@article {pmid38675875,
year = {2024},
author = {Blanchet, M and Angelo, L and Tétreault, Y and Khabir, M and Sureau, C and Vaillant, A and Labonté, P},
title = {HepG2BD: A Novel and Versatile Cell Line with Inducible HDV Replication and Constitutive HBV Expression.},
journal = {Viruses},
volume = {16},
number = {4},
pages = {},
pmid = {38675875},
issn = {1999-4915},
support = {558342-2020//Natural Sciences and Engineering Research Council/ ; },
mesh = {*Hepatitis Delta Virus/genetics/physiology ; Humans ; *Virus Replication ; *Hepatitis B virus/genetics/physiology ; Hep G2 Cells ; Hepatocytes/virology ; Hepatitis D/virology ; CRISPR-Cas Systems ; Dependovirus/genetics ; Coinfection/virology ; },
abstract = {Individuals chronically infected with hepatitis B virus (HBV) and hepatitis Delta virus (HDV) present an increased risk of developing cirrhosis and hepatocellular carcinoma in comparison to HBV mono-infected individuals. Although HDV only replicates in individuals coinfected or superinfected with HBV, there is currently no in vitro model that can stably express both viruses simultaneously, mimicking the chronic infections seen in HBV/HDV patients. Here, we present the HepG2BD cell line as a novel in vitro culture system for long-term replication of HBV and HDV. HepG2BD cells derive from HepG2.2.15 cells in which a 2 kb HDV cDNA sequence was inserted into the adeno-associated virus safe harbor integration site 1 (AAVS1) using CRISPR-Cas9. A Tet-Off promoter was placed 5' of the genomic HDV sequence for reliable initiation/repression of viral replication and secretion. HBV and HDV replication were then thoroughly characterized. Of note, non-dividing cells adopt a hepatocyte-like morphology associated with an increased production of both HDV and HBV virions. Finally, HDV seems to negatively interfere with HBV in this model system. Altogether, HepG2BD cells will be instrumental to evaluate, in vitro, the fundamental HBV-HDV interplay during simultaneous chronic replication as well as for antivirals screening targeting both viruses.},
}

*Hepatitis Delta Virus/genetics/physiology
Humans
*Virus Replication
*Hepatitis B virus/genetics/physiology
Hep G2 Cells
Hepatocytes/virology
Hepatitis D/virology
CRISPR-Cas Systems
Dependovirus/genetics
Coinfection/virology

@article {pmid38675868,
year = {2024},
author = {Li, B and Wang, D and Xie, X and Chen, X and Liang, G and Xing, D and Zhao, T and Wu, J and Zhou, X and Li, C},
title = {Mosquito E-20-Monooxygenase Gene Knockout Increases Dengue Virus Replication in Aedes aegypti Cells.},
journal = {Viruses},
volume = {16},
number = {4},
pages = {},
pmid = {38675868},
issn = {1999-4915},
support = {2019BJRC05//Young Talents Project 2019/ ; },
mesh = {Animals ; *Virus Replication/genetics ; *Aedes/virology/genetics ; *Dengue Virus/genetics/physiology ; *Gene Knockout Techniques ; Cell Line ; Cytochrome P-450 Enzyme System/genetics/metabolism ; Insect Proteins/genetics/metabolism ; Mosquito Vectors/virology/genetics ; CRISPR-Cas Systems ; Dengue/virology ; },
abstract = {E-20-monooxygenase (E20MO) is an enzymatic product of the shade (shd) locus (cytochrome p450, E20MO). Initially discovered in Drosophila, E20MO facilitates the conversion of ecdysone (E) into 20-hydroxyecdysone (20E) and is crucial for oogenesis. Prior research has implicated 20E in growth, development, and insecticide resistance. However, little attention has been given to the association between the E20MO gene and DENV2 infection. The transcriptome of Ae. aegypti cells (Aag2 cells) infected with DENV2 revealed the presence of the E20MO gene. The subsequent quantification of E20MO gene expression levels in Aag2 cells post-DENV infection was carried out. A CRISPR/Cas9 system was utilized to create an E20MO gene knockout cell line (KO), which was then subjected to DENV infection. Analyses of DENV2 copies in KO and wild-type (WT) cells were conducted at different days post-infection (dpi). Plasmids containing E20MO were constructed and transfected into KO cells, with pre- and post-transfection viral copy comparisons. Gene expression levels of E20MO increased after DENV infection. Subsequently, a successful generation of an E20MO gene knockout cell line and the verification of code-shifting mutations at both DNA and RNA levels were achieved. Furthermore, significantly elevated DENV2 RNA copies were observed in the mid-infection phase for the KO cell line. Viral RNA copies were lower in cells transfected with plasmids containing E20MO, compared to KO cells. Through knockout and plasmid complementation experiments in Aag2 cells, the role of E20MO in controlling DENV2 replication was demonstrated. These findings contribute to our understanding of the intricate biological interactions between mosquitoes and arboviruses.},
}

Animals
*Virus Replication/genetics
*Aedes/virology/genetics
*Dengue Virus/genetics/physiology
*Gene Knockout Techniques
Cell Line
Cytochrome P-450 Enzyme System/genetics/metabolism
Insect Proteins/genetics/metabolism
Mosquito Vectors/virology/genetics
CRISPR-Cas Systems
Dengue/virology

@article {pmid38674012,
year = {2024},
author = {Zhu, W and Xie, H and Chen, Y and Zhang, G},
title = {CrnnCrispr: An Interpretable Deep Learning Method for CRISPR/Cas9 sgRNA On-Target Activity Prediction.},
journal = {International journal of molecular sciences},
volume = {25},
number = {8},
pages = {},
pmid = {38674012},
issn = {1422-0067},
support = {62103249//National Natural Science Foundation of China/ ; 2022A1515011720//Guangdong Basic and Applied Basic Research Foundation/ ; NTF20032//STU Scientific Research Foundation for Talents/ ; },
mesh = {*CRISPR-Cas Systems ; *Deep Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Neural Networks, Computer ; },
abstract = {CRISPR/Cas9 is a powerful genome-editing tool in biology, but its wide applications are challenged by a lack of knowledge governing single-guide RNA (sgRNA) activity. Several deep-learning-based methods have been developed for the prediction of on-target activity. However, there is still room for improvement. Here, we proposed a hybrid neural network named CrnnCrispr, which integrates a convolutional neural network and a recurrent neural network for on-target activity prediction. We performed unbiased experiments with four mainstream methods on nine public datasets with varying sample sizes. Additionally, we incorporated a transfer learning strategy to boost the prediction power on small-scale datasets. Our results showed that CrnnCrispr outperformed existing methods in terms of accuracy and generalizability. Finally, we applied a visualization approach to investigate the generalizable nucleotide-position-dependent patterns of sgRNAs for on-target activity, which shows potential in terms of model interpretability and further helps in understanding the principles of sgRNA design.},
}

*CRISPR-Cas Systems
*Deep Learning
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
*Neural Networks, Computer

@article {pmid38672502,
year = {2024},
author = {Tan, LL and Heng, E and Leong, CY and Ng, V and Yang, LK and Seow, DCS and Koduru, L and Kanagasundaram, Y and Ng, SB and Peh, G and Lim, YH and Wong, FT},
title = {Application of Cas12j for Streptomyces Editing.},
journal = {Biomolecules},
volume = {14},
number = {4},
pages = {},
pmid = {38672502},
issn = {2218-273X},
support = {NRF-CRP19-2017-05//National Research Foundation/ ; C211917003//Agency for Science, Technology and Research/ ; C233017003//Agency for Science, Technology and Research/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Acidaminococcus/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Multigene Family ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Genome, Bacterial ; },
abstract = {In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.},
}

*Streptomyces/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Acidaminococcus/genetics
CRISPR-Associated Protein 9/genetics/metabolism
Multigene Family
Bacterial Proteins/genetics/metabolism
CRISPR-Associated Proteins/genetics/metabolism
Genome, Bacterial

@article {pmid38671524,
year = {2024},
author = {Mu, S and Chen, H and Li, Q and Gou, S and Liu, X and Wang, J and Zheng, W and Chen, M and Jin, Q and Lai, L and Wang, K and Shi, H},
title = {Enhancing prime editor flexibility with coiled-coil heterodimers.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {108},
pmid = {38671524},
issn = {1474-760X},
support = {2023YFF0724703//National Key Research and Development Program of China/ ; 2022YFA1105403//National Key Research and Development Program of China/ ; 32200418//National Natural Science Foundation of China/ ; 32200417//National Natural Science Foundation of China/ ; ZDKJ2021030//Major Science and Technology Project of Hainan Province/ ; 2019-I2M-5-025//Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences/ ; 2022M723170//China Postdoctoral Science Foundation/ ; 2023B1212060050//Science and Technology Planning Project of Guangdong Province/ ; 2021B1212040016//Science and Technology Planning Project of Guangdong Province/ ; 2021A1515110838//Science and Technology Planning Project of Guangdong Province/ ; 2023A1515011312//Science and Technology Planning Project of Guangdong Province/ ; 202201010621//Science and Technology Program of Guangzhou, China/ ; },
mesh = {*Gene Editing ; Humans ; Animals ; Mice ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Dependovirus/genetics ; },
abstract = {BACKGROUND: Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency.

RESULTS: Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol.

CONCLUSIONS: Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.},
}

*Gene Editing
Humans
Animals
Mice
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems
HEK293 Cells
Dependovirus/genetics

@article {pmid38670972,
year = {2024},
author = {Andreu-Saumell, I and Rodriguez-Garcia, A and Mühlgrabner, V and Gimenez-Alejandre, M and Marzal, B and Castellsagué, J and Brasó-Maristany, F and Calderon, H and Angelats, L and Colell, S and Nuding, M and Soria-Castellano, M and Barbao, P and Prat, A and Urbano-Ispizua, A and Huppa, JB and Guedan, S},
title = {CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3552},
pmid = {38670972},
issn = {2041-1723},
support = {LCF/PR/SP23/52950004//"la Caixa" Foundation (Caixa Foundation)/ ; LABAE20022GUED//Fundación Científica Asociación Española Contra el Cáncer (Scientific Foundation, Spanish Association Against Cancer)/ ; INVES222988RODR//Fundación Científica Asociación Española Contra el Cáncer (Scientific Foundation, Spanish Association Against Cancer)/ ; 116026//Innovative Medicines Initiative (IMI)/ ; },
mesh = {*Receptors, Chimeric Antigen/immunology/metabolism ; *Programmed Cell Death 1 Receptor/metabolism/immunology ; *B7-H1 Antigen/metabolism/immunology ; Animals ; Humans ; *Immunotherapy, Adoptive/methods ; Mice ; Cell Line, Tumor ; *T-Lymphocytes/immunology/metabolism ; Receptor, ErbB-2/metabolism/immunology ; Xenograft Model Antitumor Assays ; Female ; CRISPR-Cas Systems ; Mice, Inbred NOD ; },
abstract = {Chimeric antigen receptor (CAR)-T cell therapy for solid tumors faces significant hurdles, including T-cell inhibition mediated by the PD-1/PD-L1 axis. The effects of disrupting this pathway on T-cells are being actively explored and controversial outcomes have been reported. Here, we hypothesize that CAR-antigen affinity may be a key factor modulating T-cell susceptibility towards the PD-1/PD-L1 axis. We systematically interrogate CAR-T cells targeting HER2 with either low (LA) or high affinity (HA) in various preclinical models. Our results reveal an increased sensitivity of LA CAR-T cells to PD-L1-mediated inhibition when compared to their HA counterparts by using in vitro models of tumor cell lines and supported lipid bilayers modified to display varying PD-L1 densities. CRISPR/Cas9-mediated knockout (KO) of PD-1 enhances LA CAR-T cell cytokine secretion and polyfunctionality in vitro and antitumor effect in vivo and results in the downregulation of gene signatures related to T-cell exhaustion. By contrast, HA CAR-T cell features remain unaffected following PD-1 KO. This behavior holds true for CD28 and ICOS but not 4-1BB co-stimulated CAR-T cells, which are less sensitive to PD-L1 inhibition albeit targeting the antigen with LA. Our findings may inform CAR-T therapies involving disruption of PD-1/PD-L1 pathway tailored in particular for effective treatment of solid tumors.},
}

*Receptors, Chimeric Antigen/immunology/metabolism
*Programmed Cell Death 1 Receptor/metabolism/immunology
*B7-H1 Antigen/metabolism/immunology
Animals
Humans
*Immunotherapy, Adoptive/methods
Mice
Cell Line, Tumor
*T-Lymphocytes/immunology/metabolism
Receptor, ErbB-2/metabolism/immunology
Xenograft Model Antitumor Assays
Female
CRISPR-Cas Systems
Mice, Inbred NOD

@article {pmid38670178,
year = {2024},
author = {Bisht, D and Salave, S and Desai, N and Gogoi, P and Rana, D and Biswal, P and Sarma, G and Benival, D and Kommineni, N and Desai, D},
title = {Genome editing and its role in vaccine, diagnosis, and therapeutic advancement.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {131802},
doi = {10.1016/j.ijbiomac.2024.131802},
pmid = {38670178},
issn = {1879-0003},
abstract = {Genome editing involves precise modification of specific nucleotides in the genome using nucleases like CRISPR/Cas, ZFN, or TALEN, leading to increased efficiency of homologous recombination (HR) for gene editing, and it can result in gene disruption events via non-homologous end joining (NHEJ) or homology-driven repair (HDR). Genome editing, particularly CRISPR-Cas9, revolutionizes vaccine development by enabling precise modifications of pathogen genomes, leading to enhanced vaccine efficacy and safety. It allows for tailored antigen optimization, improved vector design, and deeper insights into host genes' impact on vaccine responses, ultimately enhancing vaccine development and manufacturing processes. This review highlights different types of genomes editing methods, their associated risks, approaches to overcome the shortcomings, and the diverse roles of genome editing.},
}

@article {pmid38589619,
year = {2024},
author = {Li, JH and Zhou, A and Lee, CD and Shah, SN and Ji, JH and Senthilkumar, V and Padilla, ET and Ball, AB and Feng, Q and Bustillos, CG and Riggan, L and Greige, A and Divakaruni, AS and Annese, F and Cooley Coleman, JA and Skinner, SA and Cowan, CW and O'Sullivan, TE},
title = {MEF2C regulates NK cell effector functions through control of lipid metabolism.},
journal = {Nature immunology},
volume = {25},
number = {5},
pages = {778-789},
pmid = {38589619},
issn = {1529-2916},
support = {R35 GM138003/GM/NIGMS NIH HHS/United States ; },
mesh = {*MEF2 Transcription Factors/metabolism/genetics ; *Killer Cells, Natural/immunology/metabolism ; Animals ; Humans ; *Lipid Metabolism ; Mice ; CRISPR-Cas Systems ; Mice, Knockout ; Interleukin-15/metabolism ; Mice, Inbred C57BL ; },
abstract = {Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR-Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism.},
}

*MEF2 Transcription Factors/metabolism/genetics
*Killer Cells, Natural/immunology/metabolism
Animals
Humans
*Lipid Metabolism
Mice
CRISPR-Cas Systems
Mice, Knockout
Interleukin-15/metabolism
Mice, Inbred C57BL

@article {pmid38579728,
year = {2024},
author = {Han, R},
title = {Hit-and-run epigenome editing durably lowers cholesterol in mice.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {5},
pages = {1190-1191},
doi = {10.1016/j.ymthe.2024.03.027},
pmid = {38579728},
issn = {1525-0024},
mesh = {Animals ; Mice ; *Gene Editing/methods ; *Cholesterol/metabolism ; CRISPR-Cas Systems ; Epigenome ; Epigenesis, Genetic ; Humans ; Disease Models, Animal ; },
}

Animals
Mice
*Gene Editing/methods
*Cholesterol/metabolism
CRISPR-Cas Systems
Epigenome
Epigenesis, Genetic
Humans
Disease Models, Animal

@article {pmid38466263,
year = {2024},
author = {Kavousinia, P and Ahmadi, MH and Sadeghian, H and Hosseini Bafghi, M},
title = {Therapeutic potential of CRISPR/CAS9 genome modification in T cell-based immunotherapy of cancer.},
journal = {Cytotherapy},
volume = {26},
number = {5},
pages = {436-443},
doi = {10.1016/j.jcyt.2024.02.014},
pmid = {38466263},
issn = {1477-2566},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/immunology/genetics ; *Gene Editing/methods ; *T-Lymphocytes/immunology ; *Immunotherapy, Adoptive/methods ; Animals ; Receptors, Chimeric Antigen/genetics/immunology ; Immunotherapy/methods ; },
abstract = {Today, genome editing technologies like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) are being used in clinical trials and the treatment of diseases like acquired immunodeficiency syndrome (AIDS) and cancer. CRISPR stands out as one of the most advanced tools for genome editing due to its simplicity and cost-effectiveness. It can selectively modify specific locations in the genome, offering new possibilities for treating human diseases. The CRISPR system uses ribonucleic acid-deoxyribonucleic acid (RNA-DNA) recognition to combat infections, regulate gene expression, and treat cancer. Chimeric antigen receptor (CAR) T-cell therapy, which uses T lymphocytes to eliminate cancer cells, can be improved by combining it with CRISPR technology. However, there are challenges in using CAR-T cells, including a lack of quantity and quality, exhaustion, neurotoxicity, cytokine release syndrome (CRS), B cell aplasia, tumor lysis syndrome, and anaphylaxis. Preclinical studies on CRISPR-edited CAR-T cells show promising results and targeting detrimental regulatory genes can enhance cancer treatment in the future.},
}

Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/therapy/immunology/genetics
*Gene Editing/methods
*T-Lymphocytes/immunology
*Immunotherapy, Adoptive/methods
Animals
Receptors, Chimeric Antigen/genetics/immunology
Immunotherapy/methods

@article {pmid38454604,
year = {2024},
author = {Locatelli, F and Cavazzana, M and Frangoul, H and Fuente, J and Algeri, M and Meisel, R},
title = {Autologous gene therapy for hemoglobinopathies: From bench to patient's bedside.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {32},
number = {5},
pages = {1202-1218},
doi = {10.1016/j.ymthe.2024.03.005},
pmid = {38454604},
issn = {1525-0024},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Hemoglobinopathies/therapy/genetics ; *Genetic Vectors/genetics/administration & dosage ; *Clinical Trials as Topic ; Anemia, Sickle Cell/therapy/genetics ; beta-Thalassemia/therapy/genetics ; Animals ; Lentivirus/genetics ; },
abstract = {In recent years, a growing number of clinical trials have been initiated to evaluate gene therapy approaches for the treatment of patients with transfusion-dependent β-thalassemia and sickle cell disease (SCD). Therapeutic modalities being assessed in these trials utilize different molecular techniques, including lentiviral vectors to add functional copies of the gene encoding the hemoglobin β subunit in defective cells and CRISPR-Cas9, transcription activator-like effector protein nuclease, and zinc finger nuclease gene editing strategies to either directly address the underlying genetic cause of disease or induce fetal hemoglobin production by gene disruption. Here, we review the mechanisms of action of these various gene addition and gene editing approaches and describe the status of clinical trials designed to evaluate the potentially for these approaches to provide one-time functional cures to patients with transfusion-dependent β-thalassemia and SCD.},
}

Humans
*Genetic Therapy/methods
*Gene Editing/methods
*CRISPR-Cas Systems
*Hemoglobinopathies/therapy/genetics
*Genetic Vectors/genetics/administration & dosage
*Clinical Trials as Topic
Anemia, Sickle Cell/therapy/genetics
beta-Thalassemia/therapy/genetics
Animals
Lentivirus/genetics

@article {pmid37788869,
year = {2024},
author = {Gratz, S and O'Connor-Giles, KM and Wildonger, J},
title = {Generating CRISPR Alleles in Drosophila.},
journal = {Cold Spring Harbor protocols},
volume = {2024},
number = {5},
pages = {pdb.prot108256},
doi = {10.1101/pdb.prot108256},
pmid = {37788869},
issn = {1559-6095},
support = {R01 NS078179/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Drosophila/genetics ; *Alleles ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR gene editing is a versatile and efficient approach for generating a wide variety of genetic reagents in flies. This unparalleled ability to manipulate the genome has revolutionized neuroscience, allowing Drosophila neurobiologists to readily generate new alleles to probe gene function, investigate the functional consequences of disease-associated variants, tag endogenous proteins to follow their dynamic localization in neurons and glia, and much more. Here, we provide a comprehensive protocol for generating heritable mutations in Drosophila We particularly focus on design considerations and tips for avoiding common errors to maximize the likelihood of successful gene editing.},
}

Animals
*Drosophila/genetics
*Alleles
*Gene Editing/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems/genetics

@article {pmid37788866,
year = {2024},
author = {Gratz, S and O'Connor-Giles, KM and Wildonger, J},
title = {Introduction to CRISPR and Its Use in Drosophila.},
journal = {Cold Spring Harbor protocols},
volume = {2024},
number = {5},
pages = {pdb.top108228},
doi = {10.1101/pdb.top108228},
pmid = {37788866},
issn = {1559-6095},
support = {R01 NS078179/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; *Drosophila/genetics ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {The preeminence of Drosophila genetics has led to key discoveries in biology across a variety of fields and disciplines. The advent of CRISPR gene editing has expanded the toolkit of genetic reagents that can be applied to manipulate and observe genes, RNAs, and proteins in an in vivo context. This review describes CRISPR and its use as a transformative gene editing tool in Drosophila We focus on the canonical pathway in which the Cas9 nuclease is directed to specific sequences by guide RNA (gRNA), where cleavage leads to DNA repair by one of two main cellular pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The error-prone NHEJ pathway can be appropriated to disrupt targeted sequences, enabling a variety of loss-of-function studies. Induction of the HDR pathway allows precise editing, including defined deletions, the introduction of specific sequence changes, and the incorporation of fluorescent and epitope tags. These approaches have increased the power of Drosophila genetics and been successfully used to conduct in vivo structure-function studies, study disease-associated variants, and follow protein dynamics.},
}

Animals
*Gene Editing/methods
*Drosophila/genetics
*CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid37487630,
year = {2024},
author = {Sánchez Rivera, FJ and Dow, LE},
title = {How CRISPR Is Revolutionizing the Generation of New Models for Cancer Research.},
journal = {Cold Spring Harbor perspectives in medicine},
volume = {14},
number = {5},
pages = {},
doi = {10.1101/cshperspect.a041384},
pmid = {37487630},
issn = {2157-1422},
mesh = {Animals ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; *Disease Models, Animal ; Mice ; Humans ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cancers arise through acquisition of mutations in genes that regulate core biological processes like cell proliferation and cell death. Decades of cancer research have led to the identification of genes and mutations causally involved in disease development and evolution, yet defining their precise function across different cancer types and how they influence therapy responses has been challenging. Mouse models have helped define the in vivo function of cancer-associated alterations, and genome-editing approaches using CRISPR have dramatically accelerated the pace at which these models are developed and studied. Here, we highlight how CRISPR technologies have impacted the development and use of mouse models for cancer research and discuss the many ways in which these rapidly evolving platforms will continue to transform our understanding of this disease.},
}

Animals
*Neoplasms/genetics/therapy
*Gene Editing/methods
*Disease Models, Animal
Mice
Humans
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid38670073,
year = {2024},
author = {Lampson, BL and Ramίrez, AS and Baro, M and He, L and Hegde, M and Koduri, V and Pfaff, JL and Hanna, RE and Kowal, J and Shirole, NH and He, Y and Doench, JG and Contessa, JN and Locher, KP and Kaelin, WG},
title = {Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.},
journal = {Cell},
volume = {187},
number = {9},
pages = {2209-2223.e16},
doi = {10.1016/j.cell.2024.03.022},
pmid = {38670073},
issn = {1097-4172},
mesh = {*Hexosyltransferases/metabolism/genetics ; *NF-kappa B/metabolism ; *Membrane Proteins/metabolism/genetics ; Humans ; *Toll-Like Receptor 4/metabolism ; *Signal Transduction ; Animals ; *CRISPR-Cas Systems/genetics ; *Lipopolysaccharides/metabolism/pharmacology ; Mice ; HEK293 Cells ; Inflammation/metabolism/genetics ; Glycosylation ; Cryoelectron Microscopy ; Catalytic Domain ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.},
}

*Hexosyltransferases/metabolism/genetics
*NF-kappa B/metabolism
*Membrane Proteins/metabolism/genetics
Humans
*Toll-Like Receptor 4/metabolism
*Signal Transduction
Animals
*CRISPR-Cas Systems/genetics
*Lipopolysaccharides/metabolism/pharmacology
Mice
HEK293 Cells
Inflammation/metabolism/genetics
Glycosylation
Cryoelectron Microscopy
Catalytic Domain
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid38670071,
year = {2024},
author = {Huang, J and He, B and Yang, X and Long, X and Wei, Y and Li, L and Tang, M and Gao, Y and Fang, Y and Ying, W and Wang, Z and Li, C and Zhou, Y and Li, S and Shi, L and Choi, S and Zhou, H and Guo, F and Yang, H and Wu, J},
title = {Generation of rat forebrain tissues in mice.},
journal = {Cell},
volume = {187},
number = {9},
pages = {2129-2142.e17},
doi = {10.1016/j.cell.2024.03.017},
pmid = {38670071},
issn = {1097-4172},
mesh = {Animals ; *Prosencephalon/metabolism/embryology ; Mice ; Rats ; Blastocyst/metabolism ; Female ; CRISPR-Cas Systems/genetics ; Transcriptome ; Organogenesis ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Male ; Mice, Inbred C57BL ; },
abstract = {Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.},
}

Animals
*Prosencephalon/metabolism/embryology
Mice
Rats
Blastocyst/metabolism
Female
CRISPR-Cas Systems/genetics
Transcriptome
Organogenesis
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Male
Mice, Inbred C57BL

@article {pmid38668730,
year = {2024},
author = {Ates, I and Stuart, C and Rathbone, T and Barzi, M and He, G and Major, AM and Shankar, V and Lyman, RA and Angner, SS and Mackay, TFC and Srinivasan, S and Farris, AB and Bissig, KD and Cottle, RN},
title = {Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.},
journal = {Hepatology communications},
volume = {8},
number = {5},
pages = {},
pmid = {38668730},
issn = {2471-254X},
mesh = {Animals ; *Tyrosinemias/therapy/genetics ; *Gene Editing/methods ; Mice ; *Hepatocytes/transplantation/metabolism ; *Hydrolases/genetics ; *Mice, Inbred C57BL ; Cell- and Tissue-Based Therapy/methods ; CRISPR-Cas Systems ; Electroporation/methods ; Mice, Knockout ; 4-Hydroxyphenylpyruvate Dioxygenase/genetics ; Disease Models, Animal ; Cyclohexanones ; Nitrobenzoates ; },
abstract = {BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1.

METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice.

RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice.

CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.},
}

Animals
*Tyrosinemias/therapy/genetics
*Gene Editing/methods
Mice
*Hepatocytes/transplantation/metabolism
*Hydrolases/genetics
*Mice, Inbred C57BL
Cell- and Tissue-Based Therapy/methods
CRISPR-Cas Systems
Electroporation/methods
Mice, Knockout
4-Hydroxyphenylpyruvate Dioxygenase/genetics
Disease Models, Animal
Cyclohexanones
Nitrobenzoates

@article {pmid38667802,
year = {2024},
author = {Græsholt, C and Brembu, T and Volpe, C and Bartosova, Z and Serif, M and Winge, P and Nymark, M},
title = {Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.},
journal = {Marine drugs},
volume = {22},
number = {4},
pages = {},
pmid = {38667802},
issn = {1660-3397},
support = {344103//The Research Council of Norway/ ; },
mesh = {*Diatoms/genetics ; *Xanthophylls/metabolism ; *Oxidoreductases/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques/methods ; Carotenoids/metabolism ; Microalgae/genetics ; Mutation ; },
abstract = {Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.},
}

*Diatoms/genetics
*Xanthophylls/metabolism
*Oxidoreductases/genetics/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques/methods
Carotenoids/metabolism
Microalgae/genetics
Mutation

@article {pmid38667326,
year = {2024},
author = {Wils, LJ and Buijze, M and Stigter-van Walsum, M and Brink, A and van Kempen, BE and Peferoen, L and Brouns, ER and de Visscher, JGAM and van der Meij, EH and Bloemena, E and Poell, JB and Brakenhoff, RH},
title = {Genomic Engineering of Oral Keratinocytes to Establish In Vitro Oral Potentially Malignant Disease Models as a Platform for Treatment Investigation.},
journal = {Cells},
volume = {13},
number = {8},
pages = {},
pmid = {38667326},
issn = {2073-4409},
support = {Unknown//Hanarth Fonds/ ; },
mesh = {Humans ; *Keratinocytes/metabolism/pathology ; *Mouth Neoplasms/genetics/pathology ; *Leukoplakia, Oral/genetics/pathology ; Telomerase/genetics/metabolism ; Genetic Engineering ; Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Tumor Suppressor Protein p53/metabolism/genetics ; Mouth Mucosa/pathology ; Precancerous Conditions/pathology/genetics ; },
abstract = {Precancerous cells in the oral cavity may appear as oral potentially malignant disorders, but they may also present as dysplasia without visual manifestation in tumor-adjacent tissue. As it is currently not possible to prevent the malignant transformation of these oral precancers, new treatments are urgently awaited. Here, we generated precancer culture models using a previously established method for the generation of oral keratinocyte cultures and incorporated CRISPR/Cas9 editing. The generated cell lines were used to investigate the efficacy of a set of small molecule inhibitors. Tumor-adjacent mucosa and oral leukoplakia biopsies were cultured and genetically characterized. Mutations were introduced in CDKN2A and TP53 using CRISPR/Cas9 and combined with the ectopic activation of telomerase to generate cell lines with prolonged proliferation. The method was tested in normal oral keratinocytes and tumor-adjacent biopsies and subsequently applied to a large set of oral leukoplakia biopsies. Finally, a subset of the immortalized cell lines was used to assess the efficacy of a set of small molecule inhibitors. Culturing and genomic engineering was highly efficient for normal and tumor-adjacent oral keratinocytes, but success rates in oral leukoplakia were remarkably low. Knock-out of CDKN2A in combination with either the activation of telomerase or knock-out of TP53 seemed a prerequisite for immortalization. Prolonged culturing was accompanied by additional genetic aberrations in these cultures. The generated cell lines were more sensitive than normal keratinocytes to small molecule inhibitors of previously identified targets. In conclusion, while very effective for normal keratinocytes and tumor-adjacent biopsies, the success rate of oral leukoplakia cell culturing methods was very low. Genomic engineering enabled the prolonged culturing of OL-derived keratinocytes but was associated with acquired genetic changes. Further studies are required to assess to what extent the immortalized cultures faithfully represent characteristics of the cells in vivo.},
}

Humans
*Keratinocytes/metabolism/pathology
*Mouth Neoplasms/genetics/pathology
*Leukoplakia, Oral/genetics/pathology
Telomerase/genetics/metabolism
Genetic Engineering
Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism
CRISPR-Cas Systems/genetics
Tumor Suppressor Protein p53/metabolism/genetics
Mouth Mucosa/pathology
Precancerous Conditions/pathology/genetics

@article {pmid38381406,
year = {2024},
author = {Guarducci, C and Nardone, A and Russo, D and Nagy, Z and Heraud, C and Grinshpun, A and Zhang, Q and Freelander, A and Leventhal, MJ and Feit, A and Cohen Feit, G and Feiglin, A and Liu, W and Hermida-Prado, F and Kesten, N and Ma, W and De Angelis, C and Morlando, A and O'Donnell, M and Naumenko, S and Huang, S and Nguyen, QD and Huang, Y and Malorni, L and Bergholz, JS and Zhao, JJ and Fraenkel, E and Lim, E and Schiff, R and Shapiro, GI and Jeselsohn, R},
title = {Selective CDK7 Inhibition Suppresses Cell Cycle Progression and MYC Signaling While Enhancing Apoptosis in Therapy-resistant Estrogen Receptor-positive Breast Cancer.},
journal = {Clinical cancer research : an official journal of the American Association for Cancer Research},
volume = {30},
number = {9},
pages = {1889-1905},
doi = {10.1158/1078-0432.CCR-23-2975},
pmid = {38381406},
issn = {1557-3265},
support = {fellowship 2017//Fondazione AIRC per la ricerca sul cancro ETS (AIRC)/ ; 19288 year 2017//Fondazione Cassa di Risparmio di Firenze (Fondazione CR Firenze)/ ; 5U01CA253547//National Cancer Institute (NCI)/ ; Barbara Weedon Fellowship//Massachusetts Institute of Technology Department of Brain and Cognitive Sciences/ ; Core Facility Award (RP170005)//Cancer Prevention and Research Institute of Texas (CPRIT)/ ; P30 Cancer Center Support Grant (NCI-CA125123)//National Cancer Institute (NCI)/ ; S10 instrument award (S10OD028648-01)//National Cancer Institute (NCI)/ ; 5RO1CA237414-02//National Cancer Institute (NCI)/ ; BARR Award//Dana-Farber Cancer Institute (DFCI)/ ; Duncan Donuts Drives Cancer Discovery Award//Duncan Donuts/ ; Post-Doctoral Research Fellowship 2018-2020//American-Italian Cancer Foundation (AICF)/ ; },
mesh = {Humans ; *Breast Neoplasms/drug therapy/pathology/genetics/metabolism ; Female ; *Drug Resistance, Neoplasm/genetics ; *Apoptosis/drug effects ; Animals ; Mice ; *Receptors, Estrogen/metabolism ; *Cyclin-Dependent Kinase-Activating Kinase ; *Cyclin-Dependent Kinases/antagonists & inhibitors/metabolism/genetics ; *Proto-Oncogene Proteins c-myc/metabolism/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; *Signal Transduction/drug effects ; *Cell Cycle/drug effects ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Cyclin-Dependent Kinase 4/antagonists & inhibitors/genetics ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms.

EXPERIMENTAL DESIGN: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models.

RESULTS: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models.

CONCLUSIONS: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments.},
}

Humans
*Breast Neoplasms/drug therapy/pathology/genetics/metabolism
Female
*Drug Resistance, Neoplasm/genetics
*Apoptosis/drug effects
Animals
Mice
*Receptors, Estrogen/metabolism
*Cyclin-Dependent Kinase-Activating Kinase
*Cyclin-Dependent Kinases/antagonists & inhibitors/metabolism/genetics
*Proto-Oncogene Proteins c-myc/metabolism/genetics
*Protein Kinase Inhibitors/pharmacology/therapeutic use
*Signal Transduction/drug effects
*Cell Cycle/drug effects
Xenograft Model Antitumor Assays
Cell Line, Tumor
Cell Proliferation/drug effects
Gene Expression Regulation, Neoplastic/drug effects
Cyclin-Dependent Kinase 4/antagonists & inhibitors/genetics
CRISPR-Cas Systems

@article {pmid38374801,
year = {2024},
author = {Zhu, X and Chen, A and Butler, NM and Zeng, Z and Xin, H and Wang, L and Lv, Z and Eshel, D and Douches, DS and Jiang, J},
title = {Molecular dissection of an intronic enhancer governing cold-induced expression of the vacuolar invertase gene in potato.},
journal = {The Plant cell},
volume = {36},
number = {5},
pages = {1985-1999},
doi = {10.1093/plcell/koae050},
pmid = {38374801},
issn = {1532-298X},
support = {1808085MC65//National Natural Science Foundation of China/ ; 2021H276//Anhui Provincial Natural Science Foundation/ ; 22532002//Scientific Research Fund for Candidates of Anhui Provincial Academic and Technical Leaders/ ; //Anhui Province Vegetable Industry Technology System Fund/ ; 2011-51181-30629//USDA-NIFA-SCRI/ ; //BARD/ ; MICL02571//AgBioResearch at Michigan State University/ ; //MSU/ ; },
mesh = {*Solanum tuberosum/genetics/enzymology ; *Introns/genetics ; *beta-Fructofuranosidase/genetics/metabolism ; *Gene Expression Regulation, Plant ; *Cold Temperature ; Plant Proteins/genetics/metabolism ; Enhancer Elements, Genetic/genetics ; Vacuoles/metabolism ; Gene Editing ; Plants, Genetically Modified ; Plant Tubers/genetics/enzymology ; CRISPR-Cas Systems ; },
abstract = {Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to minimize sprouting and losses due to disease. However, cold temperatures strongly induce the expression of the potato vacuolar invertase gene (VInv) and cause reducing sugar accumulation. This process, referred to as "cold-induced sweetening," is a major postharvest problem for the potato industry. We discovered that the cold-induced expression of VInv is controlled by a 200 bp enhancer, VInvIn2En, located in its second intron. We identified several DNA motifs in VInvIn2En that bind transcription factors involved in the plant cold stress response. Mutation of these DNA motifs abolished VInvIn2En function as a transcriptional enhancer. We developed VInvIn2En deletion lines in both diploid and tetraploid potato using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. VInv transcription in cold-stored tubers was significantly reduced in the deletion lines. Interestingly, the VInvIn2En sequence is highly conserved among distantly related Solanum species, including tomato (Solanum lycopersicum) and other non-tuber-bearing species. We conclude that the VInv gene and the VInvIn2En enhancer have adopted distinct roles in the cold stress response in tubers of tuber-bearing Solanum species.},
}

*Solanum tuberosum/genetics/enzymology
*Introns/genetics
*beta-Fructofuranosidase/genetics/metabolism
*Gene Expression Regulation, Plant
*Cold Temperature
Plant Proteins/genetics/metabolism
Enhancer Elements, Genetic/genetics
Vacuoles/metabolism
Gene Editing
Plants, Genetically Modified
Plant Tubers/genetics/enzymology
CRISPR-Cas Systems

@article {pmid36549468,
year = {2023},
author = {Yang, ZX and Fu, YW and Zhao, JJ and Zhang, F and Li, SA and Zhao, M and Wen, W and Zhang, L and Cheng, T and Zhang, JP and Zhang, XB},
title = {Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing.},
journal = {Genomics, proteomics & bioinformatics},
volume = {21},
number = {6},
pages = {1206-1220},
doi = {10.1016/j.gpb.2022.12.003},
pmid = {36549468},
issn = {2210-3244},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; K562 Cells ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/genetics/enzymology ; Staphylococcus aureus/genetics ; DNA End-Joining Repair/genetics ; },
abstract = {A series of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) systems have been engineered for genome editing. The most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus. However, a comparison of their detailed gene editing outcomes is still lacking. By characterizing the editing outcomes of 11 sites in human induced pluripotent stem cells (iPSCs) and K562 cells, we found that SaCas9 could edit the genome with greater efficiencies than SpCas9. We also compared the effects of spacer lengths of single-guide RNAs (sgRNAs; 18-21 nt for SpCas9 and 19-23 nt for SaCas9) and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9, respectively. However, the optimal spacer length for a particular sgRNA was 18-21 nt for SpCas9 and 21-22 nt for SaCas9. Furthermore, SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining (NHEJ) +1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif (PAM), indicating a characteristic of a staggered cut. Accordingly, editing with SaCas9 led to higher efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertion or homology-directed repair (HDR)-mediated adeno-associated virus serotype 6 (AAV6) donor knock-in. Finally, GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with SpCas9. Our work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
K562 Cells
*CRISPR-Associated Protein 9/genetics/metabolism
*Induced Pluripotent Stem Cells/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Streptococcus pyogenes/genetics/enzymology
Staphylococcus aureus/genetics
DNA End-Joining Repair/genetics

@article {pmid35792260,
year = {2023},
author = {Zhang, G and Zhu, C and Chen, X and Yan, J and Xue, D and Wei, Z and Chuai, G and Liu, Q},
title = {Systematic Exploration of Optimized Base Editing gRNA Design and Pleiotropic Effects with BExplorer.},
journal = {Genomics, proteomics & bioinformatics},
volume = {21},
number = {6},
pages = {1237-1245},
doi = {10.1016/j.gpb.2022.06.005},
pmid = {35792260},
issn = {2210-3244},
mesh = {*Gene Editing/methods ; Humans ; *Polymorphism, Single Nucleotide/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Human/genetics ; Genetic Pleiotropy ; CRISPR-Cas Systems/genetics ; Software ; Computational Biology/methods ; },
abstract = {Base editing technology is being increasingly applied in genome engineering, but the current strategy for designing guide RNAs (gRNAs) relies substantially on empirical experience rather than a dependable and efficient in silico design. Furthermore, the pleiotropic effect of base editing on disease treatment remains unexplored, which prevents its further clinical usage. Here, we presented BExplorer, an integrated and comprehensive computational pipeline to optimize the design of gRNAs for 26 existing types of base editors in silico. Using BExplorer, we described its results for two types of mainstream base editors, BE3 and ABE7.10, and evaluated the pleiotropic effects of the corresponding base editing loci. BExplorer revealed 524 and 900 editable pathogenic single nucleotide polymorphism (SNP) loci in the human genome together with the selected optimized gRNAs for BE3 and ABE7.10, respectively. In addition, the impact of 707 edited pathogenic SNP loci following base editing on 131 diseases was systematically explored by revealing their pleiotropic effects, indicating that base editing should be carefully utilized given the potential pleiotropic effects. Collectively, the systematic exploration of optimized base editing gRNA design and the corresponding pleiotropic effects with BExplorer provides a computational basis for applying base editing in disease treatment.},
}

*Gene Editing/methods
Humans
*Polymorphism, Single Nucleotide/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Human/genetics
Genetic Pleiotropy
CRISPR-Cas Systems/genetics
Software
Computational Biology/methods

@article {pmid35752289,
year = {2023},
author = {Quan, ZJ and Li, SA and Yang, ZX and Zhao, JJ and Li, GH and Zhang, F and Wen, W and Cheng, T and Zhang, XB},
title = {GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing.},
journal = {Genomics, proteomics & bioinformatics},
volume = {21},
number = {6},
pages = {1221-1236},
doi = {10.1016/j.gpb.2022.06.002},
pmid = {35752289},
issn = {2210-3244},
mesh = {*Gene Editing/methods ; *Nanopore Sequencing/methods ; Humans ; *Workflow ; *Polymerase Chain Reaction/methods ; *CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; DNA End-Joining Repair/genetics ; },
abstract = {To achieve the enormous potential of gene-editing technology in clinical therapies, one needs to evaluate both the on-target efficiency and unintended editing consequences comprehensively. However, there is a lack of a pipelined, large-scale, and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. Recognizing the high error rates of Oxford nanopore sequencing, we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can assess nonhomologous end-joining (NHEJ)-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also reveals a full spectrum of homology-directed repair (HDR)-mediated large gene knock-in, correlating well with the fluorescence-activated cell sorting (FACS) analysis results. Of note, we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting site. Therefore, we have established a practical workflow to evaluate various genetic changes, including quantifying insertions of short dsODNs, knock-ins of long pieces, plasmid insertions, and large fragment deletions after CRISPR/Cas9-mediated editing. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq) and the National Genomics Data Center (NGDC) BioCode (https://ngdc.cncb.ac.cn/biocode/tools/BT007293).},
}

*Gene Editing/methods
*Nanopore Sequencing/methods
Humans
*Workflow
*Polymerase Chain Reaction/methods
*CRISPR-Cas Systems/genetics
High-Throughput Nucleotide Sequencing/methods
DNA End-Joining Repair/genetics

@article {pmid38667187,
year = {2024},
author = {Shin, J and Kim, SR and Xie, Z and Jin, YS and Wang, YC},
title = {A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.},
journal = {Biosensors},
volume = {14},
number = {4},
pages = {},
pmid = {38667187},
issn = {2079-6374},
support = {ILLU-698-930//National Institute of Food and Agriculture/ ; ILLU-698-353//National Institute of Food and Agriculture/ ; R20-7704//Royal Society of Chemistry/ ; },
mesh = {*CRISPR-Cas Systems ; *Carbapenems/pharmacology ; *beta-Lactamases/genetics ; Carbapenem-Resistant Enterobacteriaceae/genetics ; Enterobacteriaceae/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; Escherichia coli/genetics/drug effects ; Biosensing Techniques ; Drug Resistance, Bacterial/genetics ; *Bacterial Proteins ; *Endodeoxyribonucleases ; *CRISPR-Associated Proteins ; },
abstract = {Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-β-lactamase (NDM) are particularly concerning due to their resistance to most β-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (blaNDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry blaNDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of blaNDM-1 carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 10[0] CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.},
}

*CRISPR-Cas Systems
*Carbapenems/pharmacology
*beta-Lactamases/genetics
Carbapenem-Resistant Enterobacteriaceae/genetics
Enterobacteriaceae/genetics/drug effects
Anti-Bacterial Agents/pharmacology
Escherichia coli/genetics/drug effects
Biosensing Techniques
Drug Resistance, Bacterial/genetics
*Bacterial Proteins
*Endodeoxyribonucleases
*CRISPR-Associated Proteins

@article {pmid38664612,
year = {2024},
author = {Lu, K and Wang, X and Zhou, Y and Zhu, Q},
title = {Genomic characterization and probiotic potential assessment of an exopolysaccharide-producing strain Pediococcus pentosaceus LL-07 isolated from fermented meat.},
journal = {BMC microbiology},
volume = {24},
number = {1},
pages = {142},
pmid = {38664612},
issn = {1471-2180},
support = {No. 32272359//National Natural Science Foundation of China/ ; No. 32272359//National Natural Science Foundation of China/ ; No. 32272359//National Natural Science Foundation of China/ ; No. 32272359//National Natural Science Foundation of China/ ; Grant No. ZK [2023] 091//Guizhou Provincial Basic Research Program (Natural Science)/ ; Grant No. ZK [2023] 091//Guizhou Provincial Basic Research Program (Natural Science)/ ; Grant No. ZK [2023] 091//Guizhou Provincial Basic Research Program (Natural Science)/ ; Grant No. ZK [2023] 091//Guizhou Provincial Basic Research Program (Natural Science)/ ; },
mesh = {*Probiotics ; *Pediococcus pentosaceus/genetics/metabolism ; *Genome, Bacterial ; *Polysaccharides, Bacterial/metabolism/biosynthesis ; *Fermentation ; Whole Genome Sequencing ; Fermented Foods/microbiology ; Meat/microbiology ; Multigene Family ; Genomics/methods ; Humans ; Plasmids/genetics ; Food Microbiology ; },
abstract = {BACKGROUND: The genomic information available for Pediococcus pentosaceus is primarily derived from fermented fruits and vegetables, with less information available from fermented meat. P. pentosaceus LL-07, a strain isolated from fermented meat, has the capability of producing exopolysaccharides (EPS). To assess the probiotic attributes of P. pentosaceus LL-07, we conducted whole-genome sequencing (WGS) using the PacBio SequelIIe and Illumina MiSeq platforms, followed by in vitro experiments to explore its probiotic potential.

RESULTS: The genome size of P. pentosaceus LL-07 is 1,782,685 bp, comprising a circular chromosome and a circular plasmid. Our investigation revealed the absence of a CRISPR/Cas system. Sugar fermentation experiments demonstrated the characteristics of carbohydrate metabolism. P. pentosaceus LL-07 contains an EPS synthesis gene cluster consisting of 13 genes, which is different from the currently known gene cluster structure. NO genes associated with hemolysis or toxin synthesis were detected. Additionally, eighty-six genes related to antibiotic resistance were identified but not present in the prophage, transposon or plasmid. In vitro experiments demonstrated that P. pentosaceus LL-07 was comparable to the reference strain P. pentosaceus ATCC25745 in terms of tolerance to artificial digestive juice and bile, autoaggregation and antioxidation, and provided corresponding genomic evidence.

CONCLUSION: This study confirmed the safety and probiotic properties of P. pentosaceus LL-07 via complete genome and phenotype analysis, supporting its characterization as a potential probiotic candidate.},
}

*Probiotics
*Pediococcus pentosaceus/genetics/metabolism
*Genome, Bacterial
*Polysaccharides, Bacterial/metabolism/biosynthesis
*Fermentation
Whole Genome Sequencing
Fermented Foods/microbiology
Meat/microbiology
Multigene Family
Genomics/methods
Humans
Plasmids/genetics
Food Microbiology

@article {pmid38664280,
year = {2024},
author = {Lin, Q and Takebayashi, K and Torigoe, N and Liu, B and Namula, Z and Hirata, M and Nagahara, M and Tanihara, F and Otoi, T},
title = {Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes.},
journal = {In vitro cellular & developmental biology. Animal},
volume = {},
number = {},
pages = {},
pmid = {38664280},
issn = {1543-706X},
support = {JP22H02499//KAKENHI/ ; JP22K19896//KAKENHI/ ; },
abstract = {The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency.},
}

@article {pmid38663232,
year = {2024},
author = {Christi, K and Hudson, J and Egan, S},
title = {Current approaches to genetic modification of marine bacteria and considerations for improved transformation efficiency.},
journal = {Microbiological research},
volume = {284},
number = {},
pages = {127729},
doi = {10.1016/j.micres.2024.127729},
pmid = {38663232},
issn = {1618-0623},
abstract = {Marine bacteria play vital roles in symbiosis, biogeochemical cycles and produce novel bioactive compounds and enzymes of interest for the pharmaceutical, biofuel and biotechnology industries. At present, investigations into marine bacterial functions and their products are primarily based on phenotypic observations, -omic type approaches and heterologous gene expression. To advance our understanding of marine bacteria and harness their full potential for industry application, it is critical that we have the appropriate tools and resources to genetically manipulate them in situ. However, current genetic tools that are largely designed for model organisms such as E. coli, produce low transformation efficiencies or have no transfer ability in marine bacteria. To improve genetic manipulation applications for marine bacteria, we need to improve transformation methods such as conjugation and electroporation in addition to identifying more marine broad host range plasmids. In this review, we aim to outline the reported methods of transformation for marine bacteria and discuss the considerations for each approach in the context of improving efficiency. In addition, we further discuss marine plasmids and future research areas including CRISPR tools and their potential applications for marine bacteria.},
}

@article {pmid38662824,
year = {2024},
author = {Sommer-Trembo, C and Santos, ME and Clark, B and Werner, M and Fages, A and Matschiner, M and Hornung, S and Ronco, F and Oliver, C and Garcia, C and Tschopp, P and Malinsky, M and Salzburger, W},
title = {The genetics of niche-specific behavioral tendencies in an adaptive radiation of cichlid fishes.},
journal = {Science (New York, N.Y.)},
volume = {384},
number = {6694},
pages = {470-475},
doi = {10.1126/science.adj9228},
pmid = {38662824},
issn = {1095-9203},
mesh = {Animals ; *Cichlids/genetics/physiology ; *Polymorphism, Single Nucleotide ; *Gene Editing ; Lakes ; Exploratory Behavior ; CRISPR-Cas Systems ; Behavior, Animal ; Receptors, AMPA/genetics/metabolism ; Genotype ; Tanzania ; Ecosystem ; Adaptation, Physiological/genetics ; },
abstract = {Behavior is critical for animal survival and reproduction, and possibly for diversification and evolutionary radiation. However, the genetics behind adaptive variation in behavior are poorly understood. In this work, we examined a fundamental and widespread behavioral trait, exploratory behavior, in one of the largest adaptive radiations on Earth, the cichlid fishes of Lake Tanganyika. By integrating quantitative behavioral data from 57 cichlid species (702 wild-caught individuals) with high-resolution ecomorphological and genomic information, we show that exploratory behavior is linked to macrohabitat niche adaptations in Tanganyikan cichlids. Furthermore, we uncovered a correlation between the genotypes at a single-nucleotide polymorphism upstream of the AMPA glutamate-receptor regulatory gene cacng5b and variation in exploratory tendency. We validated this association using behavioral predictions with a neural network approach and CRISPR-Cas9 genome editing.},
}

Animals
*Cichlids/genetics/physiology
*Polymorphism, Single Nucleotide
*Gene Editing
Lakes
Exploratory Behavior
CRISPR-Cas Systems
Behavior, Animal
Receptors, AMPA/genetics/metabolism
Genotype
Tanzania
Ecosystem
Adaptation, Physiological/genetics

@article {pmid38661236,
year = {2024},
author = {Chen, F and Zhang, C and Xue, J and Wang, F and Li, Z},
title = {Molecular mechanism for target RNA recognition and cleavage of Cas13h.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae324},
pmid = {38661236},
issn = {1362-4962},
support = {2022YFA0911800//Ministry of Science and Technology/ ; 32201004//National Natural Science Foundation of China/ ; ZRMS2022000096//Hubei Provincial Natural Science Foundation/ ; 2021BAD001//Key Science and Technology Innovation Project of Hubei Province/ ; },
abstract = {RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA knockdown efficiency. However, little is known regarding its biochemical properties and working mechanisms. Biochemical characterization of Cas13h1 indicated that it lacks in vitro pre-crRNA processing activity and adopts a central seed. The cleavage activity of Cas13h1 is enhanced by a R(G/A) 5'-PFS, and inhibited by tag:anti-tag RNA pairing. We determined the structures of Cas13h1-crRNA binary complex at 3.1 Å and Cas13h1-crRNA-target RNA ternary complex at 3.0 Å. The ternary complex adopts an elongated architecture, and encodes a nucleotide-binding pocket within Helical-2 domain to recognize the guanosine at the 5'-end of the target RNA. Base pairing between crRNA guide and target RNA disrupts Cas13h1-guide interactions, leading to dramatic movement of HEPN domains. Upon target RNA engagement, Cas13h1 adopts a complicated activation mechanism, including separation of HEPN catalytic residues and destabilization of the active site loop and NTD domain, to get activated. Collectively, these insights expand our understanding into Cas13 effectors.},
}

@article {pmid38661215,
year = {2024},
author = {Vialetto, E and Miele, S and Goren, MG and Yu, J and Yu, Y and Collias, D and Beamud, B and Osbelt, L and Lourenço, M and Strowig, T and Brisse, S and Barquist, L and Qimron, U and Bikard, D and Beisel, CL},
title = {Systematic interrogation of CRISPR antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide- and strain-dependent features influencing antimicrobial activity.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae281},
pmid = {38661215},
issn = {1362-4962},
support = {818878/ERC_/European Research Council/International ; },
abstract = {CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials using multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae as models. Comparing different Cas nucleases, DNA-targeting nucleases outperformed RNA-targeting nucleases based on the tested targets. Focusing on AsCas12a that exhibited robust targeting across different strains, we found that the elucidated modes of escape varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains, which were linked to an interplay between improper gRNA folding and strain-specific DNA repair and survival. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting.},
}

@article {pmid38660304,
year = {2024},
author = {Zhang, T and Zhang, Y and Wei, J},
title = {Overcoming the challenges encountered in CAR-T therapy: latest updates from the 2023 ASH annual conference.},
journal = {Frontiers in immunology},
volume = {15},
number = {},
pages = {1389324},
pmid = {38660304},
issn = {1664-3224},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology/genetics ; *Neoplasms/therapy/immunology ; Animals ; Combined Modality Therapy ; T-Lymphocytes/immunology ; Congresses as Topic ; },
abstract = {Chimeric antigen receptor (CAR) -T cell therapy has entered the breakthrough era, characterized by a blend of therapeutic opportunities and challenges. With the integration of genome-editing technology, CAR-T cells will be empowered to become super warriors in eradicating tumor cells and attacking various tumors, including T-cell malignancies and acute myeloid leukemia. Notably, the optimization of CAR-T cells, including efficacy, safety, and manufacturing speed, coupled with other therapeutic strategies such as radiotherapy, hematopoietic stem cell transplantation, small-molecule inhibitors, and bispecific antibodies, could revolutionize the therapeutic landscape of tumors. Consequently, next-generation cellular immunotherapy, including universal CAR-NK cells and synergistic combination approaches, are anticipated to significantly impact cancer treatment in the coming decade. Nevertheless, the failure rates of CAR-T therapy continue to be significant. The challenge lies in determining the optimal combination strategy and identifying reliable and robust biomarkers to effectively select the patients who will derive the greatest benefit from CAR-T therapy. Herein, we highlight recent innovations in CAR-T products, combination strategies and predictive biomarkers of response presented at the 2023 ASH Annual Meeting.},
}

Humans
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/immunology/genetics
*Neoplasms/therapy/immunology
Animals
Combined Modality Therapy
T-Lymphocytes/immunology
Congresses as Topic

@article {pmid38658677,
year = {2024},
author = {Pfeifer, M and Brammeld, JS and Price, S and Pilling, J and Bhavsar, D and Farcas, A and Bateson, J and Sundarrajan, A and Miragaia, RJ and Guan, N and Arnold, S and Tariq, L and Grondine, M and Talbot, S and Guerriero, ML and O'Neill, DJ and Young, J and Company, C and Dunn, S and Thorpe, H and Martin, MJ and Maratea, K and Barrell, D and Ahdesmaki, M and Mettetal, JT and , and Brownell, J and McDermott, U},
title = {Genome-wide CRISPR screens identify the YAP/TEAD axis as a driver of persister cells in EGFR mutant lung cancer.},
journal = {Communications biology},
volume = {7},
number = {1},
pages = {497},
pmid = {38658677},
issn = {2399-3642},
mesh = {Humans ; *Lung Neoplasms/genetics/drug therapy/metabolism/pathology ; *ErbB Receptors/genetics/metabolism ; *Drug Resistance, Neoplasm/genetics ; *Transcription Factors/genetics/metabolism ; *Mutation ; Cell Line, Tumor ; *Acrylamides/pharmacology/therapeutic use ; YAP-Signaling Proteins/metabolism/genetics ; Aniline Compounds/pharmacology/therapeutic use ; Gefitinib/pharmacology ; Hippo Signaling Pathway ; DNA-Binding Proteins/genetics/metabolism ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Signal Transduction ; TEA Domain Transcription Factors ; Protein Kinase Inhibitors/pharmacology ; Antineoplastic Agents/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; *Indoles ; *Pyrimidines ; },
abstract = {Most lung cancer patients with metastatic cancer eventually relapse with drug-resistant disease following treatment and EGFR mutant lung cancer is no exception. Genome-wide CRISPR screens, to either knock out or overexpress all protein-coding genes in cancer cell lines, revealed the landscape of pathways that cause resistance to the EGFR inhibitors osimertinib or gefitinib in EGFR mutant lung cancer. Among the most recurrent resistance genes were those that regulate the Hippo pathway. Following osimertinib treatment a subpopulation of cancer cells are able to survive and over time develop stable resistance. These 'persister' cells can exploit non-genetic (transcriptional) programs that enable cancer cells to survive drug treatment. Using genetic and pharmacologic tools we identified Hippo signalling as an important non-genetic mechanism of cell survival following osimertinib treatment. Further, we show that combinatorial targeting of the Hippo pathway and EGFR is highly effective in EGFR mutant lung cancer cells and patient-derived organoids, suggesting a new therapeutic strategy for EGFR mutant lung cancer patients.},
}

Humans
*Lung Neoplasms/genetics/drug therapy/metabolism/pathology
*ErbB Receptors/genetics/metabolism
*Drug Resistance, Neoplasm/genetics
*Transcription Factors/genetics/metabolism
*Mutation
Cell Line, Tumor
*Acrylamides/pharmacology/therapeutic use
YAP-Signaling Proteins/metabolism/genetics
Aniline Compounds/pharmacology/therapeutic use
Gefitinib/pharmacology
Hippo Signaling Pathway
DNA-Binding Proteins/genetics/metabolism
Adaptor Proteins, Signal Transducing/genetics/metabolism
Signal Transduction
TEA Domain Transcription Factors
Protein Kinase Inhibitors/pharmacology
Antineoplastic Agents/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
*Indoles
*Pyrimidines

@article {pmid38658578,
year = {2024},
author = {Pedrazzoli, E and Demozzi, M and Visentin, E and Ciciani, M and Bonuzzi, I and Pezzè, L and Lucchetta, L and Maule, G and Amistadi, S and Esposito, F and Lupo, M and Miccio, A and Auricchio, A and Casini, A and Segata, N and Cereseto, A},
title = {CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3478},
pmid = {38658578},
issn = {2041-1723},
support = {U01 CA230551/CA/NCI NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; Mice ; *Microbiota/genetics ; Dependovirus/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Retina/metabolism ; Clostridiales/genetics/enzymology ; HEK293 Cells ; Genetic Vectors/metabolism/genetics ; },
abstract = {The expansion of the CRISPR-Cas toolbox is highly needed to accelerate the development of therapies for genetic diseases. Here, through the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety (n = 17,173) of type II CRISPR-Cas loci. Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1004 amino acids) isolated from an uncultivated Collinsella species. CoCas9 is efficiently co-delivered with its sgRNA through adeno associated viral (AAV) vectors, obtaining efficient in vivo editing in the mouse retina. With this study we uncover a collection of previously uncharacterized Cas9 nucleases, including CoCas9, which enriches the genome editing toolbox.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
Animals
Mice
*Microbiota/genetics
Dependovirus/genetics
CRISPR-Associated Protein 9/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Retina/metabolism
Clostridiales/genetics/enzymology
HEK293 Cells
Genetic Vectors/metabolism/genetics

@article {pmid38658536,
year = {2024},
author = {Feng, X and Xu, R and Liao, J and Zhao, J and Zhang, B and Xu, X and Zhao, P and Wang, X and Yao, J and Wang, P and Wang, X and Han, W and She, Q},
title = {Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3464},
pmid = {38658536},
issn = {2041-1723},
support = {32001022//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32270040//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2020YFA0906800//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; DNA Transposable Elements/genetics ; Archaeal Proteins/metabolism/genetics ; Transposases/metabolism/genetics ; },
abstract = {TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
DNA Transposable Elements/genetics
Archaeal Proteins/metabolism/genetics
Transposases/metabolism/genetics

@article {pmid38646918,
year = {2024},
author = {Zhu, F and Yu, H and Zhao, Q},
title = {CRISPR/Cas12a-Amplified Aptamer Switch Microplate Assay for Small Molecules.},
journal = {Analytical chemistry},
volume = {96},
number = {17},
pages = {6853-6859},
doi = {10.1021/acs.analchem.4c01452},
pmid = {38646918},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; *Aflatoxin B1/analysis/chemistry ; Biosensing Techniques/methods ; Cadmium/chemistry/analysis ; Limit of Detection ; },
abstract = {The presence of small molecule contaminants such as mycotoxins and heavy metals in foods and the environment causes a serious threat to human health and huge economic losses. The development of simple, rapid, sensitive, and on-site methods for small molecule pollutant detection is highly demanded. Here, combining the advantages of structure-switchable aptamer-mediated signal conversion and CRISPR/Cas12a-based signal amplification, we developed a CRISPR/Cas12a-amplified aptamer switch assay on a microplate for sensitive small molecule detection. In this assay, a short DNA strand complementary to the aptamer (cDNA) is immobilized on a microplate, which can capture the aptamer-linked active DNA probe (Apt-acDNA) in the sample solution when the target is absent. With the addition of the Cas12a reporter system, the captured Apt-acDNA probes activate Cas12a to indiscriminately cleave fluorescent DNA substrates, producing a high fluorescence signal. When the target is present, the Apt-acDNA probe specifically binds to the target rather than hybridizing with cDNA on the microplate, and the fluorescence signal is reduced. The analytical performance of our method was demonstrated by the detection of two highly toxic pollutants, aflatoxin B1 (AFB1) and cadmium ion (Cd[2+]), as examples. The assay exhibited good selectivity and high sensitivity, with detection limits of 31 pM AFB1 and 3.9 nM Cd[2+]. It also allowed the detection of targets in the actual sample matrix. With the general signal conversion strategy, this method can be used to detect other targets by simply changing the aptamer and cDNA, showing potential practical applications in broad fields.},
}

*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems/genetics
*Aflatoxin B1/analysis/chemistry
Biosensing Techniques/methods
Cadmium/chemistry/analysis
Limit of Detection

@article {pmid38521316,
year = {2024},
author = {Li, D and Liang, P and Ling, S and Wu, Y and Lv, B},
title = {An optimized microRNA detection platform based on PAM formation-regulated CRISPR/Cas12a activation.},
journal = {International journal of biological macromolecules},
volume = {266},
number = {Pt 1},
pages = {130848},
doi = {10.1016/j.ijbiomac.2024.130848},
pmid = {38521316},
issn = {1879-0003},
mesh = {*MicroRNAs/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; },
abstract = {MicroRNAs (miRNAs) have emerged as biomarkers for the diagnosis and prognosis of various diseases, such as cancer. Recent advancements in CRISPR/Cas12a-based biosensors in combination with hybridization chain reaction (HCR) make it a promising approach for miRNA detection. To increase the compatibility of HCR and CRISPR/Cas12a, we compared two design strategies of hairpin DNA in HCR. The results showed that different arrangements of the protospacer sequence and protospacer adjacent motif (PAM) in the hairpin DNA could affect the sensing performance. The "PAM Formation" strategy, by which the duplex PAM sites are absent in the hairpin DNA and present in the long duplex DNA after HCR, exhibited advantages in detection sensitivity. By optimizing the probe sequences and reaction conditions, we developed a miRNA detection platform. With the same crRNA, this platform enables the identification of different miRNAs by simply replacing the loop region of the target recognition probe. In addition, the proposed platform can detect single-stranded DNA and distinguishing single or multiple base mutations in the target strand. The application of discriminating the target miRNA expression levels from different cell lines validated the reliability and practicability of the sensor platform, indicating its potential applications in early clinical accurate diagnosis of cancers.},
}

*MicroRNAs/genetics
*CRISPR-Cas Systems/genetics
Humans
*Biosensing Techniques/methods
Nucleic Acid Hybridization

@article {pmid38224529,
year = {2024},
author = {Liao, W and Guo, R and Qian, K and Shi, W and Whelan, J and Shou, H},
title = {The acyl-acyl carrier protein thioesterases GmFATA1 and GmFATA2 are essential for fatty acid accumulation and growth in soybean.},
journal = {The Plant journal : for cell and molecular biology},
volume = {118},
number = {3},
pages = {823-838},
doi = {10.1111/tpj.16638},
pmid = {38224529},
issn = {1365-313X},
support = {B14027//111 Project of Ministry of Education/ ; 2021YFF1001204//National Key Research and Development Program of China/ ; 2021C02057//Key Research Projects of Zhejiang Province/ ; },
mesh = {*Glycine max/genetics/growth & development/metabolism/enzymology ; *Fatty Acids/metabolism ; *Plant Proteins/metabolism/genetics ; *Thiolester Hydrolases/metabolism/genetics ; Seeds/growth & development/genetics/metabolism ; Plant Leaves/genetics/metabolism/growth & development ; Seedlings/genetics/growth & development/metabolism ; Gene Expression Regulation, Plant ; Mutation ; CRISPR-Cas Systems ; Triglycerides/metabolism ; Gene Editing ; },
abstract = {Acyl-acyl carrier protein (ACP) thioesterases (FAT) hydrolyze acyl-ACP complexes to release FA in plastids, which ultimately affects FA biosynthesis and profiles. Soybean GmFATA1 and GmFATA2 are homoeologous genes encoding oleoyl-ACP thioesterases whose role in seed oil accumulation and plant growth has not been defined. Using CRISPR/Cas9 gene editing mutation of Gmfata1 or 2 led to reduced leaf FA content and growth defect at the early seedling stage. In contrast, no homozygous double mutants were obtained. Combined this indicates that GmFATA1 and GmFATA2 display overlapping, but not complete functional redundancy. Combined transcriptomic and lipidomic analysis revealed a large number of genes involved in FA synthesis and FA chain elongation are expressed at reduced level in the Gmfata1 mutant, accompanied by a lower triacylglycerol abundance at the early seedling stage. Further analysis showed that the Gmfata1 or 2 mutants had increased composition of the beneficial FA, oleic acid. The growth defect of Gmfata1 could be at least partially attributed to reduced acetyl-CoA carboxylase activity, reduced abundance of five unsaturated monogalactosyldiacylglycerol lipids, and altered chloroplast morphology. On the other hand, overexpression of GmFATA in soybean led to significant increases in leaf FA content by 5.7%, vegetative growth, and seed yield by 26.9%, and seed FA content by 23.2%. Thus, overexpression of GmFATA is an effective strategy to enhance soybean oil content and yield.},
}

*Glycine max/genetics/growth & development/metabolism/enzymology
*Fatty Acids/metabolism
*Plant Proteins/metabolism/genetics
*Thiolester Hydrolases/metabolism/genetics
Seeds/growth & development/genetics/metabolism
Plant Leaves/genetics/metabolism/growth & development
Seedlings/genetics/growth & development/metabolism
Gene Expression Regulation, Plant
Mutation
CRISPR-Cas Systems
Triglycerides/metabolism
Gene Editing

@article {pmid38127288,
year = {2024},
author = {Abdi Ghavidel, A and Aghamiri, S and Raee, P and Mohammadi-Yeganeh, S and Noori, E and Bandehpour, M and Kazemi, B and Jajarmi, V},
title = {Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Leishmania Strains.},
journal = {Acta parasitologica},
volume = {69},
number = {1},
pages = {121-134},
pmid = {38127288},
issn = {1896-1851},
mesh = {*Leishmania/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Genome, Protozoan ; Leishmaniasis/parasitology ; Animals ; },
abstract = {BACKGROUND: Genome manipulation of Leishmania species and the creation of modified strains are widely employed strategies for various purposes, including gene function studies, the development of live attenuated vaccines, and the engineering of host cells for protein production.

OBJECTIVE: Despite the introduction of novel manipulation approaches like CRISPR/Cas9 technology with significant advancements in recent years, the development of a reliable protocol for efficiently and precisely altering the genes of Leishmania strains remains a challenging endeavor. Following the successful adaptation of the CRISPR/Cas9 system for higher eukaryotic cells, several research groups have endeavored to apply this system to manipulate the genome of Leishmania.

RESULTS: Despite the substantial differences between Leishmania and higher eukaryotes, the CRISPR/Cas9 system has been effectively tested and applied in Leishmania.  CONCLUSION: This comprehensive review summarizes all the CRISPR/Cas9 systems that have been employed in Leishmania, providing details on their methods and the expression systems for Cas9 and gRNA. The review also explores the various applications of the CRISPR system in Leishmania, including the deletion of multicopy gene families, the development of the Leishmania vaccine, complete gene deletions, investigations into chromosomal translocations, protein tagging, gene replacement, large-scale gene knockout, genome editing through cytosine base replacement, and its innovative use in the detection of Leishmania. In addition, the review offers an up-to-date overview of all double-strand break repair mechanisms in Leishmania.},
}

*Leishmania/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
Genome, Protozoan
Leishmaniasis/parasitology
Animals

@article {pmid38649187,
year = {2024},
author = {Adapa, SR and Hunter, GA and Amin, NE and Marinescu, C and Borsky, A and Sagatys, EM and Sebti, SM and Reuther, GW and Ferreira, GC and Jiang, RH},
title = {Porphyrin overdrive rewires cancer cell metabolism.},
journal = {Life science alliance},
volume = {7},
number = {7},
pages = {},
pmid = {38649187},
issn = {2575-1077},
mesh = {Humans ; *Heme/metabolism ; *Porphyrins/metabolism/pharmacology ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Neoplasms/metabolism/genetics ; Metabolic Networks and Pathways/genetics ; Cell Differentiation/genetics ; Gene Editing ; Animals ; Mice ; },
abstract = {All cancer cells reprogram metabolism to support aberrant growth. Here, we report that cancer cells employ and depend on imbalanced and dynamic heme metabolic pathways, to accumulate heme intermediates, that is, porphyrins. We coined this essential metabolic rewiring "porphyrin overdrive" and determined that it is cancer-essential and cancer-specific. Among the major drivers are genes encoding mid-step enzymes governing the production of heme intermediates. CRISPR/Cas9 editing to engineer leukemia cell lines with impaired heme biosynthetic steps confirmed our whole-genome data analyses that porphyrin overdrive is linked to oncogenic states and cellular differentiation. Although porphyrin overdrive is absent in differentiated cells or somatic stem cells, it is present in patient-derived tumor progenitor cells, demonstrated by single-cell RNAseq, and in early embryogenesis. In conclusion, we identified a dependence of cancer cells on non-homeostatic heme metabolism, and we targeted this cancer metabolic vulnerability with a novel "bait-and-kill" strategy to eradicate malignant cells.},
}

Humans
*Heme/metabolism
*Porphyrins/metabolism/pharmacology
Cell Line, Tumor
*CRISPR-Cas Systems
Neoplasms/metabolism/genetics
Metabolic Networks and Pathways/genetics
Cell Differentiation/genetics
Gene Editing
Animals
Mice

@article {pmid38507231,
year = {2024},
author = {Unnithan, D and Sartaj, A and Iqubal, MK and Ali, J and Baboota, S},
title = {A neoteric annotation on the advances in combination therapy for Parkinson's disease: nanocarrier-based combination approach and future anticipation. Part II: nanocarrier design and development in focus.},
journal = {Expert opinion on drug delivery},
volume = {21},
number = {3},
pages = {437-456},
doi = {10.1080/17425247.2024.2331216},
pmid = {38507231},
issn = {1744-7593},
mesh = {Humans ; *Parkinson Disease/drug therapy ; *Antiparkinson Agents/administration & dosage/therapeutic use/pharmacokinetics/pharmacology ; *Drug Carriers/chemistry ; Animals ; *Neuroprotective Agents/administration & dosage/therapeutic use/pharmacokinetics ; *Nanotechnology ; *Nanoparticles ; Blood-Brain Barrier/metabolism ; Drug Delivery Systems ; Precision Medicine ; Drug Therapy, Combination ; CRISPR-Cas Systems ; Drug Combinations ; Combined Modality Therapy ; Drug Development ; Drug Design ; },
abstract = {INTRODUCTION: The current treatment modalities available for Parkinson's disease (PD) prove inadequate due to the inherent constraints in effectively transporting bioactive compounds across the blood-brain barrier. The utilization of synergistic combinations of multiple drugs in conjunction with advanced nanotechnology, emerges as a promising avenue for the treatment of PD, offering potential breakthroughs in treatment efficacy, targeted therapy, and personalized medicine.

AREAS COVERED: This review provides a comprehensive analysis of the efficacy of multifactorial interventions for PD, simultaneously addressing the primary challenges of conventional therapies and highlighting how advanced technologies can help overcome these limitations. Part II focuses on the effectiveness of nanotechnology for improving pharmacokinetics of conventional therapies, through the synergistic use of dual or multiple therapeutic agents into a single nanoformulation. Significant emphasis is laid on the advancements toward innovative integrations, such as CRISPR/Cas9 with neuroprotective agents and stem cells, all effectively synergized with nanocarriers.

EXPERT OPINION: By using drug combinations, we can leverage their combined effects to enhance treatment efficacy and mitigate side effects through lower dosages. This article is meant to give nanocarrier-mediated co-delivery of drugs and the strategic incorporation of CRISPR/Cas9, either as an independent intervention or synergized with a neuroprotective agent.},
}

Humans
*Parkinson Disease/drug therapy
*Antiparkinson Agents/administration & dosage/therapeutic use/pharmacokinetics/pharmacology
*Drug Carriers/chemistry
Animals
*Neuroprotective Agents/administration & dosage/therapeutic use/pharmacokinetics
*Nanotechnology
*Nanoparticles
Blood-Brain Barrier/metabolism
Drug Delivery Systems
Precision Medicine
Drug Therapy, Combination
CRISPR-Cas Systems
Drug Combinations
Combined Modality Therapy
Drug Development
Drug Design

@article {pmid38429437,
year = {2024},
author = {Herman, L and Amo, A and Legois, B and Di Carlo, C and Veitia, RA and Todeschini, AL},
title = {A cellular model provides insights into the pathogenicity of the oncogenic FOXL2 somatic variant p.Cys134Trp.},
journal = {British journal of cancer},
volume = {130},
number = {9},
pages = {1453-1462},
pmid = {38429437},
issn = {1532-1827},
mesh = {*Forkhead Box Protein L2/genetics/metabolism ; Humans ; Female ; *Granulosa Cell Tumor/genetics/pathology ; Cell Line, Tumor ; Cell Movement/genetics ; Ovarian Neoplasms/genetics/pathology/metabolism ; Smad3 Protein/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; },
abstract = {BACKGROUND: FOXL2 is a transcription factor expressed in ovarian granulosa cells. A somatic variant of FOXL2 (c.402 C > G, p.Cys134Trp) is the hallmark of adult-type granulosa cell tumours.

METHODS: We generated KGN cell clones either heterozygous for this variant (MUT) or homozygous for the wild-type (WT) allele by CRISPR/Cas9 editing. They underwent RNA-Seq and bioinformatics analyses to uncover pathways impacted by deregulated genes. Cell morphology and migration were studied.

RESULTS: The differentially expressed genes (DEGs) between WT/MUT and WT/WT KGN cells (DEGs-WT/MUT), pointed to several dysregulated pathways, like TGF-beta pathway, cell adhesion and migration. Consistently, WT/MUT cells were rounder than WT/WT cells and displayed a different distribution of stress fibres and paxillin staining. A comparison of the DEGs-WT/MUT with those found when FOXL2 was knocked down (KD) in WT/WT KGN cells showed that most DEGs-WT/MUT cells were not so in the KD experiment, supporting a gain-of-function (GOF) scenario. MUT-FOXL2 also displayed a stronger interaction with SMAD3.

CONCLUSIONS: Our work, aiming at better understanding the GOF scenario, shows that the dysregulated genes and pathways are consistent with this idea. Besides, we propose that GOF might result from an enhanced interaction with SMAD3 that could underlie an ectopic capacity of mutated FOXL2 to bind SMAD4.},
}

*Forkhead Box Protein L2/genetics/metabolism
Humans
Female
*Granulosa Cell Tumor/genetics/pathology
Cell Line, Tumor
Cell Movement/genetics
Ovarian Neoplasms/genetics/pathology/metabolism
Smad3 Protein/genetics/metabolism
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic

@article {pmid38153113,
year = {2024},
author = {Li, M and He, Q and Li, T and Wan, W and Zhou, H},
title = {Development and evaluation of a CRISPR-Cas13a system-based diagnostic for hepatitis E virus.},
journal = {Clinical chemistry and laboratory medicine},
volume = {62},
number = {6},
pages = {1237-1247},
pmid = {38153113},
issn = {1437-4331},
mesh = {*Hepatitis E virus/genetics/isolation & purification ; Humans ; *Hepatitis E/diagnosis/virology ; *RNA, Viral/genetics/analysis ; *CRISPR-Cas Systems/genetics ; Genotype ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {OBJECTIVES: Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide. HEV RNA detection is the gold standard for HEV infection diagnosis and PCR methods are commonly used but are usually time-consuming and expensive, resulting in low detection efficiency and coverage, especially in low-income areas. Here, we developed a simpler and more accessible HEV RNA detection method based on CRISPR-Cas13a system.

METHODS: A total of 265 samples of different types and sources, including 89 positive samples and 176 negative samples, were enrolled for evaluations. The sensitivity and specificity of the Cas13a-crRNA detection system were evaluated. The World Health Organization reference panel for HEV genotypes was used to evaluate the capability for detecting different HEV genotypes. The validity of the assay was compared with RT-qPCR.

RESULTS: The 95 % limits of detection (LOD) of Cas13a-crRNA-based fluorescence assay and strip assay were 12.5 and 200 IU/mL, respectively. They did not show cross-reactivity with samples positive for hepatitis A virus, hepatitis B virus, hepatitis C virus, coxsackievirus A16, rotavirus, enterovirus 71, norovirus or enteropathic Escherichia coli. Different HEV genotypes (HEV1-4) can be detected by the assay. Compared to RT-qPCR, the positive predictive agreements of Cas13a-crRNA-based fluorescence and strip assay were 98.9 % (95 % CI: 93.9-99.8 %) and 91.0 % (95 % CI: 83.3-95.4 %), respectively. The negative predictive agreements were both 100 % (95 % CI: 97.8-100 %).

CONCLUSIONS: In conclusion, we established a rapid and convenient HEV RNA detection method with good sensitivity and specificity based on CRISPR-Cas13a system, providing a new option for HEV infection diagnosis.},
}

*Hepatitis E virus/genetics/isolation & purification
Humans
*Hepatitis E/diagnosis/virology
*RNA, Viral/genetics/analysis
*CRISPR-Cas Systems/genetics
Genotype
Sensitivity and Specificity
Limit of Detection

@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.},
}