orientia tsutsugamushi/genetics/isolation & purification ; *RNA, Ribosomal, 16S/genetics ; Humans ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Molecular Diagnostic Techniques/methods ; Polymerase Chain Reaction/methods ; },
abstract = {Scrub typhus is caused by Orientia tsutsugamushi infection and occurs frequently in an area called the Tsutsugamushi Triangle. Currently, there is no vaccine for O. tsutsugamushi, and its infection is treated with antibiotics such as doxycycline. Scrub typhus responds to effective treatment, and early treatment shortens the course of the disease, reduces mortality, and accelerates recovery. Therefore, it is important to rapidly diagnose O. tsutsugamushi infection to ensure successful outcomes. Here, we developed a CRISPR-Cas12a-based diagnostic method targeting the bacterial 16S rRNA to detect O. tsutsugamushi infection of all known genotypes. To reduce the possibility of contamination and increase field applicability, we designed the one-pot assay system in addition to conventional two-pot assay system. Using this method, we successfully detected up to 100 copies of in vitro transcribed O. tsutsugamushi 16S rRNA within 1 hour under isothermal conditions. In blood samples from patients confirmed to be infected with O. tsutsugamushi by nested PCR, the developed method exhibited a clinical sensitivity of 98% and high specificity. These data demonstrate that the presented method is applicable for the rapid and universal diagnosis of scrub typhus to facilitate timely and appropriate treatment.},
}

*Scrub Typhus/diagnosis/microbiology
*Orientia tsutsugamushi/genetics/isolation & purification
*RNA, Ribosomal, 16S/genetics
Humans
*CRISPR-Cas Systems
Sensitivity and Specificity
Molecular Diagnostic Techniques/methods
Polymerase Chain Reaction/methods

@article {pmid39817463,
year = {2025},
author = {Zuo, D and Liu, W and Zhao, R and Zhu, K and Wang, W and Xiang, H},
title = {Drosophila CG11700 may not affect male fecundity-lifespan tradeoff as previously reported.},
journal = {Molecular biology and evolution},
volume = {42},
number = {2},
pages = {},
doi = {10.1093/molbev/msaf003},
pmid = {39817463},
issn = {1537-1719},
mesh = {Animals ; Male ; *Fertility/genetics ; *Longevity/genetics ; *Drosophila Proteins/genetics ; CRISPR-Cas Systems ; Female ; Drosophila/genetics ; Drosophila melanogaster/genetics ; },
abstract = {Our recent investigations on the function of Drosophila CG11700 and CG32744 (Ubi-p5E) genes using CRISPR/Cas9 deletion technology could not repeat or confirm the results on CG11700 shown in our previous study, which was based on P-element excision assay (Zhan Z, Ding Y, Zhao R, Zhang Y, Yu H, Zhou Q, Yang S, Xiang H, Wang W. Rapid functional divergence of a newly evolved polyubiquitin gene in Drosophila and its role in the trade-off between male fecundity and lifespan. Mol Biol Evol. 2012:29(5):1407-1416. doi:10.1093/molbev/msr299). Here, by CRISPR/Cas9 editing, we generated mutants of CG32744 with the whole gene body fully deleted from the genome, and truncated mutants of CG11700 with N-terminal 103 aa deleted out of its total 301 aa peptide sequence. We carefully conducted the male fecundity assay and found that offsprings of the CG11700 mutant were not significantly more than the wild type, inconsistent with our previous report (Zhan et al. 2012). Meanwhile, we repeated the lifespan assay and did not find that the lifespan of the CG11700 mutant was significantly shorter than the wild type as reported (2012). The new results suggest that the CG11700 gene may not affect male fecundity-lifespan tradeoff as previously reported (Zhan et al. 2012). The new results are thus worthy of reporting to avoid possible misleading by the previous results to the scientific community.},
}

Animals
Male
*Fertility/genetics
*Longevity/genetics
*Drosophila Proteins/genetics
CRISPR-Cas Systems
Female
Drosophila/genetics
Drosophila melanogaster/genetics

@article {pmid39671912,
year = {2025},
author = {Zhao, D and Deshpande, R and Wu, K and Tyagi, A and Sharma, S and Wu, SY and Xing, F and O'Neill, S and Ruiz, J and Lyu, F and Watabe, K},
title = {Identification of TUBB3 as an immunotherapy target in lung cancer by genome wide in vivo CRISPR screening.},
journal = {Neoplasia (New York, N.Y.)},
volume = {60},
number = {},
pages = {101100},
pmid = {39671912},
issn = {1476-5586},
support = {P30 CA012197/CA/NCI NIH HHS/United States ; R01 CA173499/CA/NCI NIH HHS/United States ; R01 CA185650/CA/NCI NIH HHS/United States ; R01 CA205067/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Lung Neoplasms/genetics/immunology/drug therapy/therapy/pathology ; *Tubulin/genetics/metabolism/immunology ; Animals ; *Immunotherapy/methods ; Mice ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Xenograft Model Antitumor Assays ; Drug Resistance, Neoplasm/genetics ; T-Lymphocytes, Cytotoxic/immunology ; Disease Models, Animal ; Snail Family Transcription Factors/genetics/metabolism ; },
abstract = {Recent development of immune checkpoint inhibitors has revolutionized cancer immunotherapy. Although these drugs show dramatic effects on a subset of cancer patients, many other tumors are non-responsive and the pathological mechanism of the resistance is largely unknown. To identify genes underlying anti-PD-1 immunotherapy resistance using a systematic approach, we performed an in vivo genome wide CRISPR screening in lung cancer cells. We integrated our results with multi-omics clinical data and performed both in vitro and in vivo assays to evaluate the role of the top candidate in regulating cytotoxic T cell killing. We identified TUBB3 as a potential target to overcome the resistance and enhance the efficacy of anti-PD-1 immunotherapy. TUBB3 expression is upregulated in lung cancer patients, and its higher expression correlates with poorer patients' survival. We found that TUBB3 expression was significantly elevated in the non-responders compared to responders in our patient cohort that received immunotherapies. Importantly, the results of our preclinical experiments showed that inhibition of TUBB3 with a small molecule inhibitor synergized with anti-PD-1 treatment and enhanced tumor cell killing by cytotoxic T cells. Consistently, anti-PD-1 resistant cells showed significantly higher expression of TUBB3; however, TUBB3 inhibition rendered the resistant cells more susceptible to T cell killing. Mechanistic studies revealed that blocking TUBB3 suppressed the expression of PD-L1 through the EMT-related SNAI1 gene. Our results provide a rationale for a novel combination therapy consisting of the TUBB3 inhibition and anti-PD-1 immunotherapy for lung cancer.},
}

Humans
*Lung Neoplasms/genetics/immunology/drug therapy/therapy/pathology
*Tubulin/genetics/metabolism/immunology
Animals
*Immunotherapy/methods
Mice
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Immune Checkpoint Inhibitors/pharmacology/therapeutic use
Xenograft Model Antitumor Assays
Drug Resistance, Neoplasm/genetics
T-Lymphocytes, Cytotoxic/immunology
Disease Models, Animal
Snail Family Transcription Factors/genetics/metabolism

@article {pmid39668248,
year = {2025},
author = {Philip, R and Sharma, A and Matellan, L and Erpf, AC and Hsu, WH and Tkach, JM and Wyatt, HDM and Pelletier, L},
title = {qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.},
journal = {The EMBO journal},
volume = {44},
number = {3},
pages = {947-974},
pmid = {39668248},
issn = {1460-2075},
support = {187836//Canadian Government | CIHR | Institute of Cancer Research (IC)/ ; 181763//Canadian Government | CIHR | Institute of Cancer Research (IC)/ ; 156297//Canadian Government | Canadian Institutes of Health Research (CIHR)/ ; 167279//Canadian Government | Canadian Institutes of Health Research (CIHR)/ ; N/A//Krembil Foundation/ ; },
mesh = {*Plasmids/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.},
}

*Plasmids/genetics/metabolism
Humans
*CRISPR-Cas Systems
HEK293 Cells
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39893465,
year = {2025},
author = {Li, B and Shang, Y and Wang, L and Lv, J and Wu, Q and Wang, F and Chao, J and Mao, J and Ding, A and Wu, X and Xue, K and Chen, C and Cui, M and Sun, Y and Zhang, H and Dai, C},
title = {Efficient genome editing in dicot plants using calreticulin promoter-driven CRISPR/Cas system.},
journal = {Molecular horticulture},
volume = {5},
number = {1},
pages = {9},
pmid = {39893465},
issn = {2730-9401},
}

@article {pmid39893181,
year = {2025},
author = {Hu, L and Han, J and Wang, HD and Cheng, ZH and Lv, CC and Liu, DF and Yu, HQ},
title = {A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1260},
pmid = {39893181},
issn = {2041-1723},
support = {52322002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Cytosine/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Escherichia coli/genetics/metabolism ; Pseudomonas aeruginosa/genetics ; Protein Domains ; Deoxyribonuclease I/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mutation ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, editing window, and off-target effects. Here, a series of iteratively improved cytosine base editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with the unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an editing range covering 38 nucleotides upstream and downstream of the palindromic PAM, without context preference, which is 6.3 times larger than that of traditional CBEs. Additionally, lower off-target editing is achieved when incorporating high-fidelity mutations at R61A and Q964A in FrCas9n, while maintaining high editing efficiency. The final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, and Pseudomonas aeruginosa PAO1. Collectively, this tool offers robust gene editing for facilitating mechanistic studies, functional exploration, and protein evolution in microbes.},
}

*Cytosine/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
*Escherichia coli/genetics/metabolism
Pseudomonas aeruginosa/genetics
Protein Domains
Deoxyribonuclease I/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mutation
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid39893176,
year = {2025},
author = {Sulis, DB and Lavoine, N and Sederoff, H and Jiang, X and Marques, BM and Lan, K and Cofre-Vega, C and Barrangou, R and Wang, JP},
title = {Advances in lignocellulosic feedstocks for bioenergy and bioproducts.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1244},
pmid = {39893176},
issn = {2041-1723},
mesh = {*Lignin/metabolism ; *Biofuels ; *Gene Editing ; Biotechnology/methods ; Biomass ; CRISPR-Cas Systems ; },
abstract = {Lignocellulose, an abundant renewable resource, presents a promising alternative for sustainable energy and industrial applications. However, large-scale adoption of lignocellulosic feedstocks faces considerable obstacles, including scalability, bioprocessing efficiency, and resilience to climate change. This Review examines current efforts and future opportunities for leveraging lignocellulosic feedstocks in bio-based energy and products, with a focus on enhancing conversion efficiency and scalability. It also explores emerging biotechnologies such as CRISPR-based genome editing informed by machine learning, aimed at improving feedstock traits and reducing the environmental impact of fossil fuel dependence.},
}

*Lignin/metabolism
*Biofuels
*Gene Editing
Biotechnology/methods
Biomass
CRISPR-Cas Systems

@article {pmid39891928,
year = {2025},
author = {Madorsky Rowdo, FP and Martini, R and Ackermann, SE and Tang, CP and Tranquille, M and Irizarry, A and Us, I and Alawa, O and Moyer, JE and Sigouros, M and Nguyen, J and Al Assaad, M and Cheng, E and Ginter, PS and Manohar, J and Stonaker, B and Boateng, R and Oppong, JK and Adjei, EK and Awuah, B and Kyei, I and Aitpillah, FS and Adinku, MO and Ankomah, K and Osei-Bonsu, EB and Gyan, KK and Hoda, S and Newman, L and Mosquera, JM and Sboner, A and Elemento, O and Dow, LE and Davis, MB and Martin, ML},
title = {Kinome-Focused CRISPR-Cas9 Screens in African Ancestry Patient-Derived Breast Cancer Organoids Identify Essential Kinases and Synergy of EGFR and FGFR1 Inhibition.},
journal = {Cancer research},
volume = {85},
number = {3},
pages = {551-566},
doi = {10.1158/0008-5472.CAN-24-0775},
pmid = {39891928},
issn = {1538-7445},
support = {//Weill Cornell Medicine (WCM)/ ; BCRF-22-191//Breast Cancer Research Foundation (BCRF)/ ; CA259396-01//National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Organoids/pathology/metabolism ; *Breast Neoplasms/genetics/pathology/drug therapy ; Female ; *CRISPR-Cas Systems ; *Receptor, Fibroblast Growth Factor, Type 1/genetics/antagonists & inhibitors/metabolism ; *ErbB Receptors/genetics/antagonists & inhibitors/metabolism ; Protein Kinase Inhibitors/pharmacology ; Black People/genetics ; },
abstract = {Precision medicine approaches to cancer treatment aim to exploit genomic alterations that are specific to individual patients to tailor therapeutic strategies. Yet, some targetable genes and pathways are essential for tumor cell viability even in the absence of direct genomic alterations. In underrepresented populations, the mutational landscape and determinants of response to existing therapies are poorly characterized because of limited inclusion in clinical trials and studies. One way to reveal tumor essential genes is with genetic screens. Most screens are conducted on cell lines that bear little resemblance to patient tumors, after years of culture under nonphysiologic conditions. To address this problem, we aimed to develop a CRISPR screening pipeline in three-dimensionally grown patient-derived tumor organoid (PDTO) models. A breast cancer PDTO biobank that focused on underrepresented populations, including West African patients, was established and used to conduct a negative-selection kinome-focused CRISPR screen to identify kinases essential for organoid growth and potential targets for combination therapy with EGFR or MEK inhibitors. The screen identified several previously unidentified kinase targets, and the combination of FGFR1 and EGFR inhibitors synergized to block organoid proliferation. Together, these data demonstrate the feasibility of CRISPR-based genetic screens in patient-derived tumor models, including PDTOs from underrepresented patients with cancer, and identify targets for cancer therapy. Significance: Generation of a breast cancer patient-derived tumor organoid biobank focused on underrepresented populations enabled kinome-focused CRISPR screening that identified essential kinases and potential targets for combination therapy with EGFR or MEK inhibitors. See related commentary by Trembath and Spanheimer, p. 407.},
}

Humans
*Organoids/pathology/metabolism
*Breast Neoplasms/genetics/pathology/drug therapy
Female
*CRISPR-Cas Systems
*Receptor, Fibroblast Growth Factor, Type 1/genetics/antagonists & inhibitors/metabolism
*ErbB Receptors/genetics/antagonists & inhibitors/metabolism
Protein Kinase Inhibitors/pharmacology
Black People/genetics

@article {pmid39891927,
year = {2025},
author = {Trembath, HE and Spanheimer, PM},
title = {In Search of Representative Translational Cancer Model Systems.},
journal = {Cancer research},
volume = {85},
number = {3},
pages = {407-409},
doi = {10.1158/0008-5472.CAN-24-3879},
pmid = {39891927},
issn = {1538-7445},
mesh = {Humans ; *Organoids/pathology ; Female ; Breast Neoplasms/genetics/pathology/therapy ; CRISPR-Cas Systems ; Precision Medicine/methods ; Translational Research, Biomedical/methods ; Neoplasms/genetics/pathology/therapy ; Tumor Microenvironment ; },
abstract = {Racial disparities in cancer outcomes are well documented across tumor types. For patients with breast cancer, Black women are more likely to present with more aggressive molecular features and more likely to die from disease, even after accounting for those features. Recent efforts have been aimed at developing translational model systems for precision medicine strategies, and a major focus has been on patient-derived organoids. Organoids allow for robust in vitro experimental platforms, including drug and CRISPR screens while maintaining more complex cancer and tumor microenvironment subpopulations than cell lines. For results that are broadly translationally relevant, it is important that cancer models are derived from the spectrum of human disease and humans with disease. In this issue of Cancer Research, Madorsky Rowdo and colleagues derive breast cancer organoids from patients with African ancestry and use CRISPR-Cas9 screens to identify novel therapeutic vulnerabilities. These findings demonstrate the promise of representative cancer model systems to facilitate discoveries that are most likely to translate to improved therapy for all patients. See related article by Madorsky Rowdo et al., p. 551.},
}

Humans
*Organoids/pathology
Female
Breast Neoplasms/genetics/pathology/therapy
CRISPR-Cas Systems
Precision Medicine/methods
Translational Research, Biomedical/methods
Neoplasms/genetics/pathology/therapy
Tumor Microenvironment

@article {pmid39891757,
year = {2025},
author = {Roychowdhury, R and Das, SP and Das, S and Biswas, S and Patel, MK and Kumar, A and Sarker, U and Choudhary, SP and Das, R and Yogendra, K and Gangurde, SS},
title = {Advancing vegetable genetics with gene editing: a pathway to food security and nutritional resilience in climate-shifted environments.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {31},
pmid = {39891757},
issn = {1438-7948},
mesh = {*Gene Editing ; *Food Security ; *Climate Change ; *Vegetables/metabolism/genetics ; *CRISPR-Cas Systems ; Plant Breeding/methods ; Crops, Agricultural/genetics ; Humans ; },
abstract = {As global populations grow and climate change increasingly disrupts agricultural systems, ensuring food security and nutritional resilience has become a critical challenge. In addition to grains and legumes, vegetables are very important for both human and animals because they contain vitamins, minerals, and fibre. Enhancing the ability of vegetables to withstand climate change threats is essential; however, traditional breeding methods face challenges due to the complexity of the genomic clonal multiplication process. In the postgenomic era, gene editing (GE) has emerged as a powerful tool for improving vegetables. GE can help to increase traits such as abiotic stress tolerance, herbicide tolerance, and disease resistance; improve agricultural productivity; and improve nutritional content and shelf-life by fine-tuning key genes. GE technologies such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) have revolutionized vegetable breeding by enabling specific gene modifications in the genome. This review highlights recent advances in CRISPR-mediated editing across various vegetable species, highlighting successful modifications that increase their resilience to climatic stressors. Additionally, it explores the potential of GE to address malnutrition by increasing the nutrient content of vegetable crops, thereby contributing to public health and food system sustainability. Additionally, it addresses the implementation of GE-guided breeding strategies in agriculture, considering regulatory, ethical, and public acceptance issues. Enhancing vegetable genetics via GE may provide a reliable and nutritious food supply for an expanding global population under more unpredictable environmental circumstances.},
}

*Gene Editing
*Food Security
*Climate Change
*Vegetables/metabolism/genetics
*CRISPR-Cas Systems
Plant Breeding/methods
Crops, Agricultural/genetics
Humans

@article {pmid39890862,
year = {2025},
author = {Nalefski, EA and Sinan, S and Cantera, JL and Kim, AG and Kooistra, RM and Rivera, RE and Janshen, JP and Bhadra, S and Bishop, JD and Ellington, AD and Finklestein, IJ and Madan, D},
title = {Room temperature CRISPR diagnostics for low-resource settings.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {3909},
pmid = {39890862},
issn = {2045-2322},
mesh = {Humans ; *CRISPR-Cas Systems ; *Temperature ; Endodeoxyribonucleases/genetics/metabolism ; Human papillomavirus 16/genetics/isolation & purification ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins/genetics ; Female ; Recombinases/metabolism ; Nucleic Acid Amplification Techniques/methods ; Papillomavirus Infections/diagnosis/virology ; Sensitivity and Specificity ; },
abstract = {Maintaining elevated reaction temperatures and multi-step sample preparations increases the costs and complexity of diagnostics, impeding their deployment in low-resource settings. Here, we develop a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR reaction that removes these critical challenges. We show that RPA amplification is reduced by several orders of magnitude at 25 °C as compared to 37 °C. Similarly, when coupled to RPA, the performance of multiple Cas12a orthologs, including the widely used LbCas12a, is severely compromised at temperatures below 37 °C. To mitigate these limitations, we identify the ortholog TsCas12a as a highly active nuclease at 25 °C and develop a single-protocol RPA-Cas12a detection reaction with this enzyme. A quantitative kinetic analysis reveals that fast nuclease activation is more critical than higher steady-state trans-cleavage activity for room temperature diagnostic applications. RPA-TsCas12a reactions performed at 25 °C effectively detected HPV-16 in crudely prepared cervical swab samples with high sensitivity and specificity using both optical and lateral flow readouts. The reactions developed herein reduce the complexity and equipment requirements for affordable diagnostics in low- and middle-income countries.},
}

Humans
*CRISPR-Cas Systems
*Temperature
Endodeoxyribonucleases/genetics/metabolism
Human papillomavirus 16/genetics/isolation & purification
CRISPR-Associated Proteins/genetics
Bacterial Proteins/genetics
Female
Recombinases/metabolism
Nucleic Acid Amplification Techniques/methods
Papillomavirus Infections/diagnosis/virology
Sensitivity and Specificity

@article {pmid39862677,
year = {2025},
author = {Pei, J and Li, L and Li, C and Li, Z and Wu, Y and Kuang, H and Ma, P and Huang, L and Liu, J and Tian, G},
title = {Dumbbell probe-bridged CRISPR/Cas13a and nicking-mediated DNA cascade reaction for highly sensitive detection of colorectal cancer-related microRNAs.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117190},
doi = {10.1016/j.bios.2025.117190},
pmid = {39862677},
issn = {1873-4235},
mesh = {*Colorectal Neoplasms/diagnosis/genetics ; *MicroRNAs/analysis/genetics ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; DNA/chemistry/genetics ; Electrochemical Techniques/methods ; },
abstract = {Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, necessitating the development of sensitive and minimally invasive diagnostic approaches. In this study, we present a novel diagnostic strategy by integrating dumbbell probe-mediated CRISPR/Cas13a with nicking-induced DNA cascade reaction (DP-bridged Cas13a/NDCR) for highly sensitive microRNA (miRNA) detection. Target miRNA triggers Cas13a-mediated cleavage of the dumbbell probe, releasing an intermediate strand that hybridizes with a methylene blue-labeled hairpin probe on the electrode surface. Nicking enzyme cleaves the formed duplex DNA, triggering a cascade reaction that amplifies the electrochemical signal. Under optimized conditions, the method demonstrates a detection limit of 8.26 fM for miRNA-21, with reliable specificity and long-term stability. Furthermore, integration with machine learning models using multiple miRNA markers improved diagnostic accuracy, differentiating CRC from colorectal polyps and healthy controls with 100% accuracy in clinical validation cohorts. This study highlights the potential of DP-bridged Cas13a/NDCR as a sensitive and accurate diagnostic tool for CRC.},
}

*Colorectal Neoplasms/diagnosis/genetics
*MicroRNAs/analysis/genetics
Humans
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Limit of Detection
DNA/chemistry/genetics
Electrochemical Techniques/methods

@article {pmid39842078,
year = {2025},
author = {Kumar, ARK and Low, J and Lim, J and Myint, B and Sun, X and Wu, L and Cheng, HS and Yip, S and Ming Cheng, CZ and Manoharan, T and Quek, YJ and Shou, Y and Tian, JS and Ng, YY and Gascoigne, NRJ and Tan, NS and Sugimura, R and Chia, G and Sze Cheung, AM and Yawata, M and Tay, A},
title = {Non-viral, high throughput genetic engineering of primary immune cells using nanostraw-mediated transfection.},
journal = {Biomaterials},
volume = {317},
number = {},
pages = {123079},
doi = {10.1016/j.biomaterials.2024.123079},
pmid = {39842078},
issn = {1878-5905},
mesh = {Humans ; *Transfection/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; CD8-Positive T-Lymphocytes/immunology ; Electroporation/methods ; CD4-Positive T-Lymphocytes/immunology ; },
abstract = {Transfection of proteins, mRNA, and chimeric antigen receptor (CAR) transgenes into immune cells remains a critical bottleneck in cell manufacturing. Current methods, such as viruses and bulk electroporation, are hampered by low transfection efficiency, unintended transgene integration, and significant cell perturbation. The Nanostraw Electro-actuated Transfection (NExT) technology offers a solution by using high aspect-ratio nanostraws and localized electric fields to precisely deliver biomolecules into cells with minimal disruption. We demonstrate that NExT can deliver proteins, polysaccharides, and mRNA into primary human CD8[+] and CD4[+] T cells, and achieve CRISPR/Cas9 gene knockout of CXCR4 and TRAC in CD8[+] T cells. We showcase NExT's versatility across a range of primary human immune cells, including CD4[+] T cells, γδ-T cells, dendritic cells, NK cells, Treg cells, macrophages, and neutrophils. Finally, we developed a scalable, high-throughput multiwell NExT system capable of transfecting over 14 million cells and delivering diverse cargoes into multiple cell types from various donors simultaneously. This technology holds promise for streamlining high-throughput screening of allogeneic donors and reducing optimization costs for large-scale CAR-immune cell transfection.},
}

Humans
*Transfection/methods
*CRISPR-Cas Systems/genetics
*Genetic Engineering/methods
CD8-Positive T-Lymphocytes/immunology
Electroporation/methods
CD4-Positive T-Lymphocytes/immunology

@article {pmid39842058,
year = {2025},
author = {Jia, HY and Yao, SY and Li, YF and Ye, BC and Yin, BC},
title = {A conformational switch-controlled RNA sensor based on orthogonal dCas12a for RNA imaging in live cells.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117185},
doi = {10.1016/j.bios.2025.117185},
pmid = {39842058},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *RNA/chemistry/genetics ; *CRISPR-Cas Systems ; CRISPR-Associated Proteins/chemistry ; Bacterial Proteins/chemistry/genetics ; Nucleic Acid Conformation ; Endodeoxyribonucleases/chemistry ; Fluorescent Dyes/chemistry ; },
abstract = {RNA imaging technology is essential for understanding the complex RNA regulatory mechanisms and serves as a powerful tool for disease diagnosis. However, conventional RNA imaging methods often require multiple fluorescent tags for the specific labeling of individual targets, complicating both the imaging process and subsequent analysis. Herein, we develop an RNA sensor that integrates a blocked CRISPR RNA (crRNA)-based conformational switch with a controllable CRISPR activation (CRISPRa) system and apply for RNA imaging. By leveraging nuclease-inactive Cas12a (dCas12a)-mediated processing of precursor crRNA (pre-crRNA) and the orthogonality of dCas12a from different bacteria, our sensor establishes an artificial link between two unrelated RNA targets, enabling cells to sense one RNA target and image another with a single fluorescent signal. By visualizing a single target for dual-target analysis, our method significantly reduces the reliance on multiple fluorescent tags. Our sensor provides a new platform for RNA imaging, enhancing both biomedical research and the development of advanced molecular diagnostics.},
}

*Biosensing Techniques/methods
Humans
*RNA/chemistry/genetics
*CRISPR-Cas Systems
CRISPR-Associated Proteins/chemistry
Bacterial Proteins/chemistry/genetics
Nucleic Acid Conformation
Endodeoxyribonucleases/chemistry
Fluorescent Dyes/chemistry

@article {pmid39837235,
year = {2025},
author = {Ding, S and Dong, J and Shi, J and Ren, K and Cui, X and Shi, Z and Li, N and Xiang, Y and Du, F and Tang, Z},
title = {Integrating commercial personal glucose meter with peroxidase-mimic DNAzyme to develop a versatile point-of-care biosensing platform.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117171},
doi = {10.1016/j.bios.2025.117171},
pmid = {39837235},
issn = {1873-4235},
mesh = {*DNA, Catalytic/chemistry ; *Biosensing Techniques/instrumentation/methods ; Humans ; *SARS-CoV-2/isolation & purification ; *COVID-19/diagnosis/virology ; *Blood Glucose Self-Monitoring/instrumentation ; Point-of-Care Testing ; Point-of-Care Systems ; Peroxidase/chemistry ; Glucose/analysis ; Blood Glucose/analysis ; CRISPR-Cas Systems ; RNA, Viral/analysis ; Limit of Detection ; },
abstract = {The development of point-of-care testing (POCT) methods is highly desirable in molecular detection, as they enable disease diagnosis and biomarker monitoring on-site or at home. Repurposing existing POCT devices to detect diverse biomarkers is an economical way to develop new devices for POCT use. Personal glucose meter (PGM) is one of the most used off-the-shelf POCT devices that has been reused to detect non-glucose targets. However, developing a label-free, user-friendly, and cost-effective general PGM-based sensing platform remains a great challenge, primarily due to the reliance on protein enzymes in most existing signal transducing strategies. To overcome the challenges, we herein developed a DNAzyme-based signal transduction strategy that bridges non-glucose signals to PGM readouts. By integrating this strategy with CRISPR/Cas12a-mediated target sensing, we successfully established a simple and versatile platform (CaG-PGM) for biosensing. The utility of CaG-PGM in the detection of nucleic acid targets was successfully validated by detecting Monkeypox virus DNA and SARS-CoV-2 RNA with high sensitivity and specificity. We further demonstrated its generality in detecting non-nucleic acid targets including protein and small molecule. In conclusion, this study provides a cheap and effective strategy for repurposing PGM as a general biosensing platform and sheds new light on translating functional nucleic acids for POCT applications.},
}

*DNA, Catalytic/chemistry
*Biosensing Techniques/instrumentation/methods
Humans
*SARS-CoV-2/isolation & purification
*COVID-19/diagnosis/virology
*Blood Glucose Self-Monitoring/instrumentation
Point-of-Care Testing
Point-of-Care Systems
Peroxidase/chemistry
Glucose/analysis
Blood Glucose/analysis
CRISPR-Cas Systems
RNA, Viral/analysis
Limit of Detection

@article {pmid39832688,
year = {2025},
author = {Harris, H and Kittur, J},
title = {Unlocking the potential of CRISPR-Cas9 for cystic fibrosis: A systematic literature review.},
journal = {Gene},
volume = {942},
number = {},
pages = {149257},
doi = {10.1016/j.gene.2025.149257},
pmid = {39832688},
issn = {1879-0038},
mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Genetic Therapy/methods ; Animals ; Mutation ; },
abstract = {CRISPR-Cas9 technology has revolutionized genetic engineering, offering precise and efficient genome editing capabilities. This review explores the application of CRISPR-Cas9 for cystic fibrosis (CF), particularly targeting mutations in the CFTR gene. CF is a multiorgan disease primarily affecting the lungs, gastrointestinal system (e.g., CF-related diabetes (CFRD), CF-associated liver disease (CFLD)), bones (CF-bone disease), and the reproductive system. CF, a genetic disorder characterized by defective ion transport leading to thick mucus accumulation, is often caused by mutations like ΔF508 in the CFTR gene. This review employs a systematic methodology, incorporating an extensive literature search across multiple academic databases, including PubMed, Web of Science, and ScienceDirect, to identify 40 high-quality studies focused on CRISPR-Cas9 applications for CFTR gene editing. The data collection process involved predefined inclusion criteria targeting experimental approaches, gene-editing outcomes, delivery methods, and verification techniques. Data analysis synthesized findings on editing efficiency, off-target effects, and delivery system optimization to present a comprehensive overview of the field. The review highlights the historical development of CRISPR-Cas9, its mechanism, and its transformative role in genetic engineering and medicine. A detailed examination of CRISPR-Cas9's application in CFTR gene correction emphasizes the potential for therapeutic interventions while addressing challenges such as off-target effects, delivery efficiency, and ethical considerations. Future directions include optimizing delivery systems, integrating advanced editing tools like prime and base editing, and expanding personalized medicine approaches to improve treatment outcomes. By systematically analyzing the current landscape, this review provides a foundation for advancing CRISPR-Cas9 technologies for cystic fibrosis treatment and related disorders.},
}

*Cystic Fibrosis/genetics/therapy
Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*Genetic Therapy/methods
Animals
Mutation

@article {pmid39826273,
year = {2025},
author = {Pal, T and Liu, Z and Chen, J},
title = {CIMNE-CRISPR: A novel amplification-free diagnostic for rapid early detection of African Swine Fever Virus.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117154},
doi = {10.1016/j.bios.2025.117154},
pmid = {39826273},
issn = {1873-4235},
support = {R35 GM147069/GM/NIGMS NIH HHS/United States ; },
mesh = {*African Swine Fever Virus/isolation & purification/genetics ; Animals ; Swine ; *African Swine Fever/diagnosis/virology/blood ; *Biosensing Techniques/methods ; *Limit of Detection ; CRISPR-Cas Systems ; Magnetite Nanoparticles/chemistry ; DNA, Viral/analysis/genetics ; Reproducibility of Results ; },
abstract = {African Swine Fever Virus (ASFV) is a highly contagious pathogen with nearly 100% mortality in swine, causing severe global economic loss. Current detection methods rely on nucleic acid amplification, which requires specialized equipment and skilled operators, limiting accessibility in resource-constrained settings. To address these challenges, we developed the Covalently Immobilized Magnetic Nanoparticles Enhanced CRISPR (CIMNE-CRISPR) system. This amplification-free diagnostic system seamlessly combines target recognition, sequence-specific enrichment, and signal generation. This approach uses covalent immobilization of CRISPR-LbCas12a-crRNA complexes on Fe3O4@SiO2 core-shell magnetic nanoparticles, which improves enzyme specificity and robustness over traditional adsorption. The CIMNE-CRISPR assay reached a limit of detection (LOD) of 8.1 × 10[4] copies/μL and a limit of quantification (LOQ) of 4.2 × 10[5] copies/μL, with a dynamic range spanning 10[5] to 10[10] copies/μL and a matrix factor of 100.29% in porcine plasma. It maintained great specificity and accurately detecting 10[5] copies/μL of ASFV DNA even with high mutant concentrations (10[13] copies/μL). The method demonstrated decent reproducibility across different nanoparticle synthesis batches, with an RSD of 9.63% and recovery rates between 97% and 103%, and features rapid processing well-suited for field diagnostics. Overall, this system's cost-effectiveness, simplicity, and reliability highlight its potential to pave the way for advanced CRISPR-based diagnostics, particularly for diverse viral and bacterial targets in agricultural, environmental, and zoonotic disease contexts.},
}

*African Swine Fever Virus/isolation & purification/genetics
Animals
Swine
*African Swine Fever/diagnosis/virology/blood
*Biosensing Techniques/methods
*Limit of Detection
CRISPR-Cas Systems
Magnetite Nanoparticles/chemistry
DNA, Viral/analysis/genetics
Reproducibility of Results

@article {pmid39823857,
year = {2025},
author = {Fan, X and Gao, Z and Ling, D and Wang, D and Cui, Y and Du, H and Zhou, X},
title = {The dCas9/crRNA linked immunological assay (dCLISA) for sensitive, accurate, and facile drug resistance gene analysis.},
journal = {Biosensors & bioelectronics},
volume = {273},
number = {},
pages = {117147},
doi = {10.1016/j.bios.2025.117147},
pmid = {39823857},
issn = {1873-4235},
mesh = {*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics ; Humans ; *Biosensing Techniques/methods ; *Staphylococcal Infections/microbiology/immunology ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; Immunoassay/methods ; Drug Resistance, Bacterial/genetics ; },
abstract = {The rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for preventing the spread of MRSA infections and guiding therapeutic strategies. However, there is still a huge challenge in further simplifying MRSA detection procedures and improving detection selectivity to reduce false-positive results. In this study, we developed a derivative CRISPR-associated protein 9/CRISPR-derived RNA Linked Immunological Assay (dCLISA) for the sensitive and specific detection of MRSA. This technique utilizes two dCas9/crRNA complexes as specific targeting agents and employs a color reaction mediated by a hybridization chain reaction for signal output. The dCLISA method offers certain benefits compared to monoclonal antibodies in traditional immunoassays, primarily due to its capacity to selectively interact with target gene and its high sensitivity from the hybridization chain reaction process. Therefore, the minimum detectable concentration of dCLISA was 8.5 cfu/mL. Unlike traditional gene analysis approaches, target gene sequences in cell lysates can be directly detected by dCLISA within 60 min with high sensitivity without genomic material extraction. In addition, the absorbance intensity of the MRSA cell lysate was significantly higher than that of methicillin-susceptible S. aureus (MSSA) indicates the clinical application potential. This study demonstrates that the dCLISA is a simple, rapid, sensitive, and specific method, which can be directly used at the point of care to analyze drug resistance in bacteria, including MRSA. Moreover, dCLISA can be utilized for other bacteria detection by merely modifying the crRNA sequence.},
}

*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics
Humans
*Biosensing Techniques/methods
*Staphylococcal Infections/microbiology/immunology
*CRISPR-Cas Systems
CRISPR-Associated Protein 9/genetics
Immunoassay/methods
Drug Resistance, Bacterial/genetics

@article {pmid39799696,
year = {2025},
author = {Jiang, Y and Li, Y and Zheng, D and Du, X and Yang, H and Wang, C and Zhao, M and Xiao, H and Zhang, L and Li, X and Shi, S},
title = {Nano-polymeric platinum activates PAR2 gene editing to suppress tumor metastasis.},
journal = {Biomaterials},
volume = {317},
number = {},
pages = {123090},
doi = {10.1016/j.biomaterials.2025.123090},
pmid = {39799696},
issn = {1878-5905},
mesh = {*Receptor, PAR-2/metabolism/genetics ; Humans ; Animals ; *Platinum/chemistry/pharmacology/therapeutic use ; *Neoplasm Metastasis ; *Gene Editing ; Cell Line, Tumor ; Mice ; Cell Movement/drug effects ; CRISPR-Cas Systems ; Polymers/chemistry ; Mice, Nude ; Mice, Inbred BALB C ; Antineoplastic Agents/pharmacology/therapeutic use ; Female ; Nanoparticles/chemistry ; },
abstract = {Metastasis as the hallmark of cancer preferentially contributes to tumor recurrence and therapy resistance, aggrandizing the lethality of patients with cancer. Despite their robust suppressions of tumor progression, chemotherapeutics failed to attenuate cancer cell migration and even triggered pro-metastatic effects. In parallel, protease-activated receptor 2 (PAR2), a member of the G protein-coupled receptor subfamily, actively participates in cancer metastasis via multiple signal transduction pathways. CRISPR/Cas9 that is a dominating genome editing tool can evoke PAR2 knockout to inhibit cancer metastasis. However, the absence of valid delivery systems largely limits its efficacy. Herein, we nanosized polymeric platinum (NanoPt) as therapeutical drug carries to deliver CRISPR/Cas9 to elicit genome editing of PAR2, which drastically augmented anti-metastatic effects and alleviated systematic toxicity of platinum-based treatment in vitro and in vivo. More importantly, the NanoPt@Cas9-PAR2 initiated PAR2 deficiency to mechanistically attenuate EMT process and ferroptosis via RAGE/ERK signalling, consequently preventing cancer cell migration. Our findings indicate that NanoPt@Cas9-PAR2 that mitigated PAR2 signalling and cytotoxic effects of platinum could be a safe and powerful all-in-one combinatorial strategy for cancer treatment.},
}

*Receptor, PAR-2/metabolism/genetics
Humans
Animals
*Platinum/chemistry/pharmacology/therapeutic use
*Neoplasm Metastasis
*Gene Editing
Cell Line, Tumor
Mice
Cell Movement/drug effects
CRISPR-Cas Systems
Polymers/chemistry
Mice, Nude
Mice, Inbred BALB C
Antineoplastic Agents/pharmacology/therapeutic use
Female
Nanoparticles/chemistry

@article {pmid38984870,
year = {2025},
author = {Lu, J and Fu, B and Zhu, Z and Yan, C and Guan, F and Wang, P and Yu, P},
title = {Enhancing the production of L-proline in recombinant Escherichia coli BL21 by metabolic engineering.},
journal = {Preparative biochemistry & biotechnology},
volume = {55},
number = {2},
pages = {187-195},
doi = {10.1080/10826068.2024.2378104},
pmid = {38984870},
issn = {1532-2297},
mesh = {*Proline/metabolism ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Glucose/metabolism ; *CRISPR-Cas Systems ; *Fermentation ; Gene Editing/methods ; Escherichia coli Proteins/genetics/metabolism ; Batch Cell Culture Techniques ; },
abstract = {L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.},
}

*Proline/metabolism
*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
*Glucose/metabolism
*CRISPR-Cas Systems
*Fermentation
Gene Editing/methods
Escherichia coli Proteins/genetics/metabolism
Batch Cell Culture Techniques

@article {pmid39889868,
year = {2025},
author = {Zhao, Q and Wei, L and Chen, Y},
title = {From bench to bedside: Developing CRISPR/Cas-based therapy for ocular diseases.},
journal = {Pharmacological research},
volume = {},
number = {},
pages = {107638},
doi = {10.1016/j.phrs.2025.107638},
pmid = {39889868},
issn = {1096-1186},
abstract = {Vision-threatening disorders, including both hereditary and multifactorial ocular diseases, necessitate innovative therapeutic approaches. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has emerged as a promising tool for treating ocular diseases through gene editing and expression regulation. This system has contributed to the development of representative disease models, including animal models, organoids, and cell lines, thereby facilitating investigations into the pathogenesis of disease-related genes. Besides, therapeutic applications of CRISPR/Cas have been extensively explored in preclinical in vitro and in vivo studies, targeting various ocular conditions, such as retinitis pigmentosa, Leber congenital amaurosis, Usher syndrome, fundus neovascular diseases, glaucoma, and corneal diseases. Recent advancements have demonstrated the technology's potential to restore cellular homeostasis and alleviate disease phenotypes, thereby prompting a variety of clinical trials. To date, active trials include treatments for primary open angle glaucoma with MYOC mutations, refractory herpetic viral keratitis, CEP290-associated inherited retinal degenerations, neovascular age-related macular degeneration, and retinitis pigmentosa with RHO mutations. However, challenges remain, primarily concerning off-target effects, immunogenicity, ethical considerations, and regulatory particularity. To reach higher safety and efficiency before truly transitioning from bench to bedside, future research should concentrate on improving the specificity and efficacy of Cas proteins, optimizing delivery vectors, and broadening the applicability of therapeutic targets. This review summarizes the application strategies and delivery methods of CRISPR/Cas, discusses recent progress in CRISPR/Cas-based disease models and therapies, and provides an overview of the landscape of clinical trials. Current obstacles and future directions regarding the bench-to-bedside transition are also discussed.},
}

@article {pmid39887239,
year = {2025},
author = {Chen, P-R and Wei, Y and Li, X and Yu, H-Y and Wang, S-G and Yuan, X-Z and Xia, P-F},
title = {Precision engineering of the probiotic Escherichia coli Nissle 1917 with prime editing.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0003125},
doi = {10.1128/aem.00031-25},
pmid = {39887239},
issn = {1098-5336},
abstract = {CRISPR-Cas systems are transforming precision medicine with engineered probiotics as next-generation diagnostics and therapeutics. To promote human health and treat disease, engineering probiotic bacteria demands maximal versatility to enable non-natural functionalities while minimizing undesired genomic interferences. Here, we present a streamlined prime editing approach tailored for probiotic Escherichia coli Nissle 1917 utilizing only essential genetic modules, including Cas9 nickase from Streptococcus pyogenes, a codon-optimized reverse transcriptase, and a prime editing guide RNA, and an optimized workflow with longer induction. As a result, we achieved all types of prime editing in every individual round of experiments with efficiencies of 25.0%, 52.0%, and 66.7% for DNA deletion, insertion, and substitution, respectively. A comprehensive evaluation of off-target effects revealed a significant reduction in unintended mutations, particularly in comparison to two different base editing methods. Leveraging the prime editing system, we inserted a unique DNA sequence to barcode the edited strain and established an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy presents a CRISPR-Cas system that can be readily implemented in any laboratories with the basic CRISPR setups, paving the way for future innovations in engineered probiotics.IMPORTANCEOne ultimate goal of gene editing is to introduce designed DNA variations at specific loci in living organisms with minimal unintended interferences in the genome. Achieving this goal is especially critical for creating engineered probiotics as living diagnostics and therapeutics to promote human health and treat diseases. In this endeavor, we report a customized prime editing system for precision engineering of probiotic Escherichia coli Nissle 1917. With such a system, we developed a barcoding system for tracking engineered strains, and we built an antibiotic-resistance-gene-free platform to enable non-natural functionalities. We provide not only a powerful gene editing approach for probiotic bacteria but also new insights into the advancement of innovative CRISPR-Cas systems.},
}

@article {pmid39887096,
year = {2025},
author = {Brusson, M and Miccio, A},
title = {[A CRISPR/Cas approach to β-haemoglobinopathies].},
journal = {Medecine sciences : M/S},
volume = {41},
number = {1},
pages = {33-39},
doi = {10.1051/medsci/2024191},
pmid = {39887096},
issn = {1958-5381},
support = {ANR-10-IAHU-01//Agence nationale de la recherche/ ; 865797 DITSB//Conseil européen de la recherche/ ; subvention HORIZON-RIA EDITSCD n° 101057659//Commission européenne/ ; subvention 22206//AFM-Téléthon/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; *beta-Thalassemia/therapy/genetics ; Anemia, Sickle Cell/therapy/genetics ; Fetal Hemoglobin/genetics/biosynthesis ; Hematopoietic Stem Cells/metabolism/physiology ; Hemoglobinopathies/therapy/genetics ; Animals ; },
abstract = {Beta-haemoglobinopathies are severe genetic anemias caused by mutations that affect adult haemoglobin production. Many therapeutic approaches aim to reactivate the expression of the fetal hemoglobin genes. To this end, the CRISPR/Cas9 system has recently been used to genetically modify patients' hematopoietic stem/progenitor cells ex vivo and reactivate fetal hemoglobin expression in their erythroid progeny. More than 70 patients with severe β-thalassemia and sickle cell disease have been treated with the Casgevy® therapy. Most have achieved a significant improvement of clinical phenotype, with high editing efficiency in hematopoietic cells associated with normal or near normal hemoglobin levels. While the long-term safety and efficacy of this powerful approach still need to be evaluated, new strategies are being developed to further improve therapeutic outcomes, reduce potential genotoxicity and lower the costs of therapy.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Therapy/methods
*beta-Thalassemia/therapy/genetics
Anemia, Sickle Cell/therapy/genetics
Fetal Hemoglobin/genetics/biosynthesis
Hematopoietic Stem Cells/metabolism/physiology
Hemoglobinopathies/therapy/genetics
Animals

@article {pmid39806065,
year = {2025},
author = {Chen, X and Ghanizada, M and Mallajosyula, V and Sola, E and Capasso, R and Kathuria, KR and Davis, MM},
title = {Differential roles of human CD4[+] and CD8[+] regulatory T cells in controlling self-reactive immune responses.},
journal = {Nature immunology},
volume = {26},
number = {2},
pages = {230-239},
pmid = {39806065},
issn = {1529-2916},
support = {U19 AI057229/AI/NIAID NIH HHS/United States ; AI057229//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; *CD8-Positive T-Lymphocytes/immunology ; *Forkhead Transcription Factors/metabolism ; *Granzymes/metabolism ; *Autoimmunity ; Palatine Tonsil/immunology ; Animals ; Autoantigens/immunology ; Mice ; CRISPR-Cas Systems ; },
abstract = {Here we analyzed the relative contributions of CD4[+] regulatory T cells expressing Forkhead box protein P3 (FOXP3) and CD8[+] regulatory T cells expressing killer cell immunoglobulin-like receptors to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. FOXP3 and GZMB respectively encode proteins FOXP3 and granzyme B, which are critical to the suppressive functions of CD4[+] and CD8[+] regulatory T cells. Using CRISPR-Cas9 gene editing, we were able to achieve a reduction of ~90-95% in the expression of these genes. FOXP3 knockout in tonsil T cells led to production of antibodies against a variety of autoantigens and increased the affinity of influenza-specific antibodies. By contrast, GZMB knockout resulted in an increase in follicular helper T cells, consistent with the ablation of CD8[+] regulatory T cells observed in mouse models, and a marked expansion of autoreactive CD8[+] and CD4[+] T cells. These findings highlight the distinct yet complementary roles of CD8[+] and CD4[+] regulatory T cells in regulating cellular and humoral responses to prevent autoimmunity.},
}

Humans
*T-Lymphocytes, Regulatory/immunology
*CD8-Positive T-Lymphocytes/immunology
*Forkhead Transcription Factors/metabolism
*Granzymes/metabolism
*Autoimmunity
Palatine Tonsil/immunology
Animals
Autoantigens/immunology
Mice
CRISPR-Cas Systems

@article {pmid39707712,
year = {2025},
author = {Abdulsalam, L and Mordecai, J and Ahmad, I},
title = {Non-viral gene therapy for Leber's congenital amaurosis: progress and possibilities.},
journal = {Nanomedicine (London, England)},
volume = {20},
number = {3},
pages = {291-304},
doi = {10.1080/17435889.2024.2443387},
pmid = {39707712},
issn = {1748-6963},
mesh = {Humans ; *Genetic Therapy/methods ; *Leber Congenital Amaurosis/therapy/genetics ; Animals ; *Gene Transfer Techniques ; *Nanoparticles/chemistry ; Genetic Vectors/genetics ; Mutation ; CRISPR-Cas Systems/genetics ; },
abstract = {Leber's congenital amaurosis (LCA) represents a set of rare and pervasive hereditary conditions of the retina that cause severe vision loss starting in early childhood. Targeted treatment intervention has become possible thanks to recent advances in understanding LCA genetic basis. While viral vectors have shown efficacy in gene delivery, they present challenges related to safety, low cargo capacity, and the potential for random genomic integration. Non-viral gene therapy is a safer and more flexible alternative to treating the underlying genetic mutation causing LCA. Non-viral gene delivery methods, such as inorganic nanoparticles, polymer-based delivery systems, and lipid-based nanoparticles, bypass the risks of immunogenicity and genomic integration, potentially offering a more versatile and personalized treatment for patients. This review explores the genetic background of LCA, emphasizing the mutations involved, and explores diverse non-viral gene delivery methods being developed. It also highlights recent studies on non-viral gene therapy for LCA in animal models and clinical trials. It presents future perspectives for gene therapy, including integrating emerging technologies like CRISPR-Cas9, interdisciplinary collaborations, personalized medicine, and ethical considerations.},
}

Humans
*Genetic Therapy/methods
*Leber Congenital Amaurosis/therapy/genetics
Animals
*Gene Transfer Techniques
*Nanoparticles/chemistry
Genetic Vectors/genetics
Mutation
CRISPR-Cas Systems/genetics

@article {pmid39333753,
year = {2025},
author = {Tiwari, SK and Wong, WJ and Moreira, M and Pasqualini, C and Ginhoux, F},
title = {Induced pluripotent stem cell-derived macrophages as a platform for modelling human disease.},
journal = {Nature reviews. Immunology},
volume = {25},
number = {2},
pages = {108-124},
pmid = {39333753},
issn = {1474-1741},
mesh = {Humans ; *Induced Pluripotent Stem Cells/immunology/cytology ; *Macrophages/immunology ; *Cell Differentiation/immunology ; Animals ; CRISPR-Cas Systems ; Organoids/immunology ; },
abstract = {Macrophages are innate immune cells that are present in essentially all tissues, where they have vital roles in tissue development, homeostasis and pathogenesis. The importance of macrophages in tissue function is reflected by their association with various human diseases, and studying macrophage functions in both homeostasis and pathological tissue settings is a promising avenue for new targeted therapies that will improve human health. The ability to generate macrophages from induced pluripotent stem (iPS) cells has revolutionized macrophage biology, with the generation of iPS cell-derived macrophages (iMacs) providing unlimited access to genotype-specific cells that can be used to model various human diseases involving macrophage dysregulation. Such disease modelling is achieved by generating iPS cells from patient-derived cells carrying disease-related mutations or by introducing mutations into iPS cells from healthy donors using CRISPR-Cas9 technology. These iMacs that carry disease-related mutations can be used to study the aetiology of the particular disease in vitro. To achieve more physiological relevance, iMacs can be co-cultured in 2D systems with iPS cell-derived cells or in 3D systems with iPS cell-derived organoids. Here, we discuss the studies that have attempted to model various human diseases using iMacs, highlighting how these have advanced our knowledge about the role of macrophages in health and disease.},
}

Humans
*Induced Pluripotent Stem Cells/immunology/cytology
*Macrophages/immunology
*Cell Differentiation/immunology
Animals
CRISPR-Cas Systems
Organoids/immunology

@article {pmid39039203,
year = {2025},
author = {Naidoo, K and Oliver, SV},
title = {Gene drives: an alternative approach to malaria control?.},
journal = {Gene therapy},
volume = {32},
number = {1},
pages = {25-37},
pmid = {39039203},
issn = {1476-5462},
mesh = {*Malaria/prevention & control ; Animals ; *Gene Drive Technology/methods ; Humans ; *Mosquito Vectors/genetics ; *CRISPR-Cas Systems ; *Mosquito Control/methods ; Gene Editing/methods ; Culicidae/genetics ; },
abstract = {Genetic modification for the control of mosquitoes is frequently touted as a solution for a variety of vector-borne diseases. There has been some success using non-insecticidal methods like sterile or incompatible insect techniques to control arbovirus diseases. However, control by genetic modifications to reduce mosquito populations or create mosquitoes that are refractory to infection with pathogens are less developed. The advent of CRISPR-Cas9-mediated gene drives may advance this mechanism of control. In this review, use and progress of gene drives for vector control, particularly for malaria, is discussed. A brief history of population suppression and replacement gene drives in mosquitoes, rapid advancement of the field over the last decade and how genetic modification fits into the current scope of vector control are described. Mechanisms of alternative vector control by genetic modification to modulate mosquitoes' immune responses and anti-parasite effector molecules as part of a combinational strategy to combat malaria are considered. Finally, the limitations and ethics of using gene drives for mosquito control are discussed.},
}

*Malaria/prevention & control
Animals
*Gene Drive Technology/methods
Humans
*Mosquito Vectors/genetics
*CRISPR-Cas Systems
*Mosquito Control/methods
Gene Editing/methods
Culicidae/genetics

@article {pmid39885543,
year = {2025},
author = {Jiang, J and Cienfuegos-Galletd, AV and Long, T and Peirano, G and Chu, T and Pitout, JDD and Kreiswirth, BN and Chen, L},
title = {Intricate interplay of CRISPR-Cas systems, anti-CRISPR proteins, and antimicrobial resistance genes in a globally successful multi-drug resistant Klebsiella pneumoniae clone.},
journal = {Genome medicine},
volume = {17},
number = {1},
pages = {9},
pmid = {39885543},
issn = {1756-994X},
mesh = {*Klebsiella pneumoniae/genetics/drug effects ; *CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial/genetics ; *Bacterial Proteins/genetics ; Phylogeny ; Plasmids/genetics ; beta-Lactamases/genetics ; Genome, Bacterial ; Klebsiella Infections/microbiology ; Humans ; Anti-Bacterial Agents/pharmacology ; },
abstract = {BACKGROUND: Klebsiella pneumoniae is one of the most prevalent pathogens responsible for multiple infections in healthcare settings and the community. K. pneumoniae CG147, primarily including ST147 (the founder ST), ST273, and ST392, is one of the most globally successful MDR clone linked to various carbapenemases.

METHODS: One hundred and one CG147 strains were sequenced and additional 911 publicly available CG147 genome sequences were included for analysis. The molecular epidemiology, population structure, and time phylogeny were investigated. The virulome, resistome, and mobilome were analyzed, and the recombination in the capsular region was studied. The CRISPR-Cas and anti-CRISPR were identified. The interplay between CRISPR-Cas, anti-CRISPR, and carbapenemase-encoding plasmids was analyzed and experimentally validated.

RESULTS: We analyzed 1012 global CG147 genomes, with 80.4% encoding at least one carbapenemase (NDM [529/1012, 52.3%], OXA-48-like [182/1012, 17.7%], and KPC [105/1012, 10.4%]). Surprisingly, almost all CG147 strains (99.7%, 1009/1,012) harbor a chromosomal type I-E CRISPR-Cas system, with 41.8% (423/1012) containing an additional plasmid-borne type IV-A3 CRISPR-Cas system, and both target IncF plasmids, e.g., the most prevalent KPC-encoding pKpQIL-like plasmids. We found the presence of IV-A3 CRISPR-Cas system showed a negative correlation with the presence of KPC. Interestingly, a prophage-encoding anti-CRISPR AcrIE8.1 and a plasmid-borne anti-CRISPR AcrIE9.2 were detected in 40.1% (406/1012) and 54.2% (548/1012) of strains, respectively, which displayed positive correlations with the presence of a carbapenemase. Plasmid transfer experiments confirmed that the I-E and IV-A3 CRISPR-Cas systems significantly decreased (p < 0.001) KPC-encoding pKpQIL plasmid conjugation frequencies, while the AcrIE8.1 and AcrIE9.2 significantly increased (p < 0.001) pKpQIL conjugation frequencies and protected plasmids from elimination by CRISPR-Cas I-E system.

CONCLUSIONS: Our results indicated a complex interplay between CRISPR-Cas, anti-CRISPR, and mobile genetic elements that shape the evolution of CG147. Our findings advance the understanding of multi-drug resistance mechanisms and will aid in preventing the emergence of future MDR clones.},
}

*Klebsiella pneumoniae/genetics/drug effects
*CRISPR-Cas Systems
*Drug Resistance, Multiple, Bacterial/genetics
*Bacterial Proteins/genetics
Phylogeny
Plasmids/genetics
beta-Lactamases/genetics
Genome, Bacterial
Klebsiella Infections/microbiology
Humans
Anti-Bacterial Agents/pharmacology

@article {pmid39885339,
year = {2025},
author = {Low, SJ and O'Neill, M and Kerry, WJ and Wild, N and Krysiak, M and Nong, Y and Azzato, F and Hor, E and Williams, L and Taiaroa, G and Steinig, E and Pasricha, S and Williamson, DA},
title = {PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {147},
pmid = {39885339},
issn = {2399-3642},
support = {GA-F3791196-5514//State Government of Victoria (Victorian Government)/ ; PO4932//Department of Health, Australian Government (Department of Health)/ ; },
mesh = {*Genomics/methods ; *Streptococcus pyogenes/genetics ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Neisseria gonorrhoeae/genetics/isolation & purification ; Humans ; DNA Primers/genetics ; Computational Biology/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Critical to the success of CRISPR-based diagnostic assays is the selection of a diagnostic target highly specific to the organism of interest, a process often requiring iterative cycles of manual selection, optimisation, and redesign. Here we present PathoGD, a bioinformatic pipeline for rapid and high-throughput design of RPA primers and gRNAs for CRISPR-Cas12a-based pathogen detection. PathoGD is fully automated, leverages publicly available sequences and is scalable to large datasets, allowing rapid continuous monitoring and validation of primer/gRNA sets to ensure ongoing assay relevance. We designed primers and gRNAs for five clinically relevant bacterial pathogens, and experimentally validated a subset of the designs for detecting Streptococcus pyogenes and/or Neisseria gonorrhoeae in assays with and without pre-amplification. We demonstrated high specificity of primers and gRNAs designed, with minimal off-target signal observed for all combinations. We anticipate PathoGD will be an important resource for assay design for current and emerging pathogens. PathoGD is available on GitHub at https://github.com/sjlow23/pathogd .},
}

*Genomics/methods
*Streptococcus pyogenes/genetics
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
*Neisseria gonorrhoeae/genetics/isolation & purification
Humans
DNA Primers/genetics
Computational Biology/methods
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39885211,
year = {2025},
author = {Cheng, ZH and Luo, XY and Yu, SS and Min, D and Zhang, SX and Li, XF and Chen, JJ and Liu, DF and Yu, HQ},
title = {Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1166},
pmid = {39885211},
issn = {2041-1723},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/metabolism/genetics ; Influenza A virus/genetics ; COVID-19/virology/diagnosis ; Bacterial Proteins/genetics/metabolism ; Saliva/virology ; Endodeoxyribonucleases/metabolism/genetics ; Sensitivity and Specificity ; Heparin/metabolism ; Molecular Diagnostic Techniques/methods ; },
abstract = {The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal one-pot fluorescent method for the detection of epidemic pathogens, delivering results within 15-20 min. This method uses heparin sodium to precisely tunes the cis-cleavage capability of Cas12 via interference with the Cas12a-crRNA binding process, thereby generating significant fluorescence due to the accumulation of isothermal amplification products. Additionally, this universal assay accommodates both classic and suboptimal PAMs, as well as various Cas12a subtypes such as LbCas12a, AsCas12a, and AapCas12b. Such a robust method demonstrates sensitivity and specificity exceeding 95% in the detection of monkeypox pseudovirus, influenza A virus, and SARS-CoV-2 from saliva or wastewater samples, when compared with qPCR or RT-qPCR. Moreover, the cost of heparin sodium per thousand uses is $0.01 to $0.04 only. Collectively, this universal and fast one-pot approach based on heparin sodium offers potential possibilities for point-of-care testing.},
}

*SARS-CoV-2/genetics/isolation & purification
*CRISPR-Cas Systems
Humans
Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/metabolism/genetics
Influenza A virus/genetics
COVID-19/virology/diagnosis
Bacterial Proteins/genetics/metabolism
Saliva/virology
Endodeoxyribonucleases/metabolism/genetics
Sensitivity and Specificity
Heparin/metabolism
Molecular Diagnostic Techniques/methods

@article {pmid39885149,
year = {2025},
author = {Jin, W and Deng, Y and La Marca, JE and Lelliott, EJ and Diepstraten, ST and König, C and Tai, L and Snetkova, V and Dorighi, KM and Hoberecht, L and Hedditch, MG and Whelan, L and Healey, G and Fayle, D and Lau, K and Potts, MA and Chen, MZ and Johnston, APR and Liao, Y and Shi, W and Kueh, AJ and Haley, B and Fortin, JP and Herold, MJ},
title = {Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {974},
pmid = {39885149},
issn = {2041-1723},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Knock-In Techniques/methods ; CRISPR-Associated Proteins/genetics/metabolism ; Lymphoma/genetics ; Genetic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; Humans ; Gene Knockout Techniques/methods ; Red Fluorescent Protein ; Fibroblasts/metabolism ; Mice, Inbred C57BL ; },
abstract = {Cas12a is a next-generation gene editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.},
}

Animals
Mice
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Gene Knock-In Techniques/methods
CRISPR-Associated Proteins/genetics/metabolism
Lymphoma/genetics
Genetic Engineering/methods
Bacterial Proteins/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism
Humans
Gene Knockout Techniques/methods
Red Fluorescent Protein
Fibroblasts/metabolism
Mice, Inbred C57BL

@article {pmid39754719,
year = {2025},
author = {Chu, SN and Soupene, E and Sharma, D and Sinha, R and McCreary, T and Hernandez, B and Shen, H and Wienert, B and Bowman, C and Yin, H and Lesch, BJ and Jia, K and Romero, KA and Kostamo, Z and Zhang, Y and Tran, T and Cordero, M and Homma, S and Hampton, JP and Gardner, JM and Conklin, BR and MacKenzie, TC and Sheehan, VA and Porteus, MH and Cromer, MK},
title = {Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells.},
journal = {Cell reports},
volume = {44},
number = {1},
pages = {115141},
doi = {10.1016/j.celrep.2024.115141},
pmid = {39754719},
issn = {2211-1247},
support = {T32 AI125222/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *alpha-Globins/genetics/metabolism ; *Erythroid Cells/metabolism ; *alpha-Thalassemia/genetics/metabolism ; *Receptors, Erythropoietin/metabolism/genetics ; *Gene Editing/methods ; *Hemoglobins/metabolism ; Gene Knock-In Techniques ; CRISPR-Cas Systems/genetics ; Erythropoiesis/genetics ; Hematopoietic Stem Cells/metabolism ; beta-Globins/genetics/metabolism ; },
abstract = {The most severe form of α-thalassemia results from loss of all four copies of α-globin. Postnatally, patients face challenges similar to β-thalassemia, including severe anemia and erythrotoxicity due to the imbalance of β-globin and α-globin chains. Despite progress in genome editing treatments for β-thalassemia, there is no analogous curative option for α-thalassemia. To address this, we designed a Cas9/AAV6-mediated genome editing strategy that integrates a functional α-globin gene into the β-globin locus in α-thalassemia patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the α-globin integration cassette significantly increased erythropoietic output from edited HSPCs and led to the most robust production of α-globin, and consequently hemoglobin tetramers. By directing edited HSPCs toward increased production of clinically relevant erythroid cells, this approach has the potential to mitigate the limitations of current treatments for the hemoglobinopathies, including low genome editing and low engraftment rates.},
}

Humans
*alpha-Globins/genetics/metabolism
*Erythroid Cells/metabolism
*alpha-Thalassemia/genetics/metabolism
*Receptors, Erythropoietin/metabolism/genetics
*Gene Editing/methods
*Hemoglobins/metabolism
Gene Knock-In Techniques
CRISPR-Cas Systems/genetics
Erythropoiesis/genetics
Hematopoietic Stem Cells/metabolism
beta-Globins/genetics/metabolism

@article {pmid39745853,
year = {2025},
author = {Wei, R and Yu, Z and Ding, L and Lu, Z and Yao, K and Zhang, H and Huang, B and He, M and Ma, L},
title = {Improved split prime editors enable efficient in vivo genome editing.},
journal = {Cell reports},
volume = {44},
number = {1},
pages = {115144},
doi = {10.1016/j.celrep.2024.115144},
pmid = {39745853},
issn = {2211-1247},
mesh = {*Gene Editing/methods ; Animals ; *Dependovirus/genetics ; Mice ; Humans ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Efficient prime editor (PE) delivery in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), the editing efficiency at different gene loci varies among split sites. Furthermore, efficient split sites within Cas9 nickase (Cas9n) are limited. Here, we verified that 1115 (Asn) is an efficient split site when delivering PEs by dual AAVs. Additionally, we utilized a feature in which reverse transcriptase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high editing efficiency with Rma intein. To test the editing efficiency in vivo, split-ePE3-367 was packaged in AAV9 and achieved 17.5% precise editing in mice. Our findings establish an alternative split-PE architecture that enables robust editing efficiency, facilitating potential utility in disease modeling and correction.},
}

*Gene Editing/methods
Animals
*Dependovirus/genetics
Mice
Humans
CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid39721023,
year = {2025},
author = {Chen, C and Liao, Y and Zhu, M and Wang, L and Yu, X and Li, M and Peng, G},
title = {Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a.},
journal = {Cell reports},
volume = {44},
number = {1},
pages = {115105},
doi = {10.1016/j.celrep.2024.115105},
pmid = {39721023},
issn = {2211-1247},
mesh = {Animals ; *Cell Lineage ; Mice ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Cell Differentiation ; Endodeoxyribonucleases/metabolism/genetics ; Humans ; Embryoid Bodies/cytology/metabolism ; Bacterial Proteins/metabolism/genetics ; },
abstract = {Single-cell lineage tracing based on CRISPR-Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool that utilizes two orthogonal gene editing systems to record cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability to enhance lineage recording with minimized target dropouts and potentially deeper tree depths. Applying DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers, showcased by identifying transcription factor Foxb1 as a modulator in the cell fate determination of neuromesodermal progenitors. Collectively, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.},
}

Animals
*Cell Lineage
Mice
*Single-Cell Analysis/methods
*CRISPR-Cas Systems/genetics
Gene Editing/methods
CRISPR-Associated Protein 9/metabolism
CRISPR-Associated Proteins/metabolism/genetics
Cell Differentiation
Endodeoxyribonucleases/metabolism/genetics
Humans
Embryoid Bodies/cytology/metabolism
Bacterial Proteins/metabolism/genetics

@article {pmid39300230,
year = {2025},
author = {Li, G and Cheng, Y and Yu, J and Zhu, Y and Ma, H and Zhou, Y and Pu, Z and Zhu, G and Yuan, Y and Zhang, Z and Zhou, X and Tian, K and Qiao, J and Hu, X and Chen, XX and Ji, Q and Huang, X and Ma, B and Yao, Y},
title = {Compact RNA editors with natural miniature Cas13j nucleases.},
journal = {Nature chemical biology},
volume = {21},
number = {2},
pages = {280-290},
pmid = {39300230},
issn = {1552-4469},
support = {2023YFC3402402//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 02020200-K02013008//Zhejiang University (ZJU)/ ; },
mesh = {Animals ; Mice ; *Dependovirus/genetics ; Humans ; RNA Editing ; Proprotein Convertase 9/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; CRISPR-Associated Proteins/metabolism/genetics ; RNA/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats-Cas13 effectors are used for RNA editing but the adeno-associated virus (AAV) packaging limitations because of their big sizes hinder their therapeutic application. Here we report the identification of the Cas13j family, with LepCas13j (529 aa) and ChiCas13j (424 aa) being the smallest and most highly efficient variants for RNA interference. The miniaturized Cas13j proteins enable the development of compact RNA base editors. Chi-RESCUE-S, by fusing dChiCas13j with hADAR2dd, demonstrates high efficiency and specificity in A-to-G and C-to-U conversions. Importantly, this system is compatible with single-AAV packaging without the need for protein sequence truncation. It successfully corrected pathogenic mutations, such as APOC3[D65N] and SCN9A[R896Q], to the wild-type forms. In addition, we developed an optimized system, Chi-RESCUE-S-mini3, which pioneered efficient in vivo C-to-U RNA editing of PCSK9 in mice through single-AAV delivery, resulting in reduced total cholesterol levels. These results highlight the potential of Cas13j to treat human diseases.},
}

Animals
Mice
*Dependovirus/genetics
Humans
RNA Editing
Proprotein Convertase 9/genetics/metabolism
HEK293 Cells
CRISPR-Cas Systems
CRISPR-Associated Proteins/metabolism/genetics
RNA/genetics/metabolism

@article {pmid39884612,
year = {2025},
author = {He, D and Zhao, S and Wang, F and Wu, B and Wei, F and Zhao, Y and Wei, X and Ren, H and Zhang, M and Fan, Y and Zhang, J and Yu, S and Tang, Y and Diao, Y},
title = {H9N2 avian influenza virus diagnostics utilizing specific high-sensitivity enzymatic molecular system termed RPA-based CRISPR-Cas13a.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {140474},
doi = {10.1016/j.ijbiomac.2025.140474},
pmid = {39884612},
issn = {1879-0003},
abstract = {H9N2 avian influenza virus (AIV), a major pathogen causing respiratory infections in poultry, poses a significant threat to the poultry industry and human health. Early detection and control of H9N2 infections are essential for minimizing economic losses and preventing potential zoonotic transmission. A novel CRISPR-Cas family member called CRISPR-Cas13a comprises the CRISPR RNA (crRNA) and Cas13a nuclease. Through the crRNA-based reprogramming of Cas13a, a platform for sensing RNAs specifically is available. In this study, we developed a RPA-based CRISPR-Cas13a diagnostic method for rapid detection of the H9N2 AIV. The results demonstrated that at a limit of 10 copies/μL and 10[2] copies/μL could be detected within 50 min, by fluorescence detection and lateral flow strip, respectively, offering a highly sensitive method for H9N2 detection. This method exhibited excellent specificity, distinguishing H9N2 from other pathogens. Furthermore, the RPA-Cas13a-based detection system was tested on clinical samples, showing comparable performance to RT-qPCR. The detection results were visualized using either lateral flow assays or fluorescence, making it a suitable tool for on-site, field-deployable diagnostics. In a word, this RPA-Cas13a diagnostic approach offers high reliability, sensitivity, and specificity, with promising potential for rapidly detecting H9N2 and other viral pathogens in clinical and food safety applications.},
}

@article {pmid39884405,
year = {2025},
author = {S, BR and Dhar, R and Devi, A},
title = {Exosome-mediated CRISPR/Cas delivery: A cutting-edge frontier in cancer gene therapy.},
journal = {Gene},
volume = {},
number = {},
pages = {149296},
doi = {10.1016/j.gene.2025.149296},
pmid = {39884405},
issn = {1879-0038},
abstract = {Cancer is considered the second most common disease globally. In the past few decades, many approaches have been proposed for cancer treatment. One among those is targeted therapy using CRISPR-Cas system which plays an irreplaceable role in translational research through gene editing. However, due to its inability to cope with specific targeting, off-target effects, and limited tumor penetration, it is very challenging to use this approach in cancer studies. To increase its efficacy, CRISPR components are engineered into the extracellular vesicles (EVs), especially exosomes (a subpopulation of EVs). Exosomes have a significant role in cellular communication. Exosome-based CRISPR-Cas system transport for gene editing enhanced specificity, reduced off-target effects, and improved therapeutic potential. In this review, we highlighted the role of exosomes and the CRISPR-Cas system in cancer research, exosome-based CRISPR delivery for cancer treatment, and its future orientation.},
}

@article {pmid39883775,
year = {2025},
author = {Koeppel, J and Ferreira, R and Vanderstichele, T and Riedmayr, LM and Peets, EM and Girling, G and Weller, J and Murat, P and Liberante, FG and Ellis, T and Church, GM and Parts, L},
title = {Randomizing the human genome by engineering recombination between repeat elements.},
journal = {Science (New York, N.Y.)},
volume = {387},
number = {6733},
pages = {eado3979},
doi = {10.1126/science.ado3979},
pmid = {39883775},
issn = {1095-9203},
mesh = {Humans ; *Genome, Human ; *Repetitive Sequences, Nucleic Acid ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Recombination, Genetic ; Translocation, Genetic ; Sequence Deletion ; Gene Rearrangement ; Chromosome Inversion ; Genes, Essential ; Genetic Engineering/methods ; },
abstract = {We lack tools to edit DNA sequences at scales necessary to study 99% of the human genome that is noncoding. To address this gap, we applied CRISPR prime editing to insert recombination handles into repetitive sequences, up to 1697 per cell line, which enables generating large-scale deletions, inversions, translocations, and circular DNA. Recombinase induction produced more than 100 stochastic megabase-sized rearrangements in each cell. We tracked these rearrangements over time to measure selection pressures, finding a preference for shorter variants that avoided essential genes. We characterized 29 clones with multiple rearrangements, finding an impact of deletions on expression of genes in the variant but not on nearby genes. This genome-scrambling strategy enables large deletions, sequence relocations, and the insertion of regulatory elements to explore genome dispensability and organization.},
}

Humans
*Genome, Human
*Repetitive Sequences, Nucleic Acid
*Gene Editing/methods
*CRISPR-Cas Systems
*Recombination, Genetic
Translocation, Genetic
Sequence Deletion
Gene Rearrangement
Chromosome Inversion
Genes, Essential
Genetic Engineering/methods

@article {pmid39883010,
year = {2025},
author = {Zhang, W and Zhong, Y and Wang, J and Zou, G and Chen, Q and Liu, C},
title = {Direct repeat region 3' end modifications regulate Cas12a activity and expand its applications.},
journal = {Nucleic acids research},
volume = {53},
number = {3},
pages = {},
doi = {10.1093/nar/gkaf040},
pmid = {39883010},
issn = {1362-4962},
support = {22307150//National Natural Science Foundation of China/ ; A2303012//Shenzhen Medical Research Fund/ ; 2024A1515012319//Guangdong Basic and Applied Basic Research Foundation/ ; JCYJ20230807110315032//Shenzhen Science and Technology Program/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; Alkaline Phosphatase/metabolism/genetics ; alpha-Fetoproteins/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; Phosphorylation ; Immunoglobulin G ; DNA/metabolism/genetics/chemistry ; Herpesvirus 4, Human/genetics ; },
abstract = {CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing its inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3' end modifications and de-modifications, which can regulate LbCas12a's cis- and trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA modifications at the DR 3' end on LbCas12a's functionality. We find that the temporary inhibitory function of Cas12a can be reactivated by DR 3' end modification corresponding substances, such as alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), DNA exonucleases, ultraviolet radiation, and DNA glycosylases, which greatly expand the scope of application of Cas12a. Clinical applications demonstrated promising results in ALP, AFP, and trace Epstein-Barr virus detection compared to gold standard methods. Our research provides valuable insights into regulating LbCas12a activity through direct modification of DR and significantly expands its potential clinical detection targets, paving the way for future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
Humans
Alkaline Phosphatase/metabolism/genetics
alpha-Fetoproteins/genetics/metabolism
Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins/metabolism/genetics
Phosphorylation
Immunoglobulin G
DNA/metabolism/genetics/chemistry
Herpesvirus 4, Human/genetics

@article {pmid39880939,
year = {2025},
author = {Wang, W and Pan, Q and Tian, B and Yu, Z and Davidson, D and Bai, G and Akhunova, A and Trick, H and Akhunov, E},
title = {Non-additive dosage-dependent effects of TaGS3 gene editing on grain size and weight in wheat.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {2},
pages = {38},
pmid = {39880939},
issn = {1432-2242},
support = {2021-67013-34174//National Institute of Food and Agriculture/ ; 2020-67013-30906//National Institute of Food and Agriculture/ ; 2022-68013-36439//National Institute of Food and Agriculture/ ; INV-004430/GATES/Bill & Melinda Gates Foundation/United States ; },
mesh = {*Triticum/genetics/growth & development ; *Gene Editing/methods ; *Edible Grain/genetics/growth & development ; *CRISPR-Cas Systems ; Alleles ; Phenotype ; Plant Proteins/genetics ; Seeds/growth & development/genetics ; Gene Dosage ; Genes, Plant ; Plants, Genetically Modified/growth & development/genetics ; Loss of Function Mutation ; },
abstract = {Loss-of-function mutations induced by CRISPR-Cas9 in the TaGS3 gene homoeologs show non-additive dosage-dependent effects on grain size and weight and have potential utility for increasing grain yield in wheat. The grain size in cereals is one of the component traits contributing to yield. Previous studies showed that loss-of-function (LOF) mutations in GS3, encoding Gγ subunit of the multimeric G protein complex, increase grain size and weight in rice. While an association between allelic variation in the GS3 homologs of wheat and grain weight/size has been detected previously, the effects of LOF alleles at TaGS3 on these traits remain unknown. We used genome editing to create TaGS3 mutant lines with varying LOF homeo-allele dosages. Contrary to the results obtained in rice, editing all three TaGS3 homoeologous copies resulted in a significant decrease in grain length (4.4%), width (3.4%), grain area (7.3%) and weight (7.5%), without affecting the number of grains per spike. Compared to the wild type, the highest increase in grain weight (up to 9.6%) and area (up to 5.0%) was observed in homozygous mutants with one or two genomes carrying LOF homeo-alleles, suggesting non-additive suppressive effects of TaGS3 on grain size and weight in wheat. Our results suggest that the regulatory effects of GS3 homologs in wheat and rice have diverged. The newly developed LOF homeo-alleles of TaGS3 expand the set of CRISPR-Cas9-induced variants of yield component genes that have potential to increase grain weight in wheat.},
}

*Triticum/genetics/growth & development
*Gene Editing/methods
*Edible Grain/genetics/growth & development
*CRISPR-Cas Systems
Alleles
Phenotype
Plant Proteins/genetics
Seeds/growth & development/genetics
Gene Dosage
Genes, Plant
Plants, Genetically Modified/growth & development/genetics
Loss of Function Mutation

@article {pmid39880685,
year = {2025},
author = {van Dongen, JE and Segerink, LI},
title = {Building the Future of Clinical Diagnostics: An Analysis of Potential Benefits and Current Barriers in CRISPR/Cas Diagnostics.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00816},
pmid = {39880685},
issn = {2161-5063},
abstract = {Advancements in molecular diagnostics, such as polymerase chain reaction and next-generation sequencing, have revolutionized disease management and prognosis. Despite these advancements in molecular diagnostics, the field faces challenges due to high operational costs and the need for sophisticated equipment and highly trained personnel besides having several technical limitations. The emergent field of CRISPR/Cas sensing technology is showing promise as a new paradigm in clinical diagnostics, although widespread clinical adoption remains limited. This perspective paper discusses specific cases where CRISPR/Cas technology can surmount the challenges of existing diagnostic methods by stressing the significant role that CRISPR/Cas technology can play in revolutionizing clinical diagnostics. It underscores the urgency and importance of addressing the technological and regulatory hurdles that must be overcome to harness this technology effectively in clinical laboratories.},
}

@article {pmid39603170,
year = {2025},
author = {Ali, N and Singh, S and Garg, R},
title = {Unlocking crops' genetic potential: Advances in genome and epigenome editing of regulatory regions.},
journal = {Current opinion in plant biology},
volume = {83},
number = {},
pages = {102669},
doi = {10.1016/j.pbi.2024.102669},
pmid = {39603170},
issn = {1879-0356},
mesh = {*Crops, Agricultural/genetics ; *Gene Editing/methods ; *Genome, Plant/genetics ; Epigenesis, Genetic ; Epigenome/genetics ; CRISPR-Cas Systems ; Plant Breeding/methods ; },
abstract = {Genome editing tools could precisely and efficiently target plant genomes leading to the development of improved crops. Besides editing the coding regions, researchers can employ editing technologies to target specific gene regulatory elements or modify epigenetic marks associated with distal regulatory regions, thereby regulating gene expression in crops. This review outlines several prominent genome editing technologies, including CRISPR-Cas9, TALENs, and ZFNs and recent advancements. The applications for genome and epigenome editing especially of regulatory regions in crop plants is also discussed, including efforts to enhance abiotic stress tolerance, yield, disease resistance and plant phenotype. Additionally, the review addresses the potential of epigenetic modifications, such as DNA methylation and histone modifications, to alter gene expression for crop improvement. Finally, the limitations and future scope of utilizing various genome editing tools to manipulate regulatory elements for gene regulation to unlock the full potential of these tools in plant breeding has been discussed.},
}

*Crops, Agricultural/genetics
*Gene Editing/methods
*Genome, Plant/genetics
Epigenesis, Genetic
Epigenome/genetics
CRISPR-Cas Systems
Plant Breeding/methods

@article {pmid39880501,
year = {2025},
author = {Tian, Y and Chen, J and Chen, F and Xu, J and Huang, L and Peng, L and Li, H and Shi, K},
title = {Multiple gRNAs-assisted CRISPR/Cas12a-based portable aptasensor enabling glucometer readout for amplification-free and quantitative detection of malathion.},
journal = {Analytica chimica acta},
volume = {1341},
number = {},
pages = {343662},
doi = {10.1016/j.aca.2025.343662},
pmid = {39880501},
issn = {1873-4324},
mesh = {*Malathion/analysis ; *CRISPR-Cas Systems ; *Biosensing Techniques ; *Aptamers, Nucleotide/chemistry ; *RNA, Guide, CRISPR-Cas Systems ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The threat of toxic malathion residues to human health has always been a serious food safety issue. The CRISPR/Cas system represents an innovative detection technology for pesticide residues, but its application to malathion detection has not been reported yet. In addition, the multiple-guide RNA (gRNA) powered-CRISPR/Cas biosensor has the advantages of being fast, sensitive and does not require pre-amplification. However, the reported multiple-gRNA CRISPR/Cas-based biosensors are largely only used for the detection of nucleic acid targets, and there are still certain challenges in detecting non-nucleic acid targets.

RESULTS: In this work, a multiplex-gRNA-assisted CRISPR/Cas12a-based portable aptasensor (MgCPA) is developed for amplification-free and quantitative detection of malathion using a glucometer. When target malathion is present in the MgCPA strategy, it specifically binds with aptamer and then activates the trans-cleavage activity of the multiplex-gRNA CRISPR/Cas12a. The activated multiple Cas12a/gRNA complexes cut invertase-HP probes on the electrode surface to obtain glucose signals with glucometer assistance. Under optimal conditions, the developed MgCPA strategy achieves satisfactory portable quantitative and sensitive detection of malathion down to 300 fM (S/N = 3) without pre-amplification. Moreover, the satisfactory selectivity, high reproducibility, and good stability of the proposed strategy are also obtained. Due to its excellent and robust shelf life, our developed MgCPA strategy can be practically applied in detecting malathion in orange, apple, cabbage, and spinach samples.

SIGNIFICANCE: Amplification-free, sensitive, portable quantitative and selective detection of malathion in food samples is achieved by employing our developed MgCPA strategy. This strategy not only opens up a new path for the non-nucleic-acid target detection using amplification-free methods based on multiple-gRNA-assisted CRISPR/Cas12a, but also has broad application prospects in ensuring food safety.},
}

*Malathion/analysis
*CRISPR-Cas Systems
*Biosensing Techniques
*Aptamers, Nucleotide/chemistry
*RNA, Guide, CRISPR-Cas Systems
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39880493,
year = {2025},
author = {Wang, S and Shen, X and Chen, G and Zhang, W and Tan, B},
title = {Application and development of CRISPR-Cas12a methods for the molecular diagnosis of cancer: A review.},
journal = {Analytica chimica acta},
volume = {1341},
number = {},
pages = {343603},
doi = {10.1016/j.aca.2024.343603},
pmid = {39880493},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/genetics ; Molecular Diagnostic Techniques/methods ; },
abstract = {Rapid, sensitive, and specific molecular detection methods are crucial for diagnosing, treating and prognosing cancer patients. With advancements in biotechnology, molecular diagnostic technology has garnered significant attention as a fast and accurate method for cancer diagnosis. CRISPR-Cas12a (Cpf1), an important CRISPR-Cas family member, has revolutionized the field of molecular diagnosis since its introduction. CRISPR-Cas technologies are a new generation of molecular tools that are widely used in the detection of pathogens, cancers, and other diseases. Liquid biopsy methods based on CRISPR-Cas12a have demonstrated remarkable success in cancer diagnosis, encompassing the detection of DNA mutations, DNA methylation, tumor-related viruses, and non-nucleic acid molecule identification. This review systematically discusses the developmental history, key technologies, and principles of CRISPR-Cas12a-based molecular diagnostic techniques and their applications in cancer diagnosis. This review has also discussed the future development directions of CRISPR-Cas12a, aiming for it to become a reliable new technology that can be used in clinical application.},
}

Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/diagnosis/genetics
Molecular Diagnostic Techniques/methods

@article {pmid39880137,
year = {2025},
author = {Yang, S and Wei, Y and Quansah, E and Zhang, Z and Da, W and Wang, B and Wang, K and Sun, D and Tao, Z and Zhang, C},
title = {Cas12a Is Competitive for Gene Editing in the Malaria parasites.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {107340},
doi = {10.1016/j.micpath.2025.107340},
pmid = {39880137},
issn = {1096-1208},
abstract = {Malaria, caused by the Plasmodium parasites, has always been one of the worst infectious diseases that threaten human health, making it necessary for us to study the genetic function and physiological mechanisms of Plasmodium parasites from the molecular level to find more effective ways of addressing the increasingly pressing threat. The CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) is an RNA-guided adaptive immune system, which has been extensively developed and used as a genome editing tool in many organisms, including Plasmodium parasites. However, due to the physiological characteristics and special genomic characteristics of Plasmodium parasites, most of the tools currently used for genome editing of Plasmodium parasites have not met expectations. CRISPR-Cas12a (also known as Cpf1), one of the CRISPR-Cas systems, has attracted considerable attention because of its characteristics of being used for biological diagnosis and multiple genome editing. Recent studies have shown that its unique properties fit the genetic makeup of Plasmodium parasites making it a promising tool for gene editing in these parasites. In this review, we have summarized the relevant content of the Cas12 family, especially the frequently used Cas12a, its advantages for gene editing, and the application prospects in Plasmodium parasites.},
}

@article {pmid39879084,
year = {2025},
author = {Eom, H and Choi, YJ and Nandre, R and Kim, M and Oh, YL and Kim, S and Nakazawa, T and Honda, Y and Ro, HS},
title = {Targeted insertion of heterogenous DNA using Cas9-gRNA ribonucleoprotein-mediated gene editing in Ganoderma lucidum.},
journal = {Bioengineered},
volume = {16},
number = {1},
pages = {2458376},
doi = {10.1080/21655979.2025.2458376},
pmid = {39879084},
issn = {2165-5987},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Reishi/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Ribonucleoproteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA, Fungal/genetics/metabolism ; },
abstract = {Gene editing is emerging as a powerful tool for introducing novel functionalities in mushrooms. While CRISPR/Cas9-induced double-strand breaks (DSBs) typically rely on non-homologous end joining (NHEJ) for gene disruption, precise insertion of heterologous DNA in mushrooms is less explored. Here, we evaluated the efficacy of inserting donor DNAs (8-1008 bp) with or without homologous arms at Cas9-gRNA RNP-induced DSBs. Co-transformation of donor DNAs with RNP targeting the pyrG gene in Ganoderma lucidum yielded 184 transformants without homologous arms and 781 with 300-bp homologous arms (HR_donor DNAs). Restriction analysis and sequencing identified 122 hR_donor DNA transformants with complete donor DNA sequences, achieving 15.6% HDR efficiency (122/781), contrasting with 8 instances via NHEJ from the 184 transformants. These findings highlight the viability of HDR for precise genomic editing in mushrooms, enabling targeted modifications to enhance functionalities.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Reishi/genetics/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Ribonucleoproteins/genetics/metabolism
CRISPR-Associated Protein 9/genetics/metabolism
DNA Breaks, Double-Stranded
DNA, Fungal/genetics/metabolism

@article {pmid39878871,
year = {2025},
author = {Schöllkopf, AI and Almeida, L and Krammer, K and Rivero, CG and Liebl, W and Ehrenreich, A},
title = {Deletion of atypical type II restriction genes in Clostridium cellulovorans using a Cas9-based gene editing system.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {31},
pmid = {39878871},
issn = {1432-0614},
support = {161B0930//Bundesministerium für Bildung und Forschung/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Clostridium cellulovorans/genetics/metabolism ; Promoter Regions, Genetic ; Gene Deletion ; CRISPR-Associated Protein 9/genetics/metabolism ; Riboswitch/genetics ; Conjugation, Genetic ; Bacterial Proteins/genetics/metabolism ; },
abstract = {The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans. The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans. Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans. In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans-specific in vivo methylation of the conjugative plasmid in the E. coli donor strain. KEY POINTS: • Quantification of heterologous promoters enables rational choice for genetic engineering. • CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans. • Conjugation protocol and type II REase deletion enhance genetic accessibility.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Clostridium cellulovorans/genetics/metabolism
Promoter Regions, Genetic
Gene Deletion
CRISPR-Associated Protein 9/genetics/metabolism
Riboswitch/genetics
Conjugation, Genetic
Bacterial Proteins/genetics/metabolism

@article {pmid39878690,
year = {2025},
author = {I Edvard Smith, C and Zain, R and Blomberg, P},
title = {[Gene editing is changing the treatment of hereditary diseases].},
journal = {Lakartidningen},
volume = {122},
number = {},
pages = {},
pmid = {39878690},
issn = {1652-7518},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Genetic Diseases, Inborn/therapy/genetics ; *CRISPR-Cas Systems ; },
abstract = {Gene editing is a novel technology within gene therapy, which changes sequences in chromosomal DNA with precision. Even if there are alternative strategies, the Nobel Prize-winning CRISPR/Cas technology has become the dominating principle. During recent years base editing and prime editing, permitting editing without DNA double-strand breaks, have been developed. The first clinical gene editing results were reported in 2021; since then many patients have been treated, and recently the first treatment was approved as a novel therapy in the UK and later in USA and in EU. This update describes various aspects including methodological developments and safety.},
}

Humans
*Gene Editing/methods
*Genetic Therapy/methods
*Genetic Diseases, Inborn/therapy/genetics
*CRISPR-Cas Systems

@article {pmid39878523,
year = {2025},
author = {Xiao, X and Yang, S and Jiang, G and He, S},
title = {Current views and trends of nanomaterials as vectors for gene delivery since the 21st century: a bibliometric analysis.},
journal = {Nanomedicine (London, England)},
volume = {},
number = {},
pages = {1-16},
doi = {10.1080/17435889.2025.2457781},
pmid = {39878523},
issn = {1748-6963},
abstract = {BACKGROUND: Gene therapy is garnering increasing support due to its potential for a "once-delivered, lifelong benefit." The limitations of traditional gene delivery methods have spurred the advancement of bionanomaterials. Despite this progress, a thorough analysis of the evolution, current state, key contributors, focal studies, and future directions of nanomaterials in gene delivery remains absent.

METHODS: This study scrutinizes articles from the Web of Science, spanning 1 January 2 000, to 31 December 2023, employing various online tools for analysis and visualization.

RESULTS: The 21st century has witnessed consistent growth in scholarly work in this domain globally, with notable contributions from China and the US. At the same time, the Chinese Academy of Sciences (CAS), Harvard University, and Massachusetts Institute of Technology (MIT) have emerged as the most productive institutions, with CAS's academician Weihong Tan becoming the field's leading author. While drug delivery and nanoparticles (NPs) have been central themes for two decades, the research focus has shifted from modifying NPs and ultrafine particles to exploring polymer-hybrid NPs, mRNA vaccines, immune responses, green synthesis, and CRISPR/Cas tools.

CONCLUSIONS: This shift marks the transition from nanomaterials to bionanomaterials. The insights provided by this research offer a comprehensive overview of the field and valuable guidance for future investigations.},
}

@article {pmid39878334,
year = {2025},
author = {Yuan, Y and Li, Y and Li, G and Lei, L and Huang, X and Li, M and Yao, Y},
title = {Intelligent Design of Lipid Nanoparticles for Enhanced Gene Therapeutics.},
journal = {Molecular pharmaceutics},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.molpharmaceut.4c00925},
pmid = {39878334},
issn = {1543-8392},
abstract = {Lipid nanoparticles (LNPs) are an effective delivery system for gene therapeutics. By optimizing their formulation, the physiochemical properties of LNPs can be tailored to improve tissue penetration, cellular uptake, and precise targeting. The application of these targeted delivery strategies within the LNP framework ensures efficient delivery of therapeutic agents to specific organs or cell types, thereby maximizing therapeutic efficacy. In the realm of genome editing, LNPs have emerged as a potent vehicle for delivering CRISPR/Cas components, offering significant advantages such as high in vivo efficacy. The incorporation of machine learning into the optimization of LNP platforms for gene therapeutics represents a significant advancement, harnessing its predictive capabilities to substantially accelerate the research and development process. This review highlights the dynamic evolution of LNP technology, which is expected to drive transformative progress in the field of gene therapy.},
}

@article {pmid39878102,
year = {2025},
author = {Khosravi, S and Hinrichs, R and Rönspies, M and Haghi, R and Puchta, H and Houben, A},
title = {Epigenetic state and gene expression remain stable after CRISPR/Cas-mediated chromosomal inversions.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.20403},
pmid = {39878102},
issn = {1469-8137},
support = {EpiChrom 031B1220B//Bundesministerium für Bildung und Forschung/ ; },
abstract = {The epigenetic state of chromatin, gene activity and chromosomal positions are interrelated in plants. In Arabidopsis thaliana, chromosome arms are DNA-hypomethylated and enriched with the euchromatin-specific histone mark H3K4me3, while pericentromeric regions are DNA-hypermethylated and enriched with the heterochromatin-specific mark H3K9me2. We aimed to investigate how the chromosomal location affects epigenetic stability and gene expression by chromosome engineering. Two chromosomal inversions of different sizes were induced using CRISPR/Cas9 to move heterochromatic, pericentric sequences into euchromatic regions. The epigenetic status of these lines was investigated using whole-genome bisulfite sequencing and chromatin immunoprecipitation. Gene expression changes following the induction of the chromosomal inversions were studied via transcriptome analysis. Both inversions had a minimal impact on the global distribution of histone marks and DNA methylation patterns, although minor epigenetic changes were observed across the genome. Notably, the inverted chromosomal regions and their borders retained their original epigenetic profiles. Gene expression analysis showed that only 0.5-1% of genes were differentially expressed genome-wide following the induction of the inversions. CRISPR/Cas-induced chromosomal inversions minimally affect epigenetic landscape and gene expression, preserving their profiles in subsequent generations.},
}

@article {pmid39877015,
year = {2024},
author = {Fatkulin, AA and Chuksina, TA and Sorokina, NP and Smykov, IT and Kuraeva, EV and Masezhnaya, ES and Smagina, KA and Shkurnikov, MY},
title = {Comparative Analysis of Spacer Targets in CRISPR-Cas Systems of Starter Cultures.},
journal = {Acta naturae},
volume = {16},
number = {4},
pages = {81-85},
pmid = {39877015},
issn = {2075-8251},
abstract = {Dairy production facilities represent a unique ecological niche for bacteriophages of lactic acid bacteria. Throughout evolution, bacteria have developed a wide range of defense mechanisms against viral infections caused by bacteriophages. The CRISPR-Cas system is of particular interest due to its adaptive nature. It allows bacteria to acquire and maintain specific resistance to certain bacteriophages. In this study, we investigated the CRISPR-Cas systems of lactic acid bacteria. Special attention was paid to the specificity of the spacers in CRISPR cassettes. CRISPR-Cas systems were found in the genomes of 43% of the lactic acid bacteria studied. Additionally, only 13.1% of the total number of CRISPR cassette spacers matched bacteriophage genomes, indicating that many predicted spacers either lack known phage targets or are directed against other types of mobile genetic elements, such as plasmids.},
}

@article {pmid39876973,
year = {2024},
author = {de Mello, DC and Menezes, JM and de Oliveira, ATF and Cristovão, MM and Kimura, ET and Fuziwara, CS},
title = {Modulating gene expression as a strategy to investigate thyroid cancer biology.},
journal = {Archives of endocrinology and metabolism},
volume = {68},
number = {Spec Issue},
pages = {e240073},
pmid = {39876973},
issn = {2359-4292},
mesh = {Humans ; *Thyroid Neoplasms/genetics ; RNA Interference ; RNA, Small Interfering/genetics ; Gene Expression Regulation, Neoplastic/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; },
abstract = {Modulating the expression of a coding or noncoding gene is a key tool in scientific research. This strategy has evolved methodologically due to advances in cloning approaches, modeling/algorithms in short hairpin RNA (shRNA) design for knockdown efficiency, and biochemical modifications in RNA synthesis, among other developments. Overall, these modifications have improved the ways to either reduce or induce the expression of a given gene with efficiency and facility for implementation in the lab. Allied with that, the existence of various human cell line models for cancer covering different histotypes and biological behaviors, especially for thyroid cancer, has helped improve the understanding of cancer biology. In this review, we cover the most frequently used current techniques for gene modulation in the thyroid cancer field, such as RNA interference (RNAi), short hairpin RNA (shRNA), and gene editing with CRISPR/Cas9 for inhibiting a target gene, and strategies to overexpress a gene, such as plasmid cloning and CRISPRa. Exploring the possibilities for gene modulation allows the improvement of the scientific quality of the studies and the integration of clinicians and basic scientists, leading to better development of translational research.},
}

Humans
*Thyroid Neoplasms/genetics
RNA Interference
RNA, Small Interfering/genetics
Gene Expression Regulation, Neoplastic/genetics
Gene Editing/methods
CRISPR-Cas Systems

@article {pmid39875802,
year = {2025},
author = {Raudstein, M and Peñaranda, MMD and Kjærner-Semb, E and Grove, S and Morton, HC and Edvardsen, RB},
title = {Generation of IgM[+] B cell-deficient Atlantic salmon (Salmo salar) by CRISPR/Cas9-mediated IgM knockout.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {3599},
pmid = {39875802},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; *Salmo salar/genetics/immunology ; *Immunoglobulin M/genetics/immunology ; *B-Lymphocytes/immunology/metabolism ; Gene Knockout Techniques ; },
abstract = {Infectious diseases pose significant challenges to Norwegian Atlantic salmon aquaculture. Vaccines are critical for disease prevention; however, a deeper understanding of the immune system is essential to improve vaccine efficacy. Immunoglobulin M (IgM) is the main antibody involved in the systemic immune response of teleosts, including Atlantic salmon. In this study, we used CRISPR/Cas9 technology to knock out the two IgM genes in Atlantic salmon. High-throughput sequencing revealed an average mutagenesis efficiency of 97% across both loci, with a predominance of frameshift mutations (78%). Gene expression analyses demonstrated significantly reduced membrane-bound IgM mRNA levels in head kidney and spleen tissues. Flow cytometry revealed a 78% reduction in IgM[+] B cells in peripheral blood, and Western blot analyses showed decreased IgM protein levels in serum. Notably, an upregulation of IgT mRNA was observed, suggesting a potential compensatory mechanism. This work presents the first application of CRISPR/Cas9 to disrupt an immune-related gene in the F0 generation of Atlantic salmon, and lays the foundation for generating a model completely lacking IgM[+] B cells which can be used to study the role of B cells and antibodies. This study has implications for advancing immune research in teleosts and for developing strategies to improve salmon health and welfare in aquaculture.},
}

Animals
*CRISPR-Cas Systems
*Salmo salar/genetics/immunology
*Immunoglobulin M/genetics/immunology
*B-Lymphocytes/immunology/metabolism
Gene Knockout Techniques

@article {pmid39873837,
year = {2025},
author = {Asa, H and Kuwabara, C and Matsumoto, K and Shigeta, R and Yamamoto, T and Masuda, Y and Yamada, T},
title = {Simultaneous site-directed mutagenesis for soybean ß-amyrin synthase genes via DNA-free CRISPR/Cas9 system using a single gRNA.},
journal = {Plant cell reports},
volume = {44},
number = {2},
pages = {40},
pmid = {39873837},
issn = {1432-203X},
support = {JPMJSP2119//JST SPRING/ ; },
mesh = {*Glycine max/genetics/enzymology ; *Mutagenesis, Site-Directed/methods ; *CRISPR-Cas Systems ; Intramolecular Transferases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Saponins/metabolism/genetics ; Plant Proteins/genetics/metabolism ; Mutation/genetics ; Oleanolic Acid/analogs & derivatives/metabolism ; Gene Editing/methods ; },
abstract = {We generated soybean mutants related to two ß-amyrin synthase genes using DNA-free site-directed mutagenesis system. Our results suggested that one of the genes is predominant in the soyasaponin biosynthesis. Soyasaponins, which are triterpenoid saponins contained in soybean [Glycine max (L.) Merril], are responsible for the astringent aftertaste of soyfood, and their complete elimination from soybean seeds is a key challenge in the development of cultivars with improved taste. While the loss of function in the ß-amyrin synthase genes (GmBAS1 and GmBAS2) has proven effective in reducing soyasaponin content in soybean seeds, the specific functional roles of these genes remain unclear. In this study, site-directed mutagenesis was performed on two GmBAS loci using a DNA-free clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system. A complex of sgRNA targeting sequences conserved in the two loci and Cas9 protein was introduced into the shoot apical meristems of soybean embryonic axes via bombardment. Cleaved amplified polymorphic sequences (CAPS) analysis conducted 1 month post-bombardment revealed that 138 seedlings out of 1,467 screened exhibited mutations at one or both GmBAS loci. CAPS and sequencing analysis in the subsequent generation identified a total of 16 plants with inheritable mutations ranging from one to ten nucleotides. High-performance liquid chromatography (HPLC) analysis showed that site-directed mutagenesis in the GmBAS1 locus resulted in the absence of soyasaponins in mature seeds, as well as in young roots, stems, and leaves. These findings demonstrate that GmBAS1 is the predominant ß-amyrin synthase gene in soybean plants. In addition, the DNA-free CRISPR/Cas9 system was shown to be highly efficient in inducing simultaneous mutagenesis at two target loci using a single gRNA.},
}

*Glycine max/genetics/enzymology
*Mutagenesis, Site-Directed/methods
*CRISPR-Cas Systems
Intramolecular Transferases/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Saponins/metabolism/genetics
Plant Proteins/genetics/metabolism
Mutation/genetics
Oleanolic Acid/analogs & derivatives/metabolism
Gene Editing/methods

@article {pmid39780710,
year = {2025},
author = {Elkhadragy, L and Carlino, MJ and Jordan, LR and Pennix, T and Ismail, N and Guzman, G and Samuelson, JP and Schook, LB and Schachtschneider, KM and Gaba, RC},
title = {Development of a genetically tailored implantation hepatocellular carcinoma model in Oncopigs by somatic cell CRISPR editing.},
journal = {Disease models & mechanisms},
volume = {18},
number = {1},
pages = {},
doi = {10.1242/dmm.052079},
pmid = {39780710},
issn = {1754-8411},
support = {1R21CA219461//NIH NCI/ ; 1R21CA219461/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Carcinoma, Hepatocellular/genetics/pathology ; *Gene Editing ; *Liver Neoplasms/genetics/pathology ; *Disease Models, Animal ; *PTEN Phosphohydrolase/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mutation/genetics ; Animals, Genetically Modified ; Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; Cell Proliferation ; Cell Movement/genetics ; Cell Line, Tumor ; Swine ; Tumor Suppressor Protein p53/genetics/metabolism ; },
abstract = {Hepatocellular carcinoma (HCC) is an aggressive disease with poor prognosis, necessitating preclinical models for evaluating novel therapies. Large-animal models are particularly valuable for assessing locoregional therapies, which are widely employed across HCC stages. This study aimed to develop a large-animal HCC model with tailored tumor mutations. The Oncopig, a genetically engineered pig with inducible TP53R167H and KRASG12D, was used in the study. Hepatocytes were isolated from Oncopigs and exposed to Cre recombinase in vitro to create HCC cells, and additional mutations were introduced by CRISPR/Cas9 knockout of PTEN and CDKN2A. These edits increased Oncopig HCC cell proliferation and migration. Autologous HCC cells with these CRISPR edits were implanted into Oncopigs using two approaches: ultrasound-guided percutaneous liver injections, which resulted in the development of localized intrahepatic masses, and portal vein injections, which led to multifocal tumors that regressed over time. Tumors developed by both approaches harbored PTEN and CDKN2A knockout mutations. This study demonstrates the feasibility of developing genetically tailored HCC tumors in Oncopigs using somatic cell CRISPR editing and autologous implantation, providing a valuable large-animal model for in vivo therapeutic assessment.},
}

Animals
*Carcinoma, Hepatocellular/genetics/pathology
*Gene Editing
*Liver Neoplasms/genetics/pathology
*Disease Models, Animal
*PTEN Phosphohydrolase/genetics/metabolism
*CRISPR-Cas Systems/genetics
Mutation/genetics
Animals, Genetically Modified
Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism
Cell Proliferation
Cell Movement/genetics
Cell Line, Tumor
Swine
Tumor Suppressor Protein p53/genetics/metabolism

@article {pmid39641224,
year = {2025},
author = {Yu, J and Li, S and Xiong, B and Shen, Y and Guan, X and Zhu, Y and Fang, Y and Zhang, S and Ding, S and Liu, C and Yue, W and Yin, H and Xu, H},
title = {Probiotics Bi-Enzymatic Cascade Repair System for Editing the Inflammatory Microenvironment to Boost Probiotic Therapy in Inflammatory Bowel Disease.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {4},
pages = {e2412429},
doi = {10.1002/adma.202412429},
pmid = {39641224},
issn = {1521-4095},
support = {82151318//National Natural Science Foundation of China/ ; 82430064//National Natural Science Foundation of China/ ; 82302206//National Natural Science Foundation of China/ ; 82102050//National Natural Science Foundation of China/ ; 82402268//National Natural Science Foundation of China/ ; 82302197//National Natural Science Foundation of China/ ; 82102048//National Natural Science Foundation of China/ ; 2022ZSQD07//Scientific Research and Development Fund of Zhongshan Hospital of Fudan University/ ; 2019LJ21//Shanghai Municipal Health Commission/ ; SHSLCZDZK03502//Shanghai Municipal Health Commission/ ; 23YF1441600//Shanghai Sailing Program/ ; 2023TQ0073//China Postdoctoral Science Foundation/ ; },
mesh = {*Probiotics/therapeutic use ; Animals ; *Inflammatory Bowel Diseases/therapy/metabolism ; Mice ; CRISPR-Cas Systems/genetics ; Gene Editing ; Lacticaseibacillus rhamnosus ; Inflammation/metabolism ; Reactive Oxygen Species/metabolism ; Humans ; Gastrointestinal Microbiome ; },
abstract = {Inflammatory bowel disease presents significant treatment challenges owing to its complex pathology. Although probiotics have shown promise as a therapeutic option, their effectiveness is often limited by low concentrations at sites of inflammation, exacerbated by excessive reactive oxygen species and inflammatory triggers. To address this, an innovative cascade repair system is developed to enhance probiotic therapeutic impact by modulating the intestinal microenvironment. This system uses iMXene's catalytic properties to neutralize reactive oxygen species in the gut and its capacity to deliver the CRISPR/dCas9 gene editing system to activate the NLR family pyrin domain containing 12 genes, helping suppress inflammation. By promoting the colonization of Lactobacillus rhamnosus, the system inhibits inflammation pathways and supports the restoration of a balanced intestinal flora through a cascade repair mechanism. These findings demonstrate significant therapeutic benefits in experimental models, with improvements in the overall well-being of treated mice and effective repair of intestinal inflammation damage. This pioneering approach holds promise for inflammatory bowel disease treatment and opens new avenues for managing other inflammatory conditions, offering valuable insights and guidance for future research into inflammatory diseases.},
}

*Probiotics/therapeutic use
Animals
*Inflammatory Bowel Diseases/therapy/metabolism
Mice
CRISPR-Cas Systems/genetics
Gene Editing
Lacticaseibacillus rhamnosus
Inflammation/metabolism
Reactive Oxygen Species/metabolism
Humans
Gastrointestinal Microbiome

@article {pmid39630114,
year = {2025},
author = {Guan, X and Yang, R and Zhang, J and Moon, J and Hou, C and Guo, C and Avery, L and Scarola, D and Roberts, DS and LaSala, R and Liu, C},
title = {Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR-Cas12a.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {4},
pages = {e2411021},
doi = {10.1002/advs.202411021},
pmid = {39630114},
issn = {2198-3844},
support = {U01CA269147/NH/NIH HHS/United States ; U01AI148306/NH/NIH HHS/United States ; U01CA269147/NH/NIH HHS/United States ; U01AI148306/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Sensitivity and Specificity ; Papillomaviridae/genetics ; Papillomavirus Infections/diagnosis/genetics/virology ; Nucleic Acids/genetics ; Human papillomavirus 16/genetics ; Female ; Uterine Cervical Neoplasms/genetics/diagnosis ; },
abstract = {CRISPR-Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR-Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR-Cas12a enzyme, a dual-mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay. With the assay, 14 high-risk human papillomavirus (HPV) subtypes are simultaneously detected, collectively responsible for 99% of cervical cancer cases, with attomolar sensitivity. Specifically, the assay not only distinguishes HPV16 and HPV18, the two most common subtypes but also detects 12 other high-risk pooled HPV subtypes. To enable low-cost point-of-care testing, the assay is incorporated into a paper-based microfluidic chip. Furthermore, the clinical performance of the paper-based microfluidic chip is validated by testing 75 clinical swab samples, achieving performance comparable to that of PCR. This programmable multiplexed nucleic acid assay has the potential to be widely applied for sensitive, specific, and simultaneous detection of different pathogens.},
}

*CRISPR-Cas Systems/genetics
Humans
Sensitivity and Specificity
Papillomaviridae/genetics
Papillomavirus Infections/diagnosis/genetics/virology
Nucleic Acids/genetics
Human papillomavirus 16/genetics
Female
Uterine Cervical Neoplasms/genetics/diagnosis

@article {pmid39622167,
year = {2025},
author = {Maity, A and Sathyanarayanan, A and Kumar, R and Vora, J and Gawde, J and Jain, H and Bagal, B and Subramanian, PG and Sengar, M and Khattry, N and Patkar, N and Hasan, SK},
title = {RAPID-CRISPR: highly sensitive diagnostic assay for detection of PML::RARA isoforms in acute promyelocytic leukemia.},
journal = {Blood advances},
volume = {9},
number = {3},
pages = {463-472},
doi = {10.1182/bloodadvances.2024014539},
pmid = {39622167},
issn = {2473-9537},
mesh = {Humans ; *Leukemia, Promyelocytic, Acute/diagnosis/genetics ; *Oncogene Proteins, Fusion/genetics ; Male ; Female ; Protein Isoforms ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Adult ; Middle Aged ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; Retinoic Acid Receptor alpha/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Acute promyelocytic leukemia (APL), distinguished by the presence of PML::RARA fusion transcript, is a medical emergency because of its high early death rate, which is preventable when diagnosed early. Current diagnostic methods are precise and reliable but are time intensive, require sophisticated instruments, and analytical expertise. This study has redefined APL identification by CRISPR system (RAPID-CRISPR) to rapidly (<3 hours) detect PML::RARA. APL cell lines (NB4 and UF-1) and bone marrow/peripheral blood samples from 74 patients with APL (66/8, retrospective/prospective) and 48 controls were included in the study. We used a DETECTR (DNA endonuclease-targeted CRISPR transreporter) assay to identify the bcr1, bcr2, and bcr3 PML::RARA isoforms. To ensure high specificity, we used PML::RARA-specific loop-mediated isothermal amplification (LAMP) primers, synthetic protospacer-adjacent motif sites, and isoform-specific CRISPR RNAs. RAPID-CRISPR recognized APL with 100% sensitivity and 100% specificity in an ambispective cohort of patient samples. Furthermore, our blinded validation approach to detect PML::RARA in an unbiased manner provides an additional layer in the diagnostic precision of APL. RAPID-CRISPR demonstrated superior sensitivity, detecting as few as 1 copy of PML::RARA compared with 10 copies by the gold-standard reverse transcriptase qualitative and quantitative polymerase chain reaction. The nucleic acid extraction-free protocol combined with the 1-step reverse transcriptase LAMP-based DETECTR followed by lateral flow readout makes the RAPID-CRISPR assay suitable for diagnosing APL in point-of-care settings. This simple, cost-effective tool, with its easy-to-read format, is particularly valuable in underresourced regions. The assay facilitates timely diagnosis and prompt administration of lifesaving therapies such as all-trans retinoic acid and arsenic trioxide in APL.},
}

Humans
*Leukemia, Promyelocytic, Acute/diagnosis/genetics
*Oncogene Proteins, Fusion/genetics
Male
Female
Protein Isoforms
CRISPR-Cas Systems
Sensitivity and Specificity
Adult
Middle Aged
Clustered Regularly Interspaced Short Palindromic Repeats
Cell Line, Tumor
Retinoic Acid Receptor alpha/genetics
Nucleic Acid Amplification Techniques/methods

@article {pmid39445704,
year = {2025},
author = {Cohn, DM and Gurugama, P and Magerl, M and Katelaris, CH and Launay, D and Bouillet, L and Petersen, RS and Lindsay, K and Aygören-Pürsün, E and Maag, D and Butler, JS and Shah, MY and Golden, A and Xu, Y and Abdelhady, AM and Lebwohl, D and Longhurst, HJ},
title = {CRISPR-Based Therapy for Hereditary Angioedema.},
journal = {The New England journal of medicine},
volume = {392},
number = {5},
pages = {458-467},
doi = {10.1056/NEJMoa2405734},
pmid = {39445704},
issn = {1533-4406},
mesh = {Humans ; Male ; Female ; Adult ; *Angioedemas, Hereditary/drug therapy/genetics/therapy ; Middle Aged ; Double-Blind Method ; Genetic Therapy/adverse effects ; Gene Editing ; Plasma Kallikrein/antagonists & inhibitors ; Kallikreins/blood ; CRISPR-Cas Systems ; Young Adult ; Aged ; },
abstract = {BACKGROUND: Hereditary angioedema is a rare genetic disease characterized by severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy that is based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1). A single dose of NTLA-2002 may provide lifelong control of angioedema attacks.

METHODS: In this phase 2 portion of a phase 1-2 trial, we randomly assigned adults with hereditary angioedema in a 2:2:1 ratio to receive NTLA-2002 in a single dose of 25 mg or 50 mg or placebo. The primary end point was the number of angioedema attacks per month (the monthly attack rate) from week 1 through week 16. Secondary end points included safety, pharmacokinetics, and pharmacodynamics (i.e., the change from baseline in total plasma kallikrein protein level); exploratory end points included patient-reported outcomes.

RESULTS: Of the 27 patients who underwent randomization, 10 received 25 mg of NTLA-2002, 11 received 50 mg, and 6 received placebo. From week 1 through week 16, the estimated mean monthly attack rate was 0.70 (95% confidence interval [CI], 0.25 to 1.98) with 25 mg of NTLA-2002, 0.65 (95% CI, 0.24 to 1.76) with 50 mg, and 2.82 (95% CI, 0.80 to 9.89) with placebo; the difference in the estimated mean attack rate with NTLA-2002 as compared with placebo was -75% with 25 mg and -77% with 50 mg. Among patients who received NTLA-2002, 4 of the 10 patients who received 25 mg (40%) and 8 of the 11 who received 50 mg (73%) were attack-free with no additional treatment during the period from week 1 through week 16. The most common adverse events among patients who received NTLA-2002 were headache, fatigue, and nasopharyngitis. The mean percent change in total plasma kallikrein protein levels from baseline to week 16 was -55% with 25 mg and -86% with 50 mg; levels remained unchanged with placebo.

CONCLUSIONS: NTLA-2002 administered in a single dose of 25 mg or 50 mg reduced angioedema attacks and led to robust and sustained reduction in total plasma kallikrein levels in patients with hereditary angioedema. These results support continued investigation in a larger phase 3 trial. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830; EudraCT number, 2021-001693-33.).},
}

Humans
Male
Female
Adult
*Angioedemas, Hereditary/drug therapy/genetics/therapy
Middle Aged
Double-Blind Method
Genetic Therapy/adverse effects
Gene Editing
Plasma Kallikrein/antagonists & inhibitors
Kallikreins/blood
CRISPR-Cas Systems
Young Adult
Aged

@article {pmid39264585,
year = {2025},
author = {Lou, H and Wang, X and Jiang, Q and Li, X and Yao, Y and Chen, Q and Chen, L and Zhang, S and Yu, Y and Liu, C and Zhou, H},
title = {Clinical evaluation of a highly multiplexed CRISPR-based diagnostic assay for diagnosing lower respiratory tract infection: a prospective cohort study.},
journal = {Infectious diseases (London, England)},
volume = {57},
number = {2},
pages = {167-177},
doi = {10.1080/23744235.2024.2402921},
pmid = {39264585},
issn = {2374-4243},
mesh = {Humans ; Prospective Studies ; Male ; Female ; Middle Aged ; *Respiratory Tract Infections/diagnosis/microbiology ; Aged ; *Sensitivity and Specificity ; Adult ; *Multiplex Polymerase Chain Reaction/methods ; High-Throughput Nucleotide Sequencing/methods ; Molecular Diagnostic Techniques/methods ; Bacteria/genetics/isolation & purification/classification ; Young Adult ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Aged, 80 and over ; CRISPR-Cas Systems ; Bacterial Infections/diagnosis/microbiology ; Adolescent ; },
abstract = {OBJECTIVE: Accurate and rapid identification of causative pathogens is essential to guide the clinical management of lower respiratory tract infections (LRTIs). Here we conducted a single-centre prospective study in 284 patients suspected of lower respiratory tract infections to evaluate the utility of a nucleic acid test based on highly multiplexed polymerase chain reaction (PCR) and CRISPR-Cas12a.

METHODS: We determined the analytical and diagnostic performance of the CRISPR assay using a combination of reference standards, including conventional microbiological tests (CMTs), metagenomic Next-Generation Sequencing (mNGS), and clinical adjudication by a panel of experts on infectious diseases and microbiology.

RESULTS: The CRISPR assay showed a higher detection rate (63.0%) than conventional microbiological tests (38.4%) and was lower than metagenomic Next-Generation Sequencing (72.9%). In detecting polymicrobial infections, the positivity rate of the CRISPR assay (19.4%) was higher than conventional microbiological tests (3.5%) and lower than metagenomic Next-Generation Sequencing (28.9%). The overall diagnostic sensitivity of the CRISPR assay (67.8%) was higher than conventional microbiological tests (41.8%), and lower than metagenomic Next-Generation Sequencing (93.2%).

CONCLUSIONS: Considering the low cost, ease of operation, short turnaround time, and broad range of pathogens detected in a single test, the CRISPR assay has the potential to be implemented as a screening tool for the aetiological diagnosis of lower respiratory tract infections patients, especially in cases where atypical bacteria or coinfections are suspected.},
}

Humans
Prospective Studies
Male
Female
Middle Aged
*Respiratory Tract Infections/diagnosis/microbiology
Aged
*Sensitivity and Specificity
Adult
*Multiplex Polymerase Chain Reaction/methods
High-Throughput Nucleotide Sequencing/methods
Molecular Diagnostic Techniques/methods
Bacteria/genetics/isolation & purification/classification
Young Adult
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Aged, 80 and over
CRISPR-Cas Systems
Bacterial Infections/diagnosis/microbiology
Adolescent

@article {pmid39871009,
year = {2025},
author = {Sakthivel, K and Balasubramanian, R and Sampathrajan, V and Veerasamy, R and Appachi, SV and K K, K},
title = {Transforming tomatoes into GABA-rich functional foods through genome editing: A modern biotechnological approach.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {27},
pmid = {39871009},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *gamma-Aminobutyric Acid/metabolism ; *Solanum lycopersicum/genetics/metabolism ; *CRISPR-Cas Systems ; Functional Food ; Plant Breeding/methods ; Humans ; Genome, Plant ; },
abstract = {Gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter which blocks the impulses between nerve cells in the brain. Due to the increasing awareness about the health promoting benefits associated with GABA, it is also artificially synthesized and consumed as a nutritional supplement by people in some regions of the world. Though among the fresh vegetables, tomato fruits do contain a comparatively higher amount of GABA (0.07 to 2.01 mg g[-1] FW), it needs to be further enhanced to fully impart its potential health benefits. Achieving this feat through classical breeding approaches is time and resource consuming, and is also associated with linkage drag. On the other hand, precise targeting of specific sites in the genome with less off- target effects is mediated by CRISPR/Cas9 genome editing tool and is widely used to overcome the barriers associated with traditional breeding approaches. Combining genome editing with speed breeding techniques can enable the rapid development of GABA-rich tomato cultivars, paving a way to unlock a new era of functional foods, where every bite contributes to cognitive well-being and holistic health. This review highlights the significance of GABA boosted functional foods and explores the potential of CRISPR/Cas9 technology for developing GABA enriched tomatoes.},
}

*Gene Editing/methods
*gamma-Aminobutyric Acid/metabolism
*Solanum lycopersicum/genetics/metabolism
*CRISPR-Cas Systems
Functional Food
Plant Breeding/methods
Humans
Genome, Plant

@article {pmid39870618,
year = {2025},
author = {Ray-Jones, H and Sung, CK and Chan, LT and Haglund, A and Artemov, P and Della Rosa, M and Ruje, L and Burden, F and Kreuzhuber, R and Litovskikh, A and Weyenbergh, E and Brusselaers, Z and Tan, VXH and Frontini, M and Wallace, C and Malysheva, V and Bottolo, L and Vigorito, E and Spivakov, M},
title = {Genetic coupling of enhancer activity and connectivity in gene expression control.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {970},
pmid = {39870618},
issn = {2041-1723},
support = {MC-A652-5QA20//RCUK | Medical Research Council (MRC)/ ; MC_UU_00002/4//RCUK | Medical Research Council (MRC)/ ; MR/W029790/1//RCUK | Medical Research Council (MRC)/ ; MC_UU_00002/4//RCUK | Medical Research Council (MRC)/ ; FS/18/53/33863//British Heart Foundation (BHF)/ ; RE/18/1/34212//British Heart Foundation (BHF)/ ; WT220788//Wellcome Trust (Wellcome)/ ; EP/N510129/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; },
mesh = {*Enhancer Elements, Genetic ; *Gene Expression Regulation ; Humans ; Quantitative Trait Loci/genetics ; Monocytes/metabolism ; Cells, Cultured ; Chromatin/genetics ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Transcription Factors/metabolism ; CCCTC-Binding Factor/metabolism ; Protein Binding ; Base Sequence ; Male ; },
abstract = {Gene enhancers often form long-range contacts with promoters, but it remains unclear if the activity of enhancers and their chromosomal contacts are mediated by the same DNA sequences and recruited factors. Here, we study the effects of expression quantitative trait loci (eQTLs) on enhancer activity and promoter contacts in primary monocytes isolated from 34 male individuals. Using eQTL-Capture Hi-C and a Bayesian approach considering both intra- and inter-individual variation, we initially detect 19 eQTLs associated with enhancer-eGene promoter contacts, most of which also associate with enhancer accessibility and activity. Capitalising on these shared effects, we devise a multi-modality Bayesian strategy, identifying 629 "trimodal QTLs" jointly associated with enhancer accessibility, eGene promoter contact, and gene expression. Causal mediation analysis and CRISPR interference reveal causal relationships between these three modalities. Many detected QTLs overlap disease susceptibility loci and influence the predicted binding of myeloid transcription factors, including SPI1, GABPB and STAT3. Additionally, a variant associated with PCK2 promoter contact directly disrupts a CTCF binding motif and impacts promoter insulation from downstream enhancers. Jointly, our findings suggest an inherent genetic coupling of enhancer activity and connectivity in gene expression control relevant to human disease and highlight the regulatory role of genetically determined chromatin boundaries.},
}

*Enhancer Elements, Genetic
*Gene Expression Regulation
Humans
Quantitative Trait Loci/genetics
Monocytes/metabolism
Cells, Cultured
Chromatin/genetics
Promoter Regions, Genetic
CRISPR-Cas Systems
Transcription Factors/metabolism
CCCTC-Binding Factor/metabolism
Protein Binding
Base Sequence
Male

@article {pmid39870449,
year = {2025},
author = {Ai, X and Tang, Z},
title = {Aptazyme-directed A-to-I RNA editing.},
journal = {Methods in enzymology},
volume = {710},
number = {},
pages = {267-283},
doi = {10.1016/bs.mie.2024.11.022},
pmid = {39870449},
issn = {1557-7988},
mesh = {*RNA Editing ; Humans ; *Adenosine Deaminase/metabolism/genetics ; *RNA-Binding Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Inosine/genetics/metabolism ; Adenosine/metabolism/genetics ; HEK293 Cells ; Aptamers, Nucleotide/genetics ; },
abstract = {As a promising therapeutic approach, the RNA editing process can correct pathogenic mutations and is reversible and tunable, without permanently altering the genome. RNA editing mediated by human ADAR proteins offers unique advantages, including high specificity and low immunogenicity. Compared to CRISPR-based gene editing techniques, RNA editing events are temporary, which can reduce the risk of long-term unintended side effects, making off-target edits less concerning than DNA-targeting methods. Moreover, ADAR-based RNA editing tools are less likely to elicit immune reactions because ADAR proteins are of human origin, and their small size makes them relatively easy to incorporate into gene therapy vectors, such as adeno-associated virus vectors (AAVs), which have limited space. Despite the promise of RNA editing as a therapeutic approach, precise temporal and spatial control of RNA editing is still lacking. Therefore, we have developed a small molecule-inducible RNA editing strategy by incorporating aptazymes into the guide RNA of the BoxB-λN-ADAR system. This chapter provides detailed protocols for targeted RNA editing by ADAR deaminases using aptazyme-based guide RNAs controlled by exogenous small molecules, marking the earliest use of aptazymes to regulate RNA editing strategies. Once small molecules are added or removed, aptazymes trigger self-cleavage to release the guide RNA, thus achieving small molecule-controlled RNA editing. To satisfy different RNA editing applications, we have realized the conditional activation and deactivation of A-to-I RNA editing of target mRNA using switch aptazymes. We provide step-by-step protocols for constructing guide RNA plasmids for regulatory purposes and conducting small molecule-induced RNA regulatory editing experiments in cells.},
}

*RNA Editing
Humans
*Adenosine Deaminase/metabolism/genetics
*RNA-Binding Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Inosine/genetics/metabolism
Adenosine/metabolism/genetics
HEK293 Cells
Aptamers, Nucleotide/genetics

@article {pmid39870447,
year = {2025},
author = {Bhakta, S and Tsukahara, T},
title = {Restoration of G to A mutated transcripts using the MS2-ADAR1 system.},
journal = {Methods in enzymology},
volume = {710},
number = {},
pages = {229-240},
doi = {10.1016/bs.mie.2024.11.031},
pmid = {39870447},
issn = {1557-7988},
mesh = {*Adenosine Deaminase/genetics/metabolism ; Humans ; *RNA-Binding Proteins/genetics/metabolism ; RNA Editing ; Guanosine/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; Levivirus/genetics ; RNA, Messenger/genetics ; Point Mutation ; Mutation ; },
abstract = {Site-directed RNA editing (SDRE) holds significant promise for treating genetic disorders resulting from point mutations. Gene therapy, for common genetic illnesses is becoming more popular and, although viable treatments for genetic disorders are scarce, stop codon mutation-related conditions may benefit from gene editing. Effective SDRE generally depends on introducing many guideRNA molecules relative to the target gene; however, large ratios cannot be achieved in the context of gene therapy applications. Gene-encoded information can be altered, and functionally diverse proteins produced from a single gene by restoration of point-mutated RNA molecules using SDRE. Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme, that can specifically convert adenosine (A) residues to inosines (I), which are translated as guanosine (G). MS2 system along with ADAR1 deaminase domain can target a particular A and repair G to A mutations. In this study, we used the RNA binding MS2 coat protein fused with the ADAR1 deaminase domain controlled by the CMV promoter, and a 19 bp guide RNA (complementary to the target mRNA sequence) engineered with 6 × MS2 stem-loops downstream or 1 × MS2 stem-loop (double MS2) on either side, controlled by the U6 promoter. When the EGFP TGG codon (tryptophan) was altered to an amber (TAG), opal (TGA), or ochre (TAA) stop codon, the modified ADAR1 deaminase domain could convert A-to-I (G) at the edited sites. It is anticipated that successful establishment of this technique will result in a new era in gene therapy, allowing remarkably efficient gene repair, even in vivo.},
}

*Adenosine Deaminase/genetics/metabolism
Humans
*RNA-Binding Proteins/genetics/metabolism
RNA Editing
Guanosine/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Therapy/methods
Levivirus/genetics
RNA, Messenger/genetics
Point Mutation
Mutation

@article {pmid39823408,
year = {2025},
author = {Robert, NM and Ferrier-Tarin, S and Tremblay, JJ},
title = {A New Leydig Cell-Exclusive Cre Line Allows Lineage Tracing of Fetal and Adult Leydig Cell Populations in the Mouse.},
journal = {Endocrinology},
volume = {166},
number = {2},
pages = {},
doi = {10.1210/endocr/bqaf012},
pmid = {39823408},
issn = {1945-7170},
support = {MOP-81387/CAPMC/CIHR/Canada ; //Fondation du CHU de Québec-Université Laval/ ; },
mesh = {Animals ; *Leydig Cells/metabolism/cytology ; Male ; Mice ; *Integrases/genetics/metabolism ; *Cell Lineage ; *Insulin/metabolism ; *Proteins/genetics/metabolism ; Fetus/metabolism/cytology ; Mice, Transgenic ; Testis/metabolism/cytology/embryology ; CRISPR-Cas Systems ; Female ; },
abstract = {Leydig cells produce hormones that are required for male development, fertility, and health. Two Leydig cell populations produce these hormones but at different times during development: fetal Leydig cells, which are active during fetal life, and adult Leydig cells, which are functional postnatally. Historically, our ability to understand the origin and function of Leydig cells has been made difficult by the lack of genetic models to exclusively target these cells. Taking advantage of the Leydig cell-exclusive expression pattern of the Insl3 gene, we used a CRISPR/Cas9 gene-editing strategy to knock-in iCre recombinase into the mouse Insl3 locus. To demonstrate the Leydig cell-exclusive nature of our iCre line, lineage-tracing experiments were performed by crossing Insl3iCre mice with a Rosa26LoxSTOPLox-TdTomato reporter. iCre activity was restricted to male offspring. TdTomato fluorescence was detected both in fetal and adult Leydig cells and colocalized with CYP17A1, a classic Leydig cell marker. Prior to birth, fluorescence was observed in fetal Leydig cells beginning at embryonic day 13.0. Fluorescence was also detected in adult Leydig cells starting at postnatal day 5 and continuing to the mature testis. Fluorescence was not detected in any other fetal or adult tissue examined, except for the unexpected finding that the adrenal cortex contains some Insl3-expressing Leydig-like cells. Our Leydig cell-exclusive iCre line therefore constitutes an invaluable new tool to study not only the origin of Leydig cells but also to target genes that have been long-proposed to be important for the development and functioning of these critical endocrine cells.},
}

Animals
*Leydig Cells/metabolism/cytology
Male
Mice
*Integrases/genetics/metabolism
*Cell Lineage
*Insulin/metabolism
*Proteins/genetics/metabolism
Fetus/metabolism/cytology
Mice, Transgenic
Testis/metabolism/cytology/embryology
CRISPR-Cas Systems
Female

@article {pmid39814565,
year = {2025},
author = {Chau, CCC and Weckman, NE and Thomson, EE and Actis, P},
title = {Solid-State Nanopore Real-Time Assay for Monitoring Cas9 Endonuclease Reactivity.},
journal = {ACS nano},
volume = {19},
number = {3},
pages = {3839-3851},
doi = {10.1021/acsnano.4c15173},
pmid = {39814565},
issn = {1936-086X},
mesh = {*Nanopores ; *CRISPR-Cas Systems/genetics ; DNA/chemistry/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Endonucleases/metabolism/chemistry ; },
abstract = {The field of nanopore sensing is now moving beyond nucleic acid sequencing. An exciting avenue is the use of nanopore platforms for the monitoring of biochemical reactions. Biological nanopores have been used for this application, but solid-state nanopore approaches have lagged. This is due to the necessity of using higher salt conditions (e.g., 4 M LiCl) to improve the signal-to-noise ratio which completely abolish the activities of many biochemical reactions. We pioneered a polymer electrolyte solid-state nanopore approach that maintains a high signal-to-noise ratio even at a physiologically relevant salt concentration. Here, we report the monitoring of the restriction enzyme SwaI and CRISPR-Cas9 endonuclease activities under physiological salt conditions and in real time. We investigated the dsDNA cleavage activity of these enzymes in a range of digestion buffers and elucidated the off-target activity of CRISPR-Cas9 ribonucleoprotein endonuclease in the presence of single base pair mismatches. This approach enables the application of solid-state nanopores for the dynamic monitoring of biochemical reactions under physiological salt conditions.},
}

*Nanopores
*CRISPR-Cas Systems/genetics
DNA/chemistry/metabolism
CRISPR-Associated Protein 9/metabolism/genetics/chemistry
Endonucleases/metabolism/chemistry

@article {pmid39740438,
year = {2025},
author = {Shen, Y and Li, B and Hao, G and Duan, M and Zhao, Y and Liu, Z and Li, X and Jia, F},
title = {A CRISPR/Cas12a-based direct transverse relaxation time biosensor via hydrogel sol-gel transition for Salmonella detection.},
journal = {Food chemistry},
volume = {470},
number = {},
pages = {142693},
doi = {10.1016/j.foodchem.2024.142693},
pmid = {39740438},
issn = {1873-7072},
mesh = {*Biosensing Techniques/instrumentation ; *Salmonella/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Hydrogels/chemistry ; Bacterial Proteins/genetics/metabolism/chemistry ; Phase Transition ; },
abstract = {This research developed a magnetic relaxation switching (MRS) biosensor based on hydrogel sol-gel transition and the CRISPR/Cas12a system (MRS-CRISPR) to detect Salmonella. Herein, the alkaline phosphatase (ALP) labeled with streptavidin was captured by the biotin-modified DNA on magnetic nanoparticles (MNPs) surface, which generated an acidic environment via enzymatic reaction to release Ca[2+] and induced the transformation of alginate sol to hydrogels. In contrast, Salmonella activated the trans-cleavage activity of the CRISPR/Cas12a system, interrupting the capture of ALP and the subsequent sol-gel transition. Then, transverse relaxation time (T2), which was regulated by the hydrogelation process was measured for Salmonella detection. The MRS-CRISPR biosensor enables sensitive detection of Salmonella with a detection limit of 158 CFU/mL. It directly alters the state of water molecules, overcoming the disadvantages of traditional MRS sensors that rely on MNPs to produce T2 signals indirectly. This method offers innovative insights for the application of the MRS technology in food safety analysis.},
}

*Biosensing Techniques/instrumentation
*Salmonella/genetics/isolation & purification
*CRISPR-Cas Systems
*Hydrogels/chemistry
Bacterial Proteins/genetics/metabolism/chemistry
Phase Transition

@article {pmid39688405,
year = {2025},
author = {Allan-Blitz, L-T and Adams, G and Sanders, G and Shah, P and Ramesh, K and Jarolimova, J and Ard, KL and Branda, JA and Klausner, JD and Sabeti, PC and Lemieux, JE},
title = {Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens.},
journal = {mSphere},
volume = {10},
number = {1},
pages = {e0067724},
doi = {10.1128/msphere.00677-24},
pmid = {39688405},
issn = {2379-5042},
support = {U19AI110818//National Institute of Allergy and Infectious Diseases (NIAID)/ ; 2019123//Doris Duke Charitable Foundation (DDCF)/ ; 2021287//Doris Duke Charitable Foundation (DDCF)/ ; //American Sexually Transmitted Diseases Association (ASTDA)/ ; K23AI182453//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {Humans ; *Neisseria gonorrhoeae/genetics/isolation & purification ; *Gonorrhea/diagnosis/microbiology/urine ; Male ; *Nucleic Acid Amplification Techniques/methods ; DNA, Bacterial/genetics/urine ; CRISPR-Cas Systems ; Machine Learning ; Molecular Diagnostic Techniques/methods ; Urethritis/microbiology/diagnosis/urine ; Point-of-Care Testing ; Sensitivity and Specificity ; Smartphone ; Point-of-Care Systems ; },
abstract = {Nucleic acid amplification testing (NAAT) for N. gonorrhoeae is unavailable in resource-limited settings. We previously developed a CRISPR-based lateral flow assay for detecting N. gonorrhoeae. We aimed to pair that assay with point-of-care DNA extraction, assess performance in clinical urine specimens, and optimize assay kinetics. We collected urine specimens among men presenting with urethritis enrolling in a clinical trial at the Massachusetts General Hospital Sexual Health Clinic. We assessed the quantified DNA yield of detergent-based extractions with and without heat. We selected one detergent for extracting all specimens, paired with isothermal recombinase polymerase amplification for 90 minutes and lateral flow Cas13a detection, interpreted via pixel intensity analysis. We also trained a smartphone-based machine-learning model on 1,008 images to classify lateral flow results. We used the model to interpret lateral flow results from the clinical specimens. We also tested a modified amplification chemistry with a second forward primer lacking the T7-promoter to accelerate reaction kinetics. Extraction with 0.02% Triton X resulted in an average DNA yield of 2.6 × 10[6] copies/µL (SD ± 6.7 × 10[5]). We treated 40 urine specimens (n = 12 positive) with 0.02% Triton X, and using quantified pixel intensity analysis, the Cas13a-based assay correctly classified all specimens (100% agreement; 95% CI 91.2%-100%). The machine-learning model correctly classified 45/45 strips in the validation data set and all 40 lateral flow strips from clinical specimens. Including the second forward primer reduced incubation time to 60 minutes. Using point-of-care DNA extraction, our Cas13a-based lateral flow N. gonorrhoeae assay demonstrated promising performance among clinical urine specimens.IMPORTANCEUsing a CRISPR-based assay we previously developed for Neisseria gonorrhoeae detection, we developed new techniques to facilitate point-of-care use. We then demonstrated the promising performance of that assay in clinical specimens. Furthermore, we developed a smartphone-based machine learning application for assisting interpretation of lateral flow strip results. Such an assay has the potential to transform the care of sexually transmitted infections in low-resource settings where diagnostic tests are unavailable. A point-of-care pathogen-specific assay, paired with the connectivity offered by a smartphone application, can also support public health surveillance efforts in such areas.},
}

Humans
*Neisseria gonorrhoeae/genetics/isolation & purification
*Gonorrhea/diagnosis/microbiology/urine
Male
*Nucleic Acid Amplification Techniques/methods
DNA, Bacterial/genetics/urine
CRISPR-Cas Systems
Machine Learning
Molecular Diagnostic Techniques/methods
Urethritis/microbiology/diagnosis/urine
Point-of-Care Testing
Sensitivity and Specificity
Smartphone
Point-of-Care Systems

@article {pmid39687993,
year = {2025},
author = {Chen, S and Pinto Carneiro, S and Merkel, OM},
title = {Anionic polymer coating for enhanced delivery of Cas9 mRNA and sgRNA nanoplexes.},
journal = {Biomaterials science},
volume = {13},
number = {3},
pages = {659-676},
pmid = {39687993},
issn = {2047-4849},
mesh = {Humans ; *RNA, Messenger/administration & dosage/chemistry ; A549 Cells ; *CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Polyethylene Glycols/chemistry ; Proto-Oncogene Proteins p21(ras)/genetics ; Gene Editing ; Anions/chemistry ; CRISPR-Cas Systems ; Polymers/chemistry ; },
abstract = {Polymeric carriers have long been recognized as some of the most effective and promising systems for nucleic acid delivery. In this study, we utilized an anionic di-block co-polymer, PEG-PLE, to enhance the performance of lipid-modified PEI (C14-PEI) nanoplexes for delivering Cas9 mRNA and sgRNA targeting KRAS G12S mutations in lung cancer cells. Our results demonstrated that PEG-PLE, when combined with C14-PEI at a weight-to-weight ratio of 0.2, produced nanoplexes with a size of approximately 140 nm, a polydispersity index (PDI) of 0.08, and a zeta potential of around -1 mV. The PEG-PLE/C14-PEI nanoplexes at this ratio were observed to be both non-cytotoxic and effective in encapsulating Cas9 mRNA and sgRNA. Confocal microscopy imaging revealed efficient endosomal escape and intracellular distribution of the RNAs. Uptake pathway inhibition studies indicated that the internalization of PEG-PLE/C14-PEI primarily involves scavenger receptors and clathrin-mediated endocytosis. Compared to C14-PEI formulations, PEG-PLE/C14-PEI demonstrated a significant increase in luciferase mRNA expression and gene editing efficiency, as confirmed by T7EI and ddPCR, in A549 cells. Sanger sequencing identified insertions and/or deletions around the PAM sequence, with a total of 69% indels observed. Post-transfection, the KRAS-ERK pathway was downregulated, resulting in significant increases in cell apoptosis and inhibition of cell migration. Taken together, this study reveals a new and promising formulation for CRISPR delivery as potential lung cancer treatment.},
}

Humans
*RNA, Messenger/administration & dosage/chemistry
A549 Cells
*CRISPR-Associated Protein 9/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
Polyethylene Glycols/chemistry
Proto-Oncogene Proteins p21(ras)/genetics
Gene Editing
Anions/chemistry
CRISPR-Cas Systems
Polymers/chemistry

@article {pmid39637570,
year = {2025},
author = {Ding, L and Cao, S and Bai, L and He, S and He, L and Wang, Y and Wu, Y and Yu, S},
title = {Versatile fluorescence biosensors based on CRISPR/Cas12a for determination of site-specific DNA methylation from blood and tissues.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {329},
number = {},
pages = {125520},
doi = {10.1016/j.saa.2024.125520},
pmid = {39637570},
issn = {1873-3557},
mesh = {*DNA Methylation ; Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Spectrometry, Fluorescence/methods ; DNA/genetics ; },
abstract = {The identification of DNA methylation at specific sites is crucial for the early detection of cancer since DNA methylation is intimately associated to the occurrence and development of cancer. Herein, two types of sensors that can detect site-specific DNA methylation were developed to meet practical requirements using methylation sensitive restriction endonuclease and CRISPR/Cas12a. To accomplish rapid detection of target, an AciI-mediated CRISPR/Cas12a assay was developed by coupling AciI to recognize DNA methylation with Cas12a to identify site-specific DNA. Since protospacer adjacent motif (PAM)-dependent endonuclease activity and trans-cleavage activity of Cas12a, it is possible to detect site-specific DNA methylation within 2 h with high specificity and acceptable sensitivity. To satisfy the needs of trace target detection, we developed an GlaI-strand displacement amplification (SDA) assisted CRISPR/Cas12a system. The system converts double-stranded methylated DNA to abundant single-stranded by GlaI and SDA. Then, the combination of SDA and CRISPR/Cas12a enable cascades amplification of signal. The approach can therefore be used to detect methylation at different specified sites, even those without PAM, and can increase sensitivity with a detection limit down to 8.19 fM. Importantly, the assay can distinguish between colorectal cancer and precancerous tissue, as well as identify colorectal patients and healthy people. This study provides a new avenue for the development of new biosensors for methylation analysis, and the two methods devised have the potential to meet the multiple requirements of site-specific methylation testing in various clinical settings.},
}

*DNA Methylation
Humans
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Spectrometry, Fluorescence/methods
DNA/genetics

@article {pmid39872948,
year = {2023},
author = {Sun, Y and Lin, W and Kaundal, R and Chi, T},
title = {iMAPping the Perturb-Atlas.},
journal = {Life medicine},
volume = {2},
number = {1},
pages = {lnac057},
pmid = {39872948},
issn = {2755-1733},
abstract = {A key objective of the research in the postgenomic era is to decipher the functions of the mammalian genome, which has remained largely enigmatic despite intensive efforts in the functional genomics field over the past two decades. To attack this problem, we have combined the CRISPR-Cas and Cre-Lox technologies to develop iMAP (inducible Mosaic Animal for Perturbation), a transformative tool for rapidly unraveling mammalian genome function in situ. Furthermore, we have used iMAP to rapidly construct a "Perturb-Atlas" profiling the functions of 90 protein-coding genes across 39 tissues in mice, which has offered rich insights into gene functions difficult to readily obtain using conventional mouse gene-targeting models. In this research highlight, we offer a brief primer on the iMAP technology, outlining its mechanism, strengths and limitations, and pointing out future directions of research in the area.},
}

@article {pmid39870442,
year = {2025},
author = {Salvador, PJ and Dugan, NM and Ouye, R and Beal, PA},
title = {En masse evaluation of RNA guides (EMERGe) for ADARs.},
journal = {Methods in enzymology},
volume = {710},
number = {},
pages = {131-152},
doi = {10.1016/bs.mie.2024.11.030},
pmid = {39870442},
issn = {1557-7988},
mesh = {Humans ; *Adenosine Deaminase/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *High-Throughput Nucleotide Sequencing/methods ; RNA Editing ; Adenosine/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; Gene Library ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in duplex RNA, and through the delivery of guide RNAs, can be directed to edit specific adenosine sites. As ADARs are endogenously expressed in humans, their editing capacities hold therapeutic potential and allow us to target disease-relevant sequences in RNA through the rationale design of guide RNAs. However, current design principles are not suitable for difficult-to-edit target sites, posing challenges to unlocking the full therapeutic potential of this approach. This chapter discusses how we circumvent this barrier through an in vitro screening method, En Masse Evaluation of RNA Guides (EMERGe), which enables comprehensive screening of ADAR substrate libraries and facilitates the identification of editing-enabling guide strands for specific adenosines. From library generation and screening to next generation sequencing (NGS) data analysis to verification experiments, we describe how a sequence of interest can be identified through this high-throughput screening method. Furthermore, we discuss downstream applications of selected guide sequences, challenges in maximizing library coverage, and potential to couple the screen with machine learning or deep learning models.},
}

Humans
*Adenosine Deaminase/metabolism/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*High-Throughput Nucleotide Sequencing/methods
RNA Editing
Adenosine/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics
Gene Library

@article {pmid39870441,
year = {2025},
author = {Akira, A and Levanon, E and Ben Aroya, S},
title = {Leveraging Saccharomyces cerevisiae for ADAR research: From high-yield purification to high-throughput screening and therapeutic applications.},
journal = {Methods in enzymology},
volume = {710},
number = {},
pages = {1-18},
doi = {10.1016/bs.mie.2024.11.026},
pmid = {39870441},
issn = {1557-7988},
mesh = {*Saccharomyces cerevisiae/genetics ; *Adenosine Deaminase/metabolism/genetics/isolation & purification ; *High-Throughput Screening Assays/methods ; *RNA Editing ; *RNA-Binding Proteins/genetics/metabolism/isolation & purification ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Saccharomyces cerevisiae, a model eukaryotic organism with a rich history in research and industry, has become a pivotal tool for studying Adenosine Deaminase Acting on RNA (ADAR) enzymes despite lacking these enzymes endogenously. This chapter reviews the diverse methodologies harnessed using yeast to elucidate ADAR structure and function, emphasizing its role in advancing our understanding of RNA editing. Initially, Saccharomyces cerevisiae was instrumental in the high-yield purification of ADARs, addressing challenges associated with enzyme stability and activity in other systems. The chapter highlights the successful application of yeast in high-throughput screening platforms that identify key structural motifs and substrate preferences of ADARs, showcasing its utility in revealing complex enzyme mechanics. Furthermore, we discuss the development of yeast-based systems to optimize guide RNA sequences for site-directed RNA editing (SDRE), demonstrating how these systems can be employed to refine therapeutic strategies targeting genetic mutations. Additionally, exogenous expression of ADARs from various species in yeast has shed light on enzyme potency and substrate recognition across different temperatures, offering insights into evolutionary adaptations. Overall, Saccharomyces cerevisiae has proven to be an invaluable asset in ADAR research, facilitating significant advances in our understanding of RNA editing mechanisms and therapeutic applications.},
}

*Saccharomyces cerevisiae/genetics
*Adenosine Deaminase/metabolism/genetics/isolation & purification
*High-Throughput Screening Assays/methods
*RNA Editing
*RNA-Binding Proteins/genetics/metabolism/isolation & purification
Humans
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39870445,
year = {2025},
author = {Campbell, KB and Cheng, J and Mendoza, HG and Karki, A and Beal, PA and Fisher, AJ},
title = {Structural analysis of human ADAR2-RNA complexes by X-ray crystallography.},
journal = {Methods in enzymology},
volume = {710},
number = {},
pages = {19-53},
doi = {10.1016/bs.mie.2024.11.023},
pmid = {39870445},
issn = {1557-7988},
mesh = {*Adenosine Deaminase/chemistry/metabolism ; Humans ; *RNA-Binding Proteins/chemistry/metabolism ; Crystallography, X-Ray/methods ; RNA Editing ; Protein Binding ; RNA/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems/chemistry ; },
abstract = {Adenosine deaminases acting on RNAs (ADARs) are a class of RNA editing enzymes found in metazoa that catalyze the hydrolytic deamination of adenosine to inosine in duplexed RNA. Inosine is a nucleotide that can base pair with cytidine, therefore, inosine is interpreted by cellular processes as guanosine. ADARs are functionally important in RNA recoding events, RNA structure modulation, innate immunity, and can be harnessed for therapeutically-driven base editing to treat genetic disorders. Guide RNAs (gRNAs) bearing various modifications can be used to recruit ADARs to edit sites of interest in a process called site-directed RNA editing (SDRE). To help advance the rational design of gRNAs for therapeutics, characterizing the structure-to-activity relationship of ADARs' recognition and binding of substrate duplex RNA at atomic resolution is critical. In this chapter, we describe the process of determining the structure of human ADAR2 bound to duplex RNA using X-ray crystallography. Solid phase synthesis of 8-azanebularine-modified RNAs and purification for binding and crystallographic studies are described. The overexpression and purification of ADARs and assembly of the protein-RNA complex are detailed. Lastly, methods for crystallizing ADAR-RNA complexes and X-ray structure determination and data refinement strategies are outlined.},
}

*Adenosine Deaminase/chemistry/metabolism
Humans
*RNA-Binding Proteins/chemistry/metabolism
Crystallography, X-Ray/methods
RNA Editing
Protein Binding
RNA/chemistry/metabolism
RNA, Guide, CRISPR-Cas Systems/chemistry

@article {pmid39869590,
year = {2025},
author = {Brady, DJ and Saviane, A and Battistolli, M and Varponi, I and Barca, F and Shiomi, K and Cappellozza, S and Sandrelli, F},
title = {Enhanced silk production and pupal weight in Bombyx mori through CRISPR/Cas9-mediated circadian Clock gene disruption.},
journal = {PloS one},
volume = {20},
number = {1},
pages = {e0317572},
doi = {10.1371/journal.pone.0317572},
pmid = {39869590},
issn = {1932-6203},
mesh = {*Bombyx/genetics/growth & development ; Animals ; *CRISPR-Cas Systems ; *Pupa/genetics/growth & development ; *Circadian Clocks/genetics ; *Silk/genetics ; Larva/genetics/growth & development ; Insect Proteins/genetics/metabolism ; },
abstract = {The domesticated silkworm, Bombyx mori, is crucial for global silk production, which is a significant economic activity supporting millions of livelihoods worldwide. Beyond traditional silk production, the growing demand for insect larvae in cosmetics, biomedical products, and animal feed underscores the need to enhance B. mori productivity. This study investigates the role of the circadian clock gene Clock in B. mori using CRISPR/Cas9-mediated mutagenesis to establish the ClkΔ29 knock-out mutant strain. Dysregulation of the circadian clock in ClkΔ29 was demonstrated by altered temporal transcriptional profiles of core circadian clock genes in adult heads and disrupted circadian-controlled behaviors, including adult eclosion and egg hatching rhythms under constant darkness. By analysing larval development timing, as well as the weights of late instar larvae, pupae, and cocoon components in ClkΔ29 mutants and in ClkΔ1922 silkworms (carrying an independently generated Clk- null allele), we showed that CLK contributes to physiological processes regulating B. mori development and growth. Importantly, ClkΔ29 mutants reared on a standard sericulture diet exhibited significant increases in key economic traits, with silk production increasing by up to 7%, and pupal weight increasing by up to 25% compared to wild-type controls. This study highlights the potential of circadian clock gene manipulation to significantly enhance sericultural productivity. Future research should focus on elucidating the molecular mechanisms driving these phenotypes and determining whether they result from circadian clock functions or pleiotropic effects of B. mori Clk. These findings provide a foundation for advancing sustainable sericulture and developing new commercial applications for silkworm-derived products.},
}

*Bombyx/genetics/growth & development
Animals
*CRISPR-Cas Systems
*Pupa/genetics/growth & development
*Circadian Clocks/genetics
*Silk/genetics
Larva/genetics/growth & development
Insect Proteins/genetics/metabolism

@article {pmid39869587,
year = {2025},
author = {Kumari, Y and Gunathilaka, N and Amarasinghe, D},
title = {A comprehensive review of biological and genetic control approaches for leishmaniasis vector sand flies; emphasis towards promoting tools for integrated vector management.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {1},
pages = {e0012795},
doi = {10.1371/journal.pntd.0012795},
pmid = {39869587},
issn = {1935-2735},
mesh = {Animals ; *Psychodidae/genetics/parasitology ; *Leishmaniasis/prevention & control/transmission ; *Insect Vectors/genetics/parasitology ; Insect Control/methods ; Humans ; Gene Editing/methods ; Leishmania/genetics ; CRISPR-Cas Systems ; Pest Control, Biological/methods ; },
abstract = {BACKGROUND: Leishmaniasis is a health problem in many regions with poor health and poor life resources. According to the World Health Organization (WHO), an estimated 700,000-1 million new cases arise annually. Effective control of sand fly vector populations is crucial for reducing the transmission of this disease. Therefore, this review aims to comprehensively examine and evaluate the current methods for controlling sand fly populations, focusing on biological and gene drive techniques.

METHODS AND FINDINGS: A detailed, comprehensive literature search was carried out using databases including Google Scholar, PubMed, ScienceDirect, and the National Library of Medicine (NIH). These searches were done using specific keywords related to the field of study. This current review identified several promising methods, including genetically modified sand flies, using transgenic approaches by taking advanced gene editing tools like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) and genetic modification of symbiotic microorganisms for controlling sand fly populations, which appeared to be proven under laboratory and field settings.

CONCLUSION: Genetic control approaches have many benefits over chemical control, including long-lasting effects on targets, high specificity, and less environmental impact. Advances in genetic engineering technologies, particularly CRISPR/Cas9, sterile insect techniques, and gene drive insect modification, offer new avenues for precise and efficient sand fly management. Future research should prioritize optimizing rearing and sterilization techniques, conducting controlled field trials, and fostering collaboration across disciplines to realize the potential of genetic control strategies in combating leishmaniasis.},
}

Animals
*Psychodidae/genetics/parasitology
*Leishmaniasis/prevention & control/transmission
*Insect Vectors/genetics/parasitology
Insect Control/methods
Humans
Gene Editing/methods
Leishmania/genetics
CRISPR-Cas Systems
Pest Control, Biological/methods

@article {pmid39868861,
year = {2025},
author = {Shahrokhtash, A and Sivertsen, MVT and Laursen, SH and Sutherland, DS},
title = {Nanoscale Cellular Traction Force Quantification: CRISPR-Cas12a Supercharged DNA Tension Sensors in Nanoclustered Ligand Patterns.},
journal = {ACS applied materials & interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.4c18358},
pmid = {39868861},
issn = {1944-8252},
abstract = {High-throughput measurement of cellular traction forces at the nanoscale remains a significant challenge in mechanobiology, limiting our understanding of how cells interact with their microenvironment. Here, we present a novel technique for fabricating protein nanopatterns in standard multiwell microplate formats (96/384-wells), enabling the high-throughput quantification of cellular forces using DNA tension gauge tethers (TGTs) amplified by CRISPR-Cas12a. Our method employs sparse colloidal lithography to create nanopatterned surfaces with feature sizes ranging from sub 100 to 800 nm on transparent, planar, and fully PEGylated substrates. These surfaces allow for the orthogonal immobilization of two different proteins or biomolecules using click-chemistry, providing precise spatial control over cellular signaling cues. We demonstrate the robustness and versatility of this platform through imaging techniques, including total internal reflection fluorescence microscopy, confocal laser scanning microscopy, and high-throughput imaging. Applying this technology, we measured the early stage mechanical forces exerted by 3T3 fibroblasts across different nanoscale features, detecting forces ranging from 12 to 56 pN. By integrating the Mechano-Cas12a Assisted Tension Sensor (MCATS) system, we achieved rapid and high-throughput quantification of cellular traction forces, analyzing over 2 million cells within minutes. Our findings reveal that nanoscale clustering of integrin ligands significantly influences the mechanical responses of cells. This platform offers a powerful tool for mechanobiology research, facilitating the study of cellular forces and mechanotransduction pathways in a high-throughput manner compatible with standard cell culture systems.},
}

@article {pmid39868543,
year = {2025},
author = {},
title = {Correction to 'DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems'.},
journal = {Nucleic acids research},
volume = {53},
number = {3},
pages = {},
doi = {10.1093/nar/gkaf024},
pmid = {39868543},
issn = {1362-4962},
}

@article {pmid39868533,
year = {2025},
author = {Hu, Z and Ling, S and Duan, J and Yu, Z and Che, Y and Wang, S and Zhang, S and Zhang, X and Li, Z},
title = {Proximity-activated guide RNA of CRISPR-Cas12a for programmable diagnostic detection and gene regulation.},
journal = {Nucleic acids research},
volume = {53},
number = {3},
pages = {},
doi = {10.1093/nar/gkaf017},
pmid = {39868533},
issn = {1362-4962},
support = {2022YFF0710200//National Science and Technology Major Project of the Ministry of Science and Technology of China/ ; 22304007//National Natural Science Foundation of China/ ; 2252014//Beijing Municipal Natural Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *Escherichia coli/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; Gene Editing/methods ; Aptamers, Nucleotide/genetics ; Endodeoxyribonucleases/metabolism/genetics ; Gene Expression Regulation ; Theophylline ; HEK293 Cells ; Gene Knockout Techniques ; Bacterial Proteins ; },
abstract = {The flexibility and programmability of CRISPR-Cas technology have made it one of the most popular tools for biomarker diagnostics and gene regulation. Especially, the CRISPR-Cas12 system has shown exceptional clinical diagnosis and gene editing capabilities. Here, we discovered that although the top loop of the 5' handle of guide RNA can undergo central splitting, deactivating CRISPR-Cas12a, the segments can dramatically restore CRISPR function through nucleic acid self-assembly or interactions with small molecules and aptamers. This discovery forms the basis of an engineered Cas12a system with a programmable proximity-activated guide RNA (PARC-Cas12a) that links targets of interest to dsDNA. Leveraging the efficient trans- and cis-cleavage of Cas12, our findings further inspired a detection platform design for RNAs or non-nucleic acid biomarkers, enabling highly sensitive and multiplexed analysis. We further demonstrated the feasibility of RNA-controllable gene knockout/knockdown in Escherichia coli. Notably, we successfully validated the gene regulatory capabilities of the PARC-Cas12a system within mammalian cell systems by utilizing the classical theophylline molecule-aptamer system. Our results introduce a programmable toolbox for precise diagnostics and cell regulation, allowing the development of versatile diagnostic tools, complex synthetic biological circuits, and cellular biosensors.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
*Escherichia coli/genetics
CRISPR-Associated Proteins/metabolism/genetics
Gene Editing/methods
Aptamers, Nucleotide/genetics
Endodeoxyribonucleases/metabolism/genetics
Gene Expression Regulation
Theophylline
HEK293 Cells
Gene Knockout Techniques
Bacterial Proteins

@article {pmid39866497,
year = {2025},
author = {Owaid, HA and Al-Ouqaili, MTS},
title = {Molecular characterization and genome sequencing of selected highly resistant clinical isolates of Pseudomonas aeruginosa and its association with the clustered regularly interspaced palindromic repeat/Cas system.},
journal = {Heliyon},
volume = {11},
number = {1},
pages = {e41670},
pmid = {39866497},
issn = {2405-8440},
abstract = {The presence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in the superbug Pseudomonas (P) aeruginosa presents a unique opportunity to precisely target and edit bacterial genomes to modify their drug resistance. The objective was to detect the prevalence of CRISPR in extensively and pan-drug-resistant Pseudomonas aeruginosa and to determine the utility of whole-genome sequencing (WGS) for the analysis of the entire genome for such strains. The antimicrobial susceptibilities of one hundred isolates were assessed using the antibiotic susceptibility test (AST) card of the VITEK system. The presence of the CRISPR/Cas system was determined via specific primers using conventional polymerase chain reaction (PCR). Further, WGS was conducted using a DNA nanoball sequencing platform via BGI-Tech for the isolates of interest. Out of 54 resistant Pseudomonas aeruginosa isolates, 33 (33.0 %) were metallo-β-lactamase producers. Cas1, Cas3, CRISPR1, and CRISPR2 were positive in 6.0 % of isolates, while incomplete CRISPR1-Cas systems alone were found in 15.0 %. Also, CRISPR2-type was found intact in 26 % of isolates. The prevalence of resistance to antimicrobials in P. aeruginosa isolates was significantly greater in the CRISPR/Cas-negative group compared to the CRISPR/Cas-positive. Significant relationships for variables were examined using Fisher's exact tests using Chi-squared and a P-value of <0.05 as a statistical threshold. Further, on examination of CRs as a collective entity, encompassing both extensive drug resistance (XDR) and pan-drug resistance (PDR), it becomes evident that the vast majority of these strains (n = 29; 87.8 %) lacked CRISPR/Cas systems. In phylogenic analysis, PDR-P. aeruginosa revealed a very close evolutionary relationship with those originating from Kazakhstan, while XDR was globally unique. Further, the entire genome showed the presence of unique virulence and resistant pseudomonal genes. The CRISPR/Cas system and drug resistance are antagonistic to one another. XDR and PDR P. aeruginosa represent a potential threat to public health and contribute to the seriousness of associated illnesses by leading to resistant infections. Further, WGS for the two strains revealed resistance to multiple antibiotics. It is important to examine specific antimicrobial resistance (AMR) pathways, which suggests that a significant number of resistant genes in these isolates indicate a loss of CRISPR genes in the two strains. Furthermore, the WGS approach can lead to a better understanding of the genomic mechanism of pseudomonal resistance to antibiotics.},
}

@article {pmid39866228,
year = {2025},
author = {Lin, HW and Lee, PY and Chang, YS and Chang, MS},
title = {Loss of Arhgap39 facilitates cell migration and invasion in murine hepatocellular cancer cells.},
journal = {Oncology research},
volume = {33},
number = {2},
pages = {493-503},
pmid = {39866228},
issn = {1555-3906},
mesh = {Animals ; *Cell Movement ; Mice ; *GTPase-Activating Proteins/metabolism/genetics ; *Carcinoma, Hepatocellular/pathology/metabolism/genetics ; *Liver Neoplasms/pathology/metabolism/genetics ; *Neoplasm Invasiveness ; Cell Line, Tumor ; Humans ; Mice, Knockout ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Rho GTPases are essential regulators for cellular movement and intracellular membrane trafficking. Their enzymatic activities fluctuate between active GTP-bound and inactive GDP-bound states regulated by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Arhgap39/Vilse/Porf-2 is a newly identified GAP. The role of Arhgap39 in migration and invasion has not been addressed thoroughly.

METHODS: The Arhgap39 gene was knocked out by Crispr-Cas9 gene editing in mouse Hepa1-6 and Hepa-1c1c7 cells to analyze the impact of Arhgap39 depletion on migration and invasion.

RESULTS: Loss of Arhgap39 noticeably increased the migration and invasive potential. Purified Arhgap39 recombinant protein facilitated the hydrolysis of GTP in RhoA and Rac1 in vitro. RNA-seq analysis revealed that matrix metalloproteinase 13 (MMP13) and Laminin subunit beta 1 (LAMB1) were increased in Arhgap39[-/-] cells. We further crossed Arhgap39fl/fl with KrasLSL-G12D and p53fl/fl mice under the control of albumin-Cre recombinase to induce the spontaneous development of hepatocellular carcinomas. Intriguingly, the expression levels of MMP13 and the overall survival in Alb-Cre_KrasLSL-G12D_p53fl/fl_Arhgap39fl/fl (KPA) mice were comparable to control Alb-Cre_KrasLSL-G12D_p53fl/fl (KP) mice. The cell migration and invasion of KPA mice were also similar to those of control KP mice.

CONCLUSION: Arhgap39 loss could modulate the migration and invasion in some hepatocellular cancer cells, but not in those isolated from KPA mice.},
}

Animals
*Cell Movement
Mice
*GTPase-Activating Proteins/metabolism/genetics
*Carcinoma, Hepatocellular/pathology/metabolism/genetics
*Liver Neoplasms/pathology/metabolism/genetics
*Neoplasm Invasiveness
Cell Line, Tumor
Humans
Mice, Knockout
CRISPR-Cas Systems

@article {pmid39866131,
year = {2025},
author = {Redel, BK and Lee, K},
title = {Response to Cook et al. re: Novel Off-Targeting Events Identified After Genome Wide Analysis of CRISPR-Cas Edited Pig.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2025.0003},
pmid = {39866131},
issn = {2573-1602},
}

@article {pmid39862084,
year = {2025},
author = {Szabó, Á and Galla, Z and Spekker, E and Szűcs, M and Martos, D and Takeda, K and Ozaki, K and Inoue, H and Yamamoto, S and Toldi, J and Ono, E and Vécsei, L and Tanaka, M},
title = {Oxidative and Excitatory Neurotoxic Stresses in CRISPR/Cas9-Induced Kynurenine Aminotransferase Knockout Mice: A Novel Model for Despair-Based Depression and Post-Traumatic Stress Disorder.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {30},
number = {1},
pages = {25706},
doi = {10.31083/FBL25706},
pmid = {39862084},
issn = {2768-6698},
support = {K138125//National Research, Development, and Innovation Office-NKFIH/ ; 2022/5S729//SZTE SZAOK-KKA/ ; //HUN-REN Hungarian Research Network/ ; JPJSBP120203803//JSPS Joint Research Projects under Bilateral Programs/ ; },
mesh = {Animals ; *Stress Disorders, Post-Traumatic/genetics/metabolism ; *Transaminases/genetics/metabolism ; *Mice, Knockout ; Male ; *Oxidative Stress ; *Disease Models, Animal ; *CRISPR-Cas Systems ; *Depression/genetics/metabolism/etiology ; Mice ; Kynurenine/metabolism ; Kynurenic Acid/metabolism ; Tryptophan/metabolism ; Serotonin/metabolism ; Mice, Inbred C57BL ; },
abstract = {BACKGROUNDS: Memory and emotion are especially vulnerable to psychiatric disorders such as post-traumatic stress disorder (PTSD), which is linked to disruptions in serotonin (5-HT) metabolism. Over 90% of the 5-HT precursor tryptophan (Trp) is metabolized via the Trp-kynurenine (KYN) metabolic pathway, which generates a variety of bioactive molecules. Dysregulation of KYN metabolism, particularly low levels of kynurenic acid (KYNA), appears to be linked to neuropsychiatric disorders. The majority of KYNA is produced by the aadat (kat2) gene-encoded mitochondrial kynurenine aminotransferase (KAT) isotype 2. Little is known about the consequences of deleting the KYN enzyme gene.

METHODS: In CRISPR/Cas9-induced aadat knockout (kat2[-/-]) mice, we examined the effects on emotion, memory, motor function, Trp and its metabolite levels, enzyme activities in the plasma and urine of 8-week-old males compared to wild-type mice.

RESULTS: Transgenic mice showed more depressive-like behaviors in the forced swim test, but not in the tail suspension, anxiety, or memory tests. They also had fewer center field and corner entries, shorter walking distances, and fewer jumping counts in the open field test. Plasma metabolite levels are generally consistent with those of urine: antioxidant KYNs, 5-hydroxyindoleacetic acid, and indole-3-acetic acid levels were lower; enzyme activities in KATs, kynureninase, and monoamine oxidase/aldehyde dehydrogenase were lower, but kynurenine 3-monooxygenase was higher; and oxidative stress and excitotoxicity indices were higher. Transgenic mice displayed depression-like behavior in a learned helplessness model, emotional indifference, and motor deficits, coupled with a decrease in KYNA, a shift of Trp metabolism toward the KYN-3-hydroxykynurenine pathway, and a partial decrease in the gut microbial Trp-indole pathway metabolite.

CONCLUSIONS: This is the first evidence that deleting the aadat gene induces depression-like behaviors uniquely linked to experiences of despair, which appear to be associated with excitatory neurotoxic and oxidative stresses. This may lead to the development of a double-hit preclinical model in despair-based depression, a better understanding of these complex conditions, and more effective therapeutic strategies by elucidating the relationship between Trp metabolism and PTSD pathogenesis.},
}

Animals
*Stress Disorders, Post-Traumatic/genetics/metabolism
*Transaminases/genetics/metabolism
*Mice, Knockout
Male
*Oxidative Stress
*Disease Models, Animal
*CRISPR-Cas Systems
*Depression/genetics/metabolism/etiology
Mice
Kynurenine/metabolism
Kynurenic Acid/metabolism
Tryptophan/metabolism
Serotonin/metabolism
Mice, Inbred C57BL

@article {pmid39861899,
year = {2025},
author = {Sugiokto, FG and Li, R},
title = {Targeting EBV Episome for Anti-Cancer Therapy: Emerging Strategies and Challenges.},
journal = {Viruses},
volume = {17},
number = {1},
pages = {},
pmid = {39861899},
issn = {1999-4915},
support = {R01 AI141410/AI/NIAID NIH HHS/United States ; R01AI141410//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {Humans ; *Herpesvirus 4, Human/genetics ; *Neoplasms/therapy ; *Gene Editing/methods ; *Epstein-Barr Virus Infections/virology/therapy ; Plasmids/genetics ; Genome, Viral ; Animals ; CRISPR-Cas Systems ; },
abstract = {As a ubiquitous human pathogen, the Epstein-Barr virus (EBV) has established lifelong persistent infection in about 95% of the adult population. The EBV infection is associated with approximately 200,000 human cancer cases and 140,000 deaths per year. The presence of EBV in tumor cells provides a unique advantage in targeting the viral genome (also known as episome), to develop anti-cancer therapeutics. In this review, we summarize current strategies targeting the viral episome in cancer cells. We also highlight emerging technologies, such as clustered regularly interspersed short palindromic repeat (CRISPR)-based gene editing or activation, which offer promising avenues for selective targeting of the EBV episome for anti-cancer therapy. We discuss the challenges, limitations, and future perspectives associated with these strategies, including potential off-target effects, anti-cancer efficacy and safety.},
}

Humans
*Herpesvirus 4, Human/genetics
*Neoplasms/therapy
*Gene Editing/methods
*Epstein-Barr Virus Infections/virology/therapy
Plasmids/genetics
Genome, Viral
Animals
CRISPR-Cas Systems

@article {pmid39710780,
year = {2025},
author = {Cui, S and Wang, K and Yang, Y and Lv, X and Li, X},
title = {An integrated and paper-based microfluidic system employing LAMP-CRISPR and equipped with a portable device for simultaneous detection of pathogens.},
journal = {Analytical and bioanalytical chemistry},
volume = {417},
number = {4},
pages = {785-797},
pmid = {39710780},
issn = {1618-2650},
support = {JCKY2021602B009//Defense Industrial Technology Development Program/ ; },
mesh = {*Paper ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/instrumentation/methods ; Lab-On-A-Chip Devices ; Microfluidic Analytical Techniques/instrumentation/methods ; Bacteria/isolation & purification/genetics ; Equipment Design ; Limit of Detection ; },
abstract = {Point-of-care testing methods are essential for the large-scale diagnosis and monitoring of bacterial infections. This study introduces an integrated platform designed for the simultaneous detection of pathogenic bacteria. Users can simply inject samples into the system, which then conducts the entire procedure in a fully automated manner, eliminating the need for external power sources, all within 60 min or less. The innovative paper-based microfluidic system is capable of lysing bacteria and integrating loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12a system, achieving this with minimal reagent usage on a single piece of paper. The reaction reagents are pre-fabricated as freeze-dried powder on the paper, allowing for long-term storage. A portable and cost-effective detection device has been designed to provide stable temperature control and analyze fluorescent signals, complementing the paper-based microfluidic system. This compact device measures 150 × 150 × 100 mm, weighs less than 1.8 kg, has an average power consumption of under 15 W, and supports external power supply. The device utilizes non-contact QR codes for information transmission, ensuring functionality even in areas without Internet connectivity. This platform is capable of simultaneously detecting five typical pathogenic microorganisms, with a detection limit of 1 copy/μL. It boasts several advantages, including miniaturization, lightweight design, low power consumption, portability, affordability, rapid detection, and ease of operation, making it highly suitable for on-site detection.},
}

*Paper
*Nucleic Acid Amplification Techniques/methods/instrumentation
CRISPR-Cas Systems
Humans
Molecular Diagnostic Techniques/instrumentation/methods
Lab-On-A-Chip Devices
Microfluidic Analytical Techniques/instrumentation/methods
Bacteria/isolation & purification/genetics
Equipment Design
Limit of Detection

@article {pmid39701542,
year = {2025},
author = {Oppman, AM and Paradee, WJ and Narayanan, NS and Kim, YC},
title = {Generation and validation of a D1 dopamine receptor Flpo knock-in mouse.},
journal = {Journal of neuroscience methods},
volume = {415},
number = {},
pages = {110345},
doi = {10.1016/j.jneumeth.2024.110345},
pmid = {39701542},
issn = {1872-678X},
support = {R01 NS120987/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Receptors, Dopamine D1/genetics/metabolism ; Mice ; *Mice, Transgenic ; Gene Knock-In Techniques/methods ; Brain/metabolism ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Male ; Neurons/metabolism ; },
abstract = {BACKGROUND: Dopamine is a powerful neuromodulator of diverse brain functions, including movement, motivation, reward, and cognition. D1-type dopamine receptors (D1DRs) are the most prevalently expressed dopamine receptors in the brain. Neurons expressing D1DRs are heterogeneous and involve several subpopulations. Although these neurons can be studied with BAC-transgenic rodents, these models have some limitations especially when considering their integration with conditional or intersectional genetic tools.

NEW METHOD: We developed a novel Drd1-P2A-Flpo (Drd1-Flpo) mouse line in which the Flpo gene was knocked in immediately after the Drd1 gene using CRISPR-Cas9. We validated the Drd1-Flpo line by confirming Flp expression and functionality specific to D1DR+ neurons with immunohistochemistry and in situ hybridization.

The Drd1-Flpo line is a useful resource for studying subpopulations of D1DR+ neurons with intersectional genetic tools.

CONCLUSIONS: We demonstrated brain-wide GFP expression driven by Drd1-Flpo, suggesting that this mouse line may be useful for comprehensive anatomical and functional studies in many brain regions. The Drd1-Flpo model will advance the study of dopaminergic signaling by providing a new tool for investigating the diverse roles of D1DR+ neurons and their subpopulations in brain disease.},
}

Animals
*Receptors, Dopamine D1/genetics/metabolism
Mice
*Mice, Transgenic
Gene Knock-In Techniques/methods
Brain/metabolism
CRISPR-Cas Systems
Mice, Inbred C57BL
Male
Neurons/metabolism

@article {pmid39661735,
year = {2025},
author = {Baysal, C and Kausch, AP and Cody, JP and Altpeter, F and Voytas, DF},
title = {Rapid and efficient in planta genome editing in sorghum using foxtail mosaic virus-mediated sgRNA delivery.},
journal = {The Plant journal : for cell and molecular biology},
volume = {121},
number = {2},
pages = {e17196},
doi = {10.1111/tpj.17196},
pmid = {39661735},
issn = {1365-313X},
support = {DE-SC0018420//Biological and Environmental Research/ ; },
mesh = {*Sorghum/genetics/virology ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; Genome, Plant/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Lyases/genetics/metabolism ; Oxidoreductases/genetics/metabolism ; Potexvirus/genetics ; Plant Viruses ; },
abstract = {The requirement of in vitro tissue culture for the delivery of gene editing reagents limits the application of gene editing to commercially relevant varieties of many crop species. To overcome this bottleneck, plant RNA viruses have been deployed as versatile tools for in planta delivery of recombinant RNA. Viral delivery of single-guide RNAs (sgRNAs) to transgenic plants that stably express CRISPR-associated (Cas) endonuclease has been successfully used for targeted mutagenesis in several dicotyledonous and few monocotyledonous plants. Progress with this approach in monocotyledonous plants is limited so far by the availability of effective viral vectors. We engineered a set of foxtail mosaic virus (FoMV) and barley stripe mosaic virus (BSMV) vectors to deliver the fluorescent protein AmCyan to track viral infection and movement in Sorghum bicolor. We further used these viruses to deliver and express sgRNAs to Cas9 and Green Fluorescent Protein (GFP) expressing transgenic sorghum lines, targeting Phytoene desaturase (PDS), Magnesium-chelatase subunit I (MgCh), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, orthologs of maize Lemon white1 (Lw1) or GFP. The recombinant BSMV did neither infect sorghum nor deliver or express AmCyan and sgRNAs. In contrast, the recombinant FoMV systemically spread throughout sorghum plants and induced somatic mutations with frequencies reaching up to 60%. This mutagenesis led to visible phenotypic changes, demonstrating the potential of FoMV for in planta gene editing and functional genomics studies in sorghum.},
}

*Sorghum/genetics/virology
*Gene Editing/methods
*Plants, Genetically Modified/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Vectors/genetics
Genome, Plant/genetics
Green Fluorescent Proteins/genetics/metabolism
Lyases/genetics/metabolism
Oxidoreductases/genetics/metabolism
Potexvirus/genetics
Plant Viruses

@article {pmid39861013,
year = {2025},
author = {Yibar, A and Duman, M and Ay, H and Ajmi, N and Tasci, G and Gurler, F and Guler, S and Morick, D and Saticioglu, IB},
title = {Genomic Insight into Vibrio Isolates from Fresh Raw Mussels and Ready-to-Eat Stuffed Mussels.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {1},
pages = {},
doi = {10.3390/pathogens14010052},
pmid = {39861013},
issn = {2076-0817},
support = {TOA-2022-668 and TGA-2024-1841//Bursa Uludağ Üni̇versi̇tesi̇/ ; },
mesh = {Animals ; *Vibrio/genetics/isolation & purification/pathogenicity ; *Bivalvia/microbiology ; *Genome, Bacterial/genetics ; *Virulence Factors/genetics ; Genomics ; Food Microbiology ; Shellfish/microbiology ; Phylogeny ; },
abstract = {Consuming raw or undercooked mussels can lead to gastroenteritis and septicemia due to Vibrio contamination. This study analyzed the prevalence, density, species diversity, and molecular traits of Vibrio spp. in 48 fresh raw wild mussels (FRMs) and 48 ready-to-eat stuffed mussels (RTE-SMs) through genome analysis, assessing health risks. The results showed Vibrio prevalence rates of 12.5% in FRMs and 4.2% in RTE-SMs, with V. alginolyticus as the most common species (46.7%). It was determined that the seasonal distribution of Vibrio spp. prevalence in the samples was higher in the summer months. The genome sizes of the Vibrio spp. ranged from approximately 3.9 to 6.1 Mb, with the GC contents varying between 41.9% and 50.4%. A total of 22 virulence factor (VF) classes and up to six antimicrobial resistance (AMR) genes were detected in different Vibrio species. The presence of nine different biosynthetic gene clusters (BGCs), 27 prophage regions, and eight CRISPR/Cas systems in 15 Vibrio strains provides information about their potential pathogenicity, survival strategies, and adaptation to different habitats. Overall, this study provides a comprehensive understanding of the genomic diversity of Vibrio spp. isolated from FRM and RTE-SM samples, shedding light on the prevalence, pathogenicity, and toxicity mechanisms of Vibrio-induced gastroenteritis.},
}

Animals
*Vibrio/genetics/isolation & purification/pathogenicity
*Bivalvia/microbiology
*Genome, Bacterial/genetics
*Virulence Factors/genetics
Genomics
Food Microbiology
Shellfish/microbiology
Phylogeny

@article {pmid39858940,
year = {2025},
author = {Ma, J and Lu, Z},
title = {Developing a Versatile Arsenal: Novel Antimicrobials as Offensive Tools Against Pathogenic Bacteria.},
journal = {Microorganisms},
volume = {13},
number = {1},
pages = {},
doi = {10.3390/microorganisms13010172},
pmid = {39858940},
issn = {2076-2607},
support = {31970101//the National Natural Science Foundation of China/ ; },
abstract = {The pervasive and often indiscriminate use of antibiotics has accelerated the emergence of drug-resistant bacterial strains, thus presenting an acute threat to global public health. Despite a growing acknowledgment of the severity of this crisis, the current suite of strategies to mitigate antimicrobial resistance remains markedly inadequate. This paper asserts the paramount need for the swift development of groundbreaking antimicrobial strategies and provides a comprehensive review of an array of innovative techniques currently under scrutiny. Among these, nano-antimicrobials, antimicrobials derived from ribosomal proteins, CRISPR/Cas-based systems, agents that undermine bacterial bioenergetics, and antimicrobial polysaccharides hold particular promise. This analysis gives special attention to CRISPR/Cas-based antimicrobials, scrutinizing their underlying mechanisms, exploring their potential applications, delineating their distinct advantages, and noting their likely limitations. Furthermore, we extend our exploration by proposing theoretical advancements in antimicrobial technology and evaluating feasible methods for the effective delivery of these agents. This includes leveraging these advances for broader biomedical applications, potentially revolutionizing how we confront bacterial pathogens in the future, and laying a foundation for extended research in multimodal therapeutic strategies.},
}

@article {pmid39858819,
year = {2024},
author = {Zhang, Z and Wei, M and Jia, B and Yuan, Y},
title = {Recent Advances in Antimicrobial Resistance: Insights from Escherichia coli as a Model Organism.},
journal = {Microorganisms},
volume = {13},
number = {1},
pages = {},
doi = {10.3390/microorganisms13010051},
pmid = {39858819},
issn = {2076-2607},
support = {2018YFA0903700//National Natural Science Foundation of China/ ; },
abstract = {Antimicrobial resistance (AMR) represents a critical global health threat, and a thorough understanding of resistance mechanisms in Escherichia coli is needed to guide effective treatment interventions. This review explores recent advances for investigating AMR in E. coli, including machine learning for resistance pattern analysis, laboratory evolution to generate resistant mutants, mutant library construction, and genome sequencing for in-depth characterization. Key resistance mechanisms are discussed, including drug inactivation, target modification, altered transport, and metabolic adaptation. Additionally, we highlight strategies to mitigate the spread of AMR, such as dynamic resistance monitoring, innovative therapies like phage therapy and CRISPR-Cas technology, and tighter regulation of antibiotic use in animal production systems. This review provides actionable insights into E. coli resistance mechanisms and identifies promising directions for future antibiotic development and AMR management.},
}

@article {pmid39858595,
year = {2025},
author = {Dansereau, SJ and Cui, H and Dartawan, RP and Sheng, J},
title = {The Plethora of RNA-Protein Interactions Model a Basis for RNA Therapies.},
journal = {Genes},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/genes16010048},
pmid = {39858595},
issn = {2073-4425},
support = {1R01GM143749-22A1/NH/NIH HHS/United States ; },
mesh = {Humans ; *RNA/metabolism/genetics ; Animals ; CRISPR-Cas Systems ; Aptamers, Nucleotide/genetics/metabolism ; Genetic Therapy/methods ; RNA, Small Interfering/genetics/therapeutic use/metabolism ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {The notion of RNA-based therapeutics has gained wide attractions in both academic and commercial institutions. RNA is a polymer of nucleic acids that has been proven to be impressively versatile, dating to its hypothesized RNA World origins, evidenced by its enzymatic roles in facilitating DNA replication, mRNA decay, and protein synthesis. This is underscored through the activities of riboswitches, spliceosomes, ribosomes, and telomerases. Given its broad range of interactions within the cell, RNA can be targeted by a therapeutic or modified as a pharmacologic scaffold for diseases such as nucleotide repeat disorders, infectious diseases, and cancer. RNA therapeutic techniques that have been researched include, but are not limited to, CRISPR/Cas gene editing, anti-sense oligonucleotides (ASOs), siRNA, small molecule treatments, and RNA aptamers. The knowledge gleaned from studying RNA-centric mechanisms will inevitably improve the design of RNA-based therapeutics. Building on this understanding, we explore the physiological diversity of RNA functions, examine specific dysfunctions, such as splicing errors and viral interactions, and discuss their therapeutic implications.},
}

Humans
*RNA/metabolism/genetics
Animals
CRISPR-Cas Systems
Aptamers, Nucleotide/genetics/metabolism
Genetic Therapy/methods
RNA, Small Interfering/genetics/therapeutic use/metabolism
RNA-Binding Proteins/metabolism/genetics

@article {pmid39857007,
year = {2025},
author = {Lakshmanan, K and Liu, BM},
title = {Impact of Point-of-Care Testing on Diagnosis, Treatment, and Surveillance of Vaccine-Preventable Viral Infections.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {15},
number = {2},
pages = {},
doi = {10.3390/diagnostics15020123},
pmid = {39857007},
issn = {2075-4418},
support = {P30AI117970; U54AI150225/GF/NIH HHS/United States ; },
abstract = {With the advent of a variety of vaccines against viral infections, there are multiple viruses that can be prevented via vaccination. However, breakthrough infections or uncovered strains can still cause vaccine-preventable viral infections (VPVIs). Therefore, timely diagnosis, treatment, and surveillance of these viruses is critical to patient care and public health. Point-of-care (POC) viral diagnostics tools have brought significant improvements in the detection and management of VPVIs. These cutting-edge technologies enable prompt and accurate results, enhancing patient care by facilitating timely treatment decisions. This review delves into the advancements in POC testing, including antigen/antibody detection and molecular assays, while focusing on their impact on the diagnosis, treatment, and surveillance of VPVIs such as mpox, viral hepatitis, influenza, flaviviruses (dengue, Zika, and yellow fever virus), and COVID-19. The role of POC tests in monitoring viral infection is crucial for tracking disease progression and managing outbreaks. Furthermore, the application of POC diagnostics has shown to be vital for public health strategies. In this review, we also highlight emerging POC technologies such as CRISPR-based diagnostics and smartphone-integrated POC devices, which have proven particularly beneficial in resource-limited settings. We underscore the importance of continued research to optimize these diagnostic tools for wider global use for mpox, viral hepatitis, influenza, dengue, and COVID-19, while also addressing current challenges related to their sensitivity, specificity, availability, efficiency, and more.},
}

@article {pmid39856077,
year = {2025},
author = {Xu, X and Chen, J and Wang, Y and Liu, Y and Zhang, Y and Yang, J and Yang, X and Chen, B and He, Z and Champer, J},
title = {Gene drive-based population suppression in the malaria vector Anopheles stephensi.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {1007},
pmid = {39856077},
issn = {2041-1723},
support = {32302455//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Anopheles/genetics ; Female ; *Mosquito Vectors/genetics ; *Malaria/transmission/prevention & control ; Male ; *Gene Drive Technology/methods ; Mosquito Control/methods ; Animals, Genetically Modified ; Alleles ; CRISPR-Cas Systems ; },
abstract = {Gene drives are alleles that can bias the inheritance of specific traits in target populations for the purpose of modification or suppression. Here, we construct a homing suppression drive in the major urban malaria vector Anopheles stephensi targeting the female-specific exon of doublesex, incorporating two gRNAs and a nanos-Cas9 to reduce functional resistance and improve female heterozygote fitness. Our results show that the drive was recessive sterile in both females and males, with various intersex phenotypes in drive homozygotes. Both male and female drive heterozygotes show only moderate drive conversion, indicating that the nanos promoter has lower activity in A. stephensi than in Anopheles gambiae. By amplicon sequencing, we detect a very low level of resistance allele formation. Combination of the homing suppression drive and a vasa-Cas9 line boosts the drive conversion rate of the homing drive to 100%, suggesting the use of similar systems for population suppression in a continuous release strategy with a lower release rate than SIT or fsRIDL techniques. This study contributes valuable insights to the development of more efficient and environmentally friendly pest control tools aimed at disrupting disease transmission.},
}

Animals
*Anopheles/genetics
Female
*Mosquito Vectors/genetics
*Malaria/transmission/prevention & control
Male
*Gene Drive Technology/methods
Mosquito Control/methods
Animals, Genetically Modified
Alleles
CRISPR-Cas Systems

@article {pmid39856062,
year = {2025},
author = {Pena, IA and Shi, JS and Chang, SM and Yang, J and Block, S and Adelmann, CH and Keys, HR and Ge, P and Bathla, S and Witham, IH and Sienski, G and Nairn, AC and Sabatini, DM and Lewis, CA and Kory, N and Vander Heiden, MG and Heiman, M},
title = {SLC25A38 is required for mitochondrial pyridoxal 5'-phosphate (PLP) accumulation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {978},
pmid = {39856062},
issn = {2041-1723},
mesh = {*Mitochondria/metabolism ; *Pyridoxal Phosphate/metabolism ; Humans ; *Glycine Hydroxymethyltransferase/metabolism/genetics ; Cell Proliferation ; CRISPR-Cas Systems ; Mitochondrial Proteins/metabolism/genetics ; Anemia, Sideroblastic/metabolism/genetics ; Vitamin B 6/metabolism ; Cell Line, Tumor ; Mitochondrial Membrane Transport Proteins/metabolism/genetics ; Genetic Diseases, X-Linked ; },
abstract = {Many essential proteins require pyridoxal 5'-phosphate, the active form of vitamin B6, as a cofactor for their activity. These include enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian pyridoxal 5'-phosphate-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial pyridoxal 5'-phosphate levels in mammals remains unknown. In this study, we used a genome-wide CRISPR interference screen in erythroleukemia cells and organellar metabolomics to identify the mitochondrial inner membrane protein SLC25A38 as a regulator of mitochondrial pyridoxal 5'-phosphate. Loss of SLC25A38 causes depletion of mitochondrial, but not cellular, pyridoxal 5'-phosphate, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial pyridoxal 5'-phosphate-dependent enzymatic reactions, including serine to glycine conversion catalyzed by serine hydroxymethyltransferase-2 as well as ornithine aminotransferase. The proliferation defect of SLC25A38-null K562 cells in physiological and low vitamin B6 media can be explained by the loss of serine hydroxymethyltransferase-2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial pyridoxal 5'-phosphate accumulation and provides insights into the pathology of congenital sideroblastic anemia.},
}

*Mitochondria/metabolism
*Pyridoxal Phosphate/metabolism
Humans
*Glycine Hydroxymethyltransferase/metabolism/genetics
Cell Proliferation
CRISPR-Cas Systems
Mitochondrial Proteins/metabolism/genetics
Anemia, Sideroblastic/metabolism/genetics
Vitamin B 6/metabolism
Cell Line, Tumor
Mitochondrial Membrane Transport Proteins/metabolism/genetics
Genetic Diseases, X-Linked

@article {pmid39856035,
year = {2025},
author = {Padilla, MS and Mrksich, K and Wang, Y and Haley, RM and Li, JJ and Han, EL and El-Mayta, R and Kim, EH and Dias, S and Gong, N and Teerdhala, SV and Han, X and Chowdhary, V and Xue, L and Siddiqui, Z and Yamagata, HM and Kim, D and Yoon, IC and Wilson, JM and Radhakrishnan, R and Mitchell, MJ},
title = {Branched endosomal disruptor (BEND) lipids mediate delivery of mRNA and CRISPR-Cas9 ribonucleoprotein complex for hepatic gene editing and T cell engineering.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {996},
pmid = {39856035},
issn = {2041-1723},
support = {T90DE030854//U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)/ ; },
mesh = {*Gene Editing/methods ; *Endosomes/metabolism ; *Lipids/chemistry ; *RNA, Messenger/metabolism/genetics ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles/chemistry ; *T-Lymphocytes/metabolism ; Ribonucleoproteins/metabolism ; Liver/metabolism ; Animals ; Cell Engineering/methods ; Gene Transfer Techniques ; Transfection/methods ; Mice ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are the preeminent non-viral drug delivery vehicle for mRNA-based therapies. Immense effort has been placed on optimizing the ionizable lipid (IL) structure, which contains an amine core conjugated to lipid tails, as small molecular adjustments can result in substantial changes in the overall efficacy of the resulting LNPs. However, despite some advancements, a major barrier for LNP delivery is endosomal escape. Here, we develop a platform for synthesizing a class of branched ILs that improve endosomal escape. These compounds incorporate terminally branched groups that increase hepatic mRNA and ribonucleoprotein complex delivery and gene editing efficiency as well as T cell transfection compared to non-branched lipids. Through an array of complementary experiments, we determine that our lipid architecture induces greater endosomal penetration and disruption. This work provides a scheme to generate a class of ILs for both mRNA and protein delivery.},
}

*Gene Editing/methods
*Endosomes/metabolism
*Lipids/chemistry
*RNA, Messenger/metabolism/genetics
*CRISPR-Cas Systems
Humans
*Nanoparticles/chemistry
*T-Lymphocytes/metabolism
Ribonucleoproteins/metabolism
Liver/metabolism
Animals
Cell Engineering/methods
Gene Transfer Techniques
Transfection/methods
Mice
Liposomes