@article {pmid39727993,
year = {2024},
author = {Mascarenhas, MS and Nascimento, FDS and Schittino, LMP and Galinari, LB and Lino, LSM and Ramos, APS and Diniz, LEC and Mendes, TAO and Ferreira, CF and Santos-Serejo, JAD and Amorim, EP},
title = {Construction and Validation of CRISPR/Cas Vectors for Editing the PDS Gene in Banana (Musa spp.).},
journal = {Current issues in molecular biology},
volume = {46},
number = {12},
pages = {14422-14437},
pmid = {39727993},
issn = {1467-3045},
support = {OPP1093845//IITA - Accelerated Breeding of Better Bananas/ ; OPP1093845//Bill and Melinda Gates Foundation - Accelerated Breeding of Better Bananas/ ; },
abstract = {Bananas and plantains are important staple food crops affected by biotic and abiotic stresses. The gene editing technique via Clustered Regularly Interspaced Short Palindromic Repeats associated with the Cas protein (CRISPR/Cas) has been used as an important tool for development of cultivars with high tolerance to stresses. This study sought to develop a protocol for the construction of vectors for gene knockout. Here we use the phytoene desaturase (PDS) gene as a case study in Prata-Anã banana by the nonhomologous end junction (NHEJ) method. PDS is a key gene in the carotenoid production pathway in plants and its knockout leads to easily visualized phenotypes such as dwarfism and albinism in plants. Agrobacterium-mediated transformation delivered CRISPR/Cas9 constructs containing gRNAs were inserted into embryogenic cell suspension cultures. This is the first study to provide an effective method/protocol for constructing gene knockout vectors, demonstrating gene editing potential in a Brazilian banana variety. The constitutive (CaMV 35S) and root-specific vectors were successfully assembled and confirmed in transformed Agrobacterium by DNA extraction and PCR. The specificity of transformation protocols makes it possible to use the CRISPR-Cas9 technique to develop Prata-Anã banana plants with enhanced tolerance/resistance to major biotic and abiotic factors.},
}

@article {pmid39651221,
year = {2024},
author = {Yun, S and Chekuri, A and Art, J and Kondabolu, K and Slaugenhaupt, SA and Zeltner, N and Kleinstiver, BP and Morini, E and Alves, CRR},
title = {Engineered CRISPR-Base Editors as a Permanent Treatment for Familial Dysautonomia.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {39651221},
issn = {2692-8205},
abstract = {Familial dysautonomia (FD) is a fatal autosomal recessive congenital neuropathy caused by a T-to-C mutation in intron 20 of the Elongator acetyltransferase complex subunit 1 (ELP1) gene, which causes tissue-specific skipping of exon 20 and reduction of ELP1 protein. Here, we developed a base editor (BE) approach to precisely correct this mutation. By optimizing Cas9 variants and screening multiple gRNAs, we identified a combination that was able to promote up to 70% on-target editing in HEK293T cells harboring the ELP1 T-to-C mutation. These editing levels were sufficient to restore exon 20 inclusion in the ELP1 transcript. Moreover, we optimized an engineered dual intein-split system to deliver these constructs in vivo. Mediated by adeno-associated virus (AAV) delivery, this BE strategy effectively corrected the liver and brain ELP1 splicing defects in a humanized FD mouse model carrying the ELP1 T-to-C mutation and rescued the FD phenotype in iPSC-derived sympathetic neurons. Importantly, we observed minimal off-target editing demonstrating high levels of specificity with these optimized base editors. These findings establish a novel and highly precise BE-based therapeutic approach to correct the FD mutation and associated splicing defects and provide the foundation for the development of a transformative, permanent treatment for this devastating disease.},
}

@article {pmid39727898,
year = {2024},
author = {Zhang, X and Huang, Z and Zhang, Y and Wang, W and Ye, Z and Liang, P and Sun, K and Kang, W and Tang, Q and Yu, X},
title = {Mitigating Antibiotic Resistance: The Utilization of CRISPR Technology in Detection.},
journal = {Biosensors},
volume = {14},
number = {12},
pages = {},
pmid = {39727898},
issn = {2079-6374},
support = {2022C02049//the "Pioneer" and "Leading Goose" R&D Program of Zhejiang/ ; 2024SNJF044//Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; 2023SNJF066//the Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; },
mesh = {*CRISPR-Cas Systems ; *Anti-Bacterial Agents/pharmacology ; Humans ; Drug Resistance, Microbial ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Bacteria ; Biosensing Techniques ; Drug Resistance, Bacterial ; },
abstract = {Antibiotics, celebrated as some of the most significant pharmaceutical breakthroughs in medical history, are capable of eliminating or inhibiting bacterial growth, offering a primary defense against a wide array of bacterial infections. However, the rise in antimicrobial resistance (AMR), driven by the widespread use of antibiotics, has evolved into a widespread and ominous threat to global public health. Thus, the creation of efficient methods for detecting resistance genes and antibiotics is imperative for ensuring food safety and safeguarding human health. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) systems, initially recognized as an adaptive immune defense mechanism in bacteria and archaea, have unveiled their profound potential in sensor detection, transcending their notable gene-editing applications. CRISPR/Cas technology employs Cas enzymes and guides RNA to selectively target and cleave specific DNA or RNA sequences. This review offers an extensive examination of CRISPR/Cas systems, highlighting their unique attributes and applications in antibiotic detection. It outlines the current utilization and progress of the CRISPR/Cas toolkit for identifying both nucleic acid (resistance genes) and non-nucleic acid (antibiotic micromolecules) targets within the field of antibiotic detection. In addition, it examines the current challenges, such as sensitivity and specificity, and future opportunities, including the development of point-of-care diagnostics, providing strategic insights to facilitate the curbing and oversight of antibiotic-resistance proliferation.},
}

*CRISPR-Cas Systems
*Anti-Bacterial Agents/pharmacology
Humans
Drug Resistance, Microbial
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Bacteria
Biosensing Techniques
Drug Resistance, Bacterial

@article {pmid39727873,
year = {2024},
author = {Lu, Z and Ye, Z and Li, P and Jiang, Y and Han, S and Ma, L},
title = {An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection.},
journal = {Biosensors},
volume = {14},
number = {12},
pages = {},
pmid = {39727873},
issn = {2079-6374},
support = {WDZC20200821104802001//Universities Stable Funding Key Projects/ ; 2020YFA0908900//The National Key R&D Program of China/ ; n/a//State Key Laboratory of Chemical Oncogenomics/ ; },
mesh = {*DNA Methylation ; *CRISPR-Cas Systems ; Humans ; *Glycerol ; DNA Restriction Enzymes ; Biosensing Techniques ; Nasopharyngeal Carcinoma/diagnosis/genetics ; Nucleic Acid Amplification Techniques ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation detection methods relying on bisulfite conversion have limitations such as time-consuming, complex processes and sample degradation; thus, a more rapid and efficient method is needed.

METHODS: We propose a novel DNA methylation assay based on methylation-sensitive restriction endonuclease (MSRE) HhaI digestion and Glycerol-enhanced recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection (HGRC). MSRE has a fast digestion rate, and HhaI specifically cleaves unmethylated DNA at a specific locus, leaving the methylated target intact to trigger the downstream RPA-Cas12a detection step, generating a fluorescence signal. Moreover, the detection step was supplemented with glycerol for the separation of Cas12a-containing components and RPA- and template-containing components, which avoids over-consumption of the template and, thus, enhances the amplification efficiency and detection sensitivity.

RESULTS: The HGRC method exhibits excellent performance in the detection of a CNE2-specific methylation locus with a (limit of detection) LOD of 100 aM and a linear range of 100 aM to 100 fM. It also responds well to different methylation levels and is capable of distinguishing methylation levels as low as 0.1%. Moreover, this method can distinguish NPC cells from normal cells by detecting methylation in cellular genomes. This method provides a rapid and sensitive approach for NPC detection and also holds good application prospects for other cancers and diseases featuring DNA methylation as a biomarker.},
}

*DNA Methylation
*CRISPR-Cas Systems
Humans
*Glycerol
DNA Restriction Enzymes
Biosensing Techniques
Nasopharyngeal Carcinoma/diagnosis/genetics
Nucleic Acid Amplification Techniques
Endodeoxyribonucleases

@article {pmid39727179,
year = {2024},
author = {Lopez, SC and Lee, Y and Zhang, K and Shipman, SL},
title = {SspA is a transcriptional regulator of CRISPR adaptation in E. coli.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae1244},
pmid = {39727179},
issn = {1362-4962},
support = {MCB 2137692//National Science Foundation/ ; //Pew Biomedical Scholars Program/ ; //Chan Zuckerberg Biohub - San Francisco/ ; //Berkeley Fellowship for Graduate Study/ ; },
abstract = {The CRISPR integrases Cas1-Cas2 create immunological memories of viral infection by storing phage-derived DNA in CRISPR arrays, a process known as CRISPR adaptation. A number of host factors have been shown to influence adaptation, but the full pathway from infection to a fully integrated, phage-derived sequences in the array remains incomplete. Here, we deploy a new CRISPRi-based screen to identify putative host factors that participate in CRISPR adaptation in the Escherichia coli Type I-E system. Our screen and subsequent mechanistic characterization reveal that SspA, through its role as a global transcriptional regulator of cellular stress, is required for functional CRISPR adaptation. One target of SspA is H-NS, a known repressor of CRISPR interference proteins, but we find that the role of SspA on adaptation is not H-NS-dependent. We propose a new model of CRISPR-Cas defense that includes independent cellular control of adaptation and interference by SspA.},
}

@article {pmid39726635,
year = {2024},
author = {Brant, EJ and May, D and Eid, A and Altpeter, F},
title = {Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane.},
journal = {Frontiers in genome editing},
volume = {6},
number = {},
pages = {1505844},
pmid = {39726635},
issn = {2673-3439},
abstract = {Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100-130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.},
}

@article {pmid39532094,
year = {2024},
author = {Liang, WW and Müller, S and Hart, SK and Wessels, HH and Méndez-Mancilla, A and Sookdeo, A and Choi, O and Caragine, CM and Corman, A and Lu, L and Kolumba, O and Williams, B and Sanjana, NE},
title = {Transcriptome-scale RNA-targeting CRISPR screens reveal essential lncRNAs in human cells.},
journal = {Cell},
volume = {187},
number = {26},
pages = {7637-7654.e29},
doi = {10.1016/j.cell.2024.10.021},
pmid = {39532094},
issn = {1097-4172},
support = {R01 CA218668/CA/NCI NIH HHS/United States ; R01 HG012790/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Transcriptome/genetics ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Apoptosis/genetics ; Gene Expression Profiling ; Neoplasms/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Cycle/genetics ; Cell Line ; },
abstract = {Mammalian genomes host a diverse array of RNA that includes protein-coding and noncoding transcripts. However, the functional roles of most long noncoding RNAs (lncRNAs) remain elusive. Using RNA-targeting CRISPR-Cas13 screens, we probed how the loss of ∼6,200 lncRNAs impacts cell fitness across five human cell lines and identified 778 lncRNAs with context-specific or broad essentiality. We confirm their essentiality with individual perturbations and find that the majority of essential lncRNAs operate independently of their nearest protein-coding genes. Using transcriptome profiling in single cells, we discover that the loss of essential lncRNAs impairs cell-cycle progression and drives apoptosis. Many essential lncRNAs demonstrate dynamic expression across tissues during development. Using ∼9,000 primary tumors, we pinpoint those lncRNAs whose expression in tumors correlates with survival, yielding new biomarkers and potential therapeutic targets. This transcriptome-wide survey of functional lncRNAs advances our understanding of noncoding transcripts and demonstrates the potential of transcriptome-scale noncoding screens with Cas13.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Transcriptome/genetics
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
Apoptosis/genetics
Gene Expression Profiling
Neoplasms/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Cycle/genetics
Cell Line

@article {pmid39726634,
year = {2024},
author = {Azeez, SS and Hamad, RS and Hamad, BK and Shekha, MS and Bergsten, P},
title = {Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine.},
journal = {Frontiers in genome editing},
volume = {6},
number = {},
pages = {1509924},
pmid = {39726634},
issn = {2673-3439},
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.},
}

@article {pmid39726403,
year = {2024},
author = {Rasool, HMH and Chen, Q and Gong, X and Zhou, J},
title = {CRISPR/Cas system and its application in the diagnosis of animal infectious diseases.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {38},
number = {24},
pages = {e70252},
doi = {10.1096/fj.202401569R},
pmid = {39726403},
issn = {1530-6860},
support = {2024BEG02029//The Key Research and Development Program of Ningxia Hui Autonomous Region/ ; 2022YFC2304001//MOST | National Key Research and Development Program of China (NKPs)/ ; CAAS-ZDRW202410//ASTIP | The Agricultural Science and Technology Innovation Program/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Communicable Diseases/diagnosis/veterinary ; Humans ; Molecular Diagnostic Techniques/methods ; Zoonoses/diagnosis ; },
abstract = {Infectious diseases are a serious threat to the existence of animals and humans' life. In the 21st century, the emergence and re-emergence of several zoonotic and non-zoonotic global pandemic diseases of socio-economic importance has affected billions of humans and animals. The need for expensive equipment and laboratories, non-availability of on-site testing abilities, with time-consuming and low sensitivity and specificity issues of currently available diagnostic techniques to identify these pathogenic micro-organisms on a large scale highlighted the need for developing cheap, portable environment friendly diagnostic methods. In recent years, these issues have been addressed by clustered regularly interspaced palindromic repeats (CRISPR)-based diagnostic platforms that have transformed the molecular diagnostic field due to their outstanding ultra-sensitive nucleic acid detecting capabilities. In this study, we highlight the types, potential of different Cas proteins, and amplification systems. We also illuminate the application of currently available CRISPR integrated setups on the diagnosis of infectious diseases, majorly in food-producing animals (pigs, ruminants, poultry, and aquaculture), domestic pets (dogs and cats), and diseases of zoonotic importance. We conclude the challenges and future perspectives of using these systems to rapidly diagnose and treat other infectious diseases and also develop control strategies to prevent the spread of pathogenic organisms.},
}

Animals
*CRISPR-Cas Systems
*Communicable Diseases/diagnosis/veterinary
Humans
Molecular Diagnostic Techniques/methods
Zoonoses/diagnosis

@article {pmid39725739,
year = {2024},
author = {Pi, N and Xiang, R and Zhu, L and Li, Y and Wu, X},
title = {An HRP-integrated CRISPR/Cas12a biosensor towards chair-side diagnosis for Porphyromonas gingivalis.},
journal = {Biotechnology letters},
volume = {47},
number = {1},
pages = {15},
pmid = {39725739},
issn = {1573-6776},
support = {32060024//National Natural Science Foundation of China/ ; 32000088//National Natural Science Foundation of China/ ; 2022JJ40050//Natural Science Foundation of Hunan Province/ ; },
mesh = {*Porphyromonas gingivalis/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Bacteroidaceae Infections/diagnosis/microbiology ; Periodontitis/microbiology/diagnosis ; },
abstract = {Rapid diagnostic tools for Porphyromonas gingivalis (Pg), the primary microorganism responsible for the development of periodontitis, particularly those designed for chair-side applications, could provide substantial health benefits to patients. To address this issue, we developed a CRISPR/Cas12a-based rapid Pg detection method. Dual-gRNA and hairpin reporter strategies were employed to enhance CRISPR/Cas12a reaction efficiency. By modifying the hairpin reporter with HRP, the pre-amplification-free HRP-CRISPR/Cas12a reaction was enabled to produce a colorimetric output, amplifying the detection signal. This method achieved high sensitivity (as low as 33 CFU) without the risk of aerosol contamination from pre-amplification. When testing clinical samples, the method showed high consistency with the reference RT-PCR. Furthermore, compared with RT-PCR, this method only requires room temperature operation, is simpler, and has a shorter detection time of about 35 min. In conclusion, the pre-amplification-free HRP-integrated CRISPR/Cas12a detection method requires no complex equipment, making it an ideal, end-user-friendly approach for chair-side Pg detection.},
}

*Porphyromonas gingivalis/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans
Bacteroidaceae Infections/diagnosis/microbiology
Periodontitis/microbiology/diagnosis

@article {pmid39724853,
year = {2024},
author = {Zhao, L and Zhao, Z and Li, N and Wang, X},
title = {The nucleic acid detection using CRISPR/Cas biosensing system with micro-nano modality for point-of-care applications.},
journal = {Talanta},
volume = {286},
number = {},
pages = {127457},
doi = {10.1016/j.talanta.2024.127457},
pmid = {39724853},
issn = {1873-3573},
abstract = {Nucleic acid detection is considered the golden standard for diagnosing infectious diseases caused by various pathogens, including viruses, bacteria, and parasites. PCR and other amplification-based technologies are highly sensitive and specific, allowing for accurate detection and identification of low-level causative pathogens by targeting and amplifying their unique genetic segment (DNA or RNA). However, it is important to recognize that machinery-dependent diagnostic methods may only sometimes be available or practical in resource-limited settings, where direct implementation can be challenging. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based diagnostics offer a promising alternative for nucleic acid detection. These methods provide gene sequence-specific targeting, multiplexing capability, rapid result disclosure, and ease of operation, making them suitable for point-of-care (POC) applications. CRISPR-Cas-based nucleic acid detection leverages the intrinsic gene-editing capabilities of CRISPR systems to detect specific DNA or RNA sequences with high precision, ensuring high specificity in identifying pathogens. When integrated with micro- and nano-technologies, CRISPR-based diagnostics gain additional benefits, including automated microfluidic processes, enhanced multiplexed detection, improved sensitivity through nanoparticle integration, and combined detection strategies. In this review, we analyze the motivations for tailoring the CRISPR-Cas system with microfluidic formats or nanoscale materials for nucleic acid biosensing and detection. We discuss and categorize current achievements in such systems, highlighting their differences, commonalities, and opportunities for addressing challenges, particularly for POC diagnostics. Micro- and nano-technologies can significantly enhance the practical utility of the CRISPR-Cas system, enabling more comprehensive diagnostic and surveillance capabilities. By integrating these technologies, CRISPR-based diagnostics can achieve higher levels of automation, sensitivity, and multiplexing, making them invaluable tools in the global effort to diagnose and control infectious diseases.},
}

@article {pmid39724343,
year = {2025},
author = {Nakajima, T and Kuwasaki, Y and Yamamoto, S and Otabe, T and Sato, M},
title = {A Red Light-Activatable Endogenous Gene Transcription System with Red-CPTS.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2840},
number = {},
pages = {45-55},
pmid = {39724343},
issn = {1940-6029},
mesh = {Animals ; *Light ; Mice ; *CRISPR-Cas Systems ; *Optogenetics/methods ; Humans ; *Transcription, Genetic/radiation effects ; Red Light ; },
abstract = {Red light penetrates deep into mammalian tissues and has low phototoxicity. We developed a red light-activatable photoswitch (MagRed) for deep tissue optogenetics. Using MagRed, we developed a red light-activatable endogenous gene transcription system (Red-CPTS) based on CRISPR-Cas9. Here we provide a detailed protocol for endogenous gene activation using Red-CPTS in cultured mammalian cells and living mice in vivo.},
}

Animals
*Light
Mice
*CRISPR-Cas Systems
*Optogenetics/methods
Humans
*Transcription, Genetic/radiation effects
Red Light

@article {pmid39724342,
year = {2025},
author = {Renzl, C and Mayer, G},
title = {Optoribogenetic Modulation of Transcription.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2840},
number = {},
pages = {37-44},
pmid = {39724342},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Optogenetics/methods ; Humans ; Transcription, Genetic ; Light ; Transfection/methods ; HEK293 Cells ; Gene Expression Regulation ; Flow Cytometry/methods ; },
abstract = {Light can be used as a precise and reversible trigger for the activation of optogenetic tools with subcellular resolution. The interaction of the photoreceptor PAL and aptamer 53 was integrated into a CRISPR/dCas9 system, which can be applied for light-controlled activation of gene expression. Here, we describe a protocol for in vitro application of light-dependent overexpression using eBFP as a proof of concept. The experiment can be done in 3 days, which is split into cell seeding, transfection, and evaluation by flow cytometry. The method is broadly applicable including the upregulation of endogenous genes.},
}

*CRISPR-Cas Systems
*Optogenetics/methods
Humans
Transcription, Genetic
Light
Transfection/methods
HEK293 Cells
Gene Expression Regulation
Flow Cytometry/methods

@article {pmid39722550,
year = {2025},
author = {Victor Atoki, A and Aja, PM and Shinkafi, TS and Ondari, EN and Adeniyi, AI and Fasogbon, IV and Dangana, RS and Shehu, UU and Akin-Adewumi, A},
title = {Exploring the versatility of Drosophila melanogaster as a model organism in biomedical research: a comprehensive review.},
journal = {Fly},
volume = {19},
number = {1},
pages = {2420453},
doi = {10.1080/19336934.2024.2420453},
pmid = {39722550},
issn = {1933-6942},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Disease Models, Animal ; Biomedical Research ; Humans ; CRISPR-Cas Systems ; },
abstract = {Drosophila melanogaster is a highly versatile model organism that has profoundly advanced our understanding of human diseases. With more than 60% of its genes having human homologs, Drosophila provides an invaluable system for modelling a wide range of pathologies, including neurodegenerative disorders, cancer, metabolic diseases, as well as cardiac and muscular conditions. This review highlights key developments in utilizing Drosophila for disease modelling, emphasizing the genetic tools that have transformed research in this field. Technologies such as the GAL4/UAS system, RNA interference (RNAi) and CRISPR-Cas9 have enabled precise genetic manipulation, with CRISPR-Cas9 allowing for the introduction of human disease mutations into orthologous Drosophila genes. These approaches have yielded critical insights into disease mechanisms, identified novel therapeutic targets and facilitated both drug screening and toxicological studies. Articles were selected based on their relevance, impact and contribution to the field, with a particular focus on studies offering innovative perspectives on disease mechanisms or therapeutic strategies. Our findings emphasize the central role of Drosophila in studying complex human diseases, underscoring its genetic similarities to humans and its effectiveness in modelling conditions such as Alzheimer's disease, Parkinson's disease and cancer. This review reaffirms Drosophila's critical role as a model organism, highlighting its potential to drive future research and therapeutic advancements.},
}

Animals
*Drosophila melanogaster/genetics
*Disease Models, Animal
Biomedical Research
Humans
CRISPR-Cas Systems

@article {pmid39722520,
year = {2024},
author = {Deng, K and Li, M and Zhang, H and Deng, Y and Qin, Y and Qin, C},
title = {[Characterization of host factors ARF4 and ARF5 upon Zika virus infection in vivo by construction of gene knockout mice].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {40},
number = {12},
pages = {4605-4615},
doi = {10.13345/j.cjb.240307},
pmid = {39722520},
issn = {1872-2075},
mesh = {Animals ; *ADP-Ribosylation Factors/genetics/metabolism ; *Zika Virus Infection/genetics ; Mice ; *Mice, Knockout ; *Zika Virus/genetics ; *Mice, Inbred C57BL ; *CRISPR-Cas Systems ; Viral Load ; Male ; Female ; },
abstract = {The effects of host factors ADP-ribosylation factor 4 (ARF4) and ADP-ribosylation factor 5 (ARF5) upon Zika virus (ZIKV) infection in vivo were characterized by construction of gene knockout mice via CRISPR-Cas9. Firstly, ARF5 and ARF4 genes were modified by the CRISPR-Cas9 system and then microinjected into the fertilized eggs of C57BL/6JGpt mice. Fertilized eggs were transplanted to obtain ARF4 or ARF5 knockout (ARF4KO or ARF5KO) mice, and ARF4/5 double knockout mice were achieved by the mating between ARF4KO and ARF5KO mice (ARF4KO/ARF5KO). Then, the mouse genotypes were identified by PCR to identify the positive knockout mice, and RT-qPCR was employed to examine the knockout efficiency. The mice were then infected with ZIKV and the blood and tissue samples were collected after 2, 4, and 6 days. RT-qPCR was then employed to determine the virus load, and hematoxylin-eosin staining was employed to observe the pathological changes in the tissue. The results showed that expected PCR bands were detected from ARF4KO[-/+], ARF5KO[-/-], and ARF4KO[-/+]/ARF5KO[-/-] mice, respectively. The results of mRNA transcription measurement indicated the significant knockdown of ARF4 by 37.8%-50.0% but not ARF5 in ARF4KO[-/+] compared with the wild-type mice. Meanwhile, complete knockout of ARF5 and no changes in ARF4 were observed in ARF5KO[-/-] mice. Additionally, completed knockout of ARF5 and down-regulated mRNA level of ARF4 in the lung, kidney, and testis were detected in ARF4KO[-/+]/ARF5KO[-/-]mice in comparison with the wild-type mice. The virus load in the serum decreased in ARF4KO[-/+] mice, while it showed no significant change in ARF5KO[-/-] or ARF4KO[-/+]/ARF5KO[-/-] mice compared with that in the wild type. Meanwhile, ARF4KO[-/+] mice showcased no significant difference in virus load in various tissues but attenuated pathological changes in the brain and testis compared with the wild-type mice. We successfully constructed ARF4KO and ARF5KO mice by CRISPR-Cas9 in this study. ARF4 rather than ARF5 is essential for ZIKV infection in vivo. This study provided animal models for studying the roles of ARF4 and ARF5 in ZIKV infection and developing antivirals.},
}

Animals
*ADP-Ribosylation Factors/genetics/metabolism
*Zika Virus Infection/genetics
Mice
*Mice, Knockout
*Zika Virus/genetics
*Mice, Inbred C57BL
*CRISPR-Cas Systems
Viral Load
Male
Female

@article {pmid39722468,
year = {2024},
author = {Lohani, N and Singh, MB and Bhalla, PL},
title = {Deciphering the Vulnerability of Pollen to Heat Stress for Securing Crop Yields in a Warming Climate.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.15315},
pmid = {39722468},
issn = {1365-3040},
abstract = {Climate change is leading to more frequent and severe extreme temperature events, negatively impacting agricultural productivity and threatening global food security. Plant reproduction, the process fundamental to crop yield, is highly susceptible to heatwaves, which disrupt pollen development and ultimately affect seed-set and crop yields. Recent research has increasingly focused on understanding how pollen grains from various crops react to heat stress at the molecular and cellular levels. This surge in interest over the last decade has been driven by advances in genomic technologies, such as single-cell RNA sequencing, which holds significant potential for revealing the underlying regulatory reprogramming triggered by heat stress throughout the various stages of pollen development. This review focuses on how heat stress affects gene regulatory networks, including the heat stress response, the unfolded protein response, and autophagy, and discusses the impact of these changes on various stages of pollen development. It highlights the potential of pollen selection as a key strategy for improving heat tolerance in crops by leveraging the genetic variability among pollen grains. Additionally, genome-wide association studies and population screenings have shed light on the genetic underpinnings of traits in major crops that respond to high temperatures during male reproductive stages. Gene-editing tools like CRISPR/Cas systems could facilitate precise genetic modifications to boost pollen heat resilience. The information covered in this review is valuable for selecting traits and employing molecular genetic approaches to develop heat-tolerant genotypes.},
}

@article {pmid39671433,
year = {2024},
author = {Taylor, T and Zhu, HV and Moorthy, SD and Khader, N and Mitchell, JA},
title = {The cells are all-right: Regulation of the Lefty genes by separate enhancers in mouse embryonic stem cells.},
journal = {PLoS genetics},
volume = {20},
number = {12},
pages = {e1011513},
doi = {10.1371/journal.pgen.1011513},
pmid = {39671433},
issn = {1553-7404},
mesh = {Animals ; Mice ; *Enhancer Elements, Genetic ; *Mouse Embryonic Stem Cells/metabolism ; *Left-Right Determination Factors/genetics/metabolism ; *Chromatin/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Developmental ; },
abstract = {Enhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding the Lefty1 and Lefty2 paralogs in mouse embryonic stem cells. The Lefty genes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between the Lefty genes, we reveal a distance-dependent dosage effect of the Lefty2 enhancer on Lefty1 expression. These findings indicate a role for chromatin distance in insulating gene expression domains in the Lefty locus in the absence of architectural insulation.},
}

Animals
Mice
*Enhancer Elements, Genetic
*Mouse Embryonic Stem Cells/metabolism
*Left-Right Determination Factors/genetics/metabolism
*Chromatin/genetics/metabolism
CRISPR-Cas Systems
Gene Editing
Gene Expression Regulation, Developmental

@article {pmid39051993,
year = {2024},
author = {Xu, T and Cao, F and Dai, T and Liu, T},
title = {RPA-CRISPR/Cas12a-Mediated Isothermal Amplification for Rapid Detection of Phytopythium helicoides.},
journal = {Plant disease},
volume = {108},
number = {12},
pages = {3463-3472},
doi = {10.1094/PDIS-06-24-1300-SR},
pmid = {39051993},
issn = {0191-2917},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology ; CRISPR-Associated Proteins/genetics ; Recombinases/metabolism/genetics ; Sensitivity and Specificity ; Endodeoxyribonucleases/genetics ; Bacterial Proteins ; Molecular Diagnostic Techniques ; },
abstract = {Phytopythium helicoides, which belongs to algae (Chromista), Oomycota, Pythiales, Pythiaceae, and Phytophthora, is a quarantine pathogen that causes brown rot of fruits, stem rot and root rot, and other symptoms that can damage several tree species in urban landscaping. Therefore, disease management requires rapid and accurate diagnosis. The present study used recombinase polymerase amplification (RPA) in conjunction with the CRISPR/CRISPR-associated protein 12a (Cas12a) system to identify P. helicoides. The test exhibited high specificity and sensitivity and could detect 10 pg.μl[-1] of P. helicoides genomic DNA at 37°C within 20 min. The test results were visible by excitation of fluorophores by blue light. This groundbreaking test is able to detect P. helicoides in artificially inoculated rhododendron leaves. The RPA-CRISPR/Cas12a detection assay developed in this study is characterized by its sensitivity, efficiency, and convenience. Early detection and control of P. helicoides is crucial for the protection of urban green cover species.},
}

*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Plant Diseases/microbiology
CRISPR-Associated Proteins/genetics
Recombinases/metabolism/genetics
Sensitivity and Specificity
Endodeoxyribonucleases/genetics
Bacterial Proteins
Molecular Diagnostic Techniques

@article {pmid38699929,
year = {2024},
author = {Hu, R and Yang, W and Li, J and Jiang, L and Li, M and Zhang, M and Kang, Y and Cheng, X and Zhu, S and Zhao, L and He, W and Guo, M and Ding, S and Wu, H and Cheng, W},
title = {Multiple RNA Rapid In Situ Imaging Based on Cas9 Code Key System.},
journal = {Small methods},
volume = {8},
number = {12},
pages = {e2400195},
doi = {10.1002/smtd.202400195},
pmid = {38699929},
issn = {2366-9608},
support = {82372334//National Natural Science Foundation of China/ ; 81873972//National Natural Science Foundation of China/ ; 82202634//National Natural Science Foundation of China/ ; 82272432//National Natural Science Foundation of China/ ; cstc2019jcyjjqX0028//Chongqing Science Fund for Distinguished Young Scholars/ ; CXQT20013//Foundation for Innovative Research Groups of Chongqing Higher Education Institutions/ ; CQYC20210302385//Chongqing Talents-Innovation Leading Talents Project/ ; cstc2022ycjhbgzxm0001//Chongqing Talents-Innovation Leading Talents Project/ ; BJRC2022-02//The First Affiliated Hospital of Chongqing Medical University Top Talent Training Program/ ; 2023RC126//Zhejiang Provincial Health Bureau/ ; 2023ZF001//Zhejiang Provincial Health Bureau/ ; cstc2020jcyj-msxmX0237//Chongqing Natural Science Foundation/ ; 22A200151//Hainan Provincial Health Industry Research Project Fund/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics ; RNA/genetics/chemistry ; CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line, Tumor ; },
abstract = {Existing RNA in situ imaging strategies mostly utilize parallel repetitive nucleic acid self-assembly to achieve multiple analysis, with limitations of complicated systems and cumbersome steps. Here, a Cas9 code key system with key probe (KP) encoder and CRISPR/Cas9 signal exporter is developed. This system triggers T-protospacer adjacent motif (T-PAM structural transitions of multiple KP encoders to form coding products with uniform single-guide RNA (sgRNA) target sequences as tandem nodes. Only single sgRNA/Cas9 complex is required to cleave multiple coding products, enabling efficient "many-to-one" tandem signaling, and non-collateral cleavage activity-dependent automatic signaling output through active introduction of mismatched bases. Compared with conventional parallel multiple signaling analysis model, the proposed system greatly simplifies reaction process and enhances detection efficiency. Further, a rapid multiple RNA in situ imaging system is developed by combining the Cas9 code key system with a T-strand displacement amplification (T-SDA) signal amplifier. The constructed system is applied to tumor cells and clinicopathology slices, generating clear multi-mRNA imaging profiles in less than an hour with just one step. Therefore, this work provides reliable technical support for clinical tumor typing and molecular mechanism investigation.},
}

*CRISPR-Cas Systems
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
RNA, Messenger/genetics
RNA/genetics/chemistry
CRISPR-Associated Protein 9/genetics/metabolism
Cell Line, Tumor

@article {pmid39721357,
year = {2024},
author = {Petruškevičiūtė, A and Šimuliūnaitė, U and Polanco, CM and Rojas, B and Kuras, S and Valatkaitė-Rakštienė, B and Norvilas, R and Vijaya, AK and Muñoz, P and Neniškytė, U and Jakubauskas, A and Burokas, A and Nalvarte, I and Inzunza, J and Naumovas, D and Stoškus, M and Griškevičius, L and Baltriukienė, D and Arias, J},
title = {Generation of a genetically encoded voltage indicator MARINA reporter human iPS cell line using Cas9 (VULSCi002-A-2).},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103628},
doi = {10.1016/j.scr.2024.103628},
pmid = {39721357},
issn = {1876-7753},
abstract = {Fluorescent protein-based Genetically Encoded Voltage Indicators (GEVI) offer a remarkable system for high-throughput screening of membrane potential phenotypes. The GEVI MARINA is a derivative from ArcLight, which conversely to ArcLight increases its fluorescence intensity alongside depolarization. Here we created knock-in reporter human iPS cell lines carrying the MARINA reporter using SpCas9 programmable nuclease and characterize a heterozygous clone.},
}

@article {pmid39720725,
year = {2024},
author = {Wi, T and Choi, Y and Kim, J and Choi, YS and Pipkin, ME and Choi, J},
title = {Efficient gene deletion of Integrin alpha 4 in primary mouse CD4 T cells using CRISPR RNA pair-mediated fragmentation.},
journal = {Frontiers in immunology},
volume = {15},
number = {},
pages = {1445341},
pmid = {39720725},
issn = {1664-3224},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Integrin alpha4/genetics/metabolism/immunology ; *Gene Deletion ; CD4-Positive T-Lymphocytes/immunology/metabolism ; Th1 Cells/immunology ; Cell Differentiation/genetics/immunology ; Mice, Inbred C57BL ; Lymphocytic Choriomeningitis/immunology/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Lymphocytic choriomeningitis virus/immunology ; },
abstract = {The functional specialization of CD4 T lymphocytes into various subtypes, including TH1 and TFH cells, is crucial for effective immune responses. TFH cells facilitate B cell differentiation within germinal centers, while TH1 cells are vital for cell-mediated immunity against intracellular pathogens. Integrin α4, a cell surface adhesion molecule, plays significant roles in cell migration and co-stimulatory signaling. In this study, we investigated the role of Integrin α4 in regulating TFH and TH1 cell populations during acute viral infection using CRISPR-Cas9 gene editing. To effectively delete the Itga4 in primary mouse CD4 T cells, we selected various combinations of crRNAs and generated ribonucleoprotein complexes with fluorochrome-conjugated tracrRNAs and Cas9 proteins. These crRNA pairs enhanced gene deletion by generating deletions in the gene. By analyzing the effects of Itga4 deficiency on TFH and TH1 cell differentiation during acute LCMV infection, we found that optimized crRNA pairs significantly increased the TH1 cell population. Our results highlight the importance of selecting and combining appropriate crRNAs for effective CRISPR-Cas9 gene editing in primary CD4 T cells. Additionally, our study demonstrates the role of Integrin α4 in regulating the differentiation of CD4 T cells, suggesting the potential molecular mechanisms driving T cell subset differentiation through integrin targeting.},
}

Animals
Mice
*CRISPR-Cas Systems
*Gene Editing/methods
*Integrin alpha4/genetics/metabolism/immunology
*Gene Deletion
CD4-Positive T-Lymphocytes/immunology/metabolism
Th1 Cells/immunology
Cell Differentiation/genetics/immunology
Mice, Inbred C57BL
Lymphocytic Choriomeningitis/immunology/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Lymphocytic choriomeningitis virus/immunology

@article {pmid39719706,
year = {2024},
author = {Zhang, AN and Gaston, JM and Cárdenas, P and Zhao, S and Gu, X and Alm, EJ},
title = {CRISPR-Cas spacer acquisition is a rare event in human gut microbiome.},
journal = {Cell genomics},
volume = {},
number = {},
pages = {100725},
doi = {10.1016/j.xgen.2024.100725},
pmid = {39719706},
issn = {2666-979X},
abstract = {Host-parasite relationships drive the evolution of both parties. In microbe-phage dynamics, CRISPR functions as an adaptive defense mechanism, updating immunity via spacer acquisition. Here, we investigated these interactions within the human gut microbiome, uncovering low frequencies of spacer acquisition at an average rate of one spacer every ∼2.9 point mutations using isolates' whole genomes and ∼2.7 years using metagenome time series. We identified a highly prevalent CRISPR array in Bifidobacterium longum spreading via horizontal gene transfer (HGT), with six spacers found in various genomic regions in 15 persons from the United States and Europe. These spacers, targeting two prominent Bifidobacterium phages, comprised 76% of spacer occurrence of all spacers targeting these phages in all B. longum populations. This result suggests that HGT of an entire CRISPR-Cas system introduced three times more spacers than local CRISPR-Cas acquisition in B. longum. Overall, our findings identified key ecological and evolutionary factors in prokaryote adaptive immunity.},
}

@article {pmid39719647,
year = {2024},
author = {Yan, L and Song, YS and Zhou, J and Zhu, L and Shi, TW and Yu, HQ and Dong, ZQ and Wang, W and Long, T and Liu, HY and Shi, ZY and Li, JG},
title = {Expression of nicastrin, NICD1, and Hes1 in NCSTN knockout mice: implications for hidradenitis suppurativa, Alzheimer's, and liver cancer.},
journal = {European journal of medical research},
volume = {29},
number = {1},
pages = {622},
pmid = {39719647},
issn = {2047-783X},
support = {81773344//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Transcription Factor HES-1/genetics/metabolism ; *Amyloid Precursor Protein Secretases/genetics/metabolism ; *Mice, Knockout ; *Alzheimer Disease/genetics/metabolism ; Mice ; *Hidradenitis Suppurativa/genetics ; Female ; Male ; *Liver Neoplasms/genetics/pathology ; *Membrane Glycoproteins/genetics/metabolism ; Mice, Inbred C57BL ; Disease Models, Animal ; Receptor, Notch1/genetics ; },
abstract = {BACKGROUND: Nicastrin, a subunit of the γ-secretase complex, is encoded by the NCSTN gene and regulates notch signaling, it is involved in the pathogenesis of hidradenitis suppurativa (HS), Alzheimer disease (AD), and liver cancer. However, the animal models for studying HS are relatively scarce.

METHODS: CRISPR/Cas-mediated genetic engineering was used to generate targeted knockout offspring mice (C57BL/6J). Different doses (10 mg/kg, 20 mg/kg, and 30 mg/kg) and injection methods (subcutaneous/intraperitoneal/gavage injection) of tamoxifen were used to induce the construction of NCSTN knockout mice (mice model). The expressions of nicastrin, NICD1, hes1 in skin, brain, and liver tissue in mice model and wild-type (WT) mice were measured by qRT-PCR and IHC.

RESULTS: The construction of mice model was successfully induced by tamoxifen, knockout efficiency was 93%, there was no difference in knockout efficiency among three doses, injection methods, genders (P > 0.05). HS-like lesions appeared on the skin of NCSTN knockout mice after 1 month of treatment with tamoxifen, male mice had a higher number of skin lesions compared to female mice (male vs female = 76.5% vs 41.7%, P = 0.027). Compared with WT mice, the expressions of nicastrin (skin P = 0.0009, brain P = 0.0194, liver P = 0.0066), NICD1 (skin P = 0.0115, brain P = 0.0307, liver P = 0.008), hes1 (skin P = 0.0476, brain P = 0.0143, liver P = 0.0003) in mice model all decreased.

CONCLUSIONS: The NCSTN knockout mouse might be employed as HS animal model; Reducing nicastrin may affect the expression of notch1-hes1 pathway molecules in skin, brain, and liver tissues; low dose (10 mg/kg/d) tamoxifen could be used to induce the deletion of the target gene in mice.},
}

Animals
*Transcription Factor HES-1/genetics/metabolism
*Amyloid Precursor Protein Secretases/genetics/metabolism
*Mice, Knockout
*Alzheimer Disease/genetics/metabolism
Mice
*Hidradenitis Suppurativa/genetics
Female
Male
*Liver Neoplasms/genetics/pathology
*Membrane Glycoproteins/genetics/metabolism
Mice, Inbred C57BL
Disease Models, Animal
Receptor, Notch1/genetics

@article {pmid39719558,
year = {2024},
author = {Nadeem, I and Han, Z and Xiaoliang, H and Adzraku, SY and Kambey, PA and Kanwore, K and Peipei, M and Adekunle, AO and Adu-Amankwaah, J and Ayanlaja, AA and Zheng, Y and Dianshuai, G and Liu, X and Song, Y},
title = {Doublecortin regulates the mitochondrial-dependent apoptosis in glioma via Rho-A/Net-1/p38-MAPK signaling.},
journal = {Molecular medicine (Cambridge, Mass.)},
volume = {30},
number = {1},
pages = {272},
pmid = {39719558},
issn = {1528-3658},
support = {82273250//National Natural Science Foundation of China/ ; KYCX22_2868//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; },
mesh = {Humans ; *Glioma/metabolism/pathology/genetics ; *Apoptosis ; *Mitochondria/metabolism ; Animals ; *Doublecortin Domain Proteins ; *Doublecortin Protein ; Cell Line, Tumor ; *p38 Mitogen-Activated Protein Kinases/metabolism ; *Microtubule-Associated Proteins/metabolism/genetics ; Mice ; *Neuropeptides/metabolism/genetics ; Cell Proliferation ; Brain Neoplasms/metabolism/pathology/genetics ; Temozolomide/pharmacology ; Signal Transduction ; MAP Kinase Signaling System ; Reactive Oxygen Species/metabolism ; },
abstract = {Doublecortin (DCX) is a microtubule-associated protein known to be a key regulator of neuronal migration and differentiation during brain development. However, the role of DCX, particularly in regulating the survival and growth of glioma cells, remains unclear. In this study, we utilized CRISPR/Cas9 technology to knock down DCX in the human glioma cell line (U251). DCX depletion suppressed cell proliferation and enhanced the pro-apoptotic effects of temozolomide (TMZ) and γ-radiation treatment. DCX knockdown led to the translocation of Bax to the mitochondria and mitochondria dysfunction. Furthermore, DCX deficiency-induced apoptosis took place along with the generation of reactive oxygen species (ROS), which is crucial in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, the transcriptional inhibition of DCX downregulated Rho-A, Net-1, and activated p38-MAPK cue, critical for cell survival and proliferation. Subsequent treatment with TMZ and γ-radiation further increased p38-MAPK activity through the decreased expression of Rho-A/Net-1, resulting in a significant reduction in glioma cell migration and invasion. Additionally, intracranial xenograft tumors of DCX-modified U251 cells in nude mice demonstrated inhibited tumor growth. Tumor sections treated with TMZ and γ-radiation exhibited a higher number of TUNEL-positive cells compared to the control group, indicating increased apoptosis. Our finding suggests that DCX depletion reduces glioma cell proliferation and promotes mitochondria-dependent apoptosis by enhancing the chemo and radiotherapy response. Targeting DCX represents a potential therapeutic target for glioma treatment.},
}

Humans
*Glioma/metabolism/pathology/genetics
*Apoptosis
*Mitochondria/metabolism
Animals
*Doublecortin Domain Proteins
*Doublecortin Protein
Cell Line, Tumor
*p38 Mitogen-Activated Protein Kinases/metabolism
*Microtubule-Associated Proteins/metabolism/genetics
Mice
*Neuropeptides/metabolism/genetics
Cell Proliferation
Brain Neoplasms/metabolism/pathology/genetics
Temozolomide/pharmacology
Signal Transduction
MAP Kinase Signaling System
Reactive Oxygen Species/metabolism

@article {pmid39718242,
year = {2024},
author = {Dong, J and Hou, C and Deng, L and Gu, T and Zhu, S and Hou, J and Huo, D},
title = {CRISPR/Cas12a-Powered Electrochemical Platform for Dual-miRNA Detection via an AND Logic Circuit.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c06256},
pmid = {39718242},
issn = {1520-6882},
abstract = {The CRISPR/Cas technology shows great potential in molecular detection and diagnostics. However, it is still challenging to detect multiple targets simultaneously using the CRISPR-Cas system. Herein, we ingeniously leverage the synergistic effect of two short single-stranded DNA activators to construct a CRISPR/Cas12a-driven electrochemical sensing platform based on an AND logic circuit ("AND" LC-CRISPR) for the simultaneous detection of dual miRNAs. Specifically, the exponential amplification reaction products triggered by the dual-specific miRNAs are designed as binary inputs to bind with Cas12a/crRNA, forming an AND logic circuit and activating the trans-cleavage ability of the CRISPR-Cas12a system. Subsequently, the hairpin probe biogate on the surface of the functionalized electrochemical signal probe (MB@HP-Fe-MOF) is cleaved by activated Cas12a, leading to the release of the encapsulated electroactive signal molecule methylene blue, thereby generating a strong electrochemical signal. As a result, this "AND" LC-CRISPR sensing platform, requiring only a single crRNA assembled with Cas12a, achieves simultaneous detection of miRNA-155 and miRNA-21 at concentrations as low as 3.2 fM. Moreover, the platform allows easy adjustment of the AND logic circuit inputs according to different detection targets, allowing it to be easily expanded for the analysis and diagnosis of other multibiomarkers. This approach demonstrates promising potential for future applications in intelligent diagnostic medicine.},
}

@article {pmid39717575,
year = {2024},
author = {Ahmad, Z and Niyazi, S and Firdoos, A and Wang, C and Manzoor, MA and Ramakrishnan, M and Upadhyay, A and Ding, Y},
title = {Enhancing plant resilience: Nanotech solutions for sustainable agriculture.},
journal = {Heliyon},
volume = {10},
number = {23},
pages = {e40735},
pmid = {39717575},
issn = {2405-8440},
abstract = {The global population growth is driving up the demand for agricultural products, while traditional farming methods like those from the Green Revolution are becoming unsustainable due to climate change. To address these challenges and ensure agricultural sustainability, innovative techniques, such as nanotechnology, are essential to meet rising food demands and enhance agricultural sustainability. Nanotechnology, which promotes a more sustainable and resilient agricultural system while enhancing food security, is a key catalyst for the Agri-tech revolution. This review offers a progressive analysis of nanotechnology's role in managing plant stress. It explores how precision agriculture, particularly via nanosensors, is enhancing our comprehension of plant stress conditions. The integration of nanotechnology with genetic engineering methods, notably CRISPR-Cas technology, is also examined. Furthermore, the review considers the potential toxicological effects of nanoparticles (NPs) on both the environment and plants. Our review has the potential to make a significant impact on human food security by enhancing food production and availability while promoting sustainable agricultural practices. By tackling these challenges, we can contribute to a more reliable and sustainable food supply for the global population.},
}

@article {pmid39672387,
year = {2025},
author = {He, X and Li, P and Cao, H and Zhang, X and Zhang, M and Yu, X and Sun, Y and Ghonaim, AH and Ma, H and Li, Y and Shi, K and Zhu, H and He, Q and Li, W},
title = {Construction of a recombinant African swine fever virus with firefly luciferase and eGFP reporter genes and its application in high-throughput antiviral drug screening.},
journal = {Antiviral research},
volume = {233},
number = {},
pages = {106058},
doi = {10.1016/j.antiviral.2024.106058},
pmid = {39672387},
issn = {1872-9096},
mesh = {Animals ; *African Swine Fever Virus/genetics/drug effects ; Chlorocebus aethiops ; *Green Fluorescent Proteins/genetics ; *Antiviral Agents/pharmacology ; Vero Cells ; *Genes, Reporter ; Swine ; *Luciferases, Firefly/genetics ; *African Swine Fever/virology ; *Drug Evaluation, Preclinical/methods ; High-Throughput Screening Assays/methods ; Virus Replication/drug effects ; CRISPR-Cas Systems ; Gene Editing/methods ; Macrophages, Alveolar/virology/drug effects ; },
abstract = {African Swine Fever (ASF) is a highly lethal and contagious disease in pigs caused by African Swine Fever Virus (ASFV), which primarily infects domestic pigs and wild boars, with a mortality rate of up to 100%. Currently, there are no commercially available vaccines or drugs that are both safe and effective against ASFV. The ASFV 0428C strain was continuously passaged in Vero cells, and the adapted ASFV demonstrated efficient replication in Vero cells. The adapted ASFV was used as the parental virus, and an expression cassette encoding a dual reporter gene for firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP) was inserted into the ASFV genome using CRISPR/Cas9 gene editing technology to construct a recombinant ASFV variant (rASFV-FLuc-eGFP). rASFV-Fluc-eGFP was genetically stable, effectively infected porcine alveolar macrophages (PAM) and Vero cells, and expressed Fluc and eGFP concurrently. This study provides a tool for investigating the infection and pathogenic mechanisms of ASFV, as well as for screening essential host genes and antiviral drugs. Additionally, a high-throughput screening model of antiviral drugs was established based on rASFV-FLuc-eGFP in passaged cells, 218 compounds from the FDA-approved compound library were screened, and 5 candidate compounds with significant inhibitory effects in Vero cells were identified. The inhibitory effects on ASFV were further validated in both Vero and PAM cells, resulting in the identification of Salvianolic acid C (SAC), which demonstrated inhibitory effects and safety in both cell types. SAC is a candidate drug for the prevention and control of ASFV and shows promising application prospects.},
}

Animals
*African Swine Fever Virus/genetics/drug effects
Chlorocebus aethiops
*Green Fluorescent Proteins/genetics
*Antiviral Agents/pharmacology
Vero Cells
*Genes, Reporter
Swine
*Luciferases, Firefly/genetics
*African Swine Fever/virology
*Drug Evaluation, Preclinical/methods
High-Throughput Screening Assays/methods
Virus Replication/drug effects
CRISPR-Cas Systems
Gene Editing/methods
Macrophages, Alveolar/virology/drug effects

@article {pmid39666765,
year = {2024},
author = {Shao, G and Zhu, X and Hua, R and Chen, Y and Yang, G},
title = {Development of a Copro-RPA-CRISPR/Cas12a assay to detect Echinococcus granulosus nucleic acids isolated from canine feces using NaOH-based DNA extraction method.},
journal = {PLoS neglected tropical diseases},
volume = {18},
number = {12},
pages = {e0012753},
pmid = {39666765},
issn = {1935-2735},
mesh = {Animals ; Dogs ; *Echinococcus granulosus/genetics/isolation & purification ; *Feces/parasitology ; *Echinococcosis/diagnosis/veterinary/parasitology ; *Dog Diseases/diagnosis/parasitology ; *CRISPR-Cas Systems ; *DNA, Helminth/genetics/isolation & purification ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics/isolation & purification ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (E. granulosus s.l.), remains a significant zoonotic parasitic disease affecting both livestock and humans. It arises from the ingestion of food and water contaminated with canine feces containing E. granulosus eggs. The detection of these eggs in canine feces is essential for guiding effective preventative measures against the disease. Therefore, the development of a novel accurate, rapid, and visually interpretable point-of-care test is crucial for controlling CE.

METHODS: We combined recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with a CRISPR-associated protein 12a (Cas12a) system, forming the RPA-CRISPR/Cas12a assay. This assay targeted the E. granulosus mitochondrial nad2 gene and utilized a lateral flow strip for visual readout. To improve field applicability, we integrated a simple and cost-effective NaOH-Based DNA extraction method. Clinical validation included testing DNA extracted from eighteen canine fecal samples, followed by comparison with quantitative PCR (qPCR) and two commercial enzyme-linked immunosorbent assay (ELISA) kits.

RESULTS: The RPA-CRISPR/Cas12a assay showed a detection limit of 1 fg/μL DNA, without any cross-reactivity with related tapeworms such as Echinococcus multilocularis, Dipylidium caninum, Taenia hydatigera, Taenia multiceps, and Taenia pisiformis. When applied to 62 clinical fecal samples from dogs, the RPA-CRISPR/Cas12a assay demonstrated 68% sensitivity, while the developed RPA-CRISPR/Cas12a-NaOH assay exhibited 45% sensitivity. In the field performance comparison of the RPA-CRISPR/Cas12a and the RPA-CRISPR/Cas12a-NaOH assay with qPCR and two ELISA kits, the sensitivity, consistency rate, and Youden's index suggested good or fair agreement with the currently employed detection methods.

CONCLUSION: This study describes the development and validation of the RPA-CRISPR/Cas12a and RPA-CRISPR/Cas12a-NaOH assays for detecting E. granulosus in canine feces. The developed assays surpassed previous detection methods in providing enhanced diagnostic sensitivity and enabling point-of-care testing. Moreover, these assays hold potential for surveilling E. granulosus in low-income countries.},
}

Animals
Dogs
*Echinococcus granulosus/genetics/isolation & purification
*Feces/parasitology
*Echinococcosis/diagnosis/veterinary/parasitology
*Dog Diseases/diagnosis/parasitology
*CRISPR-Cas Systems
*DNA, Helminth/genetics/isolation & purification
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/genetics/isolation & purification
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39642411,
year = {2025},
author = {Li, H and Li, Y and Zhang, T and Liu, S and Song, C and Wang, K and Yan, W and Wang, Z and Yang, Q and Yang, X and Wang, H},
title = {Genome-wide CRISPR screen reveals specific role of type I interferon signaling pathway in Newcastle disease virus establishment of persistent infection.},
journal = {Veterinary microbiology},
volume = {300},
number = {},
pages = {110288},
doi = {10.1016/j.vetmic.2024.110288},
pmid = {39642411},
issn = {1873-2542},
mesh = {*Newcastle disease virus/immunology/genetics/physiology ; *Interferon Type I/genetics/metabolism/immunology ; Humans ; *Signal Transduction ; A549 Cells ; Animals ; *Newcastle Disease/virology/immunology ; Virus Replication ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Chickens/virology ; },
abstract = {Newcastle disease virus (NDV) is a potent oncolytic agent that exhibits sensitivity to a wide range of cancer cells. Unfortunately, some cancer cells are able to resist NDV-mediated oncolysis, by developing a persistent infection. The mechanism of persistency of infection remains poorly understood. In this study, a genome-wide CRISPR screen was conducted on non-small cell lung cancer cells (A549) to identify key host factors for NDV infection. Interestingly, a persistent infection was established in the surviving cells. CRISPR high-throughput screening results showed that members of the type I interferon signaling pathway (JAK1, STAT1, STAT2 and IRF9) were identified as top hits in the surviving cells. Further studies found that the type I IFN signaling pathway is intact in A549 cells, and a violent cytokine storm was induced after NDV infection. Both NDV infection and cytokine storm can induce cell death in A549 cells. We further blocked the type I interferon signaling pathway, and impaired type I interferon signaling pathway promoted NDV replication, but it did attenuate cell death induced by cytokine storm. Furthermore, persistent infection is more easily established in type I interferon signaling pathway-impaired A549 cells than in wild-type A549 cells. These findings suggest that the type I interferon signaling pathway plays a decisive role in persistent infection by regulating the antiviral immunity and cytokine storm inducing cell death.},
}

*Newcastle disease virus/immunology/genetics/physiology
*Interferon Type I/genetics/metabolism/immunology
Humans
*Signal Transduction
A549 Cells
Animals
*Newcastle Disease/virology/immunology
Virus Replication
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Chickens/virology

@article {pmid39401095,
year = {2025},
author = {Zheng, X and Tang, X and Wu, Y and Zheng, X and Zhou, J and Han, Q and Tang, Y and Fu, X and Deng, J and Wang, Y and Wang, D and Zhang, S and Zhang, T and Qi, Y and Zhang, Y},
title = {An efficient CRISPR-Cas12a-mediated MicroRNA knockout strategy in plants.},
journal = {Plant biotechnology journal},
volume = {23},
number = {1},
pages = {128-140},
doi = {10.1111/pbi.14484},
pmid = {39401095},
issn = {1467-7652},
support = {2023ZD04058//the Biological Breeding-Major Projects/ ; 2023ZD04076//the Biological Breeding-Major Projects/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Oryza/genetics ; *Gene Editing/methods ; *Gene Knockout Techniques ; RNA, Plant/genetics ; },
abstract = {In recent years, the CRISPR-Cas9 nuclease has been used to knock out MicroRNA (miRNA) genes in plants, greatly promoting the study of miRNA function. However, due to its propensity for generating small insertions and deletions, Cas9 is not well-suited for achieving a complete knockout of miRNA genes. By contrast, CRISPR-Cas12a nuclease generates larger deletions, which could significantly disrupt the secondary structure of pre-miRNA and prevent the production of mature miRNAs. Through the case study of OsMIR390 in rice, we confirmed that Cas12a is a more efficient tool than Cas9 in generating knockout mutants of a miRNA gene. To further demonstrate CRISPR-Cas12a-mediated knockout of miRNA genes in rice, we targeted nine OsMIRNA genes that have different spaciotemporal expression and have not been previously investigated via genetic knockout approaches. With CRISPR-Cas12a, up to 100% genome editing efficiency was observed at these miRNA loci. The resulting larger deletions suggest Cas12a robustly generated null alleles of miRNA genes. Transcriptome profiling of the miRNA mutants, as well as phenotypic analysis of the rice grains revealed the function of these miRNAs in controlling gene expression and regulating grain quality and seed development. This study established CRISPR-Cas12a as an efficient tool for genetic knockout of miRNA genes in plants.},
}

*MicroRNAs/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Oryza/genetics
*Gene Editing/methods
*Gene Knockout Techniques
RNA, Plant/genetics

@article {pmid39716183,
year = {2024},
author = {Cohen, S and Bergman, S and Lynn, N and Tuller, T},
title = {A tool for CRISPR-Cas9 sgRNA evaluation based on computational models of gene expression.},
journal = {Genome medicine},
volume = {16},
number = {1},
pages = {152},
pmid = {39716183},
issn = {1756-994X},
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Software ; Computational Biology/methods ; Gene Editing/methods ; Gene Knockout Techniques ; Computer Simulation ; Models, Genetic ; Gene Expression ; Gene Expression Regulation ; },
abstract = {BACKGROUND: CRISPR is widely used to silence genes by inducing mutations expected to nullify their expression. While numerous computational tools have been developed to design single-guide RNAs (sgRNAs) with high cutting efficiency and minimal off-target effects, only a few tools focus specifically on predicting gene knockouts following CRISPR. These tools consider factors like conservation, amino acid composition, and frameshift likelihood. However, they neglect the impact of CRISPR on gene expression, which can dramatically affect the success of CRISPR-induced gene silencing attempts. Furthermore, information regarding gene expression can be useful even when the objective is not to silence a gene. Therefore, a tool that considers gene expression when predicting CRISPR outcomes is lacking.

RESULTS: We developed EXPosition, the first computational tool that combines models predicting gene knockouts after CRISPR with models that forecast gene expression, offering more accurate predictions of gene knockout outcomes. EXPosition leverages deep-learning models to predict key steps in gene expression: transcription, splicing, and translation initiation. We showed our tool performs better at predicting gene knockout than existing tools across 6 datasets, 4 cell types and ~207k sgRNAs. We also validated our gene expression models using the ClinVar dataset by showing enrichment of pathogenic mutations in high-scoring mutations according to our models.

CONCLUSIONS: We believe EXPosition will enhance both the efficiency and accuracy of genome editing projects, by directly predicting CRISPR's effect on various aspects of gene expression. EXPosition is available at http://www.cs.tau.ac.il/~tamirtul/EXPosition . The source code is available at https://github.com/shaicoh3n/EXPosition .},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Software
Computational Biology/methods
Gene Editing/methods
Gene Knockout Techniques
Computer Simulation
Models, Genetic
Gene Expression
Gene Expression Regulation

@article {pmid39715238,
year = {2024},
author = {Qiu, M and Tian, Y and Wang, H and Yang, J and Qu, B and Jiang, Y and Zhao, Q and Zhang, X and Man, C},
title = {CRISPR/Cas System Meets CLICK-17 DNAzyme: A Click Chemistry-Based Fluorescence Biosensing Platform Designed for Highly Sensitive Detection of Salmonella.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c05316},
pmid = {39715238},
issn = {1520-6882},
abstract = {Salmonella is one of the most dangerous and contagious foodborne pathogens, posing a significant threat to public health and food safety. In this study, we developed a click chemistry-based fluorescence biosensing platform for highly sensitive detection of Salmonella enterica (S. enterica) by integrating the trans-cleavage activity of CRISPR/Cas12a with the CLICK17-mediated copper(II)-dependent azide-alkyne cycloaddition (Cu(II)AAC) click reaction. Herein, CLICK-17 can provide binding sites for Cu ions and high redox stability for one or much catalytically vital Cu[+] within its active sites, which facilitate the click reaction. With the existence of only Cu[2+], CLICK17 still can catalyze the click reaction between 3-butyn-1-ol and 3-azido-7-hydroxycoumarin to produce a fluorescence signal. By integrating the recombinase polymerase amplification (RPA), specific recognition, and trans-cleavage ability of the CRISPR/Cas12a system and the CLICK17-catalyzed Cu(II)AAC click reaction, the established biosensor obtained high detection sensitivity. This CLICK17-assisted CRISPR/Cas12a fluorescence biosensor was used for the detection of S. enterica with a limit of detection (LOD) as low as 1 cfu/mL in a wide linear detection range of 6 × 10[1]-6 × 10[7] cfu/mL. Moreover, the developed biosensor exhibited high specificity and anti-interference capability and had a recovery of 93%-104% in detection of S. enterica in spiked milk, infant formula, orange juice, and meat samples. This study provides a promising CRISPR/Cas12a-based fluorescence biosensor for the detection of foodborne pathogens.},
}

@article {pmid39714464,
year = {2024},
author = {Böck, D and Wilhelm, M and Mumenthaler, J and Carpanese, DF and Kulcsár, PI and d'Aquin, S and Cremonesi, A and Rassi, A and Häberle, J and Patriarchi, T and Schwank, G},
title = {Base editing of Ptbp1 in neurons alleviates symptoms in a mouse model of Parkinson's disease.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {39714464},
issn = {2050-084X},
support = {310030_185293/SNSF_/Swiss National Science Foundation/Switzerland ; 310030_196455/SNSF_/Swiss National Science Foundation/Switzerland ; FN20-0000000203//Novartis Foundation for Medical-Biological Research/ ; 196287/SNSF_/Swiss National Science Foundation/Switzerland ; 891959/ERC_/European Research Council/International ; },
mesh = {Animals ; *Polypyrimidine Tract-Binding Protein/metabolism/genetics ; Mice ; *Disease Models, Animal ; *Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; *Parkinson Disease/genetics/metabolism ; *Astrocytes/metabolism ; Dopaminergic Neurons/metabolism ; Gene Editing ; Male ; Corpus Striatum/metabolism ; Tyrosine 3-Monooxygenase/metabolism/genetics ; Mice, Inbred C57BL ; },
abstract = {Parkinson's disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported the successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, follow-up studies have questioned the validity of this astrocyte-to-DAN conversion model. Here, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, PTBP1 downregulation in neurons of the striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of these TH-positive cells co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where downregulation of neuronal, but not astrocytic, PTBP1 can mitigate symptoms in PD mice.},
}

Animals
*Polypyrimidine Tract-Binding Protein/metabolism/genetics
Mice
*Disease Models, Animal
*Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics
*Parkinson Disease/genetics/metabolism
*Astrocytes/metabolism
Dopaminergic Neurons/metabolism
Gene Editing
Male
Corpus Striatum/metabolism
Tyrosine 3-Monooxygenase/metabolism/genetics
Mice, Inbred C57BL

@article {pmid39661519,
year = {2024},
author = {Pan, Q and Zhang, Z and Xiong, Y and Bao, Y and Chen, T and Xu, P and Liu, Z and Ma, H and Yu, Y and Zhou, Z and Wei, W},
title = {Mapping functional elements of the DNA damage response through base editor screens.},
journal = {Cell reports},
volume = {43},
number = {12},
pages = {115047},
doi = {10.1016/j.celrep.2024.115047},
pmid = {39661519},
issn = {2211-1247},
mesh = {*DNA Damage ; Humans ; *DNA Repair ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Protein Serine-Threonine Kinases/metabolism/genetics ; Cisplatin/pharmacology ; Mutation/genetics ; Replication Protein A/metabolism/genetics ; HEK293 Cells ; },
abstract = {Maintaining genomic stability is vital for cellular equilibrium. In this study, we combined CRISPR-mediated base editing with pooled screening technologies to identify numerous mutations in lysine residues and protein-coding genes. The loss of these lysine residues and genes resulted in either sensitivity or resistance to DNA-damaging agents. Among the identified variants, we characterized both loss-of-function and gain-of-function mutations in response to DNA damage. Notably, we discovered that the K494 mutation of C17orf53 disrupts its interaction with RPA proteins, leading to increased sensitivity to cisplatin. Additionally, our analysis identified STK35 as a previously unrecognized gene involved in DNA damage response (DDR) pathways, suggesting that it may play a critical role in DNA repair. We believe that this resource will offer valuable insights into the broader functions of DNA damage response genes and accelerate research on variants relevant to cancer therapy.},
}

*DNA Damage
Humans
*DNA Repair
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Protein Serine-Threonine Kinases/metabolism/genetics
Cisplatin/pharmacology
Mutation/genetics
Replication Protein A/metabolism/genetics
HEK293 Cells

@article {pmid39660762,
year = {2024},
author = {Zeng, T and Wu, Q and Liu, Y and Qi, Q and Shen, W and Gu, W and Zhang, Y and Xiong, W and Xie, Z and Qi, X and Tian, T and Zhou, X},
title = {Unraveling the Cleavage Reaction of Hydroxylamines with Cyclopropenones Considering Biocompatibility.},
journal = {Journal of the American Chemical Society},
volume = {146},
number = {51},
pages = {35077-35089},
doi = {10.1021/jacs.4c09757},
pmid = {39660762},
issn = {1520-5126},
mesh = {*Cyclopropanes/chemistry/pharmacology ; Humans ; HeLa Cells ; *Hydroxylamines/chemistry ; Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Density Functional Theory ; Molecular Structure ; CRISPR-Cas Systems ; Prodrugs/chemistry/pharmacology/chemical synthesis ; },
abstract = {We develop a latent biocompatible cleavage reaction involving the hitherto unexplored interaction between hydroxylamines and cyclopropenones. Our study addresses the regioselectivity challenges commonly observed in asymmetric cyclopropenone transformations, substantiated by variations in substrate, Density Functional Theory calculations, and in situ NMR analysis. This reaction is characterized by high efficiency, broad substrate scope, stability, latent biocompatibility, and mild reaction conditions. Significantly, it facilitates fluorescence activation and functions as a controlled release mechanism for prodrugs, showing great promise in biological assays. Our success in achieving the controlled release of nitrogen mustard in HeLa cells underscores its potential application in cellular contexts. Additionally, we introduce a simple and highly efficient method for synthesizing α, β-substituted pentenolides, applicable to a variety of substrates. Moreover, we extend this cleavage reaction to the CRISPR-Cas9 system, achieving precise, on-demand regulation of guide RNA activity. The introduction of this cleavage reaction offers a promising tool for biochemical research and biotechnological applications.},
}

*Cyclopropanes/chemistry/pharmacology
Humans
HeLa Cells
*Hydroxylamines/chemistry
Biocompatible Materials/chemistry/pharmacology/chemical synthesis
Density Functional Theory
Molecular Structure
CRISPR-Cas Systems
Prodrugs/chemistry/pharmacology/chemical synthesis

@article {pmid39658506,
year = {2024},
author = {Shen, H and Weng, Z and Zhao, H and Song, H and Wang, F and Fan, C and Song, P},
title = {Random Sanitization in DNA Information Storage Using CRISPR-Cas12a.},
journal = {Journal of the American Chemical Society},
volume = {146},
number = {51},
pages = {35155-35164},
doi = {10.1021/jacs.4c11380},
pmid = {39658506},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems/genetics ; DNA/chemistry ; Information Storage and Retrieval ; DNA, Single-Stranded/chemistry ; Thermodynamics ; },
abstract = {DNA information storage provides an excellent solution for metadata storage due to its high density, programmability, and long-term stability. However, current research primarily focuses on the processes of storing and reading data, lacking comprehensive solutions for secure metadata wiping. Herein, we present a method of random sanitization in DNA information storage using CRISPR-Cas12a (RSDISC) based on precise control of the thermodynamic energy of primer-template hybridization. We utilize the collateral cleavage (trans-activity) of single-stranded DNA (ssDNA) by CRISPR-Cas12a to achieve selective sanitization of files in metadata. This method enables ssDNA degradation with different GC contents, lengths, and secondary structures to achieve a sanitization efficiency up to 99.9% for 28,258 oligonucleotides in DNA storage within one round. We demonstrate that the number of erasable files could reach 10[12] based on a model of primer-template hybridization efficiency. Overall, RSDISC provides a random sanitization approach to set the foundation of information encryption, file classification, memory deallocation, and accurate reading in DNA storage.},
}

*CRISPR-Cas Systems/genetics
DNA/chemistry
Information Storage and Retrieval
DNA, Single-Stranded/chemistry
Thermodynamics

@article {pmid36200515,
year = {2025},
author = {Abdallah, NA and Elsharawy, H and Abulela, HA and Thilmony, R and Abdelhadi, AA and Elarabi, NI},
title = {Multiplex CRISPR/Cas9-mediated genome editing to address drought tolerance in wheat.},
journal = {GM crops & food},
volume = {16},
number = {1},
pages = {1-17},
doi = {10.1080/21645698.2022.2120313},
pmid = {36200515},
issn = {2164-5701},
mesh = {*Triticum/genetics/growth & development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; *Droughts ; Genome, Plant/genetics ; Plant Proteins/genetics ; Mutation/genetics ; Plant Leaves/genetics ; Drought Resistance ; },
abstract = {Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3'(2'), 5'-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M1 progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.},
}

*Triticum/genetics/growth & development
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Plants, Genetically Modified/genetics
*Droughts
Genome, Plant/genetics
Plant Proteins/genetics
Mutation/genetics
Plant Leaves/genetics
Drought Resistance

@article {pmid39714446,
year = {2024},
author = {Petazzi, P and Ventura, T and Luongo, FP and McClafferty, H and May, A and Taylor, HA and Shipston, MJ and Romanò, N and Forrester, LM and Menendez, P and Fidanza, A},
title = {A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {39714446},
issn = {2050-084X},
support = {S002219/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; EHA RAG 2021//European Hematology Association/ ; Research Global Award//American Society for Hematology/ ; Precision Medicine PhD scholarship/MRC_/Medical Research Council/United Kingdom ; Tissue Repair PhD studentship//College of Medicine and Veterinary Medicine, University of Edinburgh/ ; Traineeship Program 2016/2017//Erasmus +/ ; PERIS program//Catalan Government/ ; RTC-2018-4603-1//MINECO/ ; },
mesh = {*Insulin-Like Growth Factor Binding Protein 2/metabolism/genetics ; Humans ; *Cell Differentiation ; *Hematopoietic Stem Cells/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Transcriptional Activation ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Pluripotent Stem Cells/metabolism ; },
abstract = {A major challenge in the stem cell biology field is the ability to produce fully functional cells from induced pluripotent stem cells (iPSCs) that are a valuable resource for cell therapy, drug screening, and disease modelling. Here, we developed a novel inducible CRISPR-mediated activation strategy (iCRISPRa) to drive the expression of multiple endogenous transcription factors (TFs) important for in vitro cell fate and differentiation of iPSCs to haematopoietic progenitor cells. This work has identified a key role for IGFBP2 in developing haematopoietic progenitors. We first identified nine candidate TFs that we predicted to be involved in blood cell emergence during development, then generated tagged gRNAs directed to the transcriptional start site of these TFs that could also be detected during single-cell RNA sequencing (scRNAseq). iCRISPRa activation of these endogenous TFs resulted in a significant expansion of arterial-fated endothelial cells expressing high levels of IGFBP2, and our analysis indicated that IGFBP2 is involved in the remodelling of metabolic activity during in vitro endothelial to haematopoietic transition. As well as providing fundamental new insights into the mechanisms of haematopoietic differentiation, the broader applicability of iCRISPRa provides a valuable tool for studying dynamic processes in development and for recapitulating abnormal phenotypes characterised by ectopic activation of specific endogenous gene expression in a wide range of systems.},
}

*Insulin-Like Growth Factor Binding Protein 2/metabolism/genetics
Humans
*Cell Differentiation
*Hematopoietic Stem Cells/metabolism
Induced Pluripotent Stem Cells/metabolism
Transcriptional Activation
CRISPR-Cas Systems
Transcription Factors/metabolism/genetics
Pluripotent Stem Cells/metabolism

@article {pmid39714100,
year = {2024},
author = {Cui, Q and Zhang, Z and Qin, L and Teng, Z and Wang, Z and Wu, W and Fan, L and Su, J and Hao, Y and Qin, J and Zhang, L and Wang, Q and Zhuang, Y and Zheng, H and Zhang, S and Geng, X and Zhu, L and Chen, Y and Lu, B and Li, Y and Zhu, X},
title = {Interleukin-37 promotes wound healing in diabetic mice by inhibiting the MAPK/NLRP3 pathway.},
journal = {Journal of diabetes investigation},
volume = {},
number = {},
pages = {},
doi = {10.1111/jdi.14389},
pmid = {39714100},
issn = {2040-1124},
support = {3030294002//Multidisciplinary Diagnosis and Treatment (MDT) Construction Project of Diabetic Foot/ ; 82400978//National Natural Science Foundation of China/ ; 82000790//National Natural Science Foundation of China/ ; 82200949//National Natural Science Foundation of China/ ; DGF501069/015//Multidisciplinary Diagnosis and Treatment (MDT) demonstration project in research hospital, Shanghai Medical College, Fudan University/ ; },
abstract = {AIMS/INTRODUCTION: Diabetic foot ulcer (DFU) is a prevalent complication of diabetes characterized by heightened inflammation and impaired wound-healing processes. Interleukin-37 (IL-37) is a natural suppressor of innate inflammation. Here, we aim to investigate the potential of IL-37 in enhancing the healing process of diabetic wounds.

MATERIALS AND METHODS: The skin samples of DFU and non-diabetic patients during foot and ankle orthopedic surgery were collected. The IL-37 transgenic mice (IL-37Tg) were created using CRISPR/Cas-mediated genome engineering. Mice were administered streptozotocin (STZ, 150 mg/kg) to induce a diabetic model. After 4 weeks, an equidistant full-thickness excisional wound measuring 8 mm was created on the central back of each mouse and allowed to heal naturally. Body weight and blood glucose levels were measured weekly. The wound area was measured, and skin samples were collected on Day 10 for further Quantitative polymerase chain reaction (qPCR) and WB detection and RNA sequencing analysis.

RESULTS: The proinflammation cytokines such as TNF-α and IL-1β and the MAPK signaling pathway were significantly increased in the wound margin of DFU patients. Compared with diabetic mice, diabetic IL-37Tg mice showed a significantly accelerated healing process. The enriched signaling pathways in RNA sequencing included cytokine-cytokine receptor interaction, TNF signaling pathway, and NOD-like receptor signaling pathway. Through QPCR and WB detection, we found that IL-37 could inhibit the activated MAPK and NOD-like signaling pathway, reducing TNF-α, IL-1β, and NLRP3 expression in the diabetic wound.

CONCLUSIONS: IL-37 promotes skin wound healing in diabetic mice, providing a new possible target for treating diabetic wounds.},
}

@article {pmid39713824,
year = {2024},
author = {Karesh, WB},
title = {Shifting from wildlife disease threats to wildlife health.},
journal = {Revue scientifique et technique (International Office of Epizootics)},
volume = {Special Edition},
number = {},
pages = {141-144},
doi = {10.20506/rst.SE.3568},
pmid = {39713824},
issn = {0253-1933},
mesh = {Animals ; *Animals, Wild ; Humans ; Zoonoses/prevention & control ; Conservation of Natural Resources ; Animal Diseases/prevention & control/epidemiology ; },
abstract = {The evolution of wildlife disease management and surveillance, as documented in the World Organisation for Animal Health's Scientific and Technical Review, reflects a deepening understanding of the links between wildlife health, ecosystem integrity and human well-being. Early work, beginning with the World Assembly of Delegates in 1954, primarily focused on diseases like rabies. This focus expanded over time to include broader concerns such as the impacts of climate change, habitat loss and increased human-wildlife interactions on wildlife health. By the late 20th century, the emphasis had shifted towards improved practices for wildlife disease control and the development of advanced diagnostic methods and vaccines. Articles in the Review highlight the growing complexity of wildlife diseases and the need for holistic management strategies. The adoption in recent years of cutting-edge technologies like CRISPR-Cas systems and metagenomics points to a future of more proactive and integrated approaches to wildlife disease management. There is still a need to address not just the consequences of wildlife diseases but also their anthropogenic drivers. The latest perspectives advocate for nature-based solutions, expanded partnerships and systems-level thinking to effectively tackle 21st-century challenges in wildlife and biodiversity conservation.},
}

Animals
*Animals, Wild
Humans
Zoonoses/prevention & control
Conservation of Natural Resources
Animal Diseases/prevention & control/epidemiology

@article {pmid39712894,
year = {2024},
author = {Wang, X and Yang, R and Tang, T and Zhou, Y and Chen, H and Jiang, Y and Zhang, S and Qin, S and Wang, M and Wang, C},
title = {One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1503356},
pmid = {39712894},
issn = {1664-302X},
abstract = {Compared to quantitative real-time PCR (q-PCR), CRISPR-Cas-mediated technology is more suitable for point-of-care testing (POCT) and has potential for wider application in the future. Generally, the operational procedure of CRISPR-Cas-mediated diagnostic method consists of two independent steps, the reaction of signal amplification and the CRISPR-Cas-mediated signal detection. Complex multi-step procedures can easily lead to cross-contamination. To develop a convenient and rapid method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we propose a MCTOP method (Multiple cross displacement amplification-CRISPR-Cas12b-based testing in one-pot), which targets the open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) gene of SARS-CoV-2. This method combines MCDA isothermal amplification and CRISPR-Cas-mediated sequence-specific detection into a one-pot reaction. The optimal reaction was achieved with isothermal amplification of 40 min and CRISPR-Cas-based detection of 15 min, both at 64°C. Then, the results can be visualized by the real-time fluorescence instrument and also lateral flow biosensor. The lowest detection limit of the proposed method is 10 copies of each of target sequences, and it has no cross-reactivity with non-SARS-CoV-2 templates. In a clinical test of 70 pharyngeal swab samples, MCTOP assay showed a specificity of 100% and sensitivities of 98 and 96% for the real-time fluorescence instrument and lateral flow biosensor, respectively. The MCTOP developed in this study is a rapid, convenient, highly sensitive, and specific method for SARS-CoV-2 nucleic acid detection. It can be used as an effective point-of-care testing (POCT) tool for clinical diagnosis and epidemiologic surveillance of SARS-CoV-2 infections, especially suitable for the basic, field and clinical laboratory.},
}

@article {pmid39710679,
year = {2024},
author = {Jia, X and Yuan, B and Wang, W and Wang, K and Ling, D and Wei, M and Hu, Y and Guo, W and Chen, Z and Du, L and Jin, Y},
title = {Gene editing tool-loaded biomimetic cationic vesicles with highly efficient bacterial internalization for in vivo eradication of pathogens.},
journal = {Journal of nanobiotechnology},
volume = {22},
number = {1},
pages = {787},
pmid = {39710679},
issn = {1477-3155},
support = {7232261//Beijing Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *Pseudomonas aeruginosa/drug effects/genetics ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Acinetobacter baumannii/drug effects/genetics ; Animals ; *Cations/chemistry ; Mice ; Humans ; Pseudomonas Infections/drug therapy ; Anti-Bacterial Agents/pharmacology/chemistry ; Drug Resistance, Multiple, Bacterial/drug effects/genetics ; Acinetobacter Infections/drug therapy ; COVID-19 ; Female ; Mice, Inbred BALB C ; Biomimetics/methods ; },
abstract = {In the post-COVID-19 era, drug-resistant bacterial infections emerge as one of major death causes, where multidrug-resistant Acinetobacter baumannii (MRAB) and drug-resistant Pseudomonas aeruginosa (DRPA) represent primary pathogens. However, the classical antibiotic strategy currently faces the bottleneck of drug resistance. We develop an antimicrobial strategy that applies the selective delivery of CRISPR/Cas9 plasmids to pathogens with biomimetic cationic hybrid vesicles (BCVs), irrelevant to bacterial drug resistance. CRISPR/Cas9 plasmids were constructed, replicating in MRAB or DRPA and expressing ribonucleic proteins, leading to irreparable chromosomal lesions; however, delivering the negatively charged plasmids with extremely large molecular weight to the pathogens at the infection site became a huge challenge. We found that the BCVs integrating the bacterial out membrane vesicles and cationic lipids efficiently delivered the plasmids in vitro/in vivo to the pathogens followed by effective internalization. The BCVs were used by intratracheal or topical hydrogel application against MRAB pulmonary infection or DRPA wound infection, and both of the two pathogens were eradicated from the lung or the wound. CRISPR/Cas9 plasmid-loaded BCVs become a promising medication for drug-resistant bacteria infections.},
}

*Gene Editing/methods
*Pseudomonas aeruginosa/drug effects/genetics
*CRISPR-Cas Systems
*Plasmids/genetics
*Acinetobacter baumannii/drug effects/genetics
Animals
*Cations/chemistry
Mice
Humans
Pseudomonas Infections/drug therapy
Anti-Bacterial Agents/pharmacology/chemistry
Drug Resistance, Multiple, Bacterial/drug effects/genetics
Acinetobacter Infections/drug therapy
COVID-19
Female
Mice, Inbred BALB C
Biomimetics/methods

@article {pmid39666461,
year = {2024},
author = {Yan, B and Liu, Y and Cai, Y and Liu, Y and Chen, Y},
title = {Protocol for establishing CRISPR-Cas12a for efficient genome editing of Pseudomonas aeruginosa phages.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103488},
doi = {10.1016/j.xpro.2024.103488},
pmid = {39666461},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Pseudomonas aeruginosa/genetics/virology ; Genome, Viral/genetics ; Pseudomonas Phages/genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {We developed an efficient type V CRISPR-Cas12a system tailored specifically for Pseudomonas aeruginosa phages, showcasing its remarkable cleavage activity and the ability to precisely introduce genetic modifications, including point mutations, deletions, and insertions, into phage genomes. Here, we present a protocol for establishing CRISPR-Cas12a for genome editing of Pseudomonas aeruginosa phages. We describe steps for the construction of pCRISPR-12a plasmid and guide RNA and the utilization of the type V CRISPR-Cas12a system for precise genetic editing of phages. For complete details on the use and execution of this protocol, please refer to Chen et al.[1].},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Pseudomonas aeruginosa/genetics/virology
Genome, Viral/genetics
Pseudomonas Phages/genetics
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39661507,
year = {2024},
author = {Ordóñez, A and Ron, D and Harding, HP},
title = {Protocol for iterative enrichment of integrated sgRNAs via derivative CRISPR-Cas9 libraries from genomic DNA of sorted fixed cells.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103493},
doi = {10.1016/j.xpro.2024.103493},
pmid = {39661507},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA/genetics ; Gene Library ; Lentivirus/genetics ; Gene Editing/methods ; },
abstract = {Here, we present a protocol for iterative enrichment of integrated single guide RNA (sgRNA) via derivative CRISPR-Cas9 from genomic DNA (gDNA) of phenotypically sorted fixed cells. We describe steps for high-scale lentiviral production, genome-wide screening, extracting gDNA from fixed cells, cloning of integrated sgRNAs, and high-scale transformation. This protocol introduces three key advantages: (1) applicability to fixed cells, (2) bypassing epigenetic drift, and (3) pause points lowering the contamination risk. We believe this approach will benefit researchers applying somatic cell genetics in cell biology. For complete details on the use and execution of this protocol, please refer to Ordoñez et al.[1].},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
DNA/genetics
Gene Library
Lentivirus/genetics
Gene Editing/methods

@article {pmid39487981,
year = {2024},
author = {Sonawala, U and Derevnina, L and Eves-van den Akker, S},
title = {Protocol for Cas9-targeted long-read sequencing in Globodera pallida and Globodera rostochiensis.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103427},
pmid = {39487981},
issn = {2666-1667},
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Tylenchoidea/genetics ; Solanum tuberosum/parasitology/genetics ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Associated Protein 9/genetics/metabolism ; Sequence Analysis, DNA/methods ; },
abstract = {We present a protocol to achieve a higher depth of long-read sequencing of region(s) of interest in potato cyst nematodes without amplification using a Cas9-based Nanopore enrichment approach. We describe steps for designing high-fidelity guide RNAs to be used with Cas9 nuclease, extracting high-molecular-weight DNA from the nematodes, and dephosphorylating genomic DNA ends. We then detail procedures for using Cas9-guide RNA complex to make targeted cleavage of the region of interest followed by a Nanopore library preparation. For complete details on the use and execution of this protocol, please refer to Sonawala et al.[1].},
}

Animals
*RNA, Guide, CRISPR-Cas Systems/genetics
Tylenchoidea/genetics
Solanum tuberosum/parasitology/genetics
CRISPR-Cas Systems/genetics
High-Throughput Nucleotide Sequencing/methods
CRISPR-Associated Protein 9/genetics/metabolism
Sequence Analysis, DNA/methods

@article {pmid39471176,
year = {2024},
author = {Bao, Y and Wei, W},
title = {Protocol for high-throughput screening of functional lysine residues in cell fitness.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103418},
pmid = {39471176},
issn = {2666-1667},
mesh = {Humans ; *Lysine/metabolism/chemistry ; *High-Throughput Screening Assays/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Genome, Human ; HEK293 Cells ; },
abstract = {Amino acid residues are crucial to protein structure and function and have links to various human diseases. Here, we present a protocol for screening functional lysine residues across the human genome. We describe steps for designing lysine codon-targeting single-guide RNAs (sgRNAs), constructing an sgRNA library, conducting cell fitness screenings, and acquiring screening results. This approach leverages base editing and high-throughput screening techniques to systematically examine functional amino acid residues. For complete details on the use and execution of this protocol, please refer to Bao et al.[1].},
}

Humans
*Lysine/metabolism/chemistry
*High-Throughput Screening Assays/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Gene Editing/methods
CRISPR-Cas Systems/genetics
Genome, Human
HEK293 Cells

@article {pmid39427312,
year = {2024},
author = {Mao, Q and Jiang, J and Ye, Q and Wang, H and Lin, CP},
title = {Protocol for the derivation and culture of murine trophoblast organoids for CRISPR-Cas9 screening.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103405},
pmid = {39427312},
issn = {2666-1667},
mesh = {Animals ; *Trophoblasts/cytology/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; *Organoids/cytology/metabolism ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; Pregnancy ; Cell Culture Techniques/methods ; Cell Differentiation/genetics ; Placenta/cytology/metabolism ; },
abstract = {Murine trophoblast organoids present a more balanced array of trophoblast subtypes, rendering them a suitable platform for CRISPR-Cas9-based screening. Here, we present a protocol for the derivation and culture of murine trophoblast organoids from trophoblast stem cells or placentae. We describe steps for establishing and differentiating murine trophoblast organoids, the characterization of trophoblast organoids in both conditions, the generation of focused single guide RNA (sgRNA) libraries, and the subsequent screening using those libraries in murine trophoblast organoids. For complete details on the use and execution of this protocol, please refer to Mao et al.[1].},
}

Animals
*Trophoblasts/cytology/metabolism
Mice
*CRISPR-Cas Systems/genetics
*Organoids/cytology/metabolism
Female
RNA, Guide, CRISPR-Cas Systems/genetics
Pregnancy
Cell Culture Techniques/methods
Cell Differentiation/genetics
Placenta/cytology/metabolism

@article {pmid39427309,
year = {2024},
author = {Xiao, YX and Wei, J and Moffat, J},
title = {Protocol for CRISPR-based endogenous protein tagging in mammalian cells.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103404},
pmid = {39427309},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Transfection/methods ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Electroporation/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Tracking the localization and proximal interaction partners of endogenous proteins provides valuable functional insight. Here, we present a protocol for CRISPR-based endogenous protein tagging in mammalian cells. We describe steps for endogenously tagging human TSC22D2 and MAP4, including designing Cas9 and Cas12a guides for knockin, modularized repair template design and cloning, and procedures for lipid transfection and electroporation. This protocol accommodates Cas nucleases in plasmid expression or ribonucleoprotein complex (RNP) formats. This "endo-tagging" approach offers flexibility and broad applicability. For complete details on the use and execution of this protocol, please refer to Xiao et al.[1].},
}

Humans
*CRISPR-Cas Systems/genetics
HEK293 Cells
Transfection/methods
Animals
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Electroporation/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid39392744,
year = {2024},
author = {Kiesler, P and Lee, SS and Norris, AL and Miller, MF and Mercado, CJ and Moyer, AL and Maragh, S},
title = {Protocol for CRISPR-Cas9 genome editing of a swine cell line via electroporation.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103385},
doi = {10.1016/j.xpro.2024.103385},
pmid = {39392744},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Electroporation/methods ; Swine ; Cell Line ; Male ; Testis/cytology/metabolism ; Transfection/methods ; },
abstract = {Genome editing technology is being used in animals for a variety of purposes, including improvement of animal and public health outcomes. Characterization of genome editing reagents and anticipated genomic alterations is an essential step toward the development of an edited animal. Here, we present a protocol for genome editing in the swine testicular (ST) cell line. We describe steps for evaluating CRISPR-Cas9 complex functionality in vitro, delivering editing molecules into cells by transfection, and assessing target editing via Sanger sequencing.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Electroporation/methods
Swine
Cell Line
Male
Testis/cytology/metabolism
Transfection/methods

@article {pmid39342621,
year = {2024},
author = {Dhaliwal, NK and Weng, OY and Li, Y},
title = {Protocol for the efficient and inducible generation of CRISPR-Cas9-edited human cortical neurons from the iCas9-iNgn2 hPSCs.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103352},
pmid = {39342621},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neurons/cytology/metabolism ; *Gene Editing/methods ; Pluripotent Stem Cells/cytology/metabolism ; Cerebral Cortex/cytology/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Nerve Tissue Proteins/genetics/metabolism ; Lentivirus/genetics ; Basic Helix-Loop-Helix Transcription Factors ; },
abstract = {Generation of CRISPR-Cas9-edited cortical neurons from human pluripotent stem cells (hPSCs) enables the study of gene functions and neural disease mechanisms. Here, we present a protocol for developing iCas9-iNgn2 hPSC, an inducible cell line that allows the simultaneous induction of the neuralizing transcription factor Ngn2 and the Cas9 nuclease to rapidly generate edited human cortical neurons. We describe the steps of the protocol from transducing iCas9-iNgn2 with guide RNA-containing lentivirus to producing edited cortical neurons in about 2 weeks. For complete details on the use and execution of this protocol, please refer to Dhaliwal et al.[1].},
}

Humans
*CRISPR-Cas Systems/genetics
*Neurons/cytology/metabolism
*Gene Editing/methods
Pluripotent Stem Cells/cytology/metabolism
Cerebral Cortex/cytology/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Nerve Tissue Proteins/genetics/metabolism
Lentivirus/genetics
Basic Helix-Loop-Helix Transcription Factors

@article {pmid39277866,
year = {2024},
author = {Hu, X and Wang, Y},
title = {Protocol to identify receptors of secreted proteins through CRISPR-Cas9 whole-genome screening technology.},
journal = {STAR protocols},
volume = {5},
number = {4},
pages = {103315},
pmid = {39277866},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; MCF-7 Cells ; Receptors, Cell Surface/genetics/metabolism ; Flow Cytometry/methods ; Lentivirus/genetics ; },
abstract = {The interaction between cell surface receptors and their ligands is crucial for intercellular communication. However, current techniques for identifying direct receptor-ligand interactions remain limited. Here, we present a protocol to identify receptors of secreted proteins using a genome-scale CRISPR-Cas9 knockout genetic screening approach. We describe steps for creating a single-guide RNA (sgRNA) lentivirus library, infecting stable Cas9-MCF7 cells, staining with tagged Cholesin, and sorting non-binding cells via flow cytometry. We then detail procedures for extracting DNA, amplifying sgRNAs, and sequencing. For complete details on the use and execution of this protocol, please refer to Hu et al.[1].},
}

*CRISPR-Cas Systems/genetics
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
MCF-7 Cells
Receptors, Cell Surface/genetics/metabolism
Flow Cytometry/methods
Lentivirus/genetics

@article {pmid38886504,
year = {2024},
author = {Luna, SE and Camarena, J and Hampton, JP and Majeti, KR and Charlesworth, CT and Soupene, E and Selvaraj, S and Jia, K and Sheehan, VA and Cromer, MK and Porteus, MH},
title = {Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells.},
journal = {Nature biomedical engineering},
volume = {8},
number = {12},
pages = {1540-1552},
pmid = {38886504},
issn = {2157-846X},
support = {Junior Faculty Scholar Award//American Society of Hematology (ASH)/ ; Minority Medical Student Award//American Society of Hematology (ASH)/ ; Medical Scientist Training Program//SU | School of Medicine, Stanford University (Stanford School of Medicine, Stanford University)/ ; Stanford Medical Scholars Research Program//SU | School of Medicine, Stanford University (Stanford School of Medicine, Stanford University)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Receptors, Erythropoietin/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Erythropoiesis/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Antigens, CD34/metabolism ; beta-Thalassemia/genetics ; },
abstract = {Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34[+] human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a β-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.},
}

Humans
*Hematopoietic Stem Cells/metabolism/cytology
*Gene Editing/methods
*Receptors, Erythropoietin/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Erythropoiesis/genetics
CRISPR-Associated Protein 9/metabolism/genetics
Antigens, CD34/metabolism
beta-Thalassemia/genetics

@article {pmid38092857,
year = {2024},
author = {Webber, BR and Johnson, MJ and Skeate, JG and Slipek, NJ and Lahr, WS and DeFeo, AP and Mills, LJ and Qiu, X and Rathmann, B and Diers, MD and Wick, B and Henley, T and Choudhry, M and Starr, TK and McIvor, RS and Moriarity, BS},
title = {Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.},
journal = {Nature biomedical engineering},
volume = {8},
number = {12},
pages = {1553-1570},
pmid = {38092857},
issn = {2157-846X},
support = {R01 AI146009/AI/NIAID NIH HHS/United States ; R01 AI161017/AI/NIAID NIH HHS/United States ; R37 CA276345/CA/NCI NIH HHS/United States ; T32HL007062-46//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Humans ; Animals ; *T-Lymphocytes/immunology ; Mice ; DNA End-Joining Repair ; CRISPR-Cas Systems/genetics ; DNA ; Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.},
}

Humans
Animals
*T-Lymphocytes/immunology
Mice
DNA End-Joining Repair
CRISPR-Cas Systems/genetics
DNA
Receptors, Antigen, T-Cell/genetics/immunology/metabolism
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid39709563,
year = {2024},
author = {Shepelev, MV and Komkov, DS and Golubev, DS and Borovikova, SE and Mazurov, DV and Kruglova, NA},
title = {[Donor DNA Modification with Cas9 Targeting Sites Improves the Efficiency of MTC34 Knock-in into the CXCR4 Locus].},
journal = {Molekuliarnaia biologiia},
volume = {58},
number = {4},
pages = {590-600},
pmid = {39709563},
issn = {0026-8984},
mesh = {Humans ; *CRISPR-Cas Systems ; *Receptors, CXCR4/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Gene Editing/methods ; Plasmids/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA/genetics/metabolism ; Cell Line ; DNA Breaks, Double-Stranded ; HIV-1/genetics/metabolism ; },
abstract = {To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA. Presumably, tCTS sites do not induce cleavage of the donor plasmid, but can support its transport into the nucleus by Cas9. However, the exact mechanisms whereby these two donor DNA modifications increase the knock-in level are unknown. In this study, the modifications were tested for effect on the knock-in efficiency of the MTC34 genetic construct encoding the HIV-1 fusion inhibitory peptide MT-C34 into the CXCR4 locus of the CEM/R5 T-cell line. When full-length CTSs were introduced into the donor plasmid DNA, the knock-in level was doubled regardless of the CTS number or position relative to the donor sequence. Modifications with tCTSs did not affect the knock-in levels. In vitro, both CTS and tCTS were efficiently cleaved by Cas9. To understand the mechanism of action of these modifications in detail, it is necessary to evaluate their cleavage both in vitro and in vivo.},
}

Humans
*CRISPR-Cas Systems
*Receptors, CXCR4/genetics/metabolism
*Gene Knock-In Techniques/methods
Gene Editing/methods
Plasmids/genetics/metabolism
CRISPR-Associated Protein 9/genetics/metabolism
DNA/genetics/metabolism
Cell Line
DNA Breaks, Double-Stranded
HIV-1/genetics/metabolism

@article {pmid39709562,
year = {2024},
author = {Golubev, DS and Komkov, DS and Shepelev, MV and Mazurov, DV and Kruglova, NA},
title = {[Methods to Increase the Efficiency of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the CXCR4 Locus in the CEM/R5 T Cell Line].},
journal = {Molekuliarnaia biologiia},
volume = {58},
number = {4},
pages = {575-589},
pmid = {39709562},
issn = {0026-8984},
mesh = {*Receptors, CXCR4/genetics/metabolism ; Humans ; *Gene Knock-In Techniques ; *HIV-1/genetics/drug effects ; HIV Fusion Inhibitors/pharmacology ; CRISPR-Cas Systems ; Cell Line ; DNA End-Joining Repair/drug effects/genetics ; Gene Editing/methods ; Nuclear Localization Signals/genetics ; Plasmids/genetics/metabolism ; T-Lymphocytes/metabolism/drug effects ; NF-kappa B/metabolism/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; HIV Infections/genetics/virology/drug therapy ; },
abstract = {The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the CXCR4 locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining (NHEJ) pathway with DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. A combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the NHEJ pathway significantly increased the level of knock-in of the HIV-1 fusion inhibitory peptide into the clinically relevant locus CXCR4. The finding can be used to develop effective gene therapy approaches for treating HIV infection.},
}

*Receptors, CXCR4/genetics/metabolism
Humans
*Gene Knock-In Techniques
*HIV-1/genetics/drug effects
HIV Fusion Inhibitors/pharmacology
CRISPR-Cas Systems
Cell Line
DNA End-Joining Repair/drug effects/genetics
Gene Editing/methods
Nuclear Localization Signals/genetics
Plasmids/genetics/metabolism
T-Lymphocytes/metabolism/drug effects
NF-kappa B/metabolism/genetics
CRISPR-Associated Protein 9/genetics/metabolism
HIV Infections/genetics/virology/drug therapy

@article {pmid39709560,
year = {2024},
author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV},
title = {[How to Shift the Equilibrium of DNA Break Repair in Favor of Homologous Recombination].},
journal = {Molekuliarnaia biologiia},
volume = {58},
number = {4},
pages = {525-548},
pmid = {39709560},
issn = {0026-8984},
mesh = {Humans ; Animals ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; *Recombinational DNA Repair ; CRISPR-Cas Systems ; Gene Editing/methods ; Cell Cycle/genetics ; Homologous Recombination ; },
abstract = {The CRISPR/Cas technology of targeted genome editing made it possible to carry out genetic engineering manipulations with eukaryotic genomes with a high efficiency. Targeted induction of site-specific DNA breaks is one of the key stages of the technology. The cell repairs the breaks via one of the two pathways, nonhomologous end joining (NHEJ) and homology-driven repair (HDR). The choice of the DNA repair pathway is determined by the architecture of the DNA break region formed as a result of terminal resection and depends on the cell cycle phase. NHEJ is the main pathway of double-strand break (DSB) repair in mammalian cells and involves a nonspecific ligation reaction. The reaction accuracy depends on the structure of break ends, and various insertions or deletions may arise as a result in the target genome region. Integration of a necessary sequence into the genome occurs via HDR, which requires a template with homology regions flanking a DSB. Introducing a genetic construct into a particular genomic locus is an important task, but is currently intricate and laborious to perform. However, the choice of the repair pathway can be of principal importance for basic research of gene functions and construction of animal models of human diseases to develop therapies. The review summarizes and systematizes the available information on strategies designed to increase the HDR efficiency. The strategies that most efficiently shift the balance towards HDR include use of NHEJ inhibitors, regulation of the key factors of homologous recombination, control of the cell cycle and chromatin status, and construction of HDR templates.},
}

Humans
Animals
*DNA Breaks, Double-Stranded
*DNA End-Joining Repair
*Recombinational DNA Repair
CRISPR-Cas Systems
Gene Editing/methods
Cell Cycle/genetics
Homologous Recombination

@article {pmid39709559,
year = {2024},
author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV},
title = {[Current Knowledge of Base Editing and Prime Editing].},
journal = {Molekuliarnaia biologiia},
volume = {58},
number = {4},
pages = {508-524},
pmid = {39709559},
issn = {0026-8984},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Animals ; Genetic Engineering/methods ; DNA/genetics ; },
abstract = {Modern genetic engineering technologies, such as base editing and prime editing (PE), have proven to provide the efficient and reliable genome editing tools that obviate the need for donor templates and double-strand breaks (DSBs) introduced in DNA. Relatively new, they quickly gained recognition for their accuracy, simplicity, and multiplexing capabilities. The review summarizes the new literature on the technologies and considers their architecture, methods to create editors, specificity, efficiency, and versatility. Advantages and disadvantages of the editors are discussed along with their prospective use in basic and applied research. The review may be useful for planning genome editing studies and analyzing their results to solve various problems of fundamental biology, biotechnology, medicine, and agriculture.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
DNA Breaks, Double-Stranded
Animals
Genetic Engineering/methods
DNA/genetics

@article {pmid39708170,
year = {2024},
author = {Feng, R and Mao, K and Zhang, H and Zhu, H and Du, W and Yang, Z and Wang, S},
title = {Portable microfluidic devices for monitoring antibiotic resistance genes in wastewater.},
journal = {Mikrochimica acta},
volume = {192},
number = {1},
pages = {19},
pmid = {39708170},
issn = {1436-5073},
support = {42377456//the National Natural Science Foundation of China/ ; 2023415//the Youth Innovation Promotion Association CAS/ ; CAS-ANSO-FS-2024-34//CAS-ANSO Fellowship/ ; Qiankehe Jichu-ZK [2022] Yiban 565, Qiankehe Platform Talents-GCC [2023] 046//Guizhou Provincial Science and Technology Projects/ ; },
mesh = {*Wastewater/microbiology ; *Lab-On-A-Chip Devices ; Nucleic Acid Amplification Techniques/methods ; Drug Resistance, Microbial/genetics ; Genes, Bacterial ; CRISPR-Cas Systems/genetics ; Anti-Bacterial Agents ; Microfluidic Analytical Techniques/instrumentation/methods ; Drug Resistance, Bacterial/genetics ; },
abstract = {Antibiotic resistance genes (ARGs) pose serious threats to environmental and public health, and monitoring ARGs in wastewater is a growing need because wastewater is an important source. Microfluidic devices can integrate basic functional units involved in sample assays on a small chip, through the precise control and manipulation of micro/nanofluids in micro/nanoscale spaces, demonstrating the great potential of ARGs detection in wastewater. Here, we (1) summarize the state of the art in microfluidics for recognizing ARGs, (2) determine the strengths and weaknesses of portable microfluidic chips, and (3) assess the potential of portable microfluidic chips to detect ARGs in wastewater. Isothermal nucleic acid amplification and CRISPR/Cas are two commonly used identification elements for the microfluidic detection of ARGs. The former has better sensitivity due to amplification, but false positives due to inappropriate primer design and contamination; the latter has better specificity. The combination of the two can achieve complementarity to a certain extent. Compared with traditional microfluidic chips, low-cost and biocompatible paper-based microfluidics is a very attractive test for ARGs, whose fluid flow in paper does not require external force, but it is weaker in terms of repeatability and high-throughput detection. Due to that only a handful of portable microfluidics detect ARGs in wastewater, fabricating high-throughput microfluidic chips, developing and optimizing recognition techniques for the highly selective and sensitive identification and quantification of a wide range of ARGs in complex wastewater matrices are needed.},
}

*Wastewater/microbiology
*Lab-On-A-Chip Devices
Nucleic Acid Amplification Techniques/methods
Drug Resistance, Microbial/genetics
Genes, Bacterial
CRISPR-Cas Systems/genetics
Anti-Bacterial Agents
Microfluidic Analytical Techniques/instrumentation/methods
Drug Resistance, Bacterial/genetics

@article {pmid39707688,
year = {2024},
author = {Garay-Novillo, JN and Ruiz-Masó, JÁ and Del Solar, G and Barra, JL},
title = {Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris.},
journal = {Microbial biotechnology},
volume = {17},
number = {12},
pages = {e70060},
pmid = {39707688},
issn = {1751-7915},
support = {CSIC 2022AEP028//Consejo Superior de Investigaciones Científicas/ ; 2023/ResMi-00000018//Secretaría de Ciencia y Tecnología - Universidad Nacional de Córdoba and Ministerio de Ciencia y Tecnología - Córdoba/ ; PICT Start-Up 2018-00811//Agencia Nacional de Promoción Científica y Tecnológica/ ; CONSOLIDAR RESOL-2023-258-E-UNC-SECYT#ACTIP//Secretaría de Ciencia y Tecnología - Universidad Nacional de Córdoba/ ; MHE200027-EMHE-CSIC-2016-program//Consejo Superior de Investigaciones Científicas/ ; },
mesh = {*Plasmids/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Lactococcus/genetics ; Hydrogen-Ion Concentration ; },
abstract = {In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity, and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.},
}

*Plasmids/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Genetic Vectors/genetics
*Lactococcus/genetics
Hydrogen-Ion Concentration

@article {pmid39626969,
year = {2024},
author = {Haussmann, IU and Dix, TC and McQuarrie, DWJ and Dezi, V and Hans, AI and Arnold, R and Soller, M},
title = {Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts.},
journal = {Genome research},
volume = {34},
number = {12},
pages = {2279-2292},
doi = {10.1101/gr.279479.124},
pmid = {39626969},
issn = {1549-5469},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; CRISPR-Associated Protein 9/metabolism/genetics ; Gene Knockout Techniques/methods ; Drosophila/genetics ; Software ; Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; },
abstract = {A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N [6] methylated adenosines (m[6]A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR-Cas9 genome editing advance the efficient use of this technique for safer applications in humans.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Humans
CRISPR-Associated Protein 9/metabolism/genetics
Gene Knockout Techniques/methods
Drosophila/genetics
Software
Drosophila Proteins/genetics/metabolism
Drosophila melanogaster/genetics

@article {pmid39533823,
year = {2024},
author = {Yamagishi, K and Ike, M and Tokuyasu, K},
title = {Construction of a genome-editing system for the thermophilic actinomycete Streptomyces thermodiastaticus K5 strain.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {89},
number = {1},
pages = {80-87},
doi = {10.1093/bbb/zbae157},
pmid = {39533823},
issn = {1347-6947},
support = {2020-2022//National Agriculture and Food Research Organization/ ; },
mesh = {*Gene Editing/methods ; *Streptomyces/genetics ; *Plasmids/genetics ; Uracil-DNA Glycosidase/genetics/metabolism ; beta-Glucosidase/genetics/metabolism ; CRISPR-Cas Systems ; Genome, Bacterial ; Mutation ; Lignin/metabolism ; },
abstract = {Thermophilic actinomycetes significantly contribute to the terrestrial carbon cycle via the rapid degradation of lignocellulosic polysaccharides in composts. In this study, a genome-editing system was constructed for the thermophilic actinomycete Streptomyces thermodiastaticus K5 strain, which was isolated from compost. The genome-editing plasmid (pGEK5) harboring nickase Cas9 was derived from the high-copy plasmid pL99 and used for the K5 strain. It was found that pGEK5 could easily be lost from the transformed clone through cultivation on apramycin-free medium and spore formation, enabling its reuse for subsequent genome-editing cycles. With the aid of this plasmid, mutations were sequentially introduced to 2 uracil-DNA glycosylase genes (Udg1 and Udg2) and 1 β-glucosidase gene (Bgl1). Thus, the genome-editing system using pGEK5 enables us to start the functional modification of this thermophilic actinomycete, especially for improved conversion of lignocellulosic biomass.},
}

*Gene Editing/methods
*Streptomyces/genetics
*Plasmids/genetics
Uracil-DNA Glycosidase/genetics/metabolism
beta-Glucosidase/genetics/metabolism
CRISPR-Cas Systems
Genome, Bacterial
Mutation
Lignin/metabolism

@article {pmid39317648,
year = {2024},
author = {Wang, S and Wang, J and Li, B and Zhang, J},
title = {Photoactivable CRISPR for Biosensing and Cancer Therapy.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {25},
number = {24},
pages = {e202400685},
doi = {10.1002/cbic.202400685},
pmid = {39317648},
issn = {1439-7633},
support = {22274072//National Natural Science Foundation of China/ ; 2024300408//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Humans ; *Neoplasms/therapy/diagnosis/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy ; Light ; },
abstract = {Photoactivable CRISPR technology represents a transformative approach in the field of genome editing, offering unprecedented control over gene editing with high spatial and temporal precision. By harnessing the power of light to modulate the activity of CRISPR components, this innovative strategy enables precise regulation of Cas proteins, guide RNAs, and ribonucleoprotein complexes. Recent advancements in optical control methodologies, including the development of photoactivable nanocarriers, have significantly expanded the potential applications of CRISPR in biomedical fields. This Concept highlights the latest developments in designing photoactivable CRISPR systems and their promising applications in biosensing and cancer therapy. Additionally, the remaining challenges and future trends are also discussed. It is expected that the photoactivable CRISPR would facilitate translating more precise gene therapies into clinical use.},
}

Humans
*Neoplasms/therapy/diagnosis/genetics
*Biosensing Techniques
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Genetic Therapy
Light

@article {pmid39298089,
year = {2024},
author = {Wu, K and Wu, Z and Li, X},
title = {Clinical diagnostic value of CRISPR-Cas13a-based molecular technology for tuberculosis.},
journal = {Irish journal of medical science},
volume = {193},
number = {6},
pages = {2889-2895},
pmid = {39298089},
issn = {1863-4362},
mesh = {Humans ; *CRISPR-Cas Systems ; Male ; Female ; Adult ; Middle Aged ; Mycobacterium tuberculosis/genetics/isolation & purification ; Tuberculosis/diagnosis ; Bronchoalveolar Lavage Fluid/microbiology ; Sensitivity and Specificity ; Tuberculosis, Pulmonary/diagnosis ; Aged ; },
abstract = {OBJECTIVE: To address the clinical diagnostic value of CRISPR-Cas13a-based molecular technology for tuberculosis (TB).

METHODS: The 189 suspected TB patients were simultaneously sent for acid-fast staining smear of bronchoalveolar lavage fluid, MGIT 960 cultures, Xpert MTB/RIF assay, and CRISPR-Cas13a assay. Using the final clinical diagnosis as the gold standard, the TB and non-TB groups were determined, and the diagnostic values of the four assays and the combined test in TB were compared. Using MGIT 960 culture as the gold standard, the diagnostic value of CRISPR-Cas13a assay was explored in TB, and the concordance between the CRISPR-Cas13a assay and MGIT 960 culture was compared.

RESULTS: The 189 preliminary diagnosed patients with suspected TB were diagnosed, with 147 in the TB group and 42 in the non-TB group. Taking the final clinical diagnosis as the gold standard, the sensitivity, negative predictive value, and accuracy of CRISPR-Cas13a assay, MGIT 960 culture, and XpertMTB/RIF assay were higher than those of acid-fast staining smear; by comparing the area under the ROC curve, the diagnostic value of the CRISPR-Cas13a assay, MGIT 960 culture, and XpertMTB/RIF assay was superior to that of acid-fast staining smear (all P < 0.05). Using the MGIT 960 culture results as the gold standard, there was a moderate concordance between the CRISPR-Cas13a assay and the MGIT 960 culture (kappa = 0.666).

CONCLUSION: Bronchoalveolar lavage fluid CRISPR-Cas13a assay has high application value in the clinical diagnosis of TB and can be recommended for the initial screening of patients with suspected TB.},
}

Humans
*CRISPR-Cas Systems
Male
Female
Adult
Middle Aged
Mycobacterium tuberculosis/genetics/isolation & purification
Tuberculosis/diagnosis
Bronchoalveolar Lavage Fluid/microbiology
Sensitivity and Specificity
Tuberculosis, Pulmonary/diagnosis
Aged

@article {pmid39147927,
year = {2025},
author = {Xiao, Q and Li, G and Han, D and Wang, H and Yao, M and Ma, T and Zhou, J and Zhang, Y and Zhang, X and He, B and Yuan, Y and Shi, L and Li, T and Yang, H and Huang, J and Zhang, H},
title = {Engineered IscB-ωRNA system with expanded target range for base editing.},
journal = {Nature chemical biology},
volume = {21},
number = {1},
pages = {100-108},
pmid = {39147927},
issn = {1552-4469},
support = {31925016//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82021001//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82271048//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Humans ; *Muscular Dystrophy, Duchenne/genetics/metabolism ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; RNA/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism/chemistry ; Dependovirus/genetics ; },
abstract = {As the evolutionary ancestor of Cas9 nuclease, IscB proteins serve as compact RNA-guided DNA endonucleases and nickases, making them strong candidates for base editing. Nevertheless, the narrow targeting scope limits the application of IscB systems; thus, it is necessary to find more IscBs that recognize different target-adjacent motifs (TAMs). Here, we identified 10 of 19 uncharacterized IscB proteins from uncultured microbes with activity in mammalian cells. Through protein and ωRNA engineering, we further enhanced the activity of IscB ortholog IscB.m16 and expanded its TAM scope from MRNRAA to NNNGNA, resulting in a variant named IscB.m16*. By fusing the deaminase domains with IscB.m16* nickase, we generated IscB.m16*-derived base editors that exhibited robust base-editing efficiency in mammalian cells and effectively restored Duchenne muscular dystrophy proteins in diseased mice through single adeno-associated virus delivery. Thus, this study establishes a set of compact base-editing tools for basic research and therapeutic applications.},
}

Animals
*Gene Editing/methods
Mice
Humans
*Muscular Dystrophy, Duchenne/genetics/metabolism
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
RNA/metabolism/chemistry/genetics
CRISPR-Cas Systems
Bacterial Proteins/genetics/metabolism/chemistry
Dependovirus/genetics

@article {pmid39707395,
year = {2024},
author = {Xu, W and Zhang, S and Qin, H and Yao, K},
title = {From bench to bedside: cutting-edge applications of base editing and prime editing in precision medicine.},
journal = {Journal of translational medicine},
volume = {22},
number = {1},
pages = {1133},
pmid = {39707395},
issn = {1479-5876},
support = {82471107//National Natural Science Foundation of China/ ; 31970930//National Natural Science Foundation of China/ ; 2020CFA069//Natural Science Foundation of Hubei Province/ ; 2018CFB434//Natural Science Foundation of Hubei Province/ ; },
mesh = {Humans ; *Precision Medicine/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Translational Research, Biomedical ; Animals ; Genetic Therapy/methods ; },
abstract = {CRISPR-based gene editing technology theoretically allows for precise manipulation of any genetic target within living cells, achieving the desired sequence modifications. This revolutionary advancement has fundamentally transformed the field of biomedicine, offering immense clinical potential for treating and correcting genetic disorders. In the treatment of most genetic diseases, precise genome editing that avoids the generation of mixed editing byproducts is considered the ideal approach. This article reviews the current progress of base editors and prime editors, elaborating on specific examples of their applications in the therapeutic field, and highlights opportunities for improvement. Furthermore, we discuss the specific performance of these technologies in terms of safety and efficacy in clinical applications, and analyze the latest advancements and potential directions that could influence the future development of genome editing technologies. Our goal is to outline the clinical relevance of this rapidly evolving scientific field and preview a roadmap for successful DNA base editing therapies for the treatment of hereditary or idiopathic diseases.},
}

Humans
*Precision Medicine/methods
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Translational Research, Biomedical
Animals
Genetic Therapy/methods

@article {pmid39706269,
year = {2024},
author = {Li, X and Wei, Y and Wang, SY and Wang, SG and Xia, PF},
title = {One-for-all gene inactivation via PAM-independent base editing in bacteria.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {108113},
doi = {10.1016/j.jbc.2024.108113},
pmid = {39706269},
issn = {1083-351X},
abstract = {Base editing is preferable for bacterial gene inactivation without generating double strand breaks, requiring homology recombination or highly efficient DNA delivery capability. However, the potential of base editing is limited by the adjoined dependence on the editing window and protospacer adjacent motif (PAM). Herein, we report an unconstrained base editing system to enable the inactivation of any genes of interest (GOIs) in bacteria. We employed a dCas9 derivative, dSpRY, and activation-induced cytidine deaminase to build a PAM-independent base editor. Then, we programmed the base editor to exclude the START codon of a GOI instead of introducing premature STOP codons to obtain a universal approach for gene inactivation, namely XSTART, with an overall efficiency approaching 100%. By using XSTART, we successfully manipulated the amino acid metabolisms in Escherichia coli, generating glutamine, arginine, and aspartate auxotrophic strains. While we observed a high frequency of off-target events as a trade-off for increased efficiency, refining the regulatory system of XSTART to limit expression levels reduced off-target events by over 60% without sacrificing efficiency, aligning our results with previously reported levels. Finally, the effectiveness of XSTART was also demonstrated in probiotic E. coli Nissle 1917 and photoautotrophic cyanobacterium Synechococcus elongatus, illustrating its potential in reprogramming diverse bacteria.},
}

@article {pmid39706164,
year = {2024},
author = {Cenik, BK and Aoi, Y and Iwanaszko, M and Howard, BC and Morgan, MA and Andersen, GD and Bartom, ET and Shilatifard, A},
title = {TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome.},
journal = {Molecular cell},
volume = {84},
number = {24},
pages = {4929-4944.e8},
doi = {10.1016/j.molcel.2024.11.007},
pmid = {39706164},
issn = {1097-4164},
mesh = {Humans ; *Promoter Regions, Genetic ; *Transcription Factors/metabolism/genetics ; *Chromatin/metabolism/genetics ; HEK293 Cells ; RNA Polymerase II/metabolism/genetics ; Protein Binding ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism/genetics ; Positive Transcriptional Elongation Factor B/metabolism/genetics ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Proto-Oncogene Proteins c-fos/genetics/metabolism ; Gene Expression Regulation ; Heat-Shock Response/genetics ; Protein Interaction Maps ; Bromodomain Containing Proteins ; },
abstract = {Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present "TurboCas," a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.},
}

Humans
*Promoter Regions, Genetic
*Transcription Factors/metabolism/genetics
*Chromatin/metabolism/genetics
HEK293 Cells
RNA Polymerase II/metabolism/genetics
Protein Binding
CRISPR-Cas Systems
Cell Cycle Proteins/metabolism/genetics
Positive Transcriptional Elongation Factor B/metabolism/genetics
Proto-Oncogene Proteins c-myc/genetics/metabolism
Proto-Oncogene Proteins c-fos/genetics/metabolism
Gene Expression Regulation
Heat-Shock Response/genetics
Protein Interaction Maps
Bromodomain Containing Proteins

@article {pmid39705367,
year = {2024},
author = {Su, Z and Zhang, W and Shi, Y and Cui, T and Xu, Y and Yang, R and Huang, M and Zhou, C and Zhang, H and Lu, T and Qu, J and He, ZG and Gan, J and Feng, Y},
title = {A bacterial methyltransferase that initiates biotin synthesis, an attractive anti-ESKAPE druggable pathway.},
journal = {Science advances},
volume = {10},
number = {51},
pages = {eadp3954},
pmid = {39705367},
issn = {2375-2548},
mesh = {*Biotin/biosynthesis ; *Methyltransferases/metabolism/chemistry ; *Acinetobacter baumannii/enzymology ; *Klebsiella pneumoniae/enzymology ; Bacterial Proteins/metabolism/genetics/chemistry ; Adenosine/analogs & derivatives/metabolism ; Anti-Bacterial Agents/pharmacology ; Models, Molecular ; Humans ; CRISPR-Cas Systems ; },
abstract = {The covalently attached cofactor biotin plays pivotal roles in central metabolism. The top-priority ESKAPE-type pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, constitute a public health challenge of global concern. Despite the fact that the late step of biotin synthesis is a validated anti-ESKAPE drug target, the primary stage remains fragmentarily understood. We report the functional definition of two BioC isoenzymes (AbBioC for A. baumannii and KpBioC for K. pneumoniae) that act as malonyl-ACP methyltransferase and initiate biotin synthesis. The physiological requirement of biotin is diverse within ESKAPE pathogens. CRISPR-Cas9-based inactivation of bioC rendered A. baumannii and K. pneumoniae biotin auxotrophic. The availability of soluble AbBioC enabled the in vitro reconstitution of DTB/biotin synthesis. We solved two crystal structures of AbBioC bound to SAM cofactor (2.54 angstroms) and sinefungin (SIN) inhibitor (1.72 angstroms). Structural and functional study provided molecular basis for SIN inhibition of BioC. We demonstrated that BioC methyltransferase plays dual roles in K. pneumoniae infection and A. baumannii colistin resistance.},
}

*Biotin/biosynthesis
*Methyltransferases/metabolism/chemistry
*Acinetobacter baumannii/enzymology
*Klebsiella pneumoniae/enzymology
Bacterial Proteins/metabolism/genetics/chemistry
Adenosine/analogs & derivatives/metabolism
Anti-Bacterial Agents/pharmacology
Models, Molecular
Humans
CRISPR-Cas Systems

@article {pmid39705306,
year = {2024},
author = {Hou, J and Guo, P and Wang, J and Han, D and Tan, W},
title = {Artificial dynamic structure ensemble-guided rational design of a universal RNA aptamer-based sensing tag.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {52},
pages = {e2414793121},
doi = {10.1073/pnas.2414793121},
pmid = {39705306},
issn = {1091-6490},
mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Biosensing Techniques/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Nucleic Acid Conformation ; RNA/chemistry ; Fluorescent Dyes/chemistry ; },
abstract = {Artificially functional RNAs, such as fluorogenic RNA aptamer (FRApt)-based biosensing tag, represent significant advancements in various biological applications but are limited by the lack of insight into dynamic structure ensembles and universal design concepts. Through the development of an artificial RNA structure ensemble, we rationally established an RNA reconstitution model, "SSPepper-Apt," to generate a universal fluorogenic RNA biosensing tag. By utilizing various target-recognizing RNA motifs, SSPepper-Apt enables the modular generation of sensing tags for low-background, highly selective imaging of metabolites, peptides, and proteins in living cells. Additionally, by employing single guide RNA (sgRNA) as the recognition RNA motif, SSPepper-Apt generates fluorescence in both CRISPR-mediated imaging and gene editing only when the Cas9-sgRNA complex is successfully assembled; therefore, it can be an effective sgRNA screening tool for gene editing. Our fluorogenic RNA-sensing tag provides a universal approach for constructing functional RNA systems, avoiding the laborious and time-consuming process of sequence combination, and expanding the application of synthetic biological tools.},
}

*Aptamers, Nucleotide/chemistry
Humans
*Biosensing Techniques/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Gene Editing/methods
Nucleic Acid Conformation
RNA/chemistry
Fluorescent Dyes/chemistry

@article {pmid39704190,
year = {2024},
author = {Fan, Z and Xu, L and Cao, Y and Liu, T and Tian, Y and Pan, Z and Mo, Y and Wang, X and Zhu, X and Gao, Y and Zhang, X and Pan, CQ and Wang, L and Ren, F},
title = {One-Pot Assay Based on CRISPR/Cas13a Technology for HEV RNA Point-of-Care Testing.},
journal = {Journal of medical virology},
volume = {96},
number = {12},
pages = {e70115},
pmid = {39704190},
issn = {1096-9071},
support = {//This study was supported by the National Natural Science Foundation of China (82002243, 82100653), Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan (KZ202010025035), Chinese Institutes for Medical Research, Beijing (Grant No. CX24PY23), Beijing Hospitals Authority Youth Programme (QML20201702), Talent Cultivation Plan of Climbing the Peak of Beijing Municipal Hospital Administration (DFL20221503), Beijing Natural Science Foundation-Changping Innovation Joint Fund (L234046), Training Fund for Open Projects at Clinical Institutes and Departments of Capital Medical University (CCMU2023ZKYXZ003), High-Level Public Health Technical Talents Project of Beijing (Subject Leaders-02-13, xuekegugan-03-48)./ ; },
mesh = {*Hepatitis E virus/genetics/isolation & purification ; *Point-of-Care Testing ; *Hepatitis E/diagnosis/virology ; *RNA, Viral/genetics ; Humans ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Animals ; Limit of Detection ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Genotype ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Hepatitis E virus (HEV) poses a serious threat to both public health and animal food safety, thereby highlighting the demands for rapid, sensitive, and easy-to-use detection. This study aimed to develop a One-Pot assay using CRISPR/Cas13a for detecting HEV RNA, suitable for point-of-care testing (POCT) in resource-limited settings. CRISPR/Cas13a combined with reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription recombinase-aided amplification (RT-RAA) was applied to a One-Pot assay device. Additionally, a large cohort of HEV-infected patient (154) and animal (104) specimens was utilized for validation. The RT-PCR/RT-RAA + CRISPR/Cas13a assays for HEV RNA detection (genotypes: HEV-1, HEV-3, and HEV-4) were established, optimized, and validated, achieving a limit of detection (LoD) of 1 copy/μL and 100% specificity. In the application validation for HEV infection, the positive rates of the RT-PCR + CRISPR and RT-RAA + CRISPR assays were 98.6% and 89.6% for patients, and 96.6% and 88.8% for animals, respectively, which were superior to those of RT-qPCR. Furthermore, sample rapid lysis, reagent lyophilization, and the One-Pot device were integrated to construct a One-Pot assay with an LoD of 10[2] copies/μL. Despite slight decreases in sensitivity, the One-Pot assay significantly reduces the assay time to 35 min, making it easy to perform, minimizing contamination, and meeting the requirements for screening. We developed a One-Pot assay of HEV RNA using the CRISPR/Cas13a which effectively realizes a POCT test and maximizes the impetus for POCT implementation and shows potential as a valuable tool for detecting and monitoring HEV infection.},
}

*Hepatitis E virus/genetics/isolation & purification
*Point-of-Care Testing
*Hepatitis E/diagnosis/virology
*RNA, Viral/genetics
Humans
*CRISPR-Cas Systems
*Sensitivity and Specificity
Animals
Limit of Detection
Reverse Transcriptase Polymerase Chain Reaction/methods
Genotype
Nucleic Acid Amplification Techniques/methods

@article {pmid39703747,
year = {2024},
author = {Fallah, T and Shafiei, M},
title = {Comprehensive Analysis of CRISPR-Cas Systems and Their Influence on Antibiotic Resistance in Salmonella enterica Strains.},
journal = {Bioinformatics and biology insights},
volume = {18},
number = {},
pages = {11779322241307984},
pmid = {39703747},
issn = {1177-9322},
abstract = {Salmonella enterica is a gram-negative bacterium that demonstrates a remarkable ability to acquire antibiotic resistance genes (ARGs). The role of the CRISPR-Cas system in influencing antibiotic resistance in S. enterica is still under investigation. This study explores the distribution and impact of CRISPR-Cas systems on antibiotic resistance by analyzing 316 S. enterica genomes. We conducted sequence alignments, phylogenetic analyses, and conservation studies on Cas genes, direct repeats (DRs), and leader sequences. Promoter predictions and RNA secondary structure analyses were also performed. ARGs were identified, and their correlation with Cas gene clusters was evaluated. Our findings revealed that 82.33% of strains possess complete CRISPR-Cas systems, while 17.66% have orphan CRISPRs. We identified 290 distinct DRs, most of which formed stable stem-loop structures, although no promoter regions were detected within the leader sequences. Most spacers were chromosome-targeting, with a smaller proportion homologous to phages and plasmids. Importantly, strains with complete CRISPR-Cas systems showed a higher incidence of ARGs compared with those with orphan or no CRISPR systems. Specifically, the incidence of ARGs was 54.3% higher in strains with complete CRISPR-Cas systems than in strains without CRISPR-Cas systems, and 15.1% higher than in strains with orphan CRISPRs. Spearman's correlation analysis confirmed a statistically significant but weak correlation between the presence of Cas genes and the frequency of ARGs (P-value = 3.892e-06). These results suggest that CRISPR-Cas systems may play a role in the acquisition of ARGs, potentially through mutations under antibiotic pressure. Future studies should investigate mutations, particularly in Cas3-the signature protein of type I CRISPR-Cas systems. In addition, experimental validation, such as culturing S. enterica strains with complete CRISPR-Cas systems under different antibiotic conditions, followed by sequencing to assess the uptake or absence of newly acquired ARGs, would help clarify the potential role of CRISPR-Cas systems in bacterial adaptation to antimicrobial pressures.},
}

@article {pmid39702810,
year = {2024},
author = {Bhagat, M and Kamal, R and Sharma, J and Kaur, K and Sharma, A and Thakur, GS and Bhatia, R and Awasthi, A},
title = {Gene Therapy: Towards a New Era of Medicine.},
journal = {AAPS PharmSciTech},
volume = {26},
number = {1},
pages = {17},
pmid = {39702810},
issn = {1530-9932},
mesh = {Humans ; *Genetic Therapy/methods/trends ; Animals ; *Genetic Vectors ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Transfer Techniques ; },
abstract = {Over the past years, many significant advances have been made in the field of gene therapy and shown promising results in clinical trials conducted. Gene therapy aims at modifying or replacing a defective, inefficient, or nonfunctional gene with a healthy, functional gene by administration of genome material into the cell to cure genetic diseases. Various methods have been devised to do this by using several viral and non-viral vectors which are either administered by in vivo or ex vivo technique. Viral vectors are best suitable for this therapy due to their potential to invade cells and deliver their genetic material whereas non-viral vectors are less efficient than viral vectors but possess some advantages such as less immunogenic response and large gene carrying capacity. Recent advances in biotechnology such as CRISPR-Cas9 mediated genome engineering and Cancer treatment with Chimeric antigen receptor (CAR) T-cell therapy are addressed in this review. This review article also delves into some recent research studies, gene therapy trials, and its applications, laying out future hopes for gene therapy in the treatment of various diseases namely haemophilia, Muscular dystrophy, SCID, Sickle cell disease, Familial Hypercholesterolemia, Cystic Fibrosis. Additionally, it also includes various nanoformulations and clinical trial data related to gene therapy.},
}

Humans
*Genetic Therapy/methods/trends
Animals
*Genetic Vectors
CRISPR-Cas Systems
Gene Editing/methods
Gene Transfer Techniques

@article {pmid39702692,
year = {2024},
author = {Du, W and Meister, LL and van Grinsven, T and Branco Dos Santos, F},
title = {Efficient Multiplex Genome Editing of the Cyanobacterium Synechocystis sp. PCC6803 via CRISPR-Cas12a.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28910},
pmid = {39702692},
issn = {1097-0290},
abstract = {Cyanobacteria have been genetically modified to convert CO2 into biochemical products, but efficient genetic engineering tools, including CRISPR-Cas systems, remain limited. This is primarily due to the polyploid nature of cyanobacteria, which hinders their effectiveness. Here, we address the latter by specifically (i) modifying the RSF1010-based replicative plasmid to simplify cloning efforts while maintaining high conjugation efficiency; (ii) improving the design of the guide RNA (gRNA) to facilitate chromosomal cleavage; (iii) introducing template DNA fragments as pure plasmids via natural transformation; and (iv) using sacB to facilitate replicative plasmid curing. With this system, the replicative plasmid containing both Cas12a and gRNA is introduced to Synechocystis sp. PCC6803 cells via conjugation to cleave the circular chromosomes. Template DNA plasmid that has meanwhile been assimilated will then repair it achieving the desired genetic modifications. This system was validated by successfully deleting various "neutral" chromosomal loci, both individually and collectively, as well as targeting an essential gene, sll1797. With the sacB-sucrose counter-selection, all deletions were simultaneously made markerless in < 4 weeks. Moreover, we also integrate YFP with various protein degradation tags into the chromosome, allowing for their characterization at the chromosomal level. We foresee this system will greatly facilitate future genome engineering in cyanobacteria.},
}

@article {pmid39702666,
year = {2024},
author = {Kou, Z and Wang, S and Luo, X and Xu, J and Tomberlin, JK and Huang, Y},
title = {Wingless strain created using binary transgenic CRISPR/Cas9 alleviates concerns about mass rearing of Hermetia illucens.},
journal = {Communications biology},
volume = {7},
number = {1},
pages = {1652},
pmid = {39702666},
issn = {2399-3642},
support = {32021001//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100381//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Animals, Genetically Modified ; *Diptera/genetics ; Female ; Male ; },
abstract = {Larvae of the black soldier fly Hermetia illucens have potential as a natural waste recycler and subsequent use as protein-rich feed for livestock. A common question about the insect-farming processes is, what about the concerns of mass escape of insects from large populations? Here, we present a binary transgenic CRISPR/Cas9 system to generate wingless strain with the potential to address this issue. We identified gonad-specific promoters in vivo and evaluated use of the two strongest promoters, nanos and exuperantia, to drive Cas9 expression. We found that crossing the Hiexu-Cas9 with transgenic sgRNA-expressing insects resulted in higher knockout efficiency of the marker gene white. The Hiexu-Cas9 strain exhibited a maternal deposition of Cas9 that caused more effective knockout in the progeny of female Cas9-expressing individuals. Using this system, we generated wingless mutants lacking mating ability, which can be maintained in colony through a genetic cross of two single strain. These insects are less likely to escape and would be unable to successfully mate if they did escape. Taken together, this study validates effective genetic tools that can be used for gene function studies and industrial applications in black soldier fly and provides an approach to alleviate the concern about massive rearing.},
}

Animals
*CRISPR-Cas Systems
*Animals, Genetically Modified
*Diptera/genetics
Female
Male

@article {pmid39607706,
year = {2024},
author = {Deng, C and Xin, R and Li, X and Zhang, J and Fan, L and Qiu, Y and Zhao, L},
title = {Optogenetic control of Corynebacterium glutamicum gene expression.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {14260-14276},
doi = {10.1093/nar/gkae1149},
pmid = {39607706},
issn = {1362-4962},
support = {32301214//National Natural Science Foundation of China/ ; //Shanghai Municipal Education Commission/ ; },
mesh = {*Corynebacterium glutamicum/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *Optogenetics/methods ; Light ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Bioreactors ; Bacterial Proteins/metabolism/genetics ; Oligosaccharides/metabolism/biosynthesis ; Metabolic Engineering/methods ; },
abstract = {Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth and product synthesis is crucial for enhancing production efficiency. Developing dynamic, broadly applicable, and minimally toxic gene regulation tools for C. glutamicum remains challenging, as optogenetic tools ideal for dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled gene expression system using light-controlled RNA-binding proteins (RBP), a first for Corynebacterium glutamicum. We established a gene expression regulation system, 'LightOnC.glu', utilizing the light-controlled RBP to construct light-controlled transcription factors in C. glutamicum. Simultaneously, we developed a high-performance light-controlled gene interference system using CRISPR/Cpf1 tools. The metabolic flow in the synthesis network was designed to enable the production of chitin oligosaccharides (CHOSs) and chondroitin sulphate oligosaccharides A (CSA) for the first time in C. glutamicum. Additionally, a light-controlled bioreactor was constructed, achieving a CHOSs production concentration of 6.2 g/L, the highest titer recorded for CHOSs biosynthesis to date. Herein, we have established a programmable light-responsive genetic circuit in C. glutamicum, advancing the theory of dynamic regulation based on light signaling. This breakthrough has potential applications in optimizing metabolic modules in other chassis cells and synthesizing other compounds.},
}

*Corynebacterium glutamicum/genetics/metabolism
*Gene Expression Regulation, Bacterial
*Optogenetics/methods
Light
CRISPR-Cas Systems
Transcription Factors/metabolism/genetics
Bioreactors
Bacterial Proteins/metabolism/genetics
Oligosaccharides/metabolism/biosynthesis
Metabolic Engineering/methods

@article {pmid39607705,
year = {2024},
author = {Kadam, A and Shilo, S and Naor, H and Wainstein, A and Brilon, Y and Feldman, T and Minden, M and Kaushansky, N and Chapal-Ilani, N and Shlush, L},
title = {Utilizing insights of DNA repair machinery to discover MMEJ deletions and novel mechanisms.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {e106},
doi = {10.1093/nar/gkae1132},
pmid = {39607705},
issn = {1362-4962},
support = {isf-nsfc 2427/18//Rising Tide Foundation/ ; //Steven B. Rubenstein research fund/ ; //Applebaum Foundation/ ; //Ernest and Bonnie Beutler Research Program/ ; //Sagol Institute for Longevity Research/ ; //Barry and Eleanore Reznik Family Cancer Research fund/ ; 1123/21//ISF-IPMP-Israel Precision Medicine Program/ ; },
mesh = {Humans ; *Sequence Deletion ; *DNA End-Joining Repair ; Algorithms ; DNA Repair/genetics ; Exome/genetics ; CRISPR-Cas Systems ; Leukemia, Myeloid, Acute/genetics ; Female ; G-Quadruplexes ; Breast Neoplasms/genetics ; },
abstract = {We developed Del-read, an algorithm targeting medium-sized deletions (6-100 bp) in short-reads, which are challenging for current variant callers relying on alignment. Our focus was on Micro-Homolog mediated End Joining deletions (MMEJ-dels), prevalent in myeloid malignancies. MMEJ-dels follow a distinct pattern, occurring between two homologies, allowing us to generate a comprehensive list of MMEJ-dels in the exome. Using Del-read, we identified numerous novel germline and somatic MMEJ-dels in BEAT-AML and TCGA-breast datasets. Validation in 672 healthy individuals confirmed their presence. These novel MMEJ-dels were linked to genomic features associated with replication stress, like G-quadruplexes and minisatellite. Additionally, we observed a new category of MMEJ-dels with an imperfect-match at the flanking sequences of the homologies, suggesting a mechanism involving mispairing in homology alignment. We demonstrated robustness of the repair system despite CRISPR/Cas9-induced mismatches in the homologies. Further analysis of the canonical ASXL1 deletion revealed a diverse array of these imperfect-matches. This suggests a potentially more flexible and error-prone MMEJ repair system than previously understood. Our findings highlight Del-read's potential in uncovering previously undetected deletions and deepen our understanding of repair mechanisms.},
}

Humans
*Sequence Deletion
*DNA End-Joining Repair
Algorithms
DNA Repair/genetics
Exome/genetics
CRISPR-Cas Systems
Leukemia, Myeloid, Acute/genetics
Female
G-Quadruplexes
Breast Neoplasms/genetics

@article {pmid39569586,
year = {2024},
author = {Kanke, KL and Rayner, RE and Bozik, J and Abel, E and Venugopalan, A and Suu, M and Nouri, R and Stack, JT and Guo, G and Vetter, TA and Cormet-Boyaka, E and Hester, ME and Vaidyanathan, S},
title = {Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {13561-13576},
doi = {10.1093/nar/gkae1069},
pmid = {39569586},
issn = {1362-4962},
support = {HL151900-03/HL/NHLBI NIH HHS/United States ; VAIDYA22A0-KB//Cystic Fibrosis Foundation/ ; UL1TR002733//Center for Clinical and Translational Science/ ; //Ohio State University/ ; MCCOY19Ro//Nationwide Children's Hospital/ ; //Comprehensive Transplant Center Human Tissue Biorepository, OSU Wexner Medical Center/ ; DK106829//National Institute of Diabetes, Digestive and Kidney Diseases/ ; HL151900-03/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *DNA, Single-Stranded/genetics/metabolism ; *Gene Knock-In Techniques ; *Induced Pluripotent Stem Cells/metabolism ; Receptors, CCR5/genetics/metabolism ; Gene Editing/methods ; Cells, Cultured ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities.},
}

*CRISPR-Cas Systems
Humans
*DNA, Single-Stranded/genetics/metabolism
*Gene Knock-In Techniques
*Induced Pluripotent Stem Cells/metabolism
Receptors, CCR5/genetics/metabolism
Gene Editing/methods
Cells, Cultured
Hematopoietic Stem Cells/metabolism

@article {pmid39569582,
year = {2024},
author = {Arantes, PR and Chen, X and Sinha, S and Saha, A and Patel, AC and Sample, M and Nierzwicki, Ł and Lapinaite, A and Palermo, G},
title = {Dimerization of the deaminase domain and locking interactions with Cas9 boost base editing efficiency in ABE8e.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {13931-13944},
doi = {10.1093/nar/gkae1066},
pmid = {39569582},
issn = {1362-4962},
support = {//Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) program/ ; CHE-2144823//National Science Foundation/ ; R01GM141329/NH/NIH HHS/United States ; BIO230007//Pittsburgh Supercomputer Center/ ; DP2 GM149550/GM/NIGMS NIH HHS/United States ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; MCB160059//San Diego Supercomputing Center/ ; FG-2023-20431//Sloan Foundation/ ; 2138259//National Science Foundation/ ; R01GM141329/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; *DNA/metabolism/genetics/chemistry ; Protein Multimerization ; Humans ; Adenosine Deaminase/metabolism/genetics/chemistry ; Protein Domains ; Deamination ; Molecular Dynamics Simulation ; Adenine/metabolism/chemistry ; },
abstract = {CRISPR-based DNA adenine base editors (ABEs) hold remarkable promises to address human genetic diseases caused by point mutations. ABEs were developed by combining CRISPR-Cas9 with a transfer RNA (tRNA) adenosine deaminase enzyme and through directed evolution, conferring the ability to deaminate DNA. However, the molecular mechanisms driving the efficient DNA deamination in the evolved ABEs remain unresolved. Here, extensive molecular simulations and biochemical experiments reveal the biophysical basis behind the astonishing base editing efficiency of ABE8e, the most efficient ABE to date. We demonstrate that the ABE8e's DNA deaminase domain, TadA8e, forms remarkably stable dimers compared to its tRNA-deaminating progenitor and that the strength of TadA dimerization is crucial for DNA deamination. The TadA8e dimer forms robust interactions involving its R98 and R129 residues, the RuvC domain of Cas9 and the DNA. These locking interactions are exclusive to ABE8e, distinguishing it from its predecessor, ABE7.10, and are indispensable to boost DNA deamination. Additionally, we identify three critical residues that drive the evolution of ABE8e toward improved base editing by balancing the enzyme's activity and stability, reinforcing the TadA8e dimer and improving the ABE8e's functionality. These insights offer new directions to engineer superior ABEs, advancing the design of safer precision genome editing tools.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism/chemistry
*DNA/metabolism/genetics/chemistry
Protein Multimerization
Humans
Adenosine Deaminase/metabolism/genetics/chemistry
Protein Domains
Deamination
Molecular Dynamics Simulation
Adenine/metabolism/chemistry

@article {pmid39530229,
year = {2024},
author = {Song, X and Chen, Z and Sun, W and Yang, H and Guo, L and Zhao, Y and Li, Y and Ren, Z and Shi, J and Liu, C and Ma, P and Huang, X and Ji, Q and Sun, B},
title = {CRISPR-AsCas12f1 couples out-of-protospacer DNA unwinding with exonuclease activity in the sequential target cleavage.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {14030-14042},
doi = {10.1093/nar/gkae989},
pmid = {39530229},
issn = {1362-4962},
support = {32271505//National Natural Science Foundation of China/ ; 22ZR1441900//Natural Science Foundation of Shanghai/ ; SLST-YS2024004//ShanghaiTech University School of Life Science and Technology Development Fund/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA/metabolism/chemistry/genetics ; *DNA Cleavage ; CRISPR-Associated Proteins/metabolism/genetics ; Gene Editing/methods ; Bacterial Proteins/metabolism/genetics/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Exonucleases/metabolism/genetics ; },
abstract = {Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present a versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two of which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate the molecular details underlying AsCas12f1-mediated DNA cleavages. We find that following the protospacer DNA unwinding and non-target strand (NTS) DNA nicking, AsCas12f1 surprisingly carries out bidirectional exonucleolytic cleavage from the nick. Subsequently, DNA unwinding is extended to the out-of-protospacer region, and AsCas12f1 gradually digests the unwound DNA beyond the protospacer. Eventually, the single endonucleolytic target-strand DNA cleavage at 3 nt downstream of the protospacer readily dissociates the ternary AsCas12f1-sgRNA-DNA complex from the protospacer adjacent motif-distal end, leaving a staggered double-strand DNA break. The coupling between the unwinding and cleavage of both protospacer and out-of-protospacer DNA is promoted by Mg2+. Kinetic analysis on the engineered AsCas12f1-v5.1 variant identifies the only accelerated step of the protospacer NTS DNA trimming within the sequential DNA cleavage. Our findings provide a dynamic view of AsCas12f1 catalyzing DNA unwinding-coupled nucleolytic cleavage and help with practical improvements of Cas12f-based genome editing tools.},
}

*CRISPR-Cas Systems
*DNA/metabolism/chemistry/genetics
*DNA Cleavage
CRISPR-Associated Proteins/metabolism/genetics
Gene Editing/methods
Bacterial Proteins/metabolism/genetics/chemistry
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Exonucleases/metabolism/genetics

@article {pmid39530221,
year = {2024},
author = {Pale, LM and Khatib, JB and Nusawardhana, A and Straka, J and Nicolae, CM and Moldovan, GL},
title = {CRISPR knockout genome-wide screens identify the HELQ-RAD52 axis in regulating the repair of cisplatin-induced single-stranded DNA gaps.},
journal = {Nucleic acids research},
volume = {52},
number = {22},
pages = {13832-13848},
doi = {10.1093/nar/gkae998},
pmid = {39530221},
issn = {1362-4962},
support = {R01 GM134681/GM/NIGMS NIH HHS/United States ; R01 ES026184/ES/NIEHS NIH HHS/United States ; R01ES026184/NH/NIH HHS/United States ; F31 CA275340/CA/NCI NIH HHS/United States ; R01 CA244417/CA/NCI NIH HHS/United States ; 4D01_2024_1002//Four Diamonds Transformative Patient-Oriented Cancer Research Project/ ; R01ES026184/NH/NIH HHS/United States ; },
mesh = {*Cisplatin/pharmacology ; *DNA, Single-Stranded/genetics/metabolism ; Humans ; *Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; *DNA Helicases/genetics/metabolism ; *DNA Repair/drug effects/genetics ; CRISPR-Cas Systems ; Antineoplastic Agents/pharmacology ; DNA Damage ; Gene Knockout Techniques ; DNA Primase/genetics/metabolism ; Cell Line, Tumor ; BRCA2 Protein/genetics/metabolism ; },
abstract = {Treatment with genotoxic agents, such as platinum compounds, is still the mainstay therapeutical approach for the majority of cancers. Our understanding of the mechanisms of action of these drugs is, however, imperfect and continuously evolving. Recent advances highlighted single-stranded DNA (ssDNA) gap accumulation as a potential determinant underlying cisplatin chemosensitivity, at least in some genetic backgrounds, such as BRCA mutations. Cisplatin-induced ssDNA gaps form upon restart of DNA synthesis downstream of cisplatin-induced lesions through repriming catalyzed by the PRIMPOL enzyme. Here, we show that PRIMPOL overexpression in otherwise wild-type cells results in accumulation of cisplatin-induced ssDNA gaps without sensitizing cells to cisplatin, suggesting that ssDNA gap accumulation does not confer cisplatin sensitivity in BRCA-proficient cells. To understand how ssDNA gaps may cause cellular sensitivity, we employed CRISPR-mediated genome-wide genetic screening to identify factors which enable the cytotoxicity of cisplatin-induced ssDNA gaps. We found that the helicase HELQ specifically suppresses cisplatin sensitivity in PRIMPOL-overexpressing cells, and this is associated with reduced ssDNA accumulation. We moreover identify RAD52 as a mediator of this pathway. RAD52 promotes ssDNA gap accumulation through a BRCA-mediated mechanism. Our work identified the HELQ-RAD52-BRCA axis as a regulator of ssDNA gap processing and cisplatin sensitization.},
}

*Cisplatin/pharmacology
*DNA, Single-Stranded/genetics/metabolism
Humans
*Rad52 DNA Repair and Recombination Protein/genetics/metabolism
*DNA Helicases/genetics/metabolism
*DNA Repair/drug effects/genetics
CRISPR-Cas Systems
Antineoplastic Agents/pharmacology
DNA Damage
Gene Knockout Techniques
DNA Primase/genetics/metabolism
Cell Line, Tumor
BRCA2 Protein/genetics/metabolism

@article {pmid39491497,
year = {2025},
author = {Pillai, A and Verma, V and Galande, S},
title = {CHARM and EvoETR: Precision epigenetic tools for gene silencing.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {1},
pages = {e2400186},
doi = {10.1002/bies.202400186},
pmid = {39491497},
issn = {1521-1878},
support = {JCB/2019/000013//Science and Engineering Research Board/ ; },
mesh = {Animals ; *Gene Silencing ; *Gene Editing/methods ; *Epigenesis, Genetic ; Mice ; Humans ; CRISPR-Cas Systems ; DNA Methylation ; Proprotein Convertase 9/genetics/metabolism ; Prions/genetics/metabolism ; },
abstract = {With the advent of gene editing technologies like CRISPR/Cas9, it has become possible to edit genomic regions of interest for research and therapeutic purposes. These technologies have also been adapted to alter gene expression without changing their DNA sequence, allowing epigenetic edits. While genetic editors make edits by cutting the genome at specified regions, epigenetic editors leverage the same targeting mechanism but act based on the epigenetic modifier fused to them, such as a methyltransferase. Here, we discuss two recently employed epigenetic editors (epi-editors) that silenced target genes involved in disease to mitigate their effects. Neumann et al. reported the construction of an epigenetic editor called CHARM that could methylate and silence the prion gene in mouse brains and subsequently switch itself off. Additionally, Capelluti et al. developed an epi-editor called EvoETR that knocked down Pcsk9 in the murine liver to reduce LDL levels. We aim to highlight the design principles underlying the design of these epi-editors to inform future editor designs.},
}

Animals
*Gene Silencing
*Gene Editing/methods
*Epigenesis, Genetic
Mice
Humans
CRISPR-Cas Systems
DNA Methylation
Proprotein Convertase 9/genetics/metabolism
Prions/genetics/metabolism

@article {pmid39441374,
year = {2025},
author = {Guccio, N and Alcaino, C and Miedzybrodzka, EL and Santos-Hernández, M and Smith, CA and Davison, A and Bany Bakar, R and Kay, RG and Reimann, F and Gribble, FM},
title = {Molecular mechanisms underlying glucose-dependent insulinotropic polypeptide secretion in human duodenal organoids.},
journal = {Diabetologia},
volume = {68},
number = {1},
pages = {217-230},
pmid = {39441374},
issn = {1432-0428},
support = {100574/Z/12/Z/WT_/Wellcome Trust/United Kingdom ; 220271/Z/20/Z/WT_/Wellcome Trust/United Kingdom ; MR/M009041/1/MRC_/Medical Research Council/United Kingdom ; MRC_MC_UU_00014/5/MRC_/Medical Research Council/United Kingdom ; MRC_MC_UU_12012/3/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *Gastric Inhibitory Polypeptide/metabolism ; *Organoids/metabolism ; *Duodenum/metabolism ; *Receptors, G-Protein-Coupled/metabolism/genetics ; *Enteroendocrine Cells/metabolism ; Glucose/metabolism ; Receptors, Calcium-Sensing/metabolism/genetics ; Calcium/metabolism ; CRISPR-Cas Systems ; Cyclic AMP/metabolism ; },
abstract = {AIMS/HYPOTHESIS: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by enteroendocrine K cells in the proximal small intestine. This study aimed to explore the function of human K cells at the molecular and cellular levels.

METHODS: CRISPR-Cas9 homology-directed repair was used to insert transgenes encoding a yellow fluorescent protein (Venus) or an Epac-based cAMP sensor (Epac-S-H187) in the GIP locus in human duodenal-derived organoids. Fluorescently labelled K cells were purified by FACS for RNA-seq and peptidomic analysis. GIP reporter organoids were employed for GIP secretion assays, live-cell imaging of Ca[2+] using Fura-2 and cAMP using Epac-S-H187, and basic electrophysiological characterisation. The G protein-coupled receptor genes GPR142 and CASR were knocked out to evaluate roles in amino acid sensing.

RESULTS: RNA-seq of human duodenal K cells revealed enrichment of several G protein-coupled receptors involved in nutrient sensing, including FFAR1, GPBAR1, GPR119, CASR and GPR142. Glucose induced action potential firing and cytosolic Ca[2+] elevation and caused a 1.8-fold increase in GIP secretion, which was inhibited by the sodium glucose co-transporter 1/2 (SGLT1/2) blocker sotagliflozin. Activation of the long-chain fatty acid receptor free fatty acid receptor 1 (FFAR1) induced a 2.7-fold increase in GIP secretion, while tryptophan and phenylalanine stimulated secretion by 2.8- and 2.1-fold, respectively. While CASR knockout blunted intracellular Ca[2+] responses, a CASR/GPR142 double knockout was needed to reduce GIP secretory responses to aromatic amino acids.

CONCLUSIONS/INTERPRETATION: The newly generated human organoid K cell model enables transcriptomic and functional characterisation of nutrient-sensing pathways involved in human GIP secretion. Both calcium-sensing receptor (CASR) and G protein-coupled receptor 142 (GPR142) contribute to protein-stimulated GIP secretion. This model will be further used to identify potential targets for modulation of native GIP secretion in diabetes and obesity.},
}

Humans
*Gastric Inhibitory Polypeptide/metabolism
*Organoids/metabolism
*Duodenum/metabolism
*Receptors, G-Protein-Coupled/metabolism/genetics
*Enteroendocrine Cells/metabolism
Glucose/metabolism
Receptors, Calcium-Sensing/metabolism/genetics
Calcium/metabolism
CRISPR-Cas Systems
Cyclic AMP/metabolism

@article {pmid39702333,
year = {2024},
author = {Liang, QZ and Chen, W and Liu, RC and Fu, QL and Fu, GH and Cheng, LF and Chen, HM and Jiang, NS and Zhu, T and Huang, Y},
title = {CRISPR/Cas12a and recombinase polymerase amplification-based rapid on-site nucleic acid detection of duck circovirus.},
journal = {Virology journal},
volume = {21},
number = {1},
pages = {322},
pmid = {39702333},
issn = {1743-422X},
support = {ZYTS202423//the Freedom Explore Program of Fujian Academy of Agricultural Sciences/ ; 2023J01363//the Natural Science Foundation Project of Fujian Province/ ; CARS-42//the National Waterfowl Industry Technology System of Modern Agriculture for China/ ; },
mesh = {*Circovirus/genetics/isolation & purification ; Animals ; *Ducks/virology ; *Circoviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Sensitivity and Specificity ; Poultry Diseases/virology/diagnosis ; Recombinases/metabolism/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: Duck circovirus (DuCV) infections commonly induce immunosuppression and secondary infections in ducks, resulting in significant economic losses in the duck breeding industry. Currently, effective vaccines and treatments for DuCV have been lacking. Therefore, rapid, specific, and sensitive detection methods are crucial for preventing and controlling DuCV.

METHODS: A lateral flow strip (LFS) detection method was developed using recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). The RPA-CRISPR/Cas12a-LFS targeted the DuCV replication protein (Rep) and was operated at 37 ℃ and allowed for visual interpretation without requiring sophisticated equipment.

RESULTS: The results revealed that the reaction time of RPA-CRISPR/Cas12a-LFS is only 45 min. This method achieved a low detection limit of 2.6 gene copies. Importantly, this method demonstrated high specificity and no cross-reactivity with six other avian viruses. In a study involving 97 waterfowl samples, the Rep RPA-CRISPR/Cas12a-LFS showed 100% consistency and agreement with real-time quantitative polymerase chain reaction.

CONCLUSION: These findings underscored the potential of this user-friendly, rapid, sensitive, and accurate detection method for on-site DuCV detection.},
}

*Circovirus/genetics/isolation & purification
Animals
*Ducks/virology
*Circoviridae Infections/veterinary/diagnosis/virology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Sensitivity and Specificity
Poultry Diseases/virology/diagnosis
Recombinases/metabolism/genetics
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39701024,
year = {2024},
author = {Lee, J and Jeong, C},
title = {Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage.},
journal = {BMB reports},
volume = {},
number = {},
pages = {},
pmid = {39701024},
issn = {1976-670X},
abstract = {CRISPR/Cas systems have emerged as powerful tools for gene editing, nucleic acid detection, and therapeutic applications. Recent advances in single-molecule techniques have provided new insights into the DNA-targeting mechanisms of CRISPR/Cas systems, in particular, Types I, II, and V. Here, we review how single-molecule approaches have expanded our understanding of key processes, namely target search, recognition, and cleavage. Furthermore, we focus on the dynamic behavior of Cas proteins, including PAM site recognition and R-loop formation, which are crucial to ensure specificity and efficiency in gene editing. Additionally, we discuss the conformational changes and interactions that drive precise DNA cleavage by different Cas proteins. This mini review provides a comprehensive overview of CRISPR/Cas molecular dynamics, offering conclusive insights into their broader potential for genome editing and biotechnological applications.},
}

@article {pmid39700796,
year = {2024},
author = {Wang, X and Ding, H and Sun, Y and Ma, Y and Wang, G and Chen, J and Choo, J and Chen, L},
title = {CRISPR/HCR-powered ratiometric fluorescence aptasensor for ochratoxin A detection.},
journal = {Food chemistry},
volume = {468},
number = {},
pages = {142437},
doi = {10.1016/j.foodchem.2024.142437},
pmid = {39700796},
issn = {1873-7072},
abstract = {To address the need for highly sensitive and reliable detection of trace ochratoxin A (OTA) in food matrices, we developed a ratiometric fluorescent aptasensor by integrating CRISPR/Cas12a, hybridization chain reaction (HCR), and horseradish peroxidase (HRP)-induced inner filter effect (IFE). The mechanism involves OTA releasing an activator that initiates CRISPR/Cas12a trans-cleavage, blocking HCR assembly. This reduces HRP levels, limiting the conversion of o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP) (emitting at 562 nm) while maintaining strong emission from 2-amino terephthalic acid (BDC-NH2) at 426 nm. The F426/F562 ratio serves as a "signal-on" indicator, enabling sensitive OTA detection over 0.1 pM to 10 nM, with a detection limit of 0.0417 pM. The method exhibits excellent reproducibility, with intra-day and inter-day relative standard deviations (RSDs) of 1.91 %-3.87 % and 1.79 %, respectively, along with recovery rates of 90.1 %-110.6 % in real samples. These advantages highlight its significant potential for CRISPR/Cas-based OTA detection.},
}

@article {pmid39700179,
year = {2024},
author = {Bowland, K and Lai, J and Skaist, A and Zhang, Y and Teh, SSK and Roberts, NJ and Thompson, E and Wheelan, SJ and Hruban, RH and Karchin, R and Bailey, MH and Iacobuzio-Donahue, CA and Eshleman, JR},
title = {Islands of genomic stability in the face of genetically unstable metastatic cancer.},
journal = {PloS one},
volume = {19},
number = {12},
pages = {e0298490},
pmid = {39700179},
issn = {1932-6203},
mesh = {Humans ; *Genomic Instability ; *Pancreatic Neoplasms/genetics/pathology ; *CRISPR-Cas Systems/genetics ; *Neoplasm Metastasis/genetics ; Carcinoma, Pancreatic Ductal/genetics/pathology/secondary ; Mutation ; Male ; Whole Genome Sequencing ; Female ; },
abstract = {INTRODUCTION: Metastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here, we test the hypothesis that metastatic cancer can be genetically targeted by exploiting single base substitution mutations unique to individual cells that occur as part of normal aging prior to transformation. These mutations are targetable because ~10% of them form novel tumor-specific "NGG" protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9.

METHODS: Whole genome sequencing was performed on five rapid autopsy cases of patient-matched primary tumor, normal and metastatic tissue from pancreatic ductal adenocarcinoma decedents. CRISPR-Cas9 PAM targets were determined by bioinformatic tumor-normal subtraction for each patient and verified in metastatic samples by high-depth capture-based sequencing.

RESULTS: We found that 90% of PAM targets were maintained between primary carcinomas and metastases overall. We identified rules that predict PAM loss or retention, where PAMs located in heterozygous regions in the primary tumor can be lost in metastases (private LOH), but PAMs occurring in regions of loss of heterozygosity (LOH) in the primary tumor were universally conserved in metastases.

CONCLUSIONS: Regions of truncal LOH are strongly retained in the presence of genetic instability and, therefore, represent genetic vulnerabilities in pancreatic adenocarcinomas. A CRISPR-based gene therapy approach targeting these regions may be a novel way to genetically target metastatic cancer.},
}

Humans
*Genomic Instability
*Pancreatic Neoplasms/genetics/pathology
*CRISPR-Cas Systems/genetics
*Neoplasm Metastasis/genetics
Carcinoma, Pancreatic Ductal/genetics/pathology/secondary
Mutation
Male
Whole Genome Sequencing
Female

@article {pmid39700052,
year = {2024},
author = {Li, X and Chen, B and Xie, Y and Luo, Y and Zhu, D and Wang, L and Su, S},
title = {Polyvalent Aptamers Structure-Mediated Fluorescent Aptasensor for the Early Diagnosis of Alzheimer's Disease by Coupling with HCR and CRISPR-Cas System.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c05329},
pmid = {39700052},
issn = {1520-6882},
abstract = {The early diagnosis of Alzheimer's disease (AD) plays a vital role in slowing the progression of AD and improving the quality of human life. However, it is still a challenge in the medical field. Herein, an ultrasensitive fluorescent aptasensor was designed for the detection of special phosphorylated tau181 (P-tau181) by coupling with polyvalent aptamers (PAs) structure, hybridization chain reaction (HCR), and the CRISPR-Cas system. Coupling with the signal amplification strategy, the specific recognition ability of the aptamer, and the high cleavage activity of Cas12a protein, the designed aptasensor showed a wide linear range (0.1-10[6] pg/mL), a low detection limit (0.069 pg/mL), high selectivity, and excellent anti-interference ability for the detection of P-tau181. Moreover, the aptasensor can efficiently analyze P-tau181 in artificial cerebro spinal fluid (aCSF) and serum, proving that it has a promising application in the early diagnosis of AD.},
}

@article {pmid39699265,
year = {2024},
author = {Li, X and Wang, L and Lin, J and Gu, Y and Liu, Z and Hu, J},
title = {Detection of CRISPR‒Cas and type I R-M systems in Klebsiella pneumoniae of human and animal origins and their relationship to antibiotic resistance and virulence.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0000924},
doi = {10.1128/spectrum.00009-24},
pmid = {39699265},
issn = {2165-0497},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)‒CRISPR-associated protein (Cas) and restriction‒modification (R-M) systems are important immune systems in bacteria. Information about the distributions of these two systems in Klebsiella pneumoniae from different hosts and their mutual effect on antibiotic resistance and virulence is still limited. In this study, the whole genomes of 520 strains of K. pneumoniae from GenBank, including 325 from humans and 195 from animals, were collected for CRISPR‒Cas systems and type I R-M systems, virulence genes, antibiotic resistance genes, and multilocus sequence typing detection. The results showed that host origin had no obvious influence on the distributions of the two systems (CRISPR‒Cas systems in 29.8% and 24.1%, type I R-M systems in 9.8% and 11.8% of human-origin and animal-origin strains, respectively) in K. pneumoniae. Identical spacer sequences from different hosts demonstrated there was a risk of human-animal transmission. All virulence genes (yersiniabactin, colibactin, aerobactin, salmochelin, rmpADC, and rmpA2) detection rates were higher when only the CRISPR‒Cas systems were present but were all reduced when coexisting with type I R-M systems. However, a lower prevalence of most antibiotic-resistance genes was found when the CRISPR‒Cas systems were alone, and when type I R-M systems were coexisting, some of the antibiotic resistance gene incidence rates were even lower (aminoglycosides, clindamycins, rifampicin-associated resistance genes, sulfonamides, methotrexates, beta-lactamases, and ultrabroad-spectrum beta-lactamases), and some of them were higher instead (quinolones, macrolides, tetracyclines, and carbapenems). The synergistic and opposed effects of the two systems on virulence and antibiotic-resistance genes need further study.IMPORTANCEK. pneumoniae is an important opportunistic pathogen responsible for both human and animal infections, and the emergence of hypervirulent and multidrug-resistant K. pneumoniae has made it difficult to control this pathogen worldwide. Here, we find that CRISPR‒Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, have synergistic and opposed effects on virulence and antibiotic resistance genes in K. pneumoniae. Moreover, this study provides insights into the distributions of the two systems in K. pneumoniae from different hosts, and there is no significant difference in the prevalence of the two systems among K. pneumoniae spp. In addition, this study also characterizes the CRISPR arrays of K. pneumoniae from different hosts, suggesting that the strains sharing the same spacer sequences have the potential to spread between humans and animals.},
}

@article {pmid39699033,
year = {2024},
author = {Paggi, RA and Ferrari, MC and Cerletti, M and Giménez, MI and Schwarz, TS and Marchfelder, A and De Castro, RE},
title = {Practical laboratory class to assess gene silencing using CRISPR interference (CRISPRi) technology in the archaeon Haloferax volcanii.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {},
number = {},
pages = {},
doi = {10.1002/bmb.21872},
pmid = {39699033},
issn = {1539-3429},
support = {//Universidad Nacional de Mar del Plata/ ; PICT 02228//Fondo para la Investigación Científica y Tecnológica (FONCYT)/ ; },
abstract = {Perturbation of gene expression using RNA interference (RNAi) or CRISPR interference (CRISPRi) is a useful strategy to explore the function of essential genes. In the archaeon Haloferax volcanii, the CRISPR-Cas system has been adapted as a CRISPRi tool to silence the expression of specific genes. We developed a laboratory class (LC) to conceptualize gene silencing through inactivation of the H. volcanii LonB protease gene, a negative regulator of carotenoid pigments biosynthesis, using CRISPRi. This LC has been successfully applied in the Biology and Biochemistry of Microorganisms course for undergraduate students of Biology in 2022 and 2023. The following objectives were proposed: (a) generate H. volcanii mutant strains with reduced expression of the lonB gene using CRISPRi; (b) examine the effect of lonB gene silencing on cell pigmentation and growth rate; (c) assess lonB gene repression by Western blotting (WB). This LC allows students to obtain and screen CRISPRi silenced-mutants by means of simple procedures using a non-pathogenic organism as well as handle basic microbiology, biochemistry and molecular biology protocols. Additionally, the LC fosters social actions through collaborative work (experimental work), the interpretation and discussion of data and the ability to communicate outcomes orally and in a written format (scientific report).},
}

@article {pmid39602282,
year = {2025},
author = {Lim, RM and Lu, A and Chuang, BM and Anaraki, C and Chu, B and Halbrook, CJ and Edinger, AL},
title = {CARMIL1-AA selectively inhibits macropinocytosis while sparing autophagy.},
journal = {Molecular biology of the cell},
volume = {36},
number = {1},
pages = {ar4},
doi = {10.1091/mbc.E24-09-0434},
pmid = {39602282},
issn = {1939-4586},
mesh = {*Pinocytosis/physiology ; Humans ; *Autophagy/physiology ; Cell Line, Tumor ; Sodium-Hydrogen Exchangers/metabolism/genetics ; Amiloride/pharmacology/analogs & derivatives ; Cell Proliferation ; CRISPR-Cas Systems ; },
abstract = {Macropinocytosis is reported to fuel tumor growth and drug resistance by allowing cancer cells to scavenge extracellular macromolecules. However, accurately defining the role of macropinocytosis in cancer depends on our ability to selectively block this process. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) is widely used to inhibit macropinocytosis but affects multiple Na[+]/H[+] exchangers (NHE) that regulate cytoplasmic and organellar pH. Consistent with this, we report that EIPA slows proliferation to a greater extent than can be accounted for by macropinocytosis inhibition and triggers conjugation of ATG8 to single membranes (CASM). Knocking down only NHE1 would not avoid macropinocytosis-independent effects on pH. Moreover, contrary to published reports, NHE1 loss did not block macropinocytosis in multiple cell lines. Knocking down CARMIL1 with CRISPR-Cas9 editing limited macropinocytosis, but only by 50%. In contrast, expressing the CARMIL1-AA mutant inhibits macropinocytosis induced by a wide range of macropinocytic stimuli to a similar extent as EIPA. CARMIL1-AA expression did not inhibit proliferation, highlighting the shortcomings of EIPA as a macropinocytosis inhibitor. Importantly, autophagy, another actin dependent, nutrient-producing process, was not affected by CARMIL1-AA expression. In sum, constitutive or inducible CARMIL1-AA expression reduced macropinocytosis without affecting proliferation, RAC activation, or autophagy, other processes that drive tumor initiation and progression.},
}

*Pinocytosis/physiology
Humans
*Autophagy/physiology
Cell Line, Tumor
Sodium-Hydrogen Exchangers/metabolism/genetics
Amiloride/pharmacology/analogs & derivatives
Cell Proliferation
CRISPR-Cas Systems

@article {pmid39565688,
year = {2024},
author = {Asano, K and Yoshimi, K and Takeshita, K and Mitsuhashi, S and Kochi, Y and Hirano, R and Tingyu, Z and Ishida, S and Mashimo, T},
title = {CRISPR Diagnostics for Quantification and Rapid Diagnosis of Myotonic Dystrophy Type 1 Repeat Expansion Disorders.},
journal = {ACS synthetic biology},
volume = {13},
number = {12},
pages = {3926-3935},
doi = {10.1021/acssynbio.4c00265},
pmid = {39565688},
issn = {2161-5063},
mesh = {*Myotonic Dystrophy/genetics/diagnosis ; Humans ; *CRISPR-Cas Systems/genetics ; *Trinucleotide Repeat Expansion/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Sensitivity and Specificity ; },
abstract = {Repeat expansion disorders, exemplified by myotonic dystrophy type 1 (DM1), present challenges in diagnostic quantification because of the variability and complexity of repeat lengths. Traditional diagnostic methods, including PCR and Southern blotting, exhibit limitations in sensitivity and specificity, necessitating the development of innovative approaches for precise and rapid diagnosis. Here, we introduce a CRISPR-based diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for the quantification and rapid diagnosis of DM1. This method, using in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity and specificity in quantifying CTG repeat expansion lengths, correlated with disease severity. We also validate the robustness and accuracy of CRISPR diagnostics in quantitatively diagnosing DM1 using patient genomes. Furthermore, we optimize a REPLICA-based assay for point-of-care-testing using lateral flow test strips, facilitating rapid screening and detection. In summary, REPLICA-based CRISPR diagnostics offer precise and rapid detection of repeat expansion disorders, promising personalized treatment strategies.},
}

*Myotonic Dystrophy/genetics/diagnosis
Humans
*CRISPR-Cas Systems/genetics
*Trinucleotide Repeat Expansion/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Sensitivity and Specificity

@article {pmid39499900,
year = {2024},
author = {Liu, Z and Chen, S and Lo, CH and Wang, Q and Sun, Y},
title = {All-in-one AAV-mediated Nrl gene inactivation rescues retinal degeneration in Pde6a mice.},
journal = {JCI insight},
volume = {9},
number = {24},
pages = {},
doi = {10.1172/jci.insight.178159},
pmid = {39499900},
issn = {2379-3708},
mesh = {Animals ; *Cyclic Nucleotide Phosphodiesterases, Type 6/genetics ; Mice ; *Dependovirus/genetics ; *Genetic Therapy/methods ; *Retinal Degeneration/therapy/genetics/pathology ; *Gene Editing/methods ; *Disease Models, Animal ; *Eye Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Retinitis Pigmentosa/genetics/therapy/pathology ; Retina/pathology/metabolism ; Humans ; Mutation ; Basic-Leucine Zipper Transcription Factors ; },
abstract = {Retinitis pigmentosa (RP) is a complex group of inherited retinal diseases characterized by progressive death of photoreceptor cells and eventual blindness. Pde6a, which encodes a cGMP-specific phosphodiesterase, is a crucial pathogenic gene for autosomal recessive RP (RP43); there is no effective therapy for this form of RP. The compact CRISPR/Staphylococcus aureus Cas9 (CRISPR/SaCas9) system, which can be packaged into a single adeno-associated virus (AAV), holds promise for simplifying effective gene therapy. Here, we demonstrated that all-in-one AAV-SaCas9-mediated Nrl gene inactivation can efficiently prevent retinal degeneration in a RP mouse model with Pde6anmf363/nmf363 mutation. We screened single-guide RNAs capable of efficiently editing the mouse Nrl gene in N2a cells and then achieved effective gene editing by using a single AAV to codeliver SaCas9 and an optimal Nrl-sg2 into the mouse retina. Excitingly, in vivo inactivation of Nrl improved photoreceptor cell survival and rescued retinal function in treated Pde6a-deficient mice. Thus, we showed that a practical, gene-independent method, AAV-SaCas9-mediated Nrl inactivation, holds promise for future therapeutic applications in patients with RP.},
}

Animals
*Cyclic Nucleotide Phosphodiesterases, Type 6/genetics
Mice
*Dependovirus/genetics
*Genetic Therapy/methods
*Retinal Degeneration/therapy/genetics/pathology
*Gene Editing/methods
*Disease Models, Animal
*Eye Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Retinitis Pigmentosa/genetics/therapy/pathology
Retina/pathology/metabolism
Humans
Mutation
Basic-Leucine Zipper Transcription Factors

@article {pmid39440484,
year = {2024},
author = {Wong, PK and Syafruddin, SE and Cheah, FC and Azmi, N and Ng, PY and Chua, EW},
title = {Introduction of a single-nucleotide variant, rs16851030, into the ADORA1 gene increased cellular susceptibility to hypoxia.},
journal = {Personalized medicine},
volume = {21},
number = {6},
pages = {353-366},
doi = {10.1080/17410541.2024.2412514},
pmid = {39440484},
issn = {1744-828X},
support = {FRGS/1/2019/SKK08/UKM/03/4//Fundamental Research Grant Scheme/ ; },
mesh = {Humans ; *Polymorphism, Single Nucleotide/genetics ; HEK293 Cells ; *Receptor, Adenosine A1/genetics ; *CRISPR-Cas Systems/genetics ; Cell Hypoxia/genetics ; Genetic Predisposition to Disease/genetics ; Gene Editing/methods ; Hypoxia/genetics ; Caffeine/pharmacology ; 3' Untranslated Regions/genetics ; },
abstract = {Aim: Rs16851030, a single-nucleotide variant located in the 3'-untranslated region of the ADORA1 gene, has been proposed as a potential marker of caffeine sensitivity in apnea of prematurity. Besides, it is associated with aspirin-induced asthma and the development of acute chest syndrome. However, its functional significance is still unconfirmed. This study aimed to elucidate the functional impact of rs16851030 by using CRISPR/Cas9 approach to induce the DNA variant and attendant physiological changes.Methods: Rs16851030 was introduced into HEK293 cells via homology-directed repair (HDR). Edited cells were fluorescence-enriched, sorted, isolated, and expanded into single-cell-derived clones. The edit was confirmed by Sanger sequencing. RNA sequencing was used to analyze affected pathways.Results: Rs16851030-mutant cells showed increased susceptibility to hypoxia, a condition related to apnea of prematurity. After 24 h of hypoxia, the viability of mutant clones 1 and 2 was low compared with wild-type cells (75.45% and 74.47% vs. 96.34%). RNA sequencing revealed transcriptomic changes linked to this increased vulnerability.Conclusion: Rs16851030 impairs cellular resistance to hypoxia, suggesting its role in conditions like apnea of prematurity. Further research should investigate the molecular mechanisms and transcriptomic alterations caused by rs16851030 under hypoxic conditions.},
}

Humans
*Polymorphism, Single Nucleotide/genetics
HEK293 Cells
*Receptor, Adenosine A1/genetics
*CRISPR-Cas Systems/genetics
Cell Hypoxia/genetics
Genetic Predisposition to Disease/genetics
Gene Editing/methods
Hypoxia/genetics
Caffeine/pharmacology
3' Untranslated Regions/genetics

@article {pmid39192154,
year = {2025},
author = {Zhang, H and Zhu, JK},
title = {Epigenetic gene regulation in plants and its potential applications in crop improvement.},
journal = {Nature reviews. Molecular cell biology},
volume = {26},
number = {1},
pages = {51-67},
pmid = {39192154},
issn = {1471-0080},
mesh = {*Epigenesis, Genetic/genetics ; *DNA Methylation/genetics ; *Crops, Agricultural/genetics ; *Gene Expression Regulation, Plant/genetics ; Gene Editing/methods ; Plant Breeding/methods ; Plants/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {DNA methylation, also known as 5-methylcytosine, is an epigenetic modification that has crucial functions in plant growth, development and adaptation. The cellular DNA methylation level is tightly regulated by the combined action of DNA methyltransferases and demethylases. Protein complexes involved in the targeting and interpretation of DNA methylation have been identified, revealing intriguing roles of methyl-DNA binding proteins and molecular chaperones. Structural studies and in vitro reconstituted enzymatic systems have provided mechanistic insights into RNA-directed DNA methylation, the main pathway catalysing de novo methylation in plants. A better understanding of the regulatory mechanisms will enable locus-specific manipulation of the DNA methylation status. CRISPR-dCas9-based epigenome editing tools are being developed for this goal. Given that DNA methylation patterns can be stably transmitted through meiosis, and that large phenotypic variations can be contributed by epimutations, epigenome editing holds great promise in crop breeding by creating additional phenotypic variability on the same genetic material.},
}

*Epigenesis, Genetic/genetics
*DNA Methylation/genetics
*Crops, Agricultural/genetics
*Gene Expression Regulation, Plant/genetics
Gene Editing/methods
Plant Breeding/methods
Plants/genetics/metabolism
CRISPR-Cas Systems/genetics

@article {pmid38377059,
year = {2024},
author = {Shamjetsabam, ND and Rana, R and Malik, P and Ganguly, NK},
title = {CRISPR/Cas9: an overview of recent developments and applications in cancer research.},
journal = {International journal of surgery (London, England)},
volume = {110},
number = {10},
pages = {6198-6213},
pmid = {38377059},
issn = {1743-9159},
mesh = {*CRISPR-Cas Systems ; Humans ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; Genetic Therapy/methods ; Biomedical Research ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) has risen as a potent gene editing method with vast potential across numerous domains, including its application in cancer research and therapy. This review article provides an extensive overview of the research that has been done so far on CRISPR-Cas9 with an emphasis on how it could be utilized in the treatment of cancer. The authors go into the underlying ideas behind CRISPR-Cas9, its mechanisms of action, and its application for the study of cancer biology. Furthermore, the authors investigate the various uses of CRISPR-Cas9 in cancer research, spanning from the discovery of genes and the disease to the creation of novel therapeutic approaches. The authors additionally discuss the challenges and limitations posed by CRISPR-Cas9 technology and offer insights into the potential applications and future directions of this cutting-edge field of research. The article intends to consolidate the present understanding and stimulate more research into CRISPR-Cas9's promise as a game-changing tool for cancer research and therapy.},
}

*CRISPR-Cas Systems
Humans
*Neoplasms/genetics/therapy
*Gene Editing/methods
Genetic Therapy/methods
Biomedical Research

@article {pmid39698811,
year = {2024},
author = {Soczek, KM and Cofsky, JC and Tuck, OT and Shi, H and Doudna, JA},
title = {CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae1192},
pmid = {39698811},
issn = {1362-4962},
support = {U19AI171110/NH/NIH HHS/United States ; U19NS132303/NS/NINDS NIH HHS/United States ; R21HL173710/HB/NHLBI NIH HHS/United States ; 2334028//National Science Foundation/ ; /HHMI/Howard Hughes Medical Institute/United States ; //Panattoni Family Foundation/ ; //National Science Foundation Graduate Research Fellowship/ ; //Helen Hay Whitney Foundation/ ; //HHMI Fellow of The Jane Coffin Childs Fund for Medical Research/ ; DE-AC02-05CH11231//Department of Energy/ ; 24 180//Apple Tree Partners/ ; //Lawrence Livermore National Laboratory/ ; //UCB-Hampton University Summer Program/ ; //Mr. Li Ka Shing/ ; //Koret-Berkeley-TAU/ ; //Innovative Genomics Institute/ ; },
abstract = {RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.},
}

@article {pmid39698318,
year = {2024},
author = {Cao, S and Ma, D and Xie, J and Wu, Z and Yan, H and Ji, S and Zhou, M and Zhu, S},
title = {Point-of-care testing diagnosis of African swine fever virus by targeting multiple genes with enzymatic recombinase amplification and CRISPR/Cas12a System.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1474825},
pmid = {39698318},
issn = {2235-2988},
mesh = {*African Swine Fever Virus/genetics/isolation & purification ; Animals ; *CRISPR-Cas Systems ; Swine ; *African Swine Fever/diagnosis/virology ; *Point-of-Care Testing ; *Sensitivity and Specificity ; *Recombinases/metabolism/genetics ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; },
abstract = {African swine fever virus (ASFV) infection is causing devastating outbreaks globally; pig farming has suffered severe economic losses due to the ASFV. Currently, strict biosecurity control measures can mitigate the incidence of ASF. Rapid, cost-effective, and sensitive detection of ASFV can significantly reduce disease transmission and mortality. CRISPR/Cas-associated proteins can detect polymorphisms with high specificity and sensitivity, making them ideal for detecting pathogens. In this study, based on CRISPR/Cas12a integrated with enzymatic recombinase amplification (ERA) technology, a CRISPR/Cas12a detection system capable of identifying ASFV E183L, K205R, and C962R gene sequences has been developed. The ERA-CRISPR/Cas12a detection system detected ASFV precisely without cross-reactivity with other porcine pathogen templates and with a sensitivity detection limit of 10 copies per reaction; it takes 60 minutes to complete the detection process. In combination with this integrated ERA pre-amplification and Cas12a/crRNA cutting assay, it provides a rapid, straightforward, sensitive, and specific method for ASFV detection in the field.},
}

*African Swine Fever Virus/genetics/isolation & purification
Animals
*CRISPR-Cas Systems
Swine
*African Swine Fever/diagnosis/virology
*Point-of-Care Testing
*Sensitivity and Specificity
*Recombinases/metabolism/genetics
Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/genetics
Endodeoxyribonucleases/genetics
Molecular Diagnostic Techniques/methods
Bacterial Proteins

@article {pmid39696686,
year = {2024},
author = {Izadifar, M and Massumi, M and Prentice, KJ and Oussenko, T and Li, B and Elbaz, J and Puri, M and Wheeler, MB and Nagy, A},
title = {Microfluidic chip systems for characterizing glucose-responsive insulin-secreting cells equipped with FailSafe kill-switch.},
journal = {Stem cell research & therapy},
volume = {15},
number = {1},
pages = {486},
pmid = {39696686},
issn = {1757-6512},
support = {143231//Canadian Institutes of Health Research Foundation Grant/ ; 950-230422//Canada Research Chairs/ ; },
mesh = {*Insulin-Secreting Cells/metabolism/drug effects ; Animals ; Mice ; *Glucose/metabolism ; *Thymidine Kinase/genetics/metabolism ; Ganciclovir/pharmacology ; CRISPR-Cas Systems ; CDC2 Protein Kinase/metabolism/genetics ; Lab-On-A-Chip Devices ; Insulin/metabolism ; Homeodomain Proteins/metabolism/genetics ; Trans-Activators ; },
abstract = {BACKGROUND: Pluripotent cell-derived islet replacement therapy offers promise for treating Type 1 diabetes (T1D), but concerns about uncontrolled cell proliferation and tumorigenicity present significant safety challenges. To address the safety concern, this study aims to establish a proof-of-concept for a glucose-responsive, insulin-secreting cell line integrated with a built-in FailSafe kill-switch.

METHOD: We generated β cell-induced progenitor-like cells (βiPLCs) from primary mouse pancreatic β cells through interrupted reprogramming. Then, we transcriptionally linked our FailSafe (FS) kill-switch, HSV-thymidine kinase (TK), to Cdk1 gene using a CRISPR/Cas9 knock-in strategy, resulting in a FailSafe βiPLC line, designated as FSβiPLCs. Subsequently we evaluated and confirmed the functionality of the drug-inducible kill-switch in FSβiPLCs at different ganciclovir (GCV) concentrations using our PDMS-based transcapillary microfluidic system. Finally, we assessed the functionality of FSβiPLCs by characterizing the dynamics of insulin secretion in response to changes in glucose concentration using our microfluidic perfusion glucose-stimulated insulin secretion (GSIS) assay-on- chip.

RESULTS: The βiPLCs exhibited Ins1, Pdx1 and Nkx6.1 expression, and glucose responsive insulin secretion, the essential properties of pancreatic beta cells. The βiPLCs were amenable to genome editing which allowed for the insertion of the kill-switch into the 3'UTR of Cdk1, confirmed by PCR genotyping. Our transcapillary microfluidic system confirmed the functionality of the drug-inducible kill-switch in FSβiPLCs, showing an effective cell ablation of dividing cells from a heterogeneous cell population at different ganciclovir (GCV) concentrations. The Ki67 expression assessment further confirmed that slow- or non-dividing cells in the FSβiPLC population were resistant to GCV. Our perfusion glucose-stimulated insulin secretion (GSIS) assay-on-chip revealed that the resistant non-dividing FSβiPLCs exhibited higher levels of insulin secretion and glucose responsiveness compared to their proliferating counterparts.

CONCLUSIONS: This study establishes a proof-of-concept for the integration of a FailSafe kill-switch system into a glucose-responsive, insulin-secreting cell line to address the safety concerns in stem cell-derived cell replacement treatment for T1D. The microfluidic systems provided valuable insights into the functionality and safety of these engineered cells, demonstrating the potential of the kill-switch to reduce the risk of tumorigenicity in pluripotent cell-derived insulin-secreting cells.},
}

*Insulin-Secreting Cells/metabolism/drug effects
Animals
Mice
*Glucose/metabolism
*Thymidine Kinase/genetics/metabolism
Ganciclovir/pharmacology
CRISPR-Cas Systems
CDC2 Protein Kinase/metabolism/genetics
Lab-On-A-Chip Devices
Insulin/metabolism
Homeodomain Proteins/metabolism/genetics
Trans-Activators

@article {pmid39696608,
year = {2024},
author = {Lin, Z and Yao, Q and Lai, K and Jiao, K and Zeng, X and Lei, G and Zhang, T and Dai, H},
title = {Cas12f1 gene drives propagate efficiently in herpesviruses and induce minimal resistance.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {311},
pmid = {39696608},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; *Herpesvirus 1, Human/genetics ; Gene Drive Technology/methods ; Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Humans ; },
abstract = {BACKGROUND: Synthetic CRISPR-Cas9 gene drive has been developed to control harmful species. However, resistance to Cas9 gene drive can be acquired easily when DNA repair mechanisms patch up the genetic insults introduced by Cas9 and incorporate mutations to the sgRNA target. Although many strategies to reduce the occurrence of resistance have been developed so far, they are difficult to implement and not always effective.

RESULTS: Here, Cas12f1, a recently developed CRISPR-Cas system with minimal potential for causing mutations within target sequences, has been explored as a potential platform for yielding low-resistance in gene drives. We construct Cas9 and Cas12f1 gene drives in a fast-replicating DNA virus, HSV1. Cas9 and Cas12f1 gene drives are able to spread among the HSV1 population with specificity towards their target sites, and their transmission among HSV1 viruses is not significantly affected by the reduced fitness incurred by the viral carriers. Cas12f1 gene drives spread similarly as Cas9 gene drives at high introduction frequency but transmit more slowly than Cas9 gene drives at low introduction frequency. However, Cas12f1 gene drives outperform Cas9 gene drives because they reach higher penetration and induce lower resistance than Cas9 gene drives in all cases.

CONCLUSIONS: Due to lower resistance and higher penetration, Cas12f1 gene drives could potentially supplant Cas9 gene drives for population control.},
}

*CRISPR-Cas Systems
*Herpesvirus 1, Human/genetics
Gene Drive Technology/methods
Animals
CRISPR-Associated Protein 9/metabolism
CRISPR-Associated Proteins/metabolism/genetics
Humans

@article {pmid39696488,
year = {2024},
author = {Koonin, EV and Makarova, KS},
title = {CRISPR in mobile genetic elements: counter-defense, inter-element competition and RNA-guided transposition.},
journal = {BMC biology},
volume = {22},
number = {1},
pages = {295},
pmid = {39696488},
issn = {1741-7007},
mesh = {*DNA Transposable Elements/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Interspersed Repetitive Sequences ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacteria/genetics/virology ; Plasmids/genetics ; },
abstract = {CRISPR are adaptive immunity systems that protect bacteria and archaea from viruses and other mobile genetic elements (MGE) via an RNA-guided interference mechanism. However, in the course of the host-parasite co-evolution, CRISPR systems have been recruited by MGE themselves for counter-defense or other functions. Some bacteriophages encode fully functional CRISPR systems that target host defense systems, and many others recruited individual components of CRISPR systems, such as single repeat units that inhibit host CRISPR systems and CRISPR mini-arrays that target related viruses contributing to inter-virus competition. Many plasmids carry type IV or subtype V-M CRISPR systems that appear to be involved in inter-plasmid competition. Numerous Tn7-like and Mu-like transposons encode CRISPR-associated transposases (CASTs) in which interference-defective CRISPR systems of type I or type V mediate RNA-guided, site-specific transposition. The recruitment of CRISPR systems and their components by MGE is a manifestation of extensive gene shuttling between host immune systems and MGE, a major trend in the coevolution of MGE with their hosts.},
}

*DNA Transposable Elements/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems
Interspersed Repetitive Sequences
RNA, Guide, CRISPR-Cas Systems/genetics
Bacteria/genetics/virology
Plasmids/genetics