@article {pmid38658526,
year = {2024},
author = {Qiao, W and Richards, CM and Kim, Y and Zengel, JR and Ding, S and Greenberg, HB and Carette, JE},
title = {MYADM binds human parechovirus 1 and is essential for viral entry.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3469},
pmid = {38658526},
issn = {2041-1723},
support = {R01 AI153169/AI/NIAID NIH HHS/United States ; R01 AI169467/AI/NIAID NIH HHS/United States ; R01 AI125249/AI/NIAID NIH HHS/United States ; },
mesh = {*Parechovirus/genetics/metabolism ; Humans ; *Virus Internalization ; *Picornaviridae Infections/virology/metabolism ; CRISPR-Cas Systems ; Protein Binding ; Receptors, Virus/metabolism/genetics ; Cell Line ; HEK293 Cells ; Organoids/virology/metabolism ; },
abstract = {Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.},
}

*Parechovirus/genetics/metabolism
Humans
*Virus Internalization
*Picornaviridae Infections/virology/metabolism
CRISPR-Cas Systems
Protein Binding
Receptors, Virus/metabolism/genetics
Cell Line
HEK293 Cells
Organoids/virology/metabolism

@article {pmid38658160,
year = {2024},
author = {Liu, Y and Ma, B and Chen, X},
title = {[Experimental teaching design of CRISPR/Cas9 technology in rice breeding application].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {40},
number = {4},
pages = {1237-1250},
doi = {10.13345/j.cjb.230380},
pmid = {38658160},
issn = {1872-2075},
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Breeding ; Teaching ; Disease Resistance/genetics ; Plant Diseases/prevention & control/microbiology/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {The CRISPR/Cas9 gene editing technology has proven to be valuable in crop breeding applications. Understanding and mastering this technology will provide a strong foundation for students majoring in biology, agronomy, and related fields to engage in scientific research and work. To incorporate CRISPR/Cas9 technology into experimental teaching courses at colleges, an innovative teaching experiment entitled "Enhancing the resistance of rice plants to bacterial blight disease using CRISPR/Cas9 technology" was designed. The experiment allows students to deepen their understanding of the basic principles of CRISPR/Cas technology, acquire proficiency in its protocol, and learn to apply the technology for targeted molecular breeding of rice. It not only expands students' knowledge and skills, but also promotes the reform and innovation of experimental teaching methods.},
}

*Oryza/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Plant Breeding
Teaching
Disease Resistance/genetics
Plant Diseases/prevention & control/microbiology/genetics
Plants, Genetically Modified/genetics

@article {pmid38658143,
year = {2024},
author = {Li, R and Li, R},
title = {[Application of CRISPR/Cas9 gene editing technology in edible fungi: a review].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {40},
number = {4},
pages = {988-1001},
doi = {10.13345/j.cjb.230725},
pmid = {38658143},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Fungi/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 gene editing system is a versatile technology for modifying gene, playing a crucial role in the study of functional genes and genetic breeding of plants, animals, fungi, and microorganisms. This review provides a comprehensive analysis of the application of this technology in gene research and genetic breeding of edible fungi. The review covers various aspects, including the delivery and expression strategies of Cas9 and sgRNA, genetic transformation methods, mutant screening, and repair strategies for target sites following DNA double-strand breaks. Additionally, the review summarizes the main challenges and optimization strategies associated with the application of this technology in edible fungi. Lastly, the future application potential of this technology in edible fungi research is discussed, drawing from the authors' personal research background.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Fungi/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid38657076,
year = {2024},
author = {Jain, I and Kolesnik, M and Kuznedelov, K and Minakhin, L and Morozova, N and Shiriaeva, A and Kirillov, A and Medvedeva, S and Livenskyi, A and Kazieva, L and Makarova, KS and Koonin, EV and Borukhov, S and Severinov, K and Semenova, E},
title = {tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector.},
journal = {Science advances},
volume = {10},
number = {17},
pages = {eadl0164},
pmid = {38657076},
issn = {2375-2548},
mesh = {*RNA, Transfer/genetics/metabolism ; *CRISPR-Cas Systems ; *Anticodon/genetics ; *Escherichia coli/genetics/metabolism ; Leptotrichia/genetics/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Bacteriophages/genetics ; RNA Cleavage ; },
abstract = {Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.},
}

*RNA, Transfer/genetics/metabolism
*CRISPR-Cas Systems
*Anticodon/genetics
*Escherichia coli/genetics/metabolism
Leptotrichia/genetics/metabolism
CRISPR-Associated Proteins/metabolism/genetics
Bacteriophages/genetics
RNA Cleavage

@article {pmid38656525,
year = {2024},
author = {Rahman, S and Ikram, AR and Azeem, F and Tahir Ul Qamar, M and Shaheen, T and Mehboob-Ur-Rahman, },
title = {Precision Genome Editing with CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2788},
number = {},
pages = {355-372},
pmid = {38656525},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA End-Joining Repair ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {The CRISPR/Cas9 system is a revolutionary technology for genome editing that allows for precise and efficient modifications of DNA sequences. The system is composed of two main components, the Cas9 enzyme and a guide RNA (gRNA). The gRNA is designed to specifically target a desired DNA sequence, while the Cas9 enzyme acts as molecular scissors to cut the DNA at that specific location. The cell then repairs the digested DNA, either through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in either indels or precise modifications of DNA sequences with broad implications in biotechnology, agriculture, and medicine. This chapter provides a practical approach for utilizing CRISPR/Cas9 in precise genome editing, including identifying the target gene sequence, designing gRNA and protein (Cas9), and delivering the CRISPR components to target cells.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
DNA End-Joining Repair
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid38656524,
year = {2024},
author = {Ahmad, N and Fatima, S and Hundleby, P and Mehboob-Ur-Rahman, },
title = {Genome Editing in Brassica juncea Using CRISPR/Cas9 Technology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2788},
number = {},
pages = {337-354},
pmid = {38656524},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Mustard Plant/genetics ; *Genome, Plant ; *Plants, Genetically Modified/genetics ; Agrobacterium/genetics ; Transformation, Genetic ; },
abstract = {Modern genome editing tools particularly CRISPR/Cas9 have revolutionized plant genome manipulation for engineering resilience against changing climatic conditions, disease infestation, as well as functional genomic studies. CRISPR-mediated genome editing allows for editing at a single as well as multiple locations in the genome simultaneously, making it an effective tool for polyploid species too. However, still, its applications are limited to the model crops only. Extending it to crop plants will help improve field crops against the changing climates more rapidly and precisely. Here we describe the protocol for editing the genome of a field crop Brassica juncea (mustard), an allotetraploid and important oilseed crop of the Indo-Pak Subcontinent region. This protocol is based on the Agrobacterium-mediated transformation for the delivery of CRISPR components into the plant genome using cotyledon as explants. We elaborate on steps for recovering genome-edited knockouts, for validation of the edits, as well as recovering the transgene-free edited plants through a commonly used segregating approach.},
}

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

@article {pmid38497408,
year = {2024},
author = {Yang, X and Xu, K and Li, S and Zhang, J and Xie, Y and Lou, Y and Xiao, X},
title = {Novel methods for the rapid and sensitive detection of Nipah virus based on a CRISPR/Cas12a system.},
journal = {The Analyst},
volume = {149},
number = {9},
pages = {2586-2593},
doi = {10.1039/d4an00027g},
pmid = {38497408},
issn = {1364-5528},
mesh = {*Nipah Virus/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Limit of Detection ; Humans ; Animals ; Nucleic Acid Amplification Techniques/methods ; DNA, Viral/genetics/analysis ; Fluorescent Dyes/chemistry ; },
abstract = {Nipah virus (NiV), a bat-borne zoonotic viral pathogen with high infectivity and lethality to humans, has caused severe outbreaks in several countries of Asia during the past two decades. Because of the worldwide distribution of the NiV natural reservoir, fruit bats, and lack of effective treatments or vaccines for NiV, routine surveillance and early detection are the key measures for containing NiV outbreaks and reducing its influence. In this study, we developed two rapid, sensitive and easy-to-conduct methods, RAA-CRISPR/Cas12a-FQ and RAA-CRISPR/Cas12a-FB, for NiV detection based on a recombinase-aided amplification (RAA) assay and a CRISPR/Cas12a system by utilizing dual-labeled fluorophore-quencher or fluorophore-biotin ssDNA probes. These two methods can be completed in 45 min and 55 min and achieve a limit of detection of 10 copies per μL and 100 copies per μL of NiV N DNA, respectively. In addition, they do not cross-react with nontarget nucleic acids extracted from the pathogens causing similar symptoms to NiV, showing high specificity for NiV N DNA detection. Meanwhile, they show satisfactory performance in the detection of spiked samples from pigs and humans. Collectively, the RAA-CRISPR/Cas12a-FQ and RAA-CRISPR/Cas12a-FB methods developed by us would be promising candidates for the early detection and routine surveillance of NiV in resource-poor areas and outdoors.},
}

*Nipah Virus/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Limit of Detection
Humans
Animals
Nucleic Acid Amplification Techniques/methods
DNA, Viral/genetics/analysis
Fluorescent Dyes/chemistry

@article {pmid38489061,
year = {2024},
author = {Zhang, Z and Zhang, S and Wong, HT and Li, D and Feng, B},
title = {Targeted Gene Insertion: The Cutting Edge of CRISPR Drug Development with Hemophilia as a Highlight.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {38},
number = {3},
pages = {369-385},
pmid = {38489061},
issn = {1179-190X},
support = {14116719//University Grants Committee/ ; 14115520 to B.F.;//University Grants Committee/ ; },
mesh = {Humans ; *Hemophilia A/genetics/therapy ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Drug Development/methods ; *Genetic Therapy/methods ; Animals ; Mutagenesis, Insertional ; },
abstract = {The remarkable advance in gene editing technology presents unparalleled opportunities for transforming medicine and finding cures for hereditary diseases. Human trials of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9)-based therapeutics have demonstrated promising results in disrupting or deleting target sequences to treat specific diseases. However, the potential of targeted gene insertion approaches, which offer distinct advantages over disruption/deletion methods, remains largely unexplored in human trials due to intricate technical obstacles and safety concerns. This paper reviews the recent advances in preclinical studies demonstrating in vivo targeted gene insertion for therapeutic benefits, targeting somatic solid tissues through systemic delivery. With a specific emphasis on hemophilia as a prominent disease model, we highlight advancements in insertion strategies, including considerations of DNA repair pathways, targeting site selection, and donor design. Furthermore, we discuss the complex challenges and recent breakthroughs that offer valuable insights for progressing towards clinical trials.},
}

Humans
*Hemophilia A/genetics/therapy
*CRISPR-Cas Systems
*Gene Editing/methods
*Drug Development/methods
*Genetic Therapy/methods
Animals
Mutagenesis, Insertional

@article {pmid38462163,
year = {2024},
author = {Munir, M and Embry, A and Doench, JG and Heaton, NS and Wilen, CB and Orchard, RC},
title = {Genome-wide CRISPR activation screen identifies JADE3 as an antiviral activator of NF-kB-dependent IFITM3 expression.},
journal = {The Journal of biological chemistry},
volume = {300},
number = {4},
pages = {107153},
pmid = {38462163},
issn = {1083-351X},
mesh = {Humans ; *NF-kappa B/metabolism/genetics ; *Membrane Proteins/metabolism/genetics ; *RNA-Binding Proteins/metabolism/genetics ; Influenza A virus ; HEK293 Cells ; Signal Transduction ; Influenza, Human/genetics/metabolism/virology ; Immunity, Innate ; Animals ; Gene Expression Regulation ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape, we conducted a gain-of-function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including Jade family PHD zinc finger 3 (JADE3) a protein involved in directing the histone acetyltransferase histone acetyltransferase binding to ORC1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Our results suggest a distinct function for JADE3 as expression of the closely related paralogs JADE1 and JADE2 does not confer resistance to influenza A virus infection. JADE3 is required for both constitutive and inducible expression of the well-characterized antiviral gene interferon-induced transmembrane protein 3 (IFITM3). Furthermore, we find JADE3 activates the NF-kB signaling pathway, which is required for the promotion of IFITM3 expression by JADE3. Therefore, we propose JADE3 activates an antiviral genetic program involving NF-kB-dependent IFITM3 expression to restrict influenza A virus infection.},
}

Humans
*NF-kappa B/metabolism/genetics
*Membrane Proteins/metabolism/genetics
*RNA-Binding Proteins/metabolism/genetics
Influenza A virus
HEK293 Cells
Signal Transduction
Influenza, Human/genetics/metabolism/virology
Immunity, Innate
Animals
Gene Expression Regulation
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems

@article {pmid38184185,
year = {2024},
author = {Adashi, EY and Gruppuso, PA and Cohen, IG},
title = {CRISPR Therapy of Sickle Cell Disease: The Dawning of the Gene Editing Era.},
journal = {The American journal of medicine},
volume = {137},
number = {5},
pages = {390-392},
doi = {10.1016/j.amjmed.2023.12.018},
pmid = {38184185},
issn = {1555-7162},
mesh = {*Anemia, Sickle Cell/therapy/genetics ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; },
}

*Anemia, Sickle Cell/therapy/genetics
Humans
*Gene Editing/methods
*Genetic Therapy/methods
*CRISPR-Cas Systems

@article {pmid38656523,
year = {2024},
author = {Satyavathi, VV and Princy, K and Gupta, N and Nizampatnam, NR and Sharma, R and Sreelakshmi, Y},
title = {A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2788},
number = {},
pages = {317-335},
pmid = {38656523},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Vectors/genetics ; Genome, Plant ; Plants, Genetically Modified/genetics ; Transformation, Genetic ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato.},
}

*Solanum lycopersicum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
*Genetic Vectors/genetics
Genome, Plant
Plants, Genetically Modified/genetics
Transformation, Genetic

@article {pmid38656522,
year = {2024},
author = {Freudhofmaier, M and Hoyle, JW and Maghuly, F},
title = {CRISPR/Cas9 Vector Construction for Gene Knockout.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2788},
number = {},
pages = {295-316},
pmid = {38656522},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Plasmids/genetics ; Medicago truncatula/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Cloning, Molecular/methods ; Promoter Regions, Genetic/genetics ; DNA End-Joining Repair/genetics ; Transformation, Genetic ; },
abstract = {This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction of double-stranded breaks (DSBs) in DNA and subsequent imprecise repair via the non-homologous end-joining (NHEJ) pathway. The gRNA expression cassettes are assembled from three components. First, the Medicago truncatula U6.6 (MtU6) promoter (352 bp) and scaffold (83 bp) sequences are amplified from a pUC-based plasmid. Additionally, a third fragment, corresponding to the target sequence, is synthesized as an oligonucleotide. The three gRNA expression fragments are then loosely assembled in a ligation-free cloning reaction and used as a template for an additional PCR step to amplify a single gRNA expression construct, ready for assembly into the transformation vector. The benefits of this design include cost efficiency, as subsequent cloning reactions only require 59 oligonucleotides and standard cloning reagents. Researchers engaged in CRISPR/Cas9-mediated genome editing in plants will find this protocol a clear and resource-efficient approach to create transformation plasmids for their experiments.},
}

*CRISPR-Cas Systems
*Genetic Vectors/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
Plasmids/genetics
Medicago truncatula/genetics
Gene Editing/methods
Plants, Genetically Modified/genetics
Cloning, Molecular/methods
Promoter Regions, Genetic/genetics
DNA End-Joining Repair/genetics
Transformation, Genetic

@article {pmid38656521,
year = {2024},
author = {Freudhofmaier, M and Hoyle, JW and Maghuly, F},
title = {In Silico Design of gRNA for CRISPR/Cas9-Mediated Gene Knockout.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2788},
number = {},
pages = {287-294},
pmid = {38656521},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; Computer Simulation ; DNA End-Joining Repair/genetics ; },
abstract = {CRISPR/Cas9 stands as a revolutionary and versatile gene editing technology. At its core, the Cas9 DNA endonuclease is guided with precision by a specifically designed single-guide RNA (gRNA). This guidance system facilitates the introduction of double-stranded breaks (DSBs) within the DNA. Subsequent imprecise repairs, mainly through the non-homologous end-joining (NHEJ) pathway, yield insertions or deletions, resulting in frameshift mutations. These mutations are instrumental in achieving the successful knockout of the target gene. In this chapter, we describe all necessary steps to create and design a gRNA for a gene knockout to a target gene before to transfer it to a target plant.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
*Gene Editing/methods
Computer Simulation
DNA End-Joining Repair/genetics

@article {pmid38656520,
year = {2024},
author = {Hinrichs, AK and Koch, A and Richter, AM},
title = {Dual-Luciferase Reporter Assay for Prescreening CRISPR (d)Cas9-Mediated Epigenetic Editing on a Plant Promoter Using Human Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2788},
number = {},
pages = {273-285},
pmid = {38656520},
issn = {1940-6029},
mesh = {*Promoter Regions, Genetic ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epigenesis, Genetic ; *Genes, Reporter ; *Luciferases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Epigenetic editing, also known as EpiEdit, offers an exciting way to control gene expression without altering the DNA sequence. In this study, we evaluate the application of EpiEdit to plant promoters, specifically the MLO (mildew locus o) gene promoter. We use a modified CRISPR-(d)Cas9 system, in which the nuclease-deficient Cas9 (dCas9) is fused to an epigenetic modifier, to experimentally demonstrate the utility of this tool for optimizing epigenetic engineering of a plant promoter prior to in vivo plant epigenome editing. Guide RNAs are used to deliver the dCas9-epigenetic modifier fusion protein to the target gene sequence, where it induces modification of MLO gene expression. We perform preliminary experiments using a plant promoter cloned into the luciferase reporter system, which is transfected into a human system and analyzed using the dual-luciferase reporter assay. The results suggest that this approach may be useful in the early stages of plant epigenome editing, as it can aid in the selection of appropriate modifications to the plant promoter prior to conducting in vivo experiments under plant system conditions. Overall, the results demonstrate the potential of CRISPR (d)Cas9-based EpiEdit for precise and controlled regulation of gene expression.},
}

*Promoter Regions, Genetic
Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Epigenesis, Genetic
*Genes, Reporter
*Luciferases/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid38656519,
year = {2024},
author = {Rojas-Vásquez, R and Hernández-Soto, A and Arrieta-Espinoza, G and Gatica-Arias, A},
title = {CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2788},
number = {},
pages = {257-271},
pmid = {38656519},
issn = {1940-6029},
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Agrobacterium tumefaciens/genetics ; Genome, Plant ; Plant Breeding/methods ; Transformation, Genetic ; Plants, Genetically Modified/genetics ; Biolistics/methods ; },
abstract = {Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.},
}

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

@article {pmid38656517,
year = {2024},
author = {Gatica-Arias, A and Pereira, LF},
title = {Agroinfiltration for Enhanced Transgene Expression in Coffee Leaves (Coffea arabica L.).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2788},
number = {},
pages = {227-241},
pmid = {38656517},
issn = {1940-6029},
mesh = {*Coffea/genetics ; *Plant Leaves/genetics/metabolism ; *Plants, Genetically Modified/genetics ; *Transgenes ; *Agrobacterium tumefaciens/genetics ; *Gene Editing/methods ; Transformation, Genetic ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; },
abstract = {The Coffea spp. plant is a significant crop in Latin America, Africa, and Asia, and recent advances in genomics and transcriptomics have opened possibilities for studying candidate genes and introducing new desirable traits through genetic engineering. While stable transformation of coffee plants has been reported using various techniques, it is a time-consuming and laborious process. To overcome this, transient transformation methods have been developed, which avoid the limitations of stable transformation. This chapter describes an ex vitro protocol for transient expression using A. tumefaciens-mediated infiltration of coffee leaves, which could be used to produce coffee plants expressing desirable traits against biotic and abiotic stresses, genes controlling biochemical and physiological traits, as well as for gene editing through CRISPR/Cas9.},
}

*Coffea/genetics
*Plant Leaves/genetics/metabolism
*Plants, Genetically Modified/genetics
*Transgenes
*Agrobacterium tumefaciens/genetics
*Gene Editing/methods
Transformation, Genetic
CRISPR-Cas Systems
Gene Expression Regulation, Plant

@article {pmid38656516,
year = {2024},
author = {Gatica-Arias, A and Villalta-Villalobos, J and Pereira, LF},
title = {Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2788},
number = {},
pages = {209-226},
pmid = {38656516},
issn = {1940-6029},
mesh = {*Coffea/genetics ; *Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Bacterial Proteins/genetics/metabolism ; Bacillus thuringiensis/genetics ; Endotoxins/genetics ; Bacillus thuringiensis Toxins ; Gene Editing/methods ; Hemolysin Proteins/genetics ; Gene Expression Regulation, Plant ; Transformation, Genetic ; Coffee/genetics ; },
abstract = {Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.},
}

*Coffea/genetics
*Agrobacterium tumefaciens/genetics
CRISPR-Cas Systems
Plants, Genetically Modified/genetics
Bacterial Proteins/genetics/metabolism
Bacillus thuringiensis/genetics
Endotoxins/genetics
Bacillus thuringiensis Toxins
Gene Editing/methods
Hemolysin Proteins/genetics
Gene Expression Regulation, Plant
Transformation, Genetic
Coffee/genetics

@article {pmid38656412,
year = {2024},
author = {Li, Y and Jiang, Y and Cao, D and Dang, B and Yang, X and Fan, S and Shen, Y and Li, G and Liu, B},
title = {Creating a zero amylose barley with high soluble sugar content by genome editing.},
journal = {Plant molecular biology},
volume = {114},
number = {3},
pages = {50},
pmid = {38656412},
issn = {1573-5028},
support = {2021-ZJ-958Q//Qinghai Provincial Department of Science and Technology/ ; },
mesh = {*Amylose/metabolism ; *Hordeum/genetics/metabolism ; *Gene Editing/methods ; *Starch Synthase/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Amylopectin/metabolism ; Sucrose/metabolism ; Sugars/metabolism ; Gene Expression Regulation, Plant ; Mutation ; beta-Glucans/metabolism ; Plants, Genetically Modified ; Solubility ; },
abstract = {Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the wild type. Sucrose and β-glucan, were 39.16% and 35.40% higher in waxy mutants than in the wild type, respectively. Transcriptome analysis identified differences between the wild type and waxy mutants that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and β-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.},
}

*Amylose/metabolism
*Hordeum/genetics/metabolism
*Gene Editing/methods
*Starch Synthase/genetics/metabolism
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Amylopectin/metabolism
Sucrose/metabolism
Sugars/metabolism
Gene Expression Regulation, Plant
Mutation
beta-Glucans/metabolism
Plants, Genetically Modified
Solubility

@article {pmid38655256,
year = {2024},
author = {Luan, H and Wang, S and Ju, L and Liu, T and Shi, H and Ge, S and Jiang, S and Wu, J and Peng, J},
title = {KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.},
journal = {Frontiers in immunology},
volume = {15},
number = {},
pages = {1358960},
pmid = {38655256},
issn = {1664-3224},
mesh = {*African Swine Fever Virus/genetics ; Animals ; Swine ; *CRISPR-Cas Systems ; *African Swine Fever/virology/diagnosis ; CRISPR-Associated Proteins/genetics ; Recombinases/genetics/metabolism ; Viral Proteins/genetics ; Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases/genetics ; Sensitivity and Specificity ; *Bacterial Proteins ; },
abstract = {INTRODUCTION: Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains.

METHODS: This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays.

RESULTS: The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/μL, and both assays were completed in 30 min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays.

DISCUSSION: The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.},
}

*African Swine Fever Virus/genetics
Animals
Swine
*CRISPR-Cas Systems
*African Swine Fever/virology/diagnosis
CRISPR-Associated Proteins/genetics
Recombinases/genetics/metabolism
Viral Proteins/genetics
Nucleic Acid Amplification Techniques/methods
Endodeoxyribonucleases/genetics
Sensitivity and Specificity
*Bacterial Proteins

@article {pmid38310456,
year = {2024},
author = {Chanchal, DK and Chaudhary, JS and Kumar, P and Agnihotri, N and Porwal, P},
title = {CRISPR-Based Therapies: Revolutionizing Drug Development and Precision Medicine.},
journal = {Current gene therapy},
volume = {24},
number = {3},
pages = {193-207},
pmid = {38310456},
issn = {1875-5631},
mesh = {Humans ; *Precision Medicine/methods ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Drug Development ; Neoplasms/therapy/genetics/drug therapy ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {With the discovery of CRISPR-Cas9, drug development and precision medicine have undergone a major change. This review article looks at the new ways that CRISPR-based therapies are being used and how they are changing the way medicine is done. CRISPR technology's ability to precisely and flexibly edit genes has opened up new ways to find, validate, and develop drug targets. Also, it has made way for personalized gene therapies, precise gene editing, and advanced screening techniques, all of which hold great promise for treating a wide range of diseases. In this article, we look at the latest research and clinical trials that show how CRISPR could be used to treat genetic diseases, cancer, infectious diseases, and other hard-to-treat conditions. However, ethical issues and problems with regulations are also discussed in relation to CRISPR-based therapies, which shows how important it is to use them safely and responsibly. As CRISPR continues to change how drugs are made and used, this review shines a light on the amazing things that have been done and what the future might hold in this rapidly changing field.},
}

Humans
*Precision Medicine/methods
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Therapy/methods
*Drug Development
Neoplasms/therapy/genetics/drug therapy
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid38655087,
year = {2024},
author = {Mancilla-Rojano, J and Flores, V and Cevallos, MA and Ochoa, SA and Parra-Flores, J and Arellano-Galindo, J and Xicohtencatl-Cortes, J and Cruz-Córdova, A},
title = {A bioinformatic approach to identify confirmed and probable CRISPR-Cas systems in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex genomes.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1335997},
pmid = {38655087},
issn = {1664-302X},
abstract = {INTRODUCTION: The Acinetobacter calcoaceticus-Acinetobacter baumannii complex, or Acb complex, consists of six species: Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii, and Acinetobacter lactucae. A. baumannii is the most clinically significant of these species and is frequently related to healthcare-associated infections (HCAIs). Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and associated genes (cas) constitute bacterial adaptive immune systems and function as variable genetic elements. This study aimed to conduct a genomic analysis of Acb complex genomes available in databases to describe and characterize CRISPR systems and cas genes.

METHODS: Acb complex genomes available in the NCBI and BV-BRC databases, the identification and characterization of CRISPR-Cas systems were performed using CRISPRCasFinder, CRISPRminer, and CRISPRDetect. Sequence types (STs) were determined using the Oxford scheme and ribosomal multilocus sequence typing (rMLST). Prophages were identified using PHASTER and Prophage Hunter.

RESULTS: A total of 293 genomes representing six Acb species exhibited CRISPR-related sequences. These genomes originate from various sources, including clinical specimens, animals, medical devices, and environmental samples. Sequence typing identified 145 ribosomal multilocus sequence types (rSTs). CRISPR-Cas systems were confirmed in 26.3% of the genomes, classified as subtypes I-Fa, I-Fb and I-Fv. Probable CRISPR arrays and cas genes associated with CRISPR-Cas subtypes III-A, I-B, and III-B were also detected. Some of the CRISPR-Cas systems are associated with genomic regions related to Cap4 proteins, and toxin-antitoxin systems. Moreover, prophage sequences were prevalent in 68.9% of the genomes. Analysis revealed a connection between these prophages and CRISPR-Cas systems, indicating an ongoing arms race between the bacteria and their bacteriophages. Furthermore, proteins associated with anti-CRISPR systems, such as AcrF11 and AcrF7, were identified in the A. baumannii and A. pittii genomes.

DISCUSSION: This study elucidates CRISPR-Cas systems and defense mechanisms within the Acb complex, highlighting their diverse distribution and interactions with prophages and other genetic elements. This study also provides valuable insights into the evolution and adaptation of these microorganisms in various environments and clinical settings.},
}

@article {pmid38654173,
year = {2024},
author = {Xiang, DL and Li, GS},
title = {Control of leaf development in the water fern Ceratopteris richardii by the auxin efflux transporter CrPINMa in the CRISPR/Cas9 analysis.},
journal = {BMC plant biology},
volume = {24},
number = {1},
pages = {322},
pmid = {38654173},
issn = {1471-2229},
support = {31260056//the National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Plant Leaves/genetics/growth & development/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Editing ; Pteridaceae/genetics/metabolism/growth & development ; Indoleacetic Acids/metabolism ; Membrane Transport Proteins/genetics/metabolism ; Phylogeny ; Gene Expression Regulation, Plant ; Ferns/genetics/growth & development/metabolism ; },
abstract = {BACKGROUND: PIN-FORMED genes (PINs) are crucial in plant development as they determine the directionality of auxin flow. They are present in almost all land plants and even in green algae. However, their role in fern development has not yet been determined. This study aims to investigate the function of CrPINMa in the quasi-model water fern Ceratopteris richardii.

RESULTS: CrPINMa possessed a long central hydrophilic loop and characteristic motifs within it, which indicated that it belonged to the canonical rather than the non-canonical PINs. CrPINMa was positioned in the lineage leading to Arabidopsis PIN6 but not that to its PIN1, and it had undergone numerous gene duplications. CRISPR/Cas9 genome editing had been performed in ferns for the first time, producing diverse mutations including local frameshifts for CrPINMa. Plants possessing disrupted CrPINMa exhibited retarded leaf emergence and reduced leaf size though they could survive and reproduce at the same time. CrPINMa transcripts were distributed in the shoot apical meristem, leaf primordia and their vasculature. Finally, CrPINMa proteins were localized to the plasma membrane rather than other cell parts.

CONCLUSIONS: CRISPR/Cas9 genome editing is feasible in ferns, and that PINs can play a role in fern leaf development.},
}

*CRISPR-Cas Systems
*Plant Leaves/genetics/growth & development/metabolism
*Plant Proteins/genetics/metabolism
Gene Editing
Pteridaceae/genetics/metabolism/growth & development
Indoleacetic Acids/metabolism
Membrane Transport Proteins/genetics/metabolism
Phylogeny
Gene Expression Regulation, Plant
Ferns/genetics/growth & development/metabolism

@article {pmid38653778,
year = {2024},
author = {Banerjee, D and Bagchi, S and Liu, Z and Chou, HC and Xu, M and Sun, M and Aloisi, S and Vaksman, Z and Diskin, SJ and Zimmerman, M and Khan, J and Gryder, B and Thiele, CJ},
title = {Lineage specific transcription factor waves reprogram neuroblastoma from self-renewal to differentiation.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3432},
pmid = {38653778},
issn = {2041-1723},
mesh = {*Neuroblastoma/metabolism/genetics/pathology ; *Tretinoin/pharmacology/metabolism ; *Cell Differentiation/drug effects/genetics ; *SOXC Transcription Factors/metabolism/genetics ; Humans ; Animals ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Mice ; Transcription Factors/metabolism/genetics ; Cell Self Renewal/drug effects/genetics ; GATA3 Transcription Factor/metabolism/genetics ; Cell Lineage/genetics ; GATA2 Transcription Factor/metabolism/genetics ; CRISPR-Cas Systems ; N-Myc Proto-Oncogene Protein/metabolism/genetics ; Cell Proliferation/drug effects/genetics ; },
abstract = {Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program.},
}

*Neuroblastoma/metabolism/genetics/pathology
*Tretinoin/pharmacology/metabolism
*Cell Differentiation/drug effects/genetics
*SOXC Transcription Factors/metabolism/genetics
Humans
Animals
Cell Line, Tumor
*Gene Expression Regulation, Neoplastic
Mice
Transcription Factors/metabolism/genetics
Cell Self Renewal/drug effects/genetics
GATA3 Transcription Factor/metabolism/genetics
Cell Lineage/genetics
GATA2 Transcription Factor/metabolism/genetics
CRISPR-Cas Systems
N-Myc Proto-Oncogene Protein/metabolism/genetics
Cell Proliferation/drug effects/genetics

@article {pmid38652440,
year = {2024},
author = {Yan, N and Hu, Z and Zhang, L},
title = {CRISPR-Cas13a-Triggered DNAzyme Signal Amplification-Based Colorimetric miRNA Detection Method and Its Application in Evaluating the Anxiety.},
journal = {Applied biochemistry and biotechnology},
volume = {},
number = {},
pages = {},
pmid = {38652440},
issn = {1559-0291},
support = {2023014010Z//he Hengshui City science and technology plan project/ ; },
abstract = {The development of a bio-sensing strategy based on CRISPR/Cas that is exceptionally sensitive is crucial for the identification of trace molecules. Colorimetric miRNA detection utilizing CRISPR/Cas13a-triggered DNAzyme signal amplification was described in this article. The developed strategy was implemented for miRNA-21 detection as a proof of concept. The cleavage activity of Cas13a was triggered when the target molecule bonded to the Cas13a-crRNA complex and cleaved uracil ribonucleotides (rU) in the substrate probe. As a consequence, the S chain was liberated from the T chain that had been modified on magnetic beads (MB). The G-rich sections were then exposed when the catalytic hairpin assembly between the H1 and H2 probes was activated by the released T@MB. G-rich section can fold into G-quadruplex. By catalyzing the formation of green ABTS[3]- via HRP-mimicking G-quadruplex/hemin complexes, colorimetric measurements of miRNA can be achieved visually through DNAzyme-mediated signal amplification. The method demonstrated a low limit of detection of 27 fM and a high selectivity towards target miRNA eventually. As a result, the developed strategy provides a clinical application platform for the detection of miRNAs that is both ultrasensitive and extremely specific.},
}

@article {pmid38621146,
year = {2024},
author = {Yang, S and Zhou, L and Fang, Z and Wang, Y and Zhou, G and Jin, X and Cao, Y and Zhao, J},
title = {Proximity-Guaranteed DNA Machine for Accurate Identification of Breast Cancer Extracellular Vesicles.},
journal = {ACS sensors},
volume = {9},
number = {4},
pages = {2194-2202},
doi = {10.1021/acssensors.4c00491},
pmid = {38621146},
issn = {2379-3694},
mesh = {*Extracellular Vesicles/chemistry ; Humans ; *Breast Neoplasms/genetics ; Female ; *DNA/chemistry/genetics ; Cell Line, Tumor ; Biomarkers, Tumor ; CRISPR-Cas Systems/genetics ; },
abstract = {Breast cancer is one of the most diagnosed cancers worldwide. Precise diagnosis and subtyping have important significance for targeted therapy and prognosis prediction of breast cancer. Herein, we design a proximity-guaranteed DNA machine for accurate identification of breast cancer extracellular vesicles (EVs), which is beneficial to explore the subtype features of breast cancer. In our design, two proximity probes are located close on the same EV through specific recognition of coexisting surface biomarkers, thus being ligated with the help of click chemistry. Then, the ligated product initiates the operation of a DNA machine involving catalytic hairpin assembly and clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas12a-mediated trans-cleavage, which finally generates a significant response that enables the identification of EVs expressing both biomarkers. Principle-of-proof studies are performed using EVs derived from the breast cancer cell line BT474 as the models, confirming the high sensitivity and specificity of the DNA machine. When further applied to clinical samples, the DNA machine is shown to be capable of not only distinguishing breast cancer patients with special subtypes but also realizing the tumor staging regarding the disease progression. Therefore, our work may provide new insights into the subtype-based diagnosis of breast cancer as well as identification of more potential therapeutic targets in the future.},
}

*Extracellular Vesicles/chemistry
Humans
*Breast Neoplasms/genetics
Female
*DNA/chemistry/genetics
Cell Line, Tumor
Biomarkers, Tumor
CRISPR-Cas Systems/genetics

@article {pmid38573977,
year = {2024},
author = {Ding, L and Cao, S and Qu, C and Wu, Y and Yu, S},
title = {Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation.},
journal = {ACS sensors},
volume = {9},
number = {4},
pages = {1877-1885},
doi = {10.1021/acssensors.3c02571},
pmid = {38573977},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA Methylation ; Humans ; DNA Restriction Enzymes/metabolism/chemistry ; Fluorescence Resonance Energy Transfer/methods ; CRISPR-Associated Proteins/chemistry/metabolism ; Biosensing Techniques/methods ; },
abstract = {The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.},
}

*CRISPR-Cas Systems/genetics
*DNA Methylation
Humans
DNA Restriction Enzymes/metabolism/chemistry
Fluorescence Resonance Energy Transfer/methods
CRISPR-Associated Proteins/chemistry/metabolism
Biosensing Techniques/methods

@article {pmid38420933,
year = {2024},
author = {Del Prado, JA and Ding, Y and Sonneville, J and der Kolk, KV and Moreno-Mateos, MA and Málaga-Trillo, E and Spaink, HP},
title = {Comparing robotic and manual injection methods in zebrafish embryos for high-throughput RNA silencing using CRISPR-RfxCas13d.},
journal = {BioTechniques},
volume = {76},
number = {5},
pages = {183-191},
doi = {10.2144/btn-2023-0062},
pmid = {38420933},
issn = {1940-9818},
mesh = {Animals ; *Zebrafish/genetics/embryology ; *CRISPR-Cas Systems/genetics ; *Microinjections/methods ; *Robotics/methods ; RNA Interference ; Embryo, Nonmammalian ; Gene Knockdown Techniques/methods ; Zebrafish Proteins/genetics ; RNA, Messenger/genetics ; },
abstract = {In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail (TBXTA) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for TBXTA. Cas9 protein exhibited a higher percentage of severe phenotypes compared with mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1.0 s per embryo as a high-throughput alternative to manual methods.},
}

Animals
*Zebrafish/genetics/embryology
*CRISPR-Cas Systems/genetics
*Microinjections/methods
*Robotics/methods
RNA Interference
Embryo, Nonmammalian
Gene Knockdown Techniques/methods
Zebrafish Proteins/genetics
RNA, Messenger/genetics

@article {pmid38261130,
year = {2024},
author = {Moustafa, K},
title = {CRISPR: Beyond the Excitement.},
journal = {Journal of bioethical inquiry},
volume = {21},
number = {1},
pages = {7-9},
pmid = {38261130},
issn = {1872-4353},
mesh = {Humans ; *Gene Editing/ethics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
}

Humans
*Gene Editing/ethics
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid38652405,
year = {2024},
author = {Zhao, S and Yin, R and Zhang, M and Zhai, Z and Shen, Z and Mou, Y and Xu, D and Zhou, L and Lai, D},
title = {Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.},
journal = {World journal of microbiology & biotechnology},
volume = {40},
number = {6},
pages = {176},
pmid = {38652405},
issn = {1573-0972},
support = {2023YFD1401400//National Key Research and Development Program of China/ ; 2023YFD1401400//National Key Research and Development Program of China/ ; 31872623//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Ascomycota/genetics/metabolism ; Endophytes/genetics/metabolism ; Melanins/biosynthesis/metabolism ; Fungal Proteins/genetics/metabolism ; Protoplasts ; },
abstract = {The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Ascomycota/genetics/metabolism
Endophytes/genetics/metabolism
Melanins/biosynthesis/metabolism
Fungal Proteins/genetics/metabolism
Protoplasts

@article {pmid38652321,
year = {2024},
author = {Wu, H and Sun, Y and Wang, Y and Luo, L and Song, Y},
title = {Advances in miniature CRISPR-Cas proteins and their applications in gene editing.},
journal = {Archives of microbiology},
volume = {206},
number = {5},
pages = {231},
pmid = {38652321},
issn = {1432-072X},
support = {32170173//National Natural Science Foundation of China/ ; ZXL2021422//Gusu Leading Talent Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The CRISPR-Cas system consists of Cas proteins and single-stranded RNAs that recruit Cas proteins and specifically target the nucleic acid. Some Cas proteins can accurately cleave the target nucleic acid under the guidance of the single-stranded RNAs. Due to its exceptionally high specificity, the CRISPR-Cas system is now widely used in various fields such as gene editing, transcription regulation, and molecular diagnosis. However, the huge size of the most frequently utilized Cas proteins (Cas9, Cas12a, and Cas13, which contain 950-1,400 amino acids) can limit their applicability, especially in eukaryotic gene editing, where larger Cas proteins are difficult to deliver into the target cells. Recently discovered miniature CRISPR-Cas proteins, consisting of only 400 to 800 amino acids, offer the possibility of overcoming this limitation. This article systematically reviews the latest research progress of several miniature CRISPR-Cas proteins (Cas12f, Cas12j, Cas12k, and Cas12m) and their practical applications in the field of gene editing.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid38651269,
year = {2024},
author = {Antony, JS and Herranz, AM and Mohammadian Gol, T and Mailand, S and Monnier, P and Rottenberger, J and Roig-Merino, A and Keller, B and Gowin, C and Milla, M and Beyer, TA and Mezger, M},
title = {Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.},
journal = {Biotechnology journal},
volume = {19},
number = {4},
pages = {e2300505},
doi = {10.1002/biot.202300505},
pmid = {38651269},
issn = {1860-7314},
support = {2485-0-0//Fortüne Tübingen/ ; 2412-0-0//Fortüne Tübingen/ ; 440-0-0//UKT-Clinician Scientist Program/ ; //University Children's Hospital of Tübingen/ ; },
mesh = {CHO Cells ; *Cricetulus ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Antibodies, Monoclonal/genetics ; Recombinant Proteins/genetics ; Gene Knockout Techniques/methods ; bcl-2-Associated X Protein/genetics/metabolism ; Tetrahydrofolate Dehydrogenase/genetics/metabolism ; Fucosyltransferases/genetics/metabolism ; Cricetinae ; Genetic Engineering/methods ; },
abstract = {Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.},
}

CHO Cells
*Cricetulus
Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Antibodies, Monoclonal/genetics
Recombinant Proteins/genetics
Gene Knockout Techniques/methods
bcl-2-Associated X Protein/genetics/metabolism
Tetrahydrofolate Dehydrogenase/genetics/metabolism
Fucosyltransferases/genetics/metabolism
Cricetinae
Genetic Engineering/methods

@article {pmid38650045,
year = {2024},
author = {Fan, XW and Gao, ZF and Ling, DD and Wang, DH and Cui, Y and Du, HQ and Li, CL and Zhou, X},
title = {CRISPR/Cas9 nickase mediated signal amplification integrating with the trans-cleavage activity of Cas12a for highly selective and sensitive detection of single base mutations.},
journal = {Military Medical Research},
volume = {11},
number = {1},
pages = {25},
pmid = {38650045},
issn = {2054-9369},
mesh = {*CRISPR-Cas Systems ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Humans ; Mutation ; Deoxyribonuclease I ; Bacterial Proteins/genetics ; },
}

*CRISPR-Cas Systems
CRISPR-Associated Proteins/genetics
Endodeoxyribonucleases/genetics
Humans
Mutation
Deoxyribonuclease I
Bacterial Proteins/genetics

@article {pmid38648198,
year = {2024},
author = {Alseth, EO and Custodio, R and Sundius, SA and Kuske, RA and Brown, SP and Westra, ER},
title = {The impact of phage and phage resistance on microbial community dynamics.},
journal = {PLoS biology},
volume = {22},
number = {4},
pages = {e3002346},
pmid = {38648198},
issn = {1545-7885},
abstract = {Where there are bacteria, there will be bacteriophages. These viruses are known to be important players in shaping the wider microbial community in which they are embedded, with potential implications for human health. On the other hand, bacteria possess a range of distinct immune mechanisms that provide protection against bacteriophages, including the mutation or complete loss of the phage receptor, and CRISPR-Cas adaptive immunity. While our previous work showed how a microbial community may impact phage resistance evolution, little is known about the inverse, namely how interactions between phages and these different phage resistance mechanisms affect the wider microbial community in which they are embedded. Here, we conducted a 10-day, fully factorial evolution experiment to examine how phage impact the structure and dynamics of an artificial four-species bacterial community that includes either Pseudomonas aeruginosa wild-type or an isogenic mutant unable to evolve phage resistance through CRISPR-Cas. Additionally, we used mathematical modelling to explore the ecological interactions underlying full community behaviour, as well as to identify general principles governing the impacts of phage on community dynamics. Our results show that the microbial community structure is drastically altered by the addition of phage, with Acinetobacter baumannii becoming the dominant species and P. aeruginosa being driven nearly extinct, whereas P. aeruginosa outcompetes the other species in the absence of phage. Moreover, we find that a P. aeruginosa strain with the ability to evolve CRISPR-based resistance generally does better when in the presence of A. baumannii, but that this benefit is largely lost over time as phage is driven extinct. Finally, we show that pairwise data alone is insufficient when modelling our microbial community, both with and without phage, highlighting the importance of higher order interactions in governing multispecies dynamics in complex communities. Combined, our data clearly illustrate how phage targeting a dominant species allows for the competitive release of the strongest competitor while also contributing to community diversity maintenance and potentially preventing the reinvasion of the target species, and underline the importance of mapping community composition before therapeutically applying phage.},
}

@article {pmid38648173,
year = {2024},
author = {Přibylová, A and Fischer, L},
title = {How to use CRISPR/Cas9 in plants - from target site selection to DNA repair.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/erae147},
pmid = {38648173},
issn = {1460-2431},
abstract = {A tool for precise, target-specific, efficient and affordable genome editing, it is a dream for many researchers, from those who do basic research to those who use it for applied research. Since 2012, we have the tool that almost fulfils such requirements; it is based on CRISPR/Cas systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight the key factors affecting its mutagenesis outcomes. Firstly, factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state or Cas9 variant, and how long it remains in place after cleavage. Secondly, factors affecting the follow-up DNA repair mechanisms include mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.},
}

@article {pmid38647391,
year = {2024},
author = {Cho, HY and Yoo, M and Pongkulapa, T and Rabie, H and Muotri, AR and Yin, PT and Choi, JW and Lee, KB},
title = {Magnetic Nanoparticle-Assisted Non-Viral CRISPR-Cas9 for Enhanced Genome Editing to Treat Rett Syndrome.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2306432},
doi = {10.1002/advs.202306432},
pmid = {38647391},
issn = {2198-3844},
support = {AARG-NTF-21-847862/ALZ/Alzheimer's Association/United States ; },
abstract = {The CRISPR-Cas9 technology has the potential to revolutionize the treatment of various diseases, including Rett syndrome, by enabling the correction of genes or mutations in human patient cells. However, several challenges need to be addressed before its widespread clinical application. These challenges include the low delivery efficiencies to target cells, the actual efficiency of the genome-editing process, and the precision with which the CRISPR-Cas system operates. Herein, the study presents a Magnetic Nanoparticle-Assisted Genome Editing (MAGE) platform, which significantly improves the transfection efficiency, biocompatibility, and genome-editing accuracy of CRISPR-Cas9 technology. To demonstrate the feasibility of the developed technology, MAGE is applied to correct the mutated MeCP2 gene in induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) from a Rett syndrome patient. By combining magnetofection and magnetic-activated cell sorting, MAGE achieves higher multi-plasmid delivery (99.3%) and repairing efficiencies (42.95%) with significantly shorter incubation times than conventional transfection agents without size limitations on plasmids. The repaired iPSC-NPCs showed similar characteristics as wild-type neurons when they differentiated into neurons, further validating MAGE and its potential for future clinical applications. In short, the developed nanobio-combined CRISPR-Cas9 technology offers the potential for various clinical applications, particularly in stem cell therapies targeting different genetic diseases.},
}

@article {pmid38647075,
year = {2024},
author = {Tan, Q and Shi, Y and Duan, C and Li, Q and Gong, T and Li, S and Duan, X and Xie, H and Li, Y and Chen, L},
title = {Simple, sensitive, and visual detection of 12 respiratory pathogens with one-pot-RPA-CRISPR/Cas12a assay.},
journal = {Journal of medical virology},
volume = {96},
number = {4},
pages = {e29624},
doi = {10.1002/jmv.29624},
pmid = {38647075},
issn = {1096-9071},
support = {22YXYJ0120//Medical Research Project of Xi'an Science and Technology Bureau/ ; 2023WGZJ-YB-39//Foreign Expert Service Project of Science and Technology Department of Shaanxi Province/ ; QCYRCXM-2022-56//Qin Chuangyuan Recruited High-level Innovation and Entrepreneurship Talents Project of Science and Technology Department of Shaanxi Province/ ; KY201904011//The Science and Technology Partnership Program, Ministry of Science and Technology of China/ ; 2021056//Sichuan Science and Technology Program/ ; 2021ZYD0085//Central Government Directed Special Funds for Local Science and Technology Development Project/ ; 2021YFH0100//The Sichuan Science and Technology Program/ ; CAMS-2021-I2M-1-060//The CAMS Initiative for Innovative Medicine/ ; },
abstract = {Respiratory infections pose a serious threat to global public health, underscoring the urgent need for rapid, accurate, and large-scale diagnostic tools. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, combined with isothermal amplification methods, has seen widespread application in nucleic acid testing (NAT). However, achieving a single-tube reaction system containing all necessary components is challenging due to the competitive effects between recombinase polymerase amplification (RPA) and CRISPR/Cas reagents. Furthermore, to enable precision medicine, distinguishing between bacterial and viral infections is essential. Here, we have developed a novel NAT method, termed one-pot-RPA-CRISPR/Cas12a, which combines RPA with CRISPR molecular diagnostic technology, enabling simultaneous detection of 12 common respiratory pathogens, including six bacteria and six viruses. RPA and CRISPR/Cas12a reactions are separated by paraffin, providing an independent platform for RPA reactions to generate sufficient target products before being mixed with the CRISPR/Cas12a system. Results can be visually observed under LED blue light. The sensitivity of the one-pot-RPA-CRISPR/Cas12a method is 2.5 × 10[0] copies/μL plasmids, with no cross-reaction with other bacteria or viruses. Additionally, the clinical utility was evaluated by testing clinical isolates of bacteria and virus throat swab samples, demonstrating favorable performance. Thus, our one-pot-RPA-CRISPR/Cas12a method shows immense potential for accurate and large-scale detection of 12 common respiratory pathogens in point-of-care testing.},
}

@article {pmid38591968,
year = {2024},
author = {Xu, J and Yang, H and Sui, Z and Yuan, X and Jia, L and Guo, L},
title = {One-pot isothermal amplification permits recycled activation of CRISPR/Cas12a for sensing terminal deoxynucleotidyl transferase activity.},
journal = {Chemical communications (Cambridge, England)},
volume = {60},
number = {35},
pages = {4683-4686},
doi = {10.1039/d4cc00825a},
pmid = {38591968},
issn = {1364-548X},
mesh = {*Nucleic Acid Amplification Techniques ; *DNA Nucleotidylexotransferase/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {This study introduces a one-pot isothermal amplification assay for ultrasensitive analysis of terminal deoxynucleotidyl transferase (TdT) activity. The system realizes recycled activation of CRISPR/Cas12a, enabling exceptional signal amplification. This approach maximizes the simplicity of the detection method, offering a promising avenue for molecular disease diagnosis.},
}

*Nucleic Acid Amplification Techniques
*DNA Nucleotidylexotransferase/metabolism
*CRISPR-Cas Systems/genetics
Humans

@article {pmid38527850,
year = {2024},
author = {Ai, N and Han, CR and Zhao, H and Cheng, SY and Ge, W},
title = {Disruption of Thyroid Hormone Receptor Thrab Leads to Female Infertility in Zebrafish.},
journal = {Endocrinology},
volume = {165},
number = {5},
pages = {},
doi = {10.1210/endocr/bqae037},
pmid = {38527850},
issn = {1945-7170},
support = {//University of Macau/ ; //UMDF/ ; //FDCT/ ; //NSFC/ ; //FDCT Funding Scheme for Postdoctoral Researchers/ ; },
mesh = {Animals ; Female ; *Zebrafish/genetics ; *Infertility, Female/genetics ; *Ovary/metabolism ; Thyroid Hormone Receptors alpha/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; Reproduction/genetics ; },
abstract = {Thyroid hormones (THs) T4 and T3 are vital for development, growth, and metabolism. Thyroid dysfunction can also cause problems in fertility, suggesting involvement of THs in reproduction. In zebrafish, there exist 2 forms of TH receptor alpha gene (thraa and thrab). Disruption of these genes by CRISPR/Cas9 showed no reproductive irregularities in the thraa mutant; however, inactivation of the thrab gene resulted in female infertility. Although young female mutants (thrabm/m) showed normal ovarian development and folliculogenesis before sexual maturation, they failed to release eggs during oviposition after sexual maturation. This spawning failure was due to oviductal blockage at the genital papilla. The obstruction of the oviduct subsequently caused an accumulation of the eggs in the ovary, resulting in severe ovarian hypertrophy, abdominal distention, and disruption of folliculogenesis. Gene expression analysis showed expression of both TH receptors and estrogen receptors in the genital papilla, suggesting a direct TH action and potential interactions between thyroid and estrogen signaling pathways in controlling genital papilla development and function. In addition to their actions in the reproductive tracts, THs may also have direct effects in the ovary, as suggested by follicle atresia and cessation of folliculogenesis in the heterozygous mutant (thrab+/m), which was normal in all aspects of female reproduction in young and sexually mature fish but exhibited premature ovarian failure in aged females. In summary, this study provides substantial evidence for roles of THs in controlling the development and functions of both reproductive tract and ovary.},
}

Animals
Female
*Zebrafish/genetics
*Infertility, Female/genetics
*Ovary/metabolism
Thyroid Hormone Receptors alpha/genetics/metabolism
Zebrafish Proteins/genetics/metabolism
Mutation
CRISPR-Cas Systems
Reproduction/genetics

@article {pmid38647045,
year = {2024},
author = {Olivi, L and Bagchus, C and Pool, V and Bekkering, E and Speckner, K and Offerhaus, H and Wu, WY and Depken, M and Martens, KJA and Staals, RHJ and Hohlbein, J},
title = {Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae283},
pmid = {38647045},
issn = {1362-4962},
support = {024.003.019//The Netherlands Organization of Scientific Research/ ; VI.Vidi.203.074//VIDI/ ; //NWO/ ; },
abstract = {CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.},
}

@article {pmid38646626,
year = {2024},
author = {Wang, Y and Pang, F},
title = {Diagnosis of bovine viral diarrhea virus: an overview of currently available methods.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1370050},
pmid = {38646626},
issn = {1664-302X},
abstract = {Bovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea (BVD), which results in significant economic losses in the global cattle industry. Fortunately, various diagnostic methods available for BVDV have been established. They include etiological methods, such as virus isolation (VI); serological methods, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA), and immunohistochemistry (IHC); molecular methods, such as reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, digital droplet PCR (ddPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR-Cas system; and biosensors. This review summarizes the current diagnostic methods for BVDV, discussing their advantages and disadvantages, and proposes future perspectives for the diagnosis of BVDV, with the intention of providing valuable guidance for effective diagnosis and control of BVD disease.},
}

@article {pmid38644403,
year = {2024},
author = {Zhang, C and Xu, J and Wu, Y and Xu, C and Xu, P},
title = {Base Editors-Mediated Gene Therapy in Hematopoietic Stem Cells for Hematologic Diseases.},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {38644403},
issn = {2629-3277},
support = {82170119//the National Natural Science Foundation of China/ ; 202310285172Y//College Student Innovation and Entrepreneurship Training Program of Jiangsu Province/ ; 2022YFC2502700，2022YFC2502701，2022YFC2502702//the National Key Research and Development Program of China/ ; },
abstract = {Base editors, developed from the CRISPR/Cas system, consist of components such as deaminase and Cas variants. Since their emergence in 2016, the precision, efficiency, and safety of base editors have been gradually optimized. The feasibility of using base editors in gene therapy has been demonstrated in several disease models. Compared with the CRISPR/Cas system, base editors have shown great potential in hematopoietic stem cells (HSCs) and HSC-based gene therapy, because they do not generate double-stranded breaks (DSBs) while achieving the precise realization of single-base substitutions. This precise editing mechanism allows for the permanent correction of genetic defects directly at their source within HSCs, thus promising a lasting therapeutic effect. Recent advances in base editors are expected to significantly increase the number of clinical trials for HSC-based gene therapies. In this review, we summarize the development and recent progress of DNA base editors, discuss their applications in HSC gene therapy, and highlight the prospects and challenges of future clinical stem cell therapies.},
}

@article {pmid38643972,
year = {2024},
author = {Kogay, R and Wolf, YI and Koonin, EV},
title = {Defence systems and horizontal gene transfer in bacteria.},
journal = {Environmental microbiology},
volume = {26},
number = {4},
pages = {e16630},
pmid = {38643972},
issn = {1462-2920},
support = {Z01 LM000073/ImNIH/Intramural NIH HHS/United States ; },
mesh = {*Gene Transfer, Horizontal ; *Bacteria/genetics/virology ; *CRISPR-Cas Systems ; Genome, Bacterial ; Interspersed Repetitive Sequences ; Bacteriophages/genetics ; Evolution, Molecular ; Prophages/genetics ; },
abstract = {Horizontal gene transfer (HGT) is a fundamental process in prokaryotic evolution, contributing significantly to diversification and adaptation. HGT is typically facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages, which often impose fitness costs on their hosts. However, a considerable number of bacterial genes are involved in defence mechanisms that limit the propagation of MGEs, suggesting they may actively restrict HGT. In our study, we investigated whether defence systems limit HGT by examining the relationship between the HGT rate and the presence of 73 defence systems across 12 bacterial species. We discovered that only six defence systems, three of which were different CRISPR-Cas subtypes, were associated with a reduced gene gain rate at the species evolution scale. Hosts of these defence systems tend to have a smaller pangenome size and fewer phage-related genes compared to genomes without these systems. This suggests that these defence mechanisms inhibit HGT by limiting prophage integration. We hypothesize that the restriction of HGT by defence systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and the fitness effect of HGT in bacterial populations.},
}

*Gene Transfer, Horizontal
*Bacteria/genetics/virology
*CRISPR-Cas Systems
Genome, Bacterial
Interspersed Repetitive Sequences
Bacteriophages/genetics
Evolution, Molecular
Prophages/genetics

@article {pmid38641067,
year = {2024},
author = {Ganguly, C and Rostami, S and Long, K and Aribam, SD and Rajan, R},
title = {Unity among the diverse RNA-guided CRISPR-Cas interference mechanisms.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {107295},
doi = {10.1016/j.jbc.2024.107295},
pmid = {38641067},
issn = {1083-351X},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are adaptive immune systems that protect bacteria and archaea from invading mobile genetic elements (MGEs). The Cas protein-CRISPR RNA (crRNA) complex uses complementarity of the crRNA "guide" region to specifically recognize the invader genome. CRISPR effectors that perform targeted destruction of the foreign genome have emerged independently as multi-subunit protein complexes (Class 1 systems) and as single multi-domain proteins (Class 2). These different CRISPR-Cas systems can cleave RNA, DNA, and protein in an RNA-guided manner to eliminate the invader, and in some cases, they initiate programmed cell death/dormancy. The versatile mechanisms of the different CRISPR-Cas systems to target and destroy nucleic acids have been adapted to develop various programmable-RNA-guided tools and have revolutionized the development of fast, accurate, and accessible genomic applications. In this review, we present the structure and interference mechanisms of different CRISPR-Cas systems and an analysis of their unified features. The three types of Class 1 systems (I, III, and IV) have a conserved right-handed helical filamentous structure that provides a backbone for sequence-specific targeting while using unique proteins with distinct mechanisms to destroy the invader. Similarly, all three Class 2 types (II, V, and VI) have a bilobed architecture that binds the RNA-DNA/RNA hybrid and uses different nuclease domains to cleave invading MGEs. Additionally, we highlight the mechanistic similarities of CRISPR-Cas enzymes with other RNA cleaving enzymes and briefly present the evolutionary routes of the different CRISPR-Cas systems.},
}

@article {pmid38640680,
year = {2024},
author = {Qiu, M and Yuan, Z and Li, N and Yang, X and Zhang, X and Jiang, Y and Zhao, Q and Man, C},
title = {Self-assembled bifunctional nanoflower-enabled CRISPR/Cas biosensing platform for dual-readout detection of Salmonella enterica.},
journal = {Journal of hazardous materials},
volume = {471},
number = {},
pages = {134323},
doi = {10.1016/j.jhazmat.2024.134323},
pmid = {38640680},
issn = {1873-3336},
abstract = {Sensitive detection and point-of-care test of bacterial pathogens is of great significance in safeguarding the public health worldwide. Inspired by the characteristics of horseradish peroxidase (HRP), we synthesized a hybrid nanoflower with peroxidase-like activity via a three-component self-assembled strategy. Interestingly, the prepared nanozyme not only could act as an alternative to HRP for colorimetric biosensing, but also function as a unique signal probe that could be recognized by a pregnancy test strip. By combining the bifunctional properties of hybrid nanoflower, isothermal amplification of LAMP, and the specific recognition and non-specific cleavage properties of CRISPR/Cas12a system, the dual-readout CRISPR/Cas12a biosensor was developed for sensitive and rapid detection of Salmonella enterica. Moreover, this platform in the detection of Salmonella enterica had limits of detection of 1 cfu/mL (colorimetric assay) in the linear range of 10[1]-10[8] cfu/mL and 10[2] cfu/mL (lateral flow assay) in the linear range of 10[2]-10[8] cfu/mL, respectively. Furthermore, the developed biosensor exhibited good recoveries in the spiked samples (lake water and milk) with varying concentrations of Salmonella enterica. This work provides new insights for the design of multifunctional nanozyme and the development of innovative dual-readout CRISPR/Cas system-based biosensing platform for the detection of pathogens.},
}

@article {pmid38639864,
year = {2024},
author = {Rahimi, A and Sameei, P and Mousavi, S and Ghaderi, K and Hassani, A and Hassani, S and Alipour, S},
title = {Application of CRISPR/Cas9 System in the Treatment of Alzheimer's Disease and Neurodegenerative Diseases.},
journal = {Molecular neurobiology},
volume = {},
number = {},
pages = {},
pmid = {38639864},
issn = {1559-1182},
abstract = {Alzheimer's, Parkinson's, and Huntington's are some of the most common neurological disorders, which affect millions of people worldwide. Although there have been many treatments for these diseases, there are still no effective treatments to treat or completely stop these disorders. Perhaps the lack of proper treatment for these diseases can be related to various reasons, but the poor results related to recent clinical research also prompted doctors to look for new treatment approaches. In this regard, various researchers from all over the world have provided many new treatments, one of which is CRISPR/Cas9. Today, the CRISPR/Cas9 system is mostly used for genetic modifications in various species. In addition, by using the abilities available in the CRISPR/Cas9 system, researchers can either remove or modify DNA sequences, which in this way can establish a suitable and useful treatment method for the treatment of genetic diseases that have undergone mutations. We conducted a non-systematic review of articles and study results from various databases, including PubMed, Medline, Web of Science, and Scopus, in recent years. and have investigated new treatment methods in neurodegenerative diseases with a focus on Alzheimer's disease. Then, in the following sections, the treatment methods were classified into three groups: anti-tau, anti-amyloid, and anti-APOE regimens. Finally, we discussed various applications of the CRISPR/Cas-9 system in Alzheimer's disease. Today, using CRISPR/Cas-9 technology, scientists create Alzheimer's disease models that have a more realistic phenotype and reveal the processes of pathogenesis; following the screening of defective genes, they establish treatments for this disease.},
}

@article {pmid38637512,
year = {2024},
author = {Schwartz, EA and Bravo, JPK and Ahsan, M and Macias, LA and McCafferty, CL and Dangerfield, TL and Walker, JN and Brodbelt, JS and Palermo, G and Fineran, PC and Fagerlund, RD and Taylor, DW},
title = {RNA targeting and cleavage by the type III-Dv CRISPR effector complex.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3324},
pmid = {38637512},
issn = {2041-1723},
support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; R35 GM138348/GM/NIGMS NIH HHS/United States ; R35 GM139658/GM/NIGMS NIH HHS/United States ; F31 CA257404/CA/NCI NIH HHS/United States ; },
mesh = {RNA/metabolism ; *RNA, Catalytic/metabolism ; CRISPR-Cas Systems/genetics ; DNA/metabolism ; Catalytic Domain ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA Cleavage ; },
abstract = {CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction[1-5]. Target RNA cleavage at regular intervals is characteristic of type III effector complexes[6-8]. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors[9,10], in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.},
}

RNA/metabolism
*RNA, Catalytic/metabolism
CRISPR-Cas Systems/genetics
DNA/metabolism
Catalytic Domain
*CRISPR-Associated Proteins/genetics/metabolism
RNA Cleavage

@article {pmid38637031,
year = {2024},
author = {Zhang, X and Zhu, L and Yang, L and Liu, G and Qiu, S and Xiong, X and Huang, K and Xiao, T and Zhu, L},
title = {A sensitive and versatile electrochemical sensor based on hybridization chain reaction and CRISPR/Cas12a system for antibiotic detection.},
journal = {Analytica chimica acta},
volume = {1304},
number = {},
pages = {342562},
doi = {10.1016/j.aca.2024.342562},
pmid = {38637031},
issn = {1873-4324},
mesh = {*Anti-Bacterial Agents ; Electrochemical Techniques/methods ; CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; },
abstract = {A sensitive electrochemical platform was constructed with NH2-Cu-MOF as electrochemical probe to detect antibiotics using CRISPR/Cas12a system triggered by hybridization chain reaction (HCR). The sensing system consists of two HCR systems. HCR1 occurred on the electrode surface independent of the target, generating long dsDNA to connect signal probes and producing a strong electrochemical signal. HCR2 was triggered by target, and the resulting dsDNA products activated the CRISPR/Cas12a, thereby resulting in effective and rapid cleavage of the trigger of HCR1, hindering the occurrence of HCR1, and reducing the number of NH2-Cu-MOF on the electrode surface. Eventually, significant signal change depended on the target was obtained. On this basis and with the help of the programmability of DNA, kanamycin and ampicillin were sensitively detected with detection limits of 60 fM and 10 fM (S/N = 3), respectively. Furthermore, the sensing platform showed good detection performance in milk and livestock wastewater samples, demonstrating its great application prospects in the detection of antibiotics in food and environmental water samples.},
}

*Anti-Bacterial Agents
Electrochemical Techniques/methods
CRISPR-Cas Systems
*Biosensing Techniques/methods
Nucleic Acid Hybridization

@article {pmid38636316,
year = {2024},
author = {Wang, X and Deng, X and Zhang, Y and Dong, W and Rao, Q and Huang, Q and Tang, F and Shen, R and Xu, H and Jin, Z and Tang, Y and Du, D},
title = {A rapid and sensitive one-pot platform integrating fluorogenic RNA aptamers and CRISPR-Cas13a for visual detection of monkeypox virus.},
journal = {Biosensors & bioelectronics},
volume = {257},
number = {},
pages = {116268},
doi = {10.1016/j.bios.2024.116268},
pmid = {38636316},
issn = {1873-4235},
abstract = {The recent global upsurge in Monkeypox virus (MPXV) outbreaks underscores the critical need for rapid and precise diagnostic solutions, particularly in resource-constrained settings. The gold standard diagnostic method, qRT-PCR, is hindered by its time-consuming nature, requirement for nucleic acid purification, expensive equipment, and the need for highly trained personnel. Traditional CRISPR/Cas fluorescence assays, relying on trans-cleavage of ssDNA/RNA reporters labeled with costly fluorophores and quenchers, pose challenges that limit their widespread application, especially for point-of-care testing (POCT). In this study, we utilized a cost-effective and stable fluorogenic RNA aptamer (Mango III), specifically binding and illuminating the fluorophore TO3-3 PEG-Biotin Fluorophore (TO3), as a reporter for Cas13a trans-cleavage activity. We propose a comprehensive strategy integrating RNA aptamer, recombinase-aided amplification (RAA), and CRISPR-Cas13a systems for the molecular detection of MPXV target. Leveraging the inherent collateral cleavage properties of the Cas13a system, we established high-sensitivity and specificity assays to distinguish MPXV from other Orthopoxviruses (OPVs). A streamlined one-pot protocol was developed to mitigate aerosol contamination risks. Our aptamer-coupled RAA-Cas13a one-pot detection method achieved a Limit of Detection (LoD) of 4 copies of target MPXV DNA in just 40 min. Validation using clinical MPX specimens confirmed the rapid and reliable application of our RAA-Cas13a-Apt assays without nucleic acid purification procedure, highlighting its potential as a point-of-care testing solution. These results underscore the user-friendliness and effectiveness of our one-pot RAA-Cas13a-Apt diagnostic platform, poised to revolutionize disease detection and management.},
}

@article {pmid38635331,
year = {2024},
author = {},
title = {Correction to: Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas by Burger et al. The CRISPR Journal, 2023;7(1):12-28; DOI: 10.1089/crispr.2023.0061.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {131},
doi = {10.1089/crispr.2023.0061.correx},
pmid = {38635331},
issn = {2573-1602},
}

@article {pmid38635329,
year = {2024},
author = {Bouchareb, A and Biggs, D and Alghadban, S and Preece, C and Davies, B},
title = {Increasing Knockin Efficiency in Mouse Zygotes by Transient Hypothermia.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {111-119},
doi = {10.1089/crispr.2023.0077},
pmid = {38635329},
issn = {2573-1602},
mesh = {Animals ; Humans ; Mice ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Zygote/metabolism ; *Hypothermia/metabolism ; Recombinational DNA Repair/genetics ; },
abstract = {Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates. Using a traffic light reporter in mouse embryonic stem cells and a fluorescence conversion reporter in human-induced pluripotent stem cells, we confirm that a transient cold shock leads to an increase in the rate of HDR, with a corresponding decrease in the rate of NHEJ repair. We then investigated whether a similar cold shock could lead to an increase in the rate of HDR in the mouse embryo. By analyzing the efficiency of gene editing using single nucleotide polymorphism changes and loxP insertion at three different genetic loci, we found that a transient reduction in temperature after zygote electroporation of CRISPR-Cas9 ribonucleoprotein with a single-stranded oligodeoxynucleotide repair template did indeed increase knockin efficiency, without affecting embryonic development. The efficiency of gene editing with and without the cold shock was first assessed by genotyping blastocysts. As a proof of concept, we then confirmed that the modified embryo culture conditions were compatible with live births by targeting the coat color gene tyrosinase and observing the repair of the albino mutation. Taken together, our data suggest that a transient cold shock could offer a simple and robust way to improve knockin outcomes in both stem cells and zygotes.},
}

Animals
Humans
Mice
*Gene Editing
CRISPR-Cas Systems/genetics
Zygote/metabolism
*Hypothermia/metabolism
Recombinational DNA Repair/genetics

@article {pmid38635328,
year = {2024},
author = {Mohamad Zamberi, NN and Abuhamad, AY and Low, TY and Mohtar, MA and Syafruddin, SE},
title = {dCas9 Tells Tales: Probing Gene Function and Transcription Regulation in Cancer.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {73-87},
doi = {10.1089/crispr.2023.0078},
pmid = {38635328},
issn = {2573-1602},
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Epigenesis, Genetic ; *Neoplasms/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing is evolving into an essential tool in the field of biological and medical research. Notably, the development of catalytically deactivated Cas9 (dCas9) enzyme has substantially broadened its traditional boundaries in gene editing or perturbation. The conjugation of dCas9 with various molecular effectors allows precise control over transcriptional processes, epigenetic modifications, visualization of chromosomal dynamics, and several other applications. This expanded repertoire of CRISPR-Cas9 applications has emerged as an invaluable molecular tool kit that empowers researchers to comprehensively interrogate and gain insights into health and diseases. This review delves into the advancements in Cas9 protein engineering, specifically on the generation of various dCas9 tools that have significantly enhanced the CRISPR-based technology capability and versatility. We subsequently discuss the multifaceted applications of dCas9, especially in interrogating the regulation and function of genes that involve in supporting cancer pathogenesis. In addition, we also delineate the designing and utilization of dCas9-based tools as well as highlighting its current constraints and transformative potentials in cancer research.},
}

*Gene Editing
CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics
Epigenesis, Genetic
*Neoplasms/genetics

@article {pmid38635327,
year = {2024},
author = {Barrangou, R},
title = {CRISPR Pigs Portend a New Era of Xenotransplantation.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {71},
doi = {10.1089/crispr.2024.29173.editorial},
pmid = {38635327},
issn = {2573-1602},
mesh = {Animals ; Swine/genetics ; Transplantation, Heterologous ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Animals, Genetically Modified/genetics ; },
}

Animals
Swine/genetics
Transplantation, Heterologous
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics
Gene Editing
Animals, Genetically Modified/genetics

@article {pmid38635326,
year = {2024},
author = {Wang, M and Rieber, L and van Baaren, J and Morgan, M and Merrett, S and McDowell, I and Bowen, T},
title = {Diverse Class 2 CRISPR Effectors as Active Nucleases with Expanded Targeting Capabilities.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {120-130},
doi = {10.1089/crispr.2023.0058},
pmid = {38635326},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing ; Endonucleases/genetics ; RNA ; },
abstract = {CRISPR-Cas systems have proven effective in a variety of applications due to their ease of use and relatively high editing efficiency. Yet, any individual CRISPR-Cas system has inherent limitations, necessitating a diversity of RNA-guided nucleases to suit applications with distinct needs. We searched through metagenomic sequences to identify RNA-guided nucleases and found enzymes from diverse CRISPR-Cas types and subtypes, the most promising of which we developed into gene-editing platforms. Based on prior annotations of the metagenomic sequences, we establish the likely taxa and sampling locations where Class 2 CRISPR-Cas systems active in eukaryotes may be found. The newly discovered systems show robust capabilities as gene editors and base editors.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing
Endonucleases/genetics
RNA

@article {pmid38633802,
year = {2024},
author = {Wang, Q and Kline, EC and Gilligan-Steinberg, SD and Lai, JJ and Hull, IT and Olanrewaju, AO and Panpradist, N and Lutz, BR},
title = {Sensitive Pathogen Detection and Drug Resistance Characterization Using Pathogen-Derived Enzyme Activity Amplified by LAMP or CRISPR-Cas.},
journal = {medRxiv : the preprint server for health sciences},
volume = {},
number = {},
pages = {},
pmid = {38633802},
support = {P30 AI027757/AI/NIAID NIH HHS/United States ; R33 AI140460/AI/NIAID NIH HHS/United States ; },
abstract = {Pathogens encapsulate or encode their own suite of enzymes to facilitate replication in the host. The pathogen-derived enzymes possess specialized activities that are essential for pathogen replication and have naturally been candidates for drug targets. Phenotypic assays detecting the activities of pathogen-derived enzymes and characterizing their inhibition under drugs offer an opportunity for pathogen detection, drug resistance testing for individual patients, and as a research tool for new drug development. Here, we used HIV as an example to develop assays targeting the reverse transcriptase (RT) enzyme encapsulated in HIV for sensitive detection and phenotypic characterization, with the potential for point-of-care (POC) applications. Specifically, we targeted the complementary (cDNA) generation activity of the HIV RT enzyme by adding engineered RNA as substrates for HIV RT enzyme to generate cDNA products, followed by cDNA amplification and detection facilitated by loop-mediated isothermal amplification (LAMP) or CRISPR-Cas systems. To guide the assay design, we first used qPCR to characterize the cDNA generation activity of HIV RT enzyme. In the LAMP-mediated Product-Amplified RT activity assay (LamPART), the cDNA generation and LAMP amplification were combined into one pot with novel assay designs. When coupled with direct immunocapture of HIV RT enzyme for sample preparation and endpoint lateral flow assays for detection, LamPART detected as few as 20 copies of HIV RT enzyme spiked into 25μL plasma (fingerstick volume), equivalent to a single virion. In the Cas-mediated Product-Amplified RT activity assay (CasPART), we tailored the substrate design to achieve a LoD of 2e4 copies (1.67fM) of HIV RT enzyme. Furthermore, with its phenotypic characterization capability, CasPART was used to characterize the inhibition of HIV RT enzyme under antiretroviral drugs and differentiate between wild-type and mutant HIV RT enzyme for potential phenotypic drug resistance testing. Moreover, the CasPART assay can be readily adapted to target the activity of other pathogen-derived enzymes. As a proof-of-concept, we successfully adapted CasPART to detect HIV integrase with a sensitivity of 83nM. We anticipate the developed approach of detecting enzyme activity with product amplification has the potential for a wide range of pathogen detection and phenotypic characterization.},
}

@article {pmid38632224,
year = {2024},
author = {De Carluccio, G and Fusco, V and di Bernardo, D},
title = {Engineering a synthetic gene circuit for high-performance inducible expression in mammalian systems.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3311},
pmid = {38632224},
issn = {2041-1723},
support = {STAR PLUS//Compagnia di San Paolo (Fondazione Compagnia di San Paolo)/ ; TIGEM//Fondazione Telethon (Telethon Foundation)/ ; 826121//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Societal Challenges | H2020 Health (H2020 Societal Challenges - Health, Demographic Change and Well-being)/ ; DICMAPI//Università degli Studi di Napoli Federico II (University of Naples Federico II)/ ; },
mesh = {Animals ; *Genes, Synthetic ; *Gene Expression Regulation ; Transcription Factors/genetics ; Gene Regulatory Networks ; Promoter Regions, Genetic ; Mammals/genetics ; CRISPR-Cas Systems ; },
abstract = {Inducible gene expression systems can be used to control the expression of a gene of interest by means of a small-molecule. One of the most common designs involves engineering a small-molecule responsive transcription factor (TF) and its cognate promoter, which often results in a compromise between minimal uninduced background expression (leakiness) and maximal induced expression. Here, we focus on an alternative strategy using quantitative synthetic biology to mitigate leakiness while maintaining high expression, without modifying neither the TF nor the promoter. Through mathematical modelling and experimental validations, we design the CASwitch, a mammalian synthetic gene circuit based on combining two well-known network motifs: the Coherent Feed-Forward Loop (CFFL) and the Mutual Inhibition (MI). The CASwitch combines the CRISPR-Cas endoribonuclease CasRx with the state-of-the-art Tet-On3G inducible gene system to achieve high performances. To demonstrate the potentialities of the CASwitch, we apply it to three different scenarios: enhancing a whole-cell biosensor, controlling expression of a toxic gene and inducible production of Adeno-Associated Virus (AAV) vectors.},
}

Animals
*Genes, Synthetic
*Gene Expression Regulation
Transcription Factors/genetics
Gene Regulatory Networks
Promoter Regions, Genetic
Mammals/genetics
CRISPR-Cas Systems

@article {pmid38632100,
year = {2024},
author = {Yan-Chun, B and Ling-Li, D and Zai-Xia, L and Feng-Ying, M and Yu, W and Yong-Bin, L and Ming-Juan, G and Ri-Su, N and Wen-Guang, Z},
title = {Progress on CRISPR/Cas9 system in the genetic improvement of livestock and poultry.},
journal = {Yi chuan = Hereditas},
volume = {46},
number = {3},
pages = {219-231},
doi = {10.16288/j.yczz.24-021},
pmid = {38632100},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems ; *Livestock/genetics ; Poultry/genetics ; Gene Editing/methods ; Gene Knock-In Techniques ; },
abstract = {CRISPR/Cas9 gene editing technology, as a highly efficient genome editing method, has been extensively employed in the realm of animal husbandry for genetic improvement. With its remarkable efficiency and precision, this technology has revolutionized the field of animal husbandry. Currently, CRISPR/Cas9-based gene knockout, gene knock-in and gene modification techniques are widely employed to achieve precise enhancements in crucial production traits of livestock and poultry species. In this review, we summarize the operational principle and development history of CRISPR/Cas9 technology. Additionally, we highlight the research advancements utilizing this technology in muscle growth and development, fiber growth, milk quality composition, disease resistance breeding, and animal welfare within the livestock and poultry sectors. Our aim is to provide a more comprehensive understanding of the application of CRISPR/Cas9 technology in gene editing for livestock and poultry.},
}

Animals
*CRISPR-Cas Systems
*Livestock/genetics
Poultry/genetics
Gene Editing/methods
Gene Knock-In Techniques

@article {pmid38631624,
year = {2024},
author = {Hu, H and Liu, L and Wei, XY and Duan, JJ and Deng, JY and Pei, DS},
title = {Revolutionizing aquatic eco-environmental monitoring: Utilizing the RPA-Cas-FQ detection platform for zooplankton.},
journal = {The Science of the total environment},
volume = {},
number = {},
pages = {172414},
doi = {10.1016/j.scitotenv.2024.172414},
pmid = {38631624},
issn = {1879-1026},
abstract = {The integration of recombinase polymerase amplification (RPA) with CRISPR/Cas technology has revolutionized molecular diagnostics and pathogen detection due to its unparalleled sensitivity and trans-cleavage ability. However, its potential in the ecological and environmental monitoring scenarios for aquatic ecosystems remains largely unexplored, particularly in accurate qualitative/quantitative detection, and its actual performance in handling complex real environmental samples. Using zooplankton as a model, we have successfully optimized the RPA-CRISPR/Cas12a fluorescence detection platform (RPA-Cas-FQ), providing several crucial "technical tips". Our findings indicate the sensitivity of CRISPR/Cas12a alone is 5 × 10[9] copies/reaction, which can be dramatically increased to 5 copies/reaction when combined with RPA. The optimized RPA-Cas-FQ enables reliable qualitative and semi-quantitative detection within 50 min, and exhibits a good linear relationship between fluorescence intensity and DNA concentration (R[2] = 0.956-0.974***). Additionally, we developed a rapid and straightforward identification procedure for single zooplankton by incorporating heat-lysis and DNA-barcode techniques. We evaluated the platform's effectiveness using real environmental DNA (eDNA) samples from the Three Gorges Reservoir, confirming its practicality. The eDNA-RPA-Cas-FQ demonstrated strong consistency (Kappa = 0.43***) with eDNA-Metabarcoding in detecting species presence/absence in the reservoir. Furthermore, the two semi-quantitative eDNA quantification technologies showed a strong positive correlation (R[2] = 0.58-0.87***). This platform also has the potential to monitor environmental pollutants by selecting appropriate indicator species. The novel insights and methodologies presented in this study represent a significant advancement in meeting the complex needs of aquatic ecosystem protection and monitoring.},
}

@article {pmid38630829,
year = {2024},
author = {Tan, E and Wan, T and Pan, Q and Duan, J and Zhang, S and Wang, R and Gao, P and Lv, J and Wang, H and Li, D and Ping, Y and Cheng, Y},
title = {Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.},
journal = {Science advances},
volume = {10},
number = {16},
pages = {eadl4336},
pmid = {38630829},
issn = {2375-2548},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics ; Gene Transfer Techniques ; Genetic Therapy ; Polymers/chemistry ; Ribonucleoproteins/genetics ; },
abstract = {Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.},
}

Animals
Mice
*CRISPR-Cas Systems
*Gene Editing
NLR Family, Pyrin Domain-Containing 3 Protein/genetics
Gene Transfer Techniques
Genetic Therapy
Polymers/chemistry
Ribonucleoproteins/genetics

@article {pmid38629716,
year = {2024},
author = {Johnson, GA and Gould, SI and Sánchez-Rivera, FJ},
title = {Deconstructing cancer with precision genome editing.},
journal = {Biochemical Society transactions},
volume = {52},
number = {2},
pages = {803-819},
doi = {10.1042/BST20230984},
pmid = {38629716},
issn = {1470-8752},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Mutation ; Animals ; Precision Medicine ; Genome, Human ; },
abstract = {Recent advances in genome editing technologies are allowing investigators to engineer and study cancer-associated mutations in their endogenous genetic contexts with high precision and efficiency. Of these, base editing and prime editing are quickly becoming gold-standards in the field due to their versatility and scalability. Here, we review the merits and limitations of these precision genome editing technologies, their application to modern cancer research, and speculate how these could be integrated to address future directions in the field.},
}

Humans
*Gene Editing/methods
*Neoplasms/genetics/therapy
*CRISPR-Cas Systems
Mutation
Animals
Precision Medicine
Genome, Human

@article {pmid38627399,
year = {2024},
author = {Bi, M and Su, W and Li, J and Mo, X},
title = {Insights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3256},
pmid = {38627399},
issn = {2041-1723},
support = {32071476//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; },
abstract = {Spacer acquisition step in CRISPR-Cas system involves the recognition and subsequent integration of protospacer by the Cas1-Cas2 complex in CRISPR-Cas systems. Here we report an anti-CRISPR protein, AcrVA5, and reveal the mechanisms by which it strongly inhibits protospacer integration. Our biochemical data shows that the integration by Cas1-Cas2 was abrogated in the presence of AcrVA5. AcrVA5 exhibits low binding affinity towards Cas2 and acetylates Cas2 at Lys[55] on the binding interface of the Cas2 and AcrVA5 N-terminal peptide complex to inhibit the Cas2-mediated endonuclease activity. Moreover, a detailed structural comparison between our crystal structure and homolog structure shows that binding of AcrVA5 to Cas2 causes steric hindrance to the neighboring protospacer resulting in the partial disassembly of the Cas1-Cas2 and protospacer complex, as demonstrated by electrophoretic mobility shift assay. Our study focuses on this mechanism of spacer acquisition inhibition and provides insights into the biology of CRISPR-Cas systems.},
}

*CRISPR-Associated Proteins/metabolism
CRISPR-Cas Systems

@article {pmid38613479,
year = {2024},
author = {Cao, H and Mao, K and Yang, J and Wu, Q and Hu, J and Zhang, H},
title = {High-Throughput μPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.},
journal = {Analytical chemistry},
volume = {96},
number = {16},
pages = {6337-6346},
doi = {10.1021/acs.analchem.3c05958},
pmid = {38613479},
issn = {1520-6882},
mesh = {*Soil/chemistry ; Horseradish Peroxidase/metabolism/chemistry ; CRISPR-Cas Systems ; Oryza/chemistry ; Soil Pollutants/analysis ; Lab-On-A-Chip Devices ; Endodeoxyribonucleases/metabolism/chemistry ; High-Throughput Screening Assays/methods ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/metabolism ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (μPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 10[6] to 6.49 × 10[7] copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.},
}

*Soil/chemistry
Horseradish Peroxidase/metabolism/chemistry
CRISPR-Cas Systems
Oryza/chemistry
Soil Pollutants/analysis
Lab-On-A-Chip Devices
Endodeoxyribonucleases/metabolism/chemistry
High-Throughput Screening Assays/methods
Bacterial Proteins/genetics/metabolism
CRISPR-Associated Proteins/metabolism
Limit of Detection
Nucleic Acid Amplification Techniques/methods

@article {pmid38608689,
year = {2024},
author = {Vos, PD and Gandadireja, AP and Rossetti, G and Siira, SJ and Mantegna, JL and Filipovska, A and Rackham, O},
title = {Mutational rescue of the activity of high-fidelity Cas9 enzymes.},
journal = {Cell reports methods},
volume = {4},
number = {4},
pages = {100756},
doi = {10.1016/j.crmeth.2024.100756},
pmid = {38608689},
issn = {2667-2375},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; Mutation ; Endonucleases/genetics/metabolism ; },
abstract = {Programmable DNA endonucleases derived from bacterial genetic defense systems, exemplified by CRISPR-Cas9, have made it significantly easier to perform genomic modifications in living cells. However, unprogrammed, off-target modifications can have serious consequences, as they often disrupt the function or regulation of non-targeted genes and compromise the safety of therapeutic gene editing applications. High-fidelity mutants of Cas9 have been established to enable more accurate gene editing, but these are typically less efficient. Here, we merge the strengths of high-fidelity Cas9 and hyperactive Cas9 variants to provide an enzyme, which we dub HyperDriveCas9, that yields the desirable properties of both parents. HyperDriveCas9 functions efficiently in mammalian cells and introduces insertion and deletion mutations into targeted genomic regions while maintaining a favorable off-target profile. HyperDriveCas9 is a precise and efficient tool for gene editing applications in science and medicine.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
HEK293 Cells
Mutation
Endonucleases/genetics/metabolism

@article {pmid38604773,
year = {2024},
author = {Li, QN and Ma, AX and Wang, DX and Dai, ZQ and Wu, SL and Lu, S and Zhu, LN and Jiang, HX and Pang, DW and Kong, DM},
title = {Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.},
journal = {Analytical chemistry},
volume = {96},
number = {16},
pages = {6426-6435},
doi = {10.1021/acs.analchem.4c00555},
pmid = {38604773},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; *MicroRNAs/analysis/metabolism ; Allosteric Regulation ; CRISPR-Associated Proteins/metabolism ; Endodeoxyribonucleases/metabolism/chemistry ; Bacterial Proteins/metabolism/chemistry/genetics ; HEK293 Cells ; },
abstract = {Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans
*MicroRNAs/analysis/metabolism
Allosteric Regulation
CRISPR-Associated Proteins/metabolism
Endodeoxyribonucleases/metabolism/chemistry
Bacterial Proteins/metabolism/chemistry/genetics
HEK293 Cells

@article {pmid38599073,
year = {2024},
author = {Li, H and Qiao, S and Zhang, H and Qiao, Y and Liu, J and Li, Y},
title = {Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.},
journal = {Biosensors & bioelectronics},
volume = {256},
number = {},
pages = {116276},
doi = {10.1016/j.bios.2024.116276},
pmid = {38599073},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; *Alpha-Ketoglutarate-Dependent Dioxygenase FTO/chemistry/metabolism ; Humans ; *DNA, Catalytic/chemistry ; *Luminescent Measurements ; *Electrochemical Techniques/methods ; *Limit of Detection ; *CRISPR-Cas Systems ; Nitriles/chemistry ; Endodeoxyribonucleases/chemistry/metabolism ; CRISPR-Associated Proteins/chemistry ; Adenosine/analogs & derivatives/analysis/chemistry ; Nanostructures/chemistry ; Ferrous Compounds/chemistry ; Bacterial Proteins/chemistry/genetics ; *Metallocenes ; },
abstract = {Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m[6]A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m[6]A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.},
}

*Biosensing Techniques
*Alpha-Ketoglutarate-Dependent Dioxygenase FTO/chemistry/metabolism
Humans
*DNA, Catalytic/chemistry
*Luminescent Measurements
*Electrochemical Techniques/methods
*Limit of Detection
*CRISPR-Cas Systems
Nitriles/chemistry
Endodeoxyribonucleases/chemistry/metabolism
CRISPR-Associated Proteins/chemistry
Adenosine/analogs & derivatives/analysis/chemistry
Nanostructures/chemistry
Ferrous Compounds/chemistry
Bacterial Proteins/chemistry/genetics
*Metallocenes

@article {pmid38574662,
year = {2024},
author = {Zhang, H and Zhang, M and Zhou, Y and Qiao, Z and Gao, L and Cao, L and Yin, H and Wang, M},
title = {Organic photoelectrochemical transistor aptasensor for dual-mode detection of DEHP with CRISPR-Cas13a assisted signal amplification.},
journal = {Journal of hazardous materials},
volume = {470},
number = {},
pages = {134175},
doi = {10.1016/j.jhazmat.2024.134175},
pmid = {38574662},
issn = {1873-3336},
mesh = {*Aptamers, Nucleotide/chemistry ; *Diethylhexyl Phthalate/chemistry/analysis ; *Biosensing Techniques/methods ; *Electrochemical Techniques/instrumentation/methods ; *CRISPR-Cas Systems ; *Transistors, Electronic ; Water Pollutants, Chemical/analysis ; Polystyrenes/chemistry ; Limit of Detection ; Nucleic Acid Amplification Techniques ; *Thiophenes ; },
abstract = {Emerging organic photoelectrochemical transistors (OPECTs) with inherent amplification capabilities, good biocompatibility and even self-powered operation have emerged as a promising detection tool, however, they are still not widely studied for pollutant detection. In this paper, a novel OPECT dual-mode aptasensor was constructed for the ultrasensitive detection of di(2-ethylhexyl) phthalate (DEHP). MXene/In2S3/In2O3 Z-scheme heterojunction was used as a light fuel for ion modulation in sensitive gated OPECT biosensing. A transistor system based on poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) converted biological events associated with photosensitive gate achieving nearly a thousand-fold higher current gain at zero bias voltage. This work quantified the target DEHP by aptamer-specific induction of CRISPR-Cas13a trans-cutting activity with target-dependent rolling circle amplification as the signal amplification unit, and incorporated the signal changes strategy of biocatalytic precipitation and TMB color development. Combining OPECT with the auxiliary validation of colorimetry (CM), high sensitivity and accurate detection of DEHP were achieved with a linear range of 0.1 pM to 200 pM and a minimum detection limit of 0.02 pM. This study not only provides a new method for the detection of DEHP, but also offers a promising prospect for the gating and application of the unique OPECT.},
}

*Aptamers, Nucleotide/chemistry
*Diethylhexyl Phthalate/chemistry/analysis
*Biosensing Techniques/methods
*Electrochemical Techniques/instrumentation/methods
*CRISPR-Cas Systems
*Transistors, Electronic
Water Pollutants, Chemical/analysis
Polystyrenes/chemistry
Limit of Detection
Nucleic Acid Amplification Techniques
*Thiophenes

@article {pmid38570687,
year = {2024},
author = {Schevenels, G and Cabochette, P and America, M and Vandenborne, A and De Grande, L and Guenther, S and He, L and Dieu, M and Christou, B and Vermeersch, M and Germano, RFV and Perez-Morga, D and Renard, P and Martin, M and Vanlandewijck, M and Betsholtz, C and Vanhollebeke, B},
title = {A brain-specific angiogenic mechanism enabled by tip cell specialization.},
journal = {Nature},
volume = {628},
number = {8009},
pages = {863-871},
pmid = {38570687},
issn = {1476-4687},
mesh = {*Zebrafish ; Animals ; *Brain/cytology/blood supply/metabolism ; *Blood-Brain Barrier/metabolism/cytology ; *Neovascularization, Physiologic ; *Endothelial Cells/metabolism/cytology ; *Basement Membrane/metabolism ; *Collagen Type IV/metabolism ; Wnt Proteins/metabolism ; CRISPR-Cas Systems/genetics ; Humans ; Organ Specificity ; Wnt Signaling Pathway ; Zebrafish Proteins/metabolism/genetics ; Cell Movement ; Meninges/cytology/blood supply/metabolism ; },
abstract = {Vertebrate organs require locally adapted blood vessels[1,2]. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals[3-5]. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-β-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.},
}

*Zebrafish
Animals
*Brain/cytology/blood supply/metabolism
*Blood-Brain Barrier/metabolism/cytology
*Neovascularization, Physiologic
*Endothelial Cells/metabolism/cytology
*Basement Membrane/metabolism
*Collagen Type IV/metabolism
Wnt Proteins/metabolism
CRISPR-Cas Systems/genetics
Humans
Organ Specificity
Wnt Signaling Pathway
Zebrafish Proteins/metabolism/genetics
Cell Movement
Meninges/cytology/blood supply/metabolism

@article {pmid38554702,
year = {2024},
author = {Köhler, AR and Haußer, J and Harsch, A and Bernhardt, S and Häußermann, L and Brenner, LM and Lungu, C and Olayioye, MA and Bashtrykov, P and Jeltsch, A},
title = {Modular dual-color BiAD sensors for locus-specific readout of epigenome modifications in single cells.},
journal = {Cell reports methods},
volume = {4},
number = {4},
pages = {100739},
doi = {10.1016/j.crmeth.2024.100739},
pmid = {38554702},
issn = {2667-2375},
mesh = {Humans ; *Epigenome ; *DNA Methylation ; *Single-Cell Analysis/methods ; *Histones/metabolism/genetics ; Epigenesis, Genetic ; Genetic Loci ; Chromatin/metabolism/genetics ; Biosensing Techniques/methods ; Color ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Dynamic changes in the epigenome at defined genomic loci play crucial roles during cellular differentiation and disease development. Here, we developed dual-color bimolecular anchor detector (BiAD) sensors for high-sensitivity readout of locus-specific epigenome modifications by fluorescence microscopy. Our BiAD sensors comprise an sgRNA/dCas9 complex as anchor and double chromatin reader domains as detector modules, both fused to complementary parts of a split IFP2.0 fluorophore, enabling its reconstitution upon binding of both parts in close proximity. In addition, a YPet fluorophore is recruited to the sgRNA to mark the genomic locus of interest. With these dual-color BiAD sensors, we detected H3K9me2/3 and DNA methylation and their dynamic changes upon RNAi or inhibitor treatment with high sensitivity at endogenous genomic regions. Furthermore, we showcased locus-specific H3K36me2/3 readout as well as H3K27me3 and H3K9me2/3 enrichment on the inactive X chromosome, highlighting the broad applicability of our dual-color BiAD sensors for single-cell epigenome studies.},
}

Humans
*Epigenome
*DNA Methylation
*Single-Cell Analysis/methods
*Histones/metabolism/genetics
Epigenesis, Genetic
Genetic Loci
Chromatin/metabolism/genetics
Biosensing Techniques/methods
Color
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid38538798,
year = {2024},
author = {Radko-Juettner, S and Yue, H and Myers, JA and Carter, RD and Robertson, AN and Mittal, P and Zhu, Z and Hansen, BS and Donovan, KA and Hunkeler, M and Rosikiewicz, W and Wu, Z and McReynolds, MG and Roy Burman, SS and Schmoker, AM and Mageed, N and Brown, SA and Mobley, RJ and Partridge, JF and Stewart, EA and Pruett-Miller, SM and Nabet, B and Peng, J and Gray, NS and Fischer, ES and Roberts, CWM},
title = {Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF.},
journal = {Nature},
volume = {628},
number = {8007},
pages = {442-449},
pmid = {38538798},
issn = {1476-4687},
mesh = {Animals ; Female ; Humans ; Male ; Mice ; Cell Line, Tumor ; CRISPR-Cas Systems ; Gene Editing ; *Mutation ; *Neoplasms/genetics/metabolism ; *SMARCB1 Protein/deficiency/genetics/metabolism ; Tumor Suppressor Proteins/deficiency/genetics/metabolism ; *Multiprotein Complexes/chemistry/metabolism ; Proteolysis ; Ubiquitin/metabolism ; },
abstract = {Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project[1-3]. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.},
}

Animals
Female
Humans
Male
Mice
Cell Line, Tumor
CRISPR-Cas Systems
Gene Editing
*Mutation
*Neoplasms/genetics/metabolism
*SMARCB1 Protein/deficiency/genetics/metabolism
Tumor Suppressor Proteins/deficiency/genetics/metabolism
*Multiprotein Complexes/chemistry/metabolism
Proteolysis
Ubiquitin/metabolism

@article {pmid38532709,
year = {2024},
author = {Sale, JE and Stoddard, BL},
title = {CRISPR in Nucleic Acids Research: the sequel.},
journal = {Nucleic acids research},
volume = {52},
number = {7},
pages = {3489-3492},
doi = {10.1093/nar/gkae159},
pmid = {38532709},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acids/genetics ; },
}

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

@article {pmid38526223,
year = {2024},
author = {Franks, SNJ and Heon-Roberts, R and Ryan, BJ},
title = {CRISPRi: a way to integrate iPSC-derived neuronal models.},
journal = {Biochemical Society transactions},
volume = {52},
number = {2},
pages = {539-551},
doi = {10.1042/BST20230190},
pmid = {38526223},
issn = {1470-8752},
mesh = {*Induced Pluripotent Stem Cells/cytology/metabolism ; Humans ; *Neurodegenerative Diseases/therapy/genetics/metabolism ; *Neurons/metabolism/cytology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; Gene Editing ; },
abstract = {The genetic landscape of neurodegenerative diseases encompasses genes affecting multiple cellular pathways which exert effects in an array of neuronal and glial cell-types. Deconvolution of the roles of genes implicated in disease and the effects of disease-associated variants remains a vital step in the understanding of neurodegeneration and the development of therapeutics. Disease modelling using patient induced pluripotent stem cells (iPSCs) has enabled the generation of key cell-types associated with disease whilst maintaining the genomic variants that predispose to neurodegeneration. The use of CRISPR interference (CRISPRi), alongside other CRISPR-perturbations, allows the modelling of the effects of these disease-associated variants or identifying genes which modify disease phenotypes. This review summarises the current applications of CRISPRi in iPSC-derived neuronal models, such as fluorescence-activated cell sorting (FACS)-based screens, and discusses the future opportunities for disease modelling, identification of disease risk modifiers and target/drug discovery in neurodegeneration.},
}

*Induced Pluripotent Stem Cells/cytology/metabolism
Humans
*Neurodegenerative Diseases/therapy/genetics/metabolism
*Neurons/metabolism/cytology
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Animals
Gene Editing

@article {pmid38499498,
year = {2024},
author = {Gawlitt, S and Collins, SP and Yu, Y and Blackman, SA and Barquist, L and Beisel, CL},
title = {Expanding the flexibility of base editing for high-throughput genetic screens in bacteria.},
journal = {Nucleic acids research},
volume = {52},
number = {7},
pages = {4079-4097},
doi = {10.1093/nar/gkae174},
pmid = {38499498},
issn = {1362-4962},
support = {1R35GM119561/NH/NIH HHS/United States ; 865973/ERC_/European Research Council/International ; 1R35GM119561/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Escherichia coli/genetics ; High-Throughput Screening Assays/methods ; Genome, Bacterial/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Streptococcus/genetics ; Streptococcus pyogenes/genetics/enzymology ; Machine Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genome-wide screens have become powerful tools for elucidating genotype-to-phenotype relationships in bacteria. Of the varying techniques to achieve knockout and knockdown, CRISPR base editors are emerging as promising options. However, the limited number of available, efficient target sites hampers their use for high-throughput screening. Here, we make multiple advances to enable flexible base editing as part of high-throughput genetic screening in bacteria. We first co-opt the Streptococcus canis Cas9 that exhibits more flexible protospacer-adjacent motif recognition than the traditional Streptococcus pyogenes Cas9. We then expand beyond introducing premature stop codons by mutating start codons. Next, we derive guide design rules by applying machine learning to an essentiality screen conducted in Escherichia coli. Finally, we rescue poorly edited sites by combining base editing with Cas9-induced cleavage of unedited cells, thereby enriching for intended edits. The efficiency of this dual system was validated through a conditional essentiality screen based on growth in minimal media. Overall, expanding the scope of genome-wide knockout screens with base editors could further facilitate the investigation of new gene functions and interactions in bacteria.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Escherichia coli/genetics
High-Throughput Screening Assays/methods
Genome, Bacterial/genetics
CRISPR-Associated Protein 9/genetics/metabolism
Streptococcus/genetics
Streptococcus pyogenes/genetics/enzymology
Machine Learning
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid38351238,
year = {2024},
author = {Asanomi, Y and Kimura, T and Shimoda, N and Shigemizu, D and Niida, S and Ozaki, K},
title = {CRISPR/Cas9-mediated knock-in cells of the late-onset Alzheimer's disease-risk variant, SHARPIN G186R, reveal reduced NF-κB pathway and accelerated Aβ secretion.},
journal = {Journal of human genetics},
volume = {69},
number = {5},
pages = {171-176},
pmid = {38351238},
issn = {1435-232X},
support = {JP21dk0207045//Japan Agency for Medical Research and Development (AMED)/ ; JP22dk0207060//Japan Agency for Medical Research and Development (AMED)/ ; JP21de0107002//Japan Agency for Medical Research and Development (AMED)/ ; JP21dk0207052//Japan Agency for Medical Research and Development (AMED)/ ; JP18kk0205009//Japan Agency for Medical Research and Development (AMED)/ ; JP18kk0205012//Japan Agency for Medical Research and Development (AMED)/ ; 21-23//National Center for Geriatrics and Gerontology (NCGG)/ ; 21-24//National Center for Geriatrics and Gerontology (NCGG)/ ; 23-7//National Center for Geriatrics and Gerontology (NCGG)/ ; },
mesh = {Humans ; *Alzheimer Disease/genetics/metabolism/pathology ; *CRISPR-Cas Systems ; *NF-kappa B/metabolism/genetics ; *Amyloid beta-Peptides/metabolism/genetics ; Signal Transduction/genetics ; Gene Knock-In Techniques ; },
}

Humans
*Alzheimer Disease/genetics/metabolism/pathology
*CRISPR-Cas Systems
*NF-kappa B/metabolism/genetics
*Amyloid beta-Peptides/metabolism/genetics
Signal Transduction/genetics
Gene Knock-In Techniques

@article {pmid38627378,
year = {2024},
author = {Wang, Y and Chen, H and Lin, K and Han, Y and Gu, Z and Wei, H and Mu, K and Wang, D and Liu, L and Jin, R and Song, R and Rong, Z and Wang, S},
title = {Ultrasensitive single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3279},
pmid = {38627378},
issn = {2041-1723},
mesh = {Humans ; Male ; *Monkeypox virus ; Biological Assay ; Cell Death ; *Exanthema ; Nucleotidyltransferases ; Nucleic Acid Amplification Techniques ; Sensitivity and Specificity ; CRISPR-Cas Systems ; Recombinases ; },
abstract = {The emerging monkeypox virus (MPXV) has raised global health concern, thereby highlighting the need for rapid, sensitive, and easy-to-use diagnostics. Here, we develop a single-step CRISPR-based diagnostic platform, termed SCOPE (Streamlined CRISPR On Pod Evaluation platform), for field-deployable ultrasensitive detection of MPXV in resource-limited settings. The viral nucleic acids are rapidly released from the rash fluid swab, oral swab, saliva, and urine samples in 2 min via a streamlined viral lysis protocol, followed by a 10-min single-step recombinase polymerase amplification (RPA)-CRISPR/Cas13a reaction. A pod-shaped vest-pocket analysis device achieves the whole process for reaction execution, signal acquisition, and result interpretation. SCOPE can detect as low as 0.5 copies/µL (2.5 copies/reaction) of MPXV within 15 min from the sample input to the answer. We validate the developed assay on 102 clinical samples from male patients / volunteers, and the testing results are 100% concordant with the real-time PCR. SCOPE achieves a single-molecular level sensitivity in minutes with a simplified procedure performed on a miniaturized wireless device, which is expected to spur substantial progress to enable the practice application of CRISPR-based diagnostics techniques in a point-of-care setting.},
}

Humans
Male
*Monkeypox virus
Biological Assay
Cell Death
*Exanthema
Nucleotidyltransferases
Nucleic Acid Amplification Techniques
Sensitivity and Specificity
CRISPR-Cas Systems
Recombinases

@article {pmid38623982,
year = {2024},
author = {Volodina, OV and Fabrichnikova, AR and Anuchina, AA and Mishina, OS and Lavrov, AV and Smirnikhina, SA},
title = {Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases.},
journal = {Current gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.2174/0115665232295117240405070809},
pmid = {38623982},
issn = {1875-5631},
abstract = {The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing - in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.},
}

@article {pmid38514901,
year = {2024},
author = {Zhai, D and Li, L and Chen, C and Wang, X and Liu, R and Shan, Y},
title = {INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells.},
journal = {Journal of clinical laboratory analysis},
volume = {38},
number = {7},
pages = {e25031},
pmid = {38514901},
issn = {1098-2825},
mesh = {*Cilia/metabolism ; *Phosphoric Monoester Hydrolases/metabolism/genetics ; Humans ; Cell Line ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Retinal Pigment Epithelium/metabolism/cytology ; Phosphatidylinositol Phosphates/metabolism ; CRISPR-Cas Systems ; Phospholipids/metabolism ; },
abstract = {BACKGROUND: Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function.

METHODS: The EGFP-2xP4M[SidM], PH[PLCδ1]-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ.

RESULTS: In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P2 is located in the ciliary membrane labeled by SMO-tRFP.

CONCLUSIONS: INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P2, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.},
}

*Cilia/metabolism
*Phosphoric Monoester Hydrolases/metabolism/genetics
Humans
Cell Line
Phosphatidylinositol 4,5-Diphosphate/metabolism
Retinal Pigment Epithelium/metabolism/cytology
Phosphatidylinositol Phosphates/metabolism
CRISPR-Cas Systems
Phospholipids/metabolism

@article {pmid38355914,
year = {2024},
author = {McLaurin, KA and Li, H and Khalili, K and Mactutus, CF and Booze, RM},
title = {HIV-1 mRNA knockdown with CRISPR/CAS9 enhances neurocognitive function.},
journal = {Journal of neurovirology},
volume = {30},
number = {1},
pages = {71-85},
pmid = {38355914},
issn = {1538-2443},
support = {DA059310/DA/NIDA NIH HHS/United States ; DA056288/DA/NIDA NIH HHS/United States ; MH106392/MH/NIMH NIH HHS/United States ; NS100624/NS/NINDS NIH HHS/United States ; DA059310/DA/NIDA NIH HHS/United States ; DA056288/DA/NIDA NIH HHS/United States ; MH106392/MH/NIMH NIH HHS/United States ; NS100624/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *HIV-1/genetics/physiology ; Rats ; *CRISPR-Cas Systems ; *Rats, Transgenic ; *RNA, Messenger/genetics/metabolism ; *Gene Editing/methods ; Neuroglia/virology/metabolism ; Dependovirus/genetics ; HIV Infections/virology/genetics ; Gene Knockdown Techniques ; RNA, Viral/genetics ; Cognition/physiology ; Humans ; },
abstract = {Mixed glia are infiltrated with HIV-1 virus early in the course of infection leading to the development of a persistent viral reservoir in the central nervous system. Modification of the HIV-1 genome using gene editing techniques, including CRISPR/Cas9, has shown great promise towards eliminating HIV-1 viral reservoirs; whether these techniques are capable of removing HIV-1 viral proteins from mixed glia, however, has not been systematically evaluated. Herein, the efficacy of adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing for eliminating HIV-1 messenger RNA (mRNA) from cortical mixed glia was evaluated in vitro and in vivo. In vitro, a within-subjects experimental design was utilized to treat mixed glia isolated from neonatal HIV-1 transgenic (Tg) rats with varying doses (0, 0.9, 1.8, 2.7, 3.6, 4.5, or 5.4 µL corresponding to a physical titer of 0, 4.23 × 10[9], 8.46 × 10[9], 1.269 × 10[10], 1.692 × 10[10], 2.115 × 10[10], and 2.538 × 10[10] gc/µL) of CRISPR/Cas9 for 72 h. Dose-dependent decreases in the number of HIV-1 mRNA, quantified using an innovative in situ hybridization technique, were observed in a subset (i.e., n = 5 out of 8) of primary mixed glia. In vivo, HIV-1 Tg rats were retro-orbitally inoculated with CRISPR/Cas9 for two weeks, whereby treatment resulted in profound excision (i.e., approximately 53.2%) of HIV-1 mRNA from the medial prefrontal cortex. Given incomplete excision of the HIV-1 viral genome, the clinical relevance of HIV-1 mRNA knockdown for eliminating neurocognitive impairments was evaluated via examination of temporal processing, a putative neurobehavioral mechanism underlying HIV-1-associated neurocognitive disorders (HAND). Indeed, treatment with CRISPR/Cas9 protractedly, albeit not permanently, restored the developmental trajectory of temporal processing. Proof-of-concept studies, therefore, support the susceptibility of mixed glia to gene editing and the potential of CRISPR/Cas9 to serve as a novel therapeutic strategy for HAND, even in the absence of full viral eradication.},
}

Animals
*HIV-1/genetics/physiology
Rats
*CRISPR-Cas Systems
*Rats, Transgenic
*RNA, Messenger/genetics/metabolism
*Gene Editing/methods
Neuroglia/virology/metabolism
Dependovirus/genetics
HIV Infections/virology/genetics
Gene Knockdown Techniques
RNA, Viral/genetics
Cognition/physiology
Humans

@article {pmid38624901,
year = {2022},
author = {Mondal, S and Halder, SK and Mondal, KC},
title = {Tailoring in fungi for next generation cellulase production with special reference to CRISPR/CAS system.},
journal = {Systems microbiology and biomanufacturing},
volume = {2},
number = {1},
pages = {113-129},
pmid = {38624901},
issn = {2662-7663},
abstract = {Cellulose is the utmost plenteous source of biopolymer in our earth, and fungi are the most efficient and ubiquitous organism in degrading the cellulosic biomass by synthesizing cellulases. Tailoring through genetic manipulation has played a substantial role in constructing novel fungal strains towards improved cellulase production of desired traits. However, the traditional methods of genetic manipulation of fungi are time-consuming and tedious. With the availability of the full-genome sequences of several industrially relevant filamentous fungi, CRISPR-CAS (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) technology has come into the focus for the proficient development of manipulated strains of filamentous fungi. This review summarizes the mode of action of cellulases, transcription level regulation for cellulase expression, various traditional strategies of genetic manipulation with CRISPR-CAS technology to develop modified fungal strains for a preferred level of cellulase production, and the futuristic trend in this arena of research.},
}

@article {pmid38622798,
year = {2024},
author = {Peng, W and Gao, M and Zhu, X and Liu, X and Yang, G and Li, S and Liu, Y and Bai, L and Yang, J and Bao, J},
title = {Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.},
journal = {Biotechnology journal},
volume = {19},
number = {4},
pages = {e2300691},
doi = {10.1002/biot.202300691},
pmid = {38622798},
issn = {1860-7314},
support = {82270662//National Natural Science Foundation of China/ ; 82070640//National Natural Science Foundation of China/ ; 2022-YF05-01255-SN//Technology Innovation R&D Project of Chengdu Science and Technology Bureau/ ; 22ZDYF1406//Key R&D Project of Sichuan Science and Technology Department/ ; ZYJC21014//1.3.5 project for disciplines of excellence, West China Hospital Sichuan University/ ; },
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Knockout Techniques ; Genome ; RNA, Messenger/genetics ; },
abstract = {CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.},
}

Animals
Swine
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Gene Knockout Techniques
Genome
RNA, Messenger/genetics

@article {pmid38622404,
year = {2024},
author = {Freen-van Heeren, JJ},
title = {Employing CRISPR-Cas9 to Enhance T Cell Effector Function.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2782},
number = {},
pages = {195-208},
pmid = {38622404},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes ; Gene Editing/methods ; Genetic Engineering/methods ; Cytokines/genetics ; },
abstract = {As part of the adaptive immune system, T cells are critical to maintain immune homeostasis. T cells provide protective immunity by killing infected cells and combatting cancerous cells. To do so, T cells produce and secrete effector molecules, such as granzymes, perforin, and cytokines such as tumor necrosis factor α and interferon γ. However, in immune suppressive environments, such as tumors, T cells gradually lose the capacity to perform their effector function. One way T cell effector function can be enhanced is through genetic engineering with tools such as clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9). This protocol explains in a step-by-step fashion how to perform a controlled electroporation-based CRISPR experiment to enhance human T cell effector function. Of note, these steps are suitable for CRISPR-mediated genome editing in T cells in general and can thus also be used to study proteins of interest that do not influence T cell effector function.},
}

Humans
*CRISPR-Cas Systems/genetics
*T-Lymphocytes
Gene Editing/methods
Genetic Engineering/methods
Cytokines/genetics

@article {pmid38622403,
year = {2024},
author = {Liao, X and Li, L},
title = {CRISPR-Cas9-Induced Gene Editing in Primary Human Monocytes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2782},
number = {},
pages = {189-193},
pmid = {38622403},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Monocytes/metabolism ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Monocytes play important and diverse roles in both homeostatic and inflammatory immune responses. The CRISPR-Cas9 system in lentiviral vectors has been widely used to manipulate specific genes of immortal monocyte cell lines to study monocyte functions. However, human primary monocytes are refractory to this method with low gene knockout (KO) efficiency. In this chapter, we developed an in vitro gene-editing procedure for primary human monocytes with a consistent and high-gene KO efficiency via a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single-guide RNA (sgRNA). This method can be adapted to study the functions of targeted signaling molecules involved in modulating monocyte polarization in primary human monocytes.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems
Monocytes/metabolism
CRISPR-Associated Protein 9/genetics

@article {pmid38622379,
year = {2024},
author = {Prostova, M and Kanevskaya, A and Panteleev, V and Lisitskaya, L and Perfilova Tugaeva, KV and Sluchanko, NN and Esyunina, D and Kulbachinskiy, A},
title = {DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {38622379},
issn = {2058-5276},
support = {22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; },
abstract = {Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.},
}

@article {pmid38622229,
year = {2024},
author = {Xiang, X and Yang, H and Yuan, X and Dong, X and Mai, S and Zhang, Q and Chen, L and Cao, D and Chen, H and Guo, W and Li, L},
title = {CRISPR/Cas9-mediated editing of GmDWF1 brassinosteroid biosynthetic gene induces dwarfism in soybean.},
journal = {Plant cell reports},
volume = {43},
number = {5},
pages = {116},
pmid = {38622229},
issn = {1432-203X},
support = {32071926//The National Natural Science Foundation of China/ ; 32072087//The National Natural Science Foundation of China/ ; CAAS-OCRI-XKPY-202103//Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences/ ; },
mesh = {*Brassinosteroids/metabolism ; Glycine max/genetics ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; *Arabidopsis/metabolism ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; },
abstract = {The study on the GmDWF1-deficient mutant dwf1 showed that GmDWF1 plays a crucial role in determining soybean plant height and yield by influencing the biosynthesis of brassinosteroids. Soybean has not adopted the Green Revolution, such as reduced height for increased planting density, which have proven beneficial for cereal crops. Our research identified the soybean genes GmDWF1a and GmDWF1b, homologous to Arabidopsis AtDWF1, and found that they are widely expressed, especially in leaves, and linked to the cellular transport system, predominantly within the endoplasmic reticulum and intracellular vesicles. These genes are essential for the synthesis of brassinosteroids (BR). Single mutants of GmDWF1a and GmDWF1b, as well as double mutants of both genes generated through CRISPR/Cas9 genome editing, exhibit a dwarf phenotype. The single-gene mutant exhibits moderate dwarfism, while the double mutant shows more pronounced dwarfism. Despite the reduced stature, all types of mutants preserve their node count. Notably, field tests have shown that the single GmDWF1a mutant produced significantly more pods than wild-type plants. Spraying exogenous brassinolide (BL) can compensate for the loss in plant height induced by the decrease in endogenous BRs. Comparing transcriptome analyses of the GmDWF1a mutant and wild-type plants revealed a significant impact on the expression of many genes that influence soybean growth. Identifying the GmDWF1a and GmDWF1b genes could aid in the development of compact, densely planted soybean varieties, potentially boosting productivity.},
}

*Brassinosteroids/metabolism
Glycine max/genetics
CRISPR-Cas Systems/genetics
Mutation/genetics
*Arabidopsis/metabolism
Gene Editing
Gene Expression Regulation, Plant/genetics

@article {pmid38622170,
year = {2024},
author = {Lee, SH and Wang, CY and Li, IJ and Abe, G and Ota, KG},
title = {Exploring the origin of a unique mutant allele in twin-tail goldfish using CRISPR/Cas9 mutants.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8716},
pmid = {38622170},
issn = {2045-2322},
support = {112-2311-B-001-033//National Science and Technology Council/ ; },
mesh = {Animals ; *Goldfish/genetics ; Alleles ; *CRISPR-Cas Systems ; Biological Evolution ; Mutation ; Phenotype ; Animals, Domestic/genetics ; },
abstract = {Artificial selection has been widely applied to genetically fix rare phenotypic features in ornamental domesticated animals. For many of these animals, the mutated loci and alleles underlying rare phenotypes are known. However, few studies have explored whether these rare genetic mutations might have been fixed due to competition among related mutated alleles or if the fixation occurred due to contingent stochastic events. Here, we performed genetic crossing with twin-tail ornamental goldfish and CRISPR/Cas9-mutated goldfish to investigate why only a single mutated allele-chdS with a E127X stop codon (also called chdA[E127X])-gives rise to the twin-tail phenotype in the modern domesticated goldfish population. Two closely related chdS mutants were generated with CRISPR/Cas9 and compared with the E127X allele in F2 and F3 generations. Both of the CRISPR/Cas9-generated alleles were equivalent to the E127X allele in terms of penetrance/expressivity of the twin-tail phenotype and viability of carriers. These findings indicate that multiple truncating mutations could have produced viable twin-tail goldfish. Therefore, the absence of polymorphic alleles for the twin-tail phenotype in modern goldfish likely stems from stochastic elimination or a lack of competing alleles in the common ancestor. Our study is the first experimental comparison of a singular domestication-derived allele with CRISPR/Cas9-generated alleles to understand how genetic fixation of a unique genotype and phenotype may have occurred. Thus, our work may provide a conceptual framework for future investigations of rare evolutionary events in domesticated animals.},
}

Animals
*Goldfish/genetics
Alleles
*CRISPR-Cas Systems
Biological Evolution
Mutation
Phenotype
Animals, Domestic/genetics

@article {pmid38621214,
year = {2024},
author = {Srivastava, R and Davison, CW and Krull, AG and Entriken, SM and Zumbrock, A and Cortes Hidalgo, MD and Adair, KJ and Escherich, AM and Lara, JN and Neverman, EC and Hodnefield, M and McElligtot, E and Sandquist, EJ and Ogilvie, C and Lafontant, P and Essner, JJ},
title = {An Undergraduate Course in CRISPR/Cas9-Mediated Gene Editing in Zebrafish.},
journal = {Zebrafish},
volume = {21},
number = {2},
pages = {162-170},
doi = {10.1089/zeb.2023.0091},
pmid = {38621214},
issn = {1557-8542},
mesh = {Humans ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Zebrafish/genetics ; RNA, Guide, CRISPR-Cas Systems ; Students ; },
abstract = {We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.},
}

Humans
Animals
*Gene Editing/methods
*CRISPR-Cas Systems
Zebrafish/genetics
RNA, Guide, CRISPR-Cas Systems
Students

@article {pmid38621205,
year = {2024},
author = {Davison, C and Harzman, H and Nicholson, J and Entriken, S and Mobley, K and Krull, A and Singhal, M and Skow, C and Matthews, N and Kopp, L and Gillette, B and Weide, TJ and Hukvari, JR and Stumpf, SCP and Feldmann, OM and McGrail, M and Srivastava, R and Essner, JJ},
title = {Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms.},
journal = {Zebrafish},
volume = {21},
number = {2},
pages = {191-197},
doi = {10.1089/zeb.2023.0096},
pmid = {38621205},
issn = {1557-8542},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; Red Fluorescent Protein ; Biotin/genetics ; Endothelial Cells ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFP[is86] line to examine the behavior and interactions between cells during vascular formation in zebrafish.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
Red Fluorescent Protein
Biotin/genetics
Endothelial Cells
RNA, Guide, CRISPR-Cas Systems

@article {pmid38616491,
year = {2024},
author = {Tang, X and Ren, Q and Yan, X and Zhang, R and Liu, L and Han, Q and Zheng, X and Qi, Y and Song, H and Zhang, Y},
title = {Boosting genome editing in plants with single transcript unit surrogate reporter systems.},
journal = {Plant communications},
volume = {},
number = {},
pages = {100921},
doi = {10.1016/j.xplc.2024.100921},
pmid = {38616491},
issn = {2590-3462},
abstract = {CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low editing activity complicates the identification of edited events. In this study, we introduce multiple Single Transcript Unit Surrogate Reporter (STU-SR) systems to enhance the selection of genome-edited plants. These systems utilize the same sgRNAs designed for endogenous genes to edit reporter genes, establishing a direct link between reporter gene editing activity and that of endogenous genes. Various strategies are employed to restore functional reporter genes post-genome editing, including efficient single strand annealing (SSA) for homologous recombination in STU-SR-SSA systems. STU-SR-BE systems leverage base editing to reinstate the start codon, enriching C-to-T and A-to-G base editing events. Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine base editing, and adenine base editing. The systems exhibit compatibility with Cas9 variants, such as the PAM-less SpRY, and are demonstrated to boost genome editing in Brassica oleracea, a dicot vegetable crop. In summary, we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.},
}

@article {pmid38583236,
year = {2024},
author = {Wong, BL and Mendoza, HG and Jacobsen, CS and Beal, PA},
title = {RNA sequences that direct selective ADAR editing from a SELEX library bearing 8-azanebularine.},
journal = {Bioorganic & medicinal chemistry},
volume = {104},
number = {},
pages = {117700},
doi = {10.1016/j.bmc.2024.117700},
pmid = {38583236},
issn = {1464-3391},
mesh = {Humans ; Adenosine ; *Adenosine Deaminase/metabolism ; Base Sequence ; *Purine Nucleosides ; *Ribonucleosides ; RNA, Double-Stranded ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs' ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.},
}

Humans
Adenosine
*Adenosine Deaminase/metabolism
Base Sequence
*Purine Nucleosides
*Ribonucleosides
RNA, Double-Stranded
RNA, Guide, CRISPR-Cas Systems

@article {pmid38527624,
year = {2024},
author = {Piñón Hofbauer, J and Guttmann-Gruber, C and Wally, V and Sharma, A and Gratz, IK and Koller, U},
title = {Challenges and progress related to gene editing in rare skin diseases.},
journal = {Advanced drug delivery reviews},
volume = {208},
number = {},
pages = {115294},
doi = {10.1016/j.addr.2024.115294},
pmid = {38527624},
issn = {1872-8294},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Quality of Life ; Skin ; *Skin Diseases/genetics/therapy ; },
abstract = {Genodermatoses represent a large group of inherited skin disorders encompassing clinically-heterogeneous conditions that manifest in the skin and other organs. Depending on disease variant, associated clinical manifestations and secondary complications can severely impact patients' quality of life and currently available treatments are transient and not curative. Multiple emerging approaches using CRISPR-based technologies offer promising prospects for therapy. Here, we explore current advances and challenges related to gene editing in rare skin diseases, including different strategies tailored to mutation type and structural organization of the affected gene, considerations for in vivo and ex vivo applications, the critical issue of delivery into the skin, and immune aspects of therapy. Against the backdrop of a landmark FDA approval for the first re-dosable gene replacement therapy for a rare genetic skin disorder, gene editing approaches are inching closer to the clinics and the possibility of a local permanent cure for patients affected by these disorders.},
}

Humans
*Gene Editing
CRISPR-Cas Systems/genetics
Quality of Life
Skin
*Skin Diseases/genetics/therapy

@article {pmid38517011,
year = {2024},
author = {Lau, MSH and Madika, A and Zhang, Y and Minton, NP},
title = {Parageobacillus thermoglucosidasius Strain Engineering Using a Theophylline Responsive RiboCas for Controlled Gene Expression.},
journal = {ACS synthetic biology},
volume = {13},
number = {4},
pages = {1237-1245},
doi = {10.1021/acssynbio.3c00735},
pmid = {38517011},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Theophylline ; Gene Editing ; Gene Expression ; *Bacillaceae ; },
abstract = {The relentless increase in atmospheric greenhouse gas concentrations as a consequence of the exploitation of fossil resources compels the adoption of sustainable routes to chemical and fuel manufacture based on biological fermentation processes. The use of thermophilic chassis in such processes is an attractive proposition; however, their effective exploitation will require improved genome editing tools. In the case of the industrially relevant chassis Parageobacillus thermoglucosidasius, CRISPR/Cas9-based gene editing has been demonstrated. The constitutive promoter used, however, accentuates the deleterious nature of Cas9, causing decreased transformation and low editing efficiencies, together with an increased likelihood of off-target effects or alternative mutations. Here, we rectify this issue by controlling the expression of Cas9 through the use of a synthetic riboswitch that is dependent on the nonmetabolized, nontoxic, and cheap inducer, theophylline. We demonstrate that the riboswitches are dose-dependent, allowing for controlled expression of the target gene. Through their use, we were then able to address the deleterious nature of Cas9 and produce an inducible system, RiboCas93. The benefits of RiboCas93 were demonstrated by increased transformation efficiency of the editing vectors, improved efficiency in mutant generation (100%), and a reduction of Cas9 toxicity, as indicated by a reduction in the number of single nucleotide polymorphisms (SNPs) observed. This new system provides a quick and efficient way to produce mutants in P. thermoglucosidasius.},
}

*CRISPR-Cas Systems/genetics
*Theophylline
Gene Editing
Gene Expression
*Bacillaceae

@article {pmid38508753,
year = {2024},
author = {Carvajal-Moreno, J and Wang, X and Hernandez, VA and Mondal, M and Zhao, X and Yalowich, JC and Elton, TS},
title = {Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIβ in Human Leukemia K562 Cells: Generation of a Resistance Phenotype.},
journal = {The Journal of pharmacology and experimental therapeutics},
volume = {389},
number = {2},
pages = {186-196},
doi = {10.1124/jpet.123.002038},
pmid = {38508753},
issn = {1521-0103},
mesh = {Humans ; Etoposide/pharmacology ; K562 Cells ; DNA Topoisomerases, Type II/genetics/metabolism ; Mitoxantrone ; CRISPR-Cas Systems/genetics ; Codon, Nonsense ; *Antineoplastic Agents/pharmacology ; *Leukemia ; DNA ; Phenotype ; },
abstract = {DNA topoisomerase IIβ (TOP2β/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2β/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2β/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2β/180, a premature stop codon was generated at the TOP2β/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2β/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2β/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2β/180 gene-edited clones, with one or all four TOP2β/180 alleles mutated, revealed partial or complete loss of TOP2β mRNA/protein, respectively. The loss of TOP2β/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}p
ropionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2β/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.},
}

Humans
Etoposide/pharmacology
K562 Cells
DNA Topoisomerases, Type II/genetics/metabolism
Mitoxantrone
CRISPR-Cas Systems/genetics
Codon, Nonsense
*Antineoplastic Agents/pharmacology
*Leukemia
DNA
Phenotype

@article {pmid38493007,
year = {2024},
author = {Dimitrievska, M and Bansal, D and Vitale, M and Strouboulis, J and Miccio, A and Nicolaides, KH and El Hoss, S and Shangaris, P and Jacków-Malinowska, J},
title = {Revolutionising healing: Gene Editing's breakthrough against sickle cell disease.},
journal = {Blood reviews},
volume = {65},
number = {},
pages = {101185},
doi = {10.1016/j.blre.2024.101185},
pmid = {38493007},
issn = {1532-1681},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems ; *Anemia, Sickle Cell/genetics/therapy ; *Hemoglobinopathies/genetics ; Fetal Hemoglobin/genetics ; },
abstract = {Recent advancements in gene editing illuminate new potential therapeutic approaches for Sickle Cell Disease (SCD), a debilitating monogenic disorder caused by a point mutation in the β-globin gene. Despite the availability of several FDA-approved medications for symptomatic relief, allogeneic hematopoietic stem cell transplantation (HSCT) remains the sole curative option, underscoring a persistent need for novel treatments. This review delves into the growing field of gene editing, particularly the extensive research focused on curing haemoglobinopathies like SCD. We examine the use of techniques such as CRISPR-Cas9 and homology-directed repair, base editing, and prime editing to either correct the pathogenic variant into a non-pathogenic or wild-type one or augment fetal haemoglobin (HbF) production. The article elucidates ways to optimize these tools for efficacious gene editing with minimal off-target effects and offers insights into their effective delivery into cells. Furthermore, we explore clinical trials involving alternative SCD treatment strategies, such as LentiGlobin therapy and autologous HSCT, distilling the current findings. This review consolidates vital information for the clinical translation of gene editing for SCD, providing strategic insights for investigators eager to further the development of gene editing for SCD.},
}

Humans
Gene Editing/methods
CRISPR-Cas Systems
*Anemia, Sickle Cell/genetics/therapy
*Hemoglobinopathies/genetics
Fetal Hemoglobin/genetics

@article {pmid38479385,
year = {2024},
author = {Ma, X and Yin, J and Qiao, L and Wan, H and Liu, X and Zhou, Y and Wu, J and Niu, L and Wu, M and Wang, X and Ye, H},
title = {A programmable targeted protein-degradation platform for versatile applications in mammalian cells and mice.},
journal = {Molecular cell},
volume = {84},
number = {8},
pages = {1585-1600.e7},
doi = {10.1016/j.molcel.2024.02.019},
pmid = {38479385},
issn = {1097-4164},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; CRISPR-Associated Protein 9/genetics/metabolism ; Proteins/metabolism ; Proteolysis ; Mammals/metabolism ; },
abstract = {Myriad physiological and pathogenic processes are governed by protein levels and modifications. Controlled protein activity perturbation is essential to studying protein function in cells and animals. Based on Trim-Away technology, we screened for truncation variants of E3 ubiquitinase Trim21 with elevated efficiency (ΔTrim21) and developed multiple ΔTrim21-based targeted protein-degradation systems (ΔTrim-TPD) that can be transfected into host cells. Three ΔTrim-TPD variants are developed to enable chemical and light-triggered programmable activation of TPD in cells and animals. Specifically, we used ΔTrim-TPD for (1) red-light-triggered inhibition of HSV-1 virus proliferation by degrading the packaging protein gD, (2) for chemical-triggered control of the activity of Cas9/dCas9 protein for gene editing, and (3) for blue-light-triggered degradation of two tumor-associated proteins for spatiotemporal inhibition of melanoma tumor growth in mice. Our study demonstrates that multiple ΔTrim21-based controllable TPD systems provide powerful tools for basic biology research and highlight their potential biomedical applications.},
}

Mice
Animals
*CRISPR-Cas Systems
*Gene Editing
CRISPR-Associated Protein 9/genetics/metabolism
Proteins/metabolism
Proteolysis
Mammals/metabolism

@article {pmid38460553,
year = {2024},
author = {Ren, QW and Liu, TY and Lan, HJ and Li, ZC and Huang, MJ and Zhao, YT and Chen, Y and Liao, LN and Ma, XH and Liu, JZ},
title = {Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {343},
number = {},
pages = {112057},
doi = {10.1016/j.plantsci.2024.112057},
pmid = {38460553},
issn = {1873-2259},
mesh = {*Nicotiana/genetics ; *Arabidopsis/genetics/metabolism ; Indoleacetic Acids/metabolism ; CRISPR-Cas Systems ; Protein Kinases/genetics ; Plants/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.},
}

*Nicotiana/genetics
*Arabidopsis/genetics/metabolism
Indoleacetic Acids/metabolism
CRISPR-Cas Systems
Protein Kinases/genetics
Plants/metabolism
Gene Expression Regulation, Plant

@article {pmid38613390,
year = {2024},
author = {Cardiff, RAL and Faulkner, ID and Beall, JG and Carothers, JM and Zalatan, JG},
title = {CRISPR-Cas tools for simultaneous transcription & translation control in bacteria.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae275},
pmid = {38613390},
issn = {1362-4962},
support = {MCB 2225632//National Science Foundation/ ; },
abstract = {Robust control over gene translation at arbitrary mRNA targets is an outstanding challenge in microbial synthetic biology. The development of tools that can regulate translation will greatly expand our ability to precisely control genes across the genome. In Escherichia coli, most genes are contained in multi-gene operons, which are subject to polar effects where targeting one gene for repression leads to silencing of other genes in the same operon. These effects pose a challenge for independently regulating individual genes in multi-gene operons. Here, we use CRISPR-dCas13 to address this challenge. We find dCas13-mediated repression exhibits up to 6-fold lower polar effects compared to dCas9. We then show that we can selectively activate single genes in a synthetic multi-gene operon by coupling dCas9 transcriptional activation of an operon with dCas13 translational repression of individual genes within the operon. We also show that dCas13 and dCas9 can be multiplexed for improved biosynthesis of a medically-relevant human milk oligosaccharide. Taken together, our findings suggest that combining transcriptional and translational control can access effects that are difficult to achieve with either mode independently. These combined tools for gene regulation will expand our abilities to precisely engineer bacteria for biotechnology and perform systematic genetic screens.},
}

@article {pmid38612794,
year = {2024},
author = {Stahl, F and Evert, BO and Han, X and Breuer, P and Wüllner, U},
title = {Spinocerebellar Ataxia Type 3 Pathophysiology-Implications for Translational Research and Clinical Studies.},
journal = {International journal of molecular sciences},
volume = {25},
number = {7},
pages = {},
pmid = {38612794},
issn = {1422-0067},
mesh = {Humans ; Animals ; *Machado-Joseph Disease/genetics ; Translational Research, Biomedical ; *Spinocerebellar Ataxias/genetics ; Translational Science, Biomedical ; Animals, Genetically Modified ; },
abstract = {The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado-Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP-T2A-luciferase (LUC) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed.},
}

Humans
Animals
*Machado-Joseph Disease/genetics
Translational Research, Biomedical
*Spinocerebellar Ataxias/genetics
Translational Science, Biomedical
Animals, Genetically Modified

@article {pmid38609947,
year = {2024},
author = {Im, SH and Jang, M and Park, JH and Chung, HJ},
title = {Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing.},
journal = {Journal of nanobiotechnology},
volume = {22},
number = {1},
pages = {175},
pmid = {38609947},
issn = {1477-3155},
support = {2021R1A2C2011763//National Research Foundation of Korea/ ; 2021R1A2C2011763//National Research Foundation of Korea/ ; HI22C2010//Ministry of Health and Welfare/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Cell Line ; Ribonucleoproteins/genetics ; *Liposomes ; *Nanoparticles ; },
abstract = {Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.},
}

*Gene Editing
*CRISPR-Cas Systems
Cell Line
Ribonucleoproteins/genetics
*Liposomes
*Nanoparticles

@article {pmid38609863,
year = {2024},
author = {Chen, X and Zhong, S and Zhan, Y and Zhang, X},
title = {CRISPR-Cas9 applications in T cells and adoptive T cell therapies.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {52},
pmid = {38609863},
issn = {1689-1392},
support = {82203862//National Natural Science Foundation of China/ ; 222300420344//Natural Science Foundation of Henan Province/ ; SBGJ202102140//Medical Science Technology Program of Henan/ ; LHGJ20200273//Medical Science Technology Program of Henan/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes ; Cell Differentiation ; Inflammation ; Lymphocyte Activation ; },
abstract = {T cell immunity is central to contemporary cancer and autoimmune therapies, encompassing immune checkpoint blockade and adoptive T cell therapies. Their diverse characteristics can be reprogrammed by different immune challenges dependent on antigen stimulation levels, metabolic conditions, and the degree of inflammation. T cell-based therapeutic strategies are gaining widespread adoption in oncology and treating inflammatory conditions. Emerging researches reveal that clustered regularly interspaced palindromic repeats-associated protein 9 (CRISPR-Cas9) genome editing has enabled T cells to be more adaptable to specific microenvironments, opening the door to advanced T cell therapies in preclinical and clinical trials. CRISPR-Cas9 can edit both primary T cells and engineered T cells, including CAR-T and TCR-T, in vivo and in vitro to regulate T cell differentiation and activation states. This review first provides a comprehensive summary of the role of CRISPR-Cas9 in T cells and its applications in preclinical and clinical studies for T cell-based therapies. We also explore the application of CRISPR screen high-throughput technology in editing T cells and anticipate the current limitations of CRISPR-Cas9, including off-target effects and delivery challenges, and envisioned improvements in related technologies for disease screening, diagnosis, and treatment.},
}

Humans
*CRISPR-Cas Systems/genetics
*T-Lymphocytes
Cell Differentiation
Inflammation
Lymphocyte Activation

@article {pmid38609574,
year = {2024},
author = {De Castro, V and Galaine, J and Loyon, R and Godet, Y},
title = {CRISPR-Cas gene knockouts to optimize engineered T cells for cancer immunotherapy.},
journal = {Cancer gene therapy},
volume = {},
number = {},
pages = {},
pmid = {38609574},
issn = {1476-5500},
support = {2022-0047//Ligue Contre le Cancer/ ; },
abstract = {While CAR-T and tgTCR-T therapies have exhibited noteworthy and promising outcomes in hematologic and solid tumors respectively, a set of distinct challenges remains. Consequently, the quest for novel strategies has become imperative to safeguard and more effectively release the full functions of engineered T cells. These factors are intricately linked to the success of adoptive cell therapy. Recently, CRISPR-based technologies have emerged as a major breakthrough for maintaining T cell functions. These technologies have allowed the discovery of T cells' negative regulators such as specific cell-surface receptors, cell-signaling proteins, and transcription factors that are involved in the development or maintenance of T cell dysfunction. By employing a CRISPR-genic invalidation approach to target these negative regulators, it has become possible to prevent the emergence of hypofunctional T cells. This review revisits the establishment of the dysfunctional profile of T cells before delving into a comprehensive summary of recent CRISPR-gene invalidations, with each invalidation contributing to the enhancement of engineered T cells' antitumor capacities. The narrative unfolds as we explore how these advancements were discovered and identified, marking a significant advancement in the pursuit of superior adoptive cell therapy.},
}

@article {pmid38609487,
year = {2024},
author = {Naveed, M and Tahir, F and Aziz, T and Waseem, M and Makhdoom, SI and Ali, N and Alharbi, M and Albekairi, TH and Alasmari, AF},
title = {Molecular identification of Proteus mirabilis, Vibrio species leading to CRISPR-Cas9 modification of tcpA and UreC genes causing cholera and UTI.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8563},
pmid = {38609487},
issn = {2045-2322},
mesh = {Humans ; *Cholera ; Proteus mirabilis/genetics ; Cadmium/toxicity ; CRISPR-Cas Systems/genetics ; RNA, Ribosomal, 16S ; Wastewater ; RNA, Guide, CRISPR-Cas Systems ; *Vibrio/genetics ; *Urinary Tract Infections ; },
abstract = {Heavy metal accumulation increases rapidly in the environment due to anthropogenic activities and industrialization. The leather and surgical industry produces many contaminants containing heavy metals. Cadmium, a prominent contaminant, is linked to severe health risks, notably kidney and liver damage, especially among individuals exposed to contaminated wastewater. This study aims to leverage the natural cadmium resistance mechanisms in bacteria for bioaccumulation purposes. The industrial wastewater samples, characterized by an alarming cadmium concentration of 29.6 ppm, 52 ppm, and 76.4 ppm-far exceeding the recommended limit of 0.003 ppm-were subjected to screening for cadmium-resistant bacteria using cadmium-supplemented media with CdCl2. 16S rRNA characterization identified Vibrio cholerae and Proteus mirabilis as cadmium-resistant bacteria in the collected samples. Subsequently, the cadmium resistance-associated cadA gene was successfully amplified in Vibrio species and Proteus mirabilis, revealing a product size of 623 bp. Further analysis of the identified bacteria included the examination of virulent genes, specifically the tcpA gene (472 bp) associated with cholera and the UreC gene (317 bp) linked to urinary tract infections. To enhance the bioaccumulation of cadmium, the study proposes the potential suppression of virulent gene expression through in-silico gene-editing tools such as CRISPR-Cas9. A total of 27 gRNAs were generated for UreC, with five selected for expression. Similarly, 42 gRNA sequences were generated for tcpA, with eight chosen for expression analysis. The selected gRNAs were integrated into the lentiCRISPR v2 expression vector. This strategic approach aims to facilitate precise gene editing of disease-causing genes (tcpA and UreC) within the bacterial genome. In conclusion, this study underscores the potential utility of Vibrio species and Proteus mirabilis as effective candidates for the removal of cadmium from industrial wastewater, offering insights for future environmental remediation strategies.},
}

Humans
*Cholera
Proteus mirabilis/genetics
Cadmium/toxicity
CRISPR-Cas Systems/genetics
RNA, Ribosomal, 16S
Wastewater
RNA, Guide, CRISPR-Cas Systems
*Vibrio/genetics
*Urinary Tract Infections

@article {pmid38609352,
year = {2024},
author = {McLean, ZL and Gao, D and Correia, K and Roy, JCL and Shibata, S and Farnum, IN and Valdepenas-Mellor, Z and Kovalenko, M and Rapuru, M and Morini, E and Ruliera, J and Gillis, T and Lucente, D and Kleinstiver, BP and Lee, JM and MacDonald, ME and Wheeler, VC and Mouro Pinto, R and Gusella, JF},
title = {Splice modulators target PMS1 to reduce somatic expansion of the Huntington's disease-associated CAG repeat.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3182},
pmid = {38609352},
issn = {2041-1723},
support = {NS091161//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS105709//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS119471//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS049206//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS126420//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; DP2-CA281401//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Huntington Disease/genetics ; Exons/genetics ; Gene Expression Profiling ; Heterozygote ; Homozygote ; MutL Proteins ; Neoplasm Proteins ; },
abstract = {Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.},
}

Humans
*Huntington Disease/genetics
Exons/genetics
Gene Expression Profiling
Heterozygote
Homozygote
MutL Proteins
Neoplasm Proteins