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

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

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

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

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

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

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

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

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

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

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

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

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

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

@article {pmid39696352,
year = {2024},
author = {Zheng, S and Luo, M and Huang, H and Huang, X and Peng, Z and Zheng, S and Tan, J},
title = {New insights into the role of mitophagy related gene affecting the metastasis of osteosarcoma through scRNA-seq and CRISPR-Cas9 genome editing.},
journal = {Cell communication and signaling : CCS},
volume = {22},
number = {1},
pages = {592},
pmid = {39696352},
issn = {1478-811X},
support = {82460548//National Natural Science Foundation of China/ ; 20242BAB20368//Natural Science Foundation of Jiangxi Province of China/ ; },
mesh = {*Osteosarcoma/genetics/pathology ; *Mitophagy/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Bone Neoplasms/genetics/pathology/metabolism ; Neoplasm Metastasis/genetics ; Cell Line, Tumor ; Mice ; Single-Cell Analysis ; Single-Cell Gene Expression Analysis ; Mitochondrial Precursor Protein Import Complex Proteins ; },
abstract = {BACKGROUND: Osteosarcoma (OSA), the most common primary bone malignancy, poses significant challenges due to its aggressive nature and propensity for metastasis, especially in adolescents. Mitophagy analysis can help identify new therapeutic targets and combined treatment strategies.

METHODS: This study integrates single-cell sequencing (scRNA-seq) data and bulk-seq to identify mitophagy-related genes (MRGs) associated with the progression of OSA metastasis and analyze their clinical significance. scRNA-seq data elucidates the relationship between mitophagy and OSA metastasis, employing "CellChat" R package to explore intercellular communications and report on hundreds of ligand-receptor interactions. Subsequently, the combination of bulk-seq and CRISPR-Cas9 gene editing identifies mitophagy-related biomarker associated with metastatic prognosis. Finally, validation of the relationship between mitophagy and OSA metastasis is achieved through cellular biology experiments and animal studies.

RESULTS: The distinct mitophagy activity of various mitochondria manifests in diverse spatial localization, cellular developmental trajectories, and intercellular interactions. OSA tissue exhibits notable heterogeneity in mitophagy within osteoblastic OSA cells. However, high mitophagy activity correlates consistently with high metastatic potential. Subsequently, we identified three critical genes associated with mitophagy in OSA, namely RPS27A, TOMM20 and UBB. According to the aforementioned queue of genes, we have constructed a mitophagy_score (MIP_score). We observed that it consistently predicts patient prognosis in both internal and external datasets, demonstrating strong robustness and stability. Furthermore, we have found that MIP_score can also guide chemotherapy, with varying sensitivities to chemotherapeutic agents based on different MIP_score. It is noteworthy that, through the integration of CRISPR-Cas9 genome-wide screening and validation via cellular and animal experiments, we have identified RPS27A as a potential novel biomarker for OSA.

CONCLUSIONS: Our comprehensive analysis elucidated the profile of mitophagy throughout the OSA metastasis process, forming the basis for a mitophagy-related prognostic model that addresses clinical outcomes and drug sensitivity following OSA metastasis. Additionally, an online interactive platform was established to assist clinicians in decision-making (https://mip-score.shinyapps.io/labtan/). These findings lay the groundwork for developing targeted therapies aimed at improving the prognosis of OSA patients.},
}

*Osteosarcoma/genetics/pathology
*Mitophagy/genetics
Humans
*CRISPR-Cas Systems/genetics
*Gene Editing
Animals
*Bone Neoplasms/genetics/pathology/metabolism
Neoplasm Metastasis/genetics
Cell Line, Tumor
Mice
Single-Cell Analysis
Single-Cell Gene Expression Analysis
Mitochondrial Precursor Protein Import Complex Proteins

@article {pmid39695426,
year = {2024},
author = {Prado, MB and Coelho, BP and Iglesia, RP and Alves, RN and Boccacino, JM and Fernandes, CFL and Melo-Escobar, MI and Ayyadhury, S and Cruz, MC and Santos, TG and Beraldo, FH and Fan, J and Ferreira, FM and Nakaya, HI and Prado, MAM and Prado, VF and Duennwald, ML and Lopes, MH},
title = {Prion protein regulates invasiveness in glioblastoma stem cells.},
journal = {BMC cancer},
volume = {24},
number = {1},
pages = {1539},
pmid = {39695426},
issn = {1471-2407},
support = {2017/26158‑0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2019/14952‑0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2020/03714-8//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2022/08198-3//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2020/07450‑5//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2019/14741‑9//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2019/11097‑1//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2017/20271‑0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 101796/2020‑0//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 409941/2021‑2//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 03592-2021 RGPIN//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {*Glioblastoma/pathology/metabolism/genetics ; Humans ; *Neoplastic Stem Cells/metabolism/pathology ; Cell Line, Tumor ; *Neoplasm Invasiveness ; *Brain Neoplasms/pathology/metabolism/genetics ; Cell Proliferation ; Gene Knockout Techniques ; PrPC Proteins/metabolism/genetics ; Prion Proteins/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor driven by glioblastoma stem cells (GSCs), which represent an appealing target for therapeutic interventions. The cellular prion protein (PrP[C]), a scaffold protein involved in diverse cellular processes, interacts with various membrane and extracellular matrix molecules, influencing tumor biology. Herein, we investigate the impact of PrP[C] expression on GBM.

METHODS: To address this goal, we employed CRISPR-Cas9 technology to generate PrP[C] knockout (KO) glioblastoma cell lines, enabling detailed loss-of-function studies. Bulk RNA sequencing followed by differentially expressed gene and pathway enrichment analyses between U87 or U251 PrP[C]-wild-type (WT) cells and PrP[C]-knockout (KO) cells were used to identify pathways regulated by PrP[C]. Immunofluorescence assays were used to evaluate cellular morphology and protein distribution. For assessment of protein levels, Western blot and flow cytometry assays were employed. Transwell and growth curve assays were used to determine the impact of loss-of-PrP[C] in GBM invasiveness and proliferation, respectively. Single-cell RNA sequencing analysis of data from patient tumors from The Cancer Genome Atlas (TCGA) and the Broad Institute of Single-Cell Data Portal were used to evaluate the correspondence between our in vitro results and patient samples.

RESULTS: Transcriptome analysis of PrP[C]-KO GBM cell lines revealed altered expression of genes associated with crucial tumor progression pathways, including migration, proliferation, and stemness. These findings were corroborated by assays that revealed impaired invasion, migration, proliferation, and self-renewal in PrP[C]-KO GBM cells, highlighting its critical role in sustaining tumor growth. Notably, loss-of-PrP[C] disrupted the expression and localization of key stemness markers, particularly CD44. Additionally, the modulation of PrP[C] levels through CD44 overexpression further emphasizes their regulatory role in these processes.

CONCLUSIONS: These findings establish PrP[C] as a modulator of essential molecules on the cell surface of GSCs, highlighting its potential as a therapeutic target for GBM.},
}

*Glioblastoma/pathology/metabolism/genetics
Humans
*Neoplastic Stem Cells/metabolism/pathology
Cell Line, Tumor
*Neoplasm Invasiveness
*Brain Neoplasms/pathology/metabolism/genetics
Cell Proliferation
Gene Knockout Techniques
PrPC Proteins/metabolism/genetics
Prion Proteins/metabolism/genetics
Gene Expression Regulation, Neoplastic
Cell Movement
CRISPR-Cas Systems

@article {pmid39694744,
year = {2024},
author = {Zhu, Y and Yu, X and Wu, J},
title = {CRISPR/Cas: a toolkit for plant disease diagnostics.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2024.11.011},
pmid = {39694744},
issn = {1878-4372},
abstract = {Genetic factors and infectious pathogens that cause plant diseases have a major impact on agricultural production. In recent years, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system in nucleic acid analysis and plant disease diagnostics has been demonstrated. We highlight progress of CRISPR/Cas technology that is significant for monitoring plant growth and preventing diseases.},
}

@article {pmid39694477,
year = {2024},
author = {Harding, KR and Malone, LM and Kyte, NAP and Jackson, SA and Smith, LM and Fineran, PC},
title = {Genome-wide identification of bacterial genes contributing to nucleus-forming jumbo phage infection.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae1194},
pmid = {39694477},
issn = {1362-4962},
support = {//Marsden Fund/ ; //Royal Society of New Zealand/ ; //Bioprotection Aotearoa/ ; //James Cook Research Fellowship/ ; //University of Otago Doctoral Scholarships/ ; //Division of Health Sciences Career Development Postdoctoral Fellowship/ ; //EMBO Postdoctoral Fellowship/ ; },
abstract = {The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage 'nucleus' compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.},
}

@article {pmid39693940,
year = {2024},
author = {Baranova, SV and Zhdanova, PV and Golyshev, VM and Lomzov, AA and Pestryakov, PE and Chernonosov, AA and Koval, VV},
title = {Thermodynamic parameters obtained for the formation of the Cas12a-RNA/DNA complex.},
journal = {Biochemical and biophysical research communications},
volume = {743},
number = {},
pages = {151176},
doi = {10.1016/j.bbrc.2024.151176},
pmid = {39693940},
issn = {1090-2104},
abstract = {The thermodynamics of interactions between Cas12a, RNA, and DNA are important to understanding the molecular mechanisms governing CRISPR-Cas12a's specificity and function. In this study, we employed isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations to investigate the binding properties and energetic contributions of Cas12a-crRNA complexes with single-stranded (ssDNA) and double-stranded (dsDNA) DNA substrates. ITC analyses revealed significant thermal effects during the interaction of Cas12a-crRNA with ssDNA but no detectable effects with dsDNA. The binding to ssDNA was characterized by an enthalpy change (ΔH°) of -243 ± 18 kcal/mol and a stoichiometry of ∼0.3, indicating partial binding due to structural hindrances such as intramolecular secondary structures in RNA and DNA. MD simulations further supported these findings, highlighting the stability and dynamic behavior of Cas12a-crRNA complexes with both DNA substrates. Binding free energy calculations (MM-GBSA) revealed stronger stabilization of the Cas12a-crRNA complex by dsDNA compared to ssDNA, likely driven by additional electrostatic interactions and protein-DNA contacts. However, these interactions did not produce measurable heat effects in ITC experiments. The combined experimental and computational findings demonstrate that the CRISPR-Cas12a system's interactions with nucleic acids are predominantly governed by their structural characteristics and conformational flexibility. These results deepen our understanding of the thermodynamic and structural principles underlying Cas12a-mediated target recognition and cleavage.},
}

@article {pmid39693439,
year = {2024},
author = {Wang, Q and Xu, X and Chen, S and Lu, R and Li, L and Lo, CH and Liu, Z and Ning, K and Li, T and Kowal, TJ and Wang, B and Hartnett, ME and Wang, S and Qi, LS and Sun, Y},
title = {dCasMINI-mediated therapy rescues photoreceptors degeneration in a mouse model of retinitis pigmentosa.},
journal = {Science advances},
volume = {10},
number = {51},
pages = {eadn7540},
doi = {10.1126/sciadv.adn7540},
pmid = {39693439},
issn = {2375-2548},
mesh = {Animals ; *Retinitis Pigmentosa/therapy/genetics/pathology/metabolism ; *Disease Models, Animal ; *Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism ; Mice ; *Genetic Therapy/methods ; *Dependovirus/genetics ; Electroretinography ; Retinal Degeneration/therapy/genetics/pathology ; CRISPR-Cas Systems ; Humans ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Eye Proteins/metabolism/genetics ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; },
abstract = {Retinitis pigmentosa (RP) is characterized by degeneration of rod and cone photoreceptors that progresses to irreversible blindness. Now, there are no mutation-agnostic approaches to treat RP. Here, we utilized a single adeno-associated virus (AAV)-based CRISPR activation system to activate phosphodiesterase 6B (Pde6b) to mitigate the severe degeneration in Pde6a[nmf363] mice. We demonstrate that transcriptional activation of Pde6b can rescue the loss of Pde6a, with preservation of retinal structure, restoration of electroretinography responses, and improvement of visual function as assessed by optokinetic response and looming-induced escape behaviors. These findings demonstrate the therapeutic potential of a dCasMINI-mediated activation strategy that provides a mutation-independent treatment for retinal degeneration. This study offers a promising therapeutic approach for RP and potentially other forms of genetic diseases.},
}

Animals
*Retinitis Pigmentosa/therapy/genetics/pathology/metabolism
*Disease Models, Animal
*Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism
Mice
*Genetic Therapy/methods
*Dependovirus/genetics
Electroretinography
Retinal Degeneration/therapy/genetics/pathology
CRISPR-Cas Systems
Humans
Retinal Rod Photoreceptor Cells/metabolism/pathology
Eye Proteins/metabolism/genetics
Retinal Cone Photoreceptor Cells/metabolism/pathology

@article {pmid39693429,
year = {2024},
author = {Shembrey, C and Yang, R and Casan, J and Hu, W and Chen, H and Singh, GJ and Sadras, T and Prasad, K and Shortt, J and Johnstone, RW and Trapani, JA and Ekert, PG and Fareh, M},
title = {Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision.},
journal = {Science advances},
volume = {10},
number = {51},
pages = {eadl0731},
doi = {10.1126/sciadv.adl0731},
pmid = {39693429},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Point Mutation ; Gene Silencing ; Gene Editing/methods ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins p21(ras)/genetics ; Base Pair Mismatch ; Membrane Proteins/genetics ; Oncogenes ; RNA, Neoplasm/genetics/metabolism ; GTP Phosphohydrolases ; Proto-Oncogene Proteins B-raf ; },
abstract = {Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we show that introducing synthetic mismatches at precise positions of the spacer sequence enables de novo design of guide RNAs [CRISPR RNAs (crRNAs)] with strong preferential silencing of point-mutated transcripts. We applied these design principles to effectively silence the oncogenic KRAS G12 hotspot, NRAS G12D and BRAF V600E transcripts with minimal off-target silencing of the wild-type transcripts, underscoring the adaptability of this platform to silence various SNVs. Unexpectedly, the SNV-selective crRNAs harboring mismatched nucleotides reduce the promiscuous collateral activity of the RfxCas13d ortholog. These findings demonstrate that the CRISPR-Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing.},
}

*CRISPR-Cas Systems
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
Point Mutation
Gene Silencing
Gene Editing/methods
Polymorphism, Single Nucleotide
Proto-Oncogene Proteins p21(ras)/genetics
Base Pair Mismatch
Membrane Proteins/genetics
Oncogenes
RNA, Neoplasm/genetics/metabolism
GTP Phosphohydrolases
Proto-Oncogene Proteins B-raf

@article {pmid39693343,
year = {2024},
author = {Casagrande Raffi, G and Chen, J and Feng, X and Chen, Z and Lieftink, C and Deng, S and Mo, J and Zeng, C and Steur, M and Wang, J and Bleijerveld, OB and Hoekman, L and van der Wel, N and Wang, F and Beijersbergen, R and Zheng, J and Bernards, R and Wang, L},
title = {An antibiotic that mediates immune destruction of senescent cancer cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {52},
pages = {e2417724121},
doi = {10.1073/pnas.2417724121},
pmid = {39693343},
issn = {1091-6490},
support = {787925//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; LSH-TKI-LSHM20083//Health Holland/ ; 19-051-ASP//Mark Foundation For Cancer Research (The Mark Foundation for Cancer Research)/ ; 12539//Dutch Cancer Society KWF/ ; W2432050; 82372695//National Natural Science Foundation of China-Guangdong Joint Fund (NSFC)/ ; 2024A04J6484//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; *Cellular Senescence/drug effects/immunology ; *Pyrans/pharmacology ; *Killer Cells, Natural/immunology/drug effects/metabolism ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism ; Necroptosis/drug effects ; Neoplasms/immunology/drug therapy/metabolism ; CD8-Positive T-Lymphocytes/immunology/drug effects/metabolism ; Anti-Bacterial Agents/pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism/genetics ; Pyroptosis/drug effects ; Apoptosis/drug effects ; Animals ; CRISPR-Cas Systems ; Polyether Polyketides ; },
abstract = {Drugs that eliminate senescent cells, senolytics, can be powerful when combined with prosenescence cancer therapies. Using a CRISPR/Cas9-based genetic screen, we identify here SLC25A23 as a vulnerability of senescent cancer cells. Suppressing SLC25A23 disrupts cellular calcium homeostasis, impairs oxidative phosphorylation, and interferes with redox signaling, leading to death of senescent cells. These effects can be replicated by salinomycin, a cation ionophore antibiotic. Salinomycin prompts a pyroptosis-apoptosis-necroptosis (PAN)optosis-like cell death in senescent cells, including apoptosis and two forms of immunogenic cell death: necroptosis and pyroptosis. Notably, we observed that salinomycin treatment or SLC25A23 suppression elevates reactive oxygen species, upregulating death receptor 5 via Jun N-terminal protein kinase (JNK) pathway activation. We show that a combination of a death receptor 5 (DR5) agonistic antibody and salinomycin is a robust senolytic cocktail. We provide evidence that this drug combination provokes a potent natural killer (NK) and CD8+ T cell-mediated immune destruction of senescent cancer cells, mediated by the pyroptotic cytokine interleukin 18 (IL18).},
}

Humans
*Cellular Senescence/drug effects/immunology
*Pyrans/pharmacology
*Killer Cells, Natural/immunology/drug effects/metabolism
Cell Line, Tumor
Reactive Oxygen Species/metabolism
Necroptosis/drug effects
Neoplasms/immunology/drug therapy/metabolism
CD8-Positive T-Lymphocytes/immunology/drug effects/metabolism
Anti-Bacterial Agents/pharmacology
Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism/genetics
Pyroptosis/drug effects
Apoptosis/drug effects
Animals
CRISPR-Cas Systems
Polyether Polyketides

@article {pmid39667940,
year = {2024},
author = {Yang, B and Wu, C and Teng, Y and Chou, KJ and Guarnieri, MT and Xiong, W},
title = {Tailoring microbial fitness through computational steering and CRISPRi-driven robustness regulation.},
journal = {Cell systems},
volume = {15},
number = {12},
pages = {1133-1147.e4},
doi = {10.1016/j.cels.2024.11.012},
pmid = {39667940},
issn = {2405-4720},
mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Computer Simulation ; Genetic Fitness/genetics ; Escherichia coli/genetics/metabolism ; Organisms, Genetically Modified ; },
abstract = {The widespread application of genetically modified microorganisms (GMMs) across diverse sectors underscores the pressing need for robust strategies to mitigate the risks associated with their potential uncontrolled escape. This study merges computational modeling with CRISPR interference (CRISPRi) to refine GMM metabolic robustness. Utilizing ensemble modeling, we achieved high-throughput in silico screening for enzymatic targets susceptible to expression alterations. Translating these insights, we developed functional CRISPRi, boosting fitness control via multiplexed gene knockdown. Our method, enhanced by an insulator-improved gRNA structure and an off-switch circuit controlling a compact Cas12m, resulted in rationally engineered strains with escape frequencies below National Institutes of Health standards. The effectiveness of this approach was confirmed under various conditions, showcasing its ability for secure GMM management. This research underscores the resilience of microbial metabolism, strategically modifying key nodes to halt growth without provoking significant resistance, thereby enabling more reliable and precise GMM control. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Computer Simulation
Genetic Fitness/genetics
Escherichia coli/genetics/metabolism
Organisms, Genetically Modified

@article {pmid39589913,
year = {2024},
author = {Wang, Q and Yang, G and Jia, R and Wang, F and Wang, G and Xu, Z and Li, J and Li, B and Yu, L and Zhang, Y and Alariqi, M and Cao, J and Liang, S and Zhang, X and Nie, X and Jin, S},
title = {Utilizing the mutant library to investigate the functional characterization of GhGLR3.4 regulating jasmonic acid to defense pest infestation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {120},
number = {6},
pages = {2889-2903},
doi = {10.1111/tpj.17152},
pmid = {39589913},
issn = {1365-313X},
support = {32272128//National Natural Science Foundation of China/ ; 32401780//National Natural Science Foundation of China/ ; 027Y2022-021//Science and Technology Project of Hubei Province of China National Tobacco Corporation/ ; 2022YFF1001403//National Key Research and Development Program of China/ ; 32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; },
mesh = {*Cyclopentanes/metabolism ; *Oxylipins/metabolism ; *Gossypium/genetics/parasitology ; *Plant Proteins/genetics/metabolism ; Animals ; Receptors, Glutamate/genetics/metabolism ; Gene Expression Regulation, Plant ; Signal Transduction ; Plant Growth Regulators/metabolism ; Arabidopsis/genetics/physiology ; Mutation ; CRISPR-Cas Systems ; Plant Diseases/parasitology/genetics/immunology ; },
abstract = {The glutamate receptor (GLR) serves as a ligand-gated ion channel that plays a vital role in plant growth, development, and stress response. Nevertheless, research on GLRs in cotton is still very limited. The present study conducted a comprehensive analysis of GLRs gene family in cotton. In total, 41 members of the GLR family were identified in cotton unveiling distinct subgroups in comparison to Arabidopsis. Among these members, the third subgroup highlights its pivotal role in cotton's defense against insect infestation. Furthermore, the CRISPR/Cas9 system was utilized to create a mutant library of GLR members, which consisted of a total of 135 independent mutant lines, resulting in the production of novel cotton materials with valuable breeding potential for pest control. Further, this study elucidates the influence of GhGLR3.4 on jasmonic acid (JA) pathway signal transduction and demonstrated its participation in the influx of intracellular Ca[2+], which regulates "calcium transients" following stimulation, thereby influencing multiple intracellular reactions. The study also found that GhGLR3.4 influences the synthesis of the JA pathway and actively partakes in long-distance signal transmission among plants, facilitating the transfer of defense signals to neighbor leaves and thereby triggering systemic defense. Consequently, this research advances our knowledge of plants' comprehensive defense mechanism against insect pest infestation.},
}

*Cyclopentanes/metabolism
*Oxylipins/metabolism
*Gossypium/genetics/parasitology
*Plant Proteins/genetics/metabolism
Animals
Receptors, Glutamate/genetics/metabolism
Gene Expression Regulation, Plant
Signal Transduction
Plant Growth Regulators/metabolism
Arabidopsis/genetics/physiology
Mutation
CRISPR-Cas Systems
Plant Diseases/parasitology/genetics/immunology

@article {pmid39581190,
year = {2025},
author = {Egawa, M and Uno, N and Komazaki, R and Ohkame, Y and Yamazaki, K and Yoshimatsu, C and Ishizu, Y and Okano, Y and Miyamoto, H and Osaki, M and Suzuki, T and Hosomichi, K and Aizawa, Y and Kazuki, Y and Tomizuka, K},
title = {Generation of Monosomy 21q Human iPS Cells by CRISPR/Cas9-Mediated Interstitial Megabase Deletion.},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
volume = {30},
number = {1},
pages = {e13184},
doi = {10.1111/gtc.13184},
pmid = {39581190},
issn = {1365-2443},
support = {21-101//Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences/ ; JP24bm1123038//Japan Agency for Medical Research and Development/ ; JP24ama121046//Japan Agency for Medical Research and Development/ ; JP24gm0010010//Japan Agency for Medical Research and Development/ ; JPMJCR18S4//CREST, Japan Science and Technology Agency/ ; JPMJSP2134//SPRING, Japan Science and Technology Agency/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Chromosomes, Human, Pair 21/genetics ; *Chromosome Deletion ; Monosomy/genetics ; Cell Line ; },
abstract = {Missing an entire chromosome or chromosome arm in normal diploid cells has a deleterious impact on cell viability, which may contribute to the development of specific birth defects. Nevertheless, the effects of chromosome loss in human cells have remained unexplored due to the lack of suitable model systems. Here, we developed an efficient, selection-free approach to generate partial monosomy in human induced pluripotent stem cells (iPSCs). The introduction of Cas9 proteins and a pair of gRNAs induces over megabase-sized interstitial chromosomal deletions. Using human chromosome 21 (HSA21) as a model, partial monosomy 21q (PM21q) iPSC lines with deletions ranging from 4.5 to 27.9 Mb were isolated. A 33.6 Mb deletion, encompassing all protein-coding genes on 21q, was also achieved, establishing the first 21q monosomy human iPSC line. Transcriptome and proteome analyses revealed that the abundances of mRNA and protein encoded by the majority of genes in the monosomic regions are half of the diploid expression level, indicating an absence of dosage compensation. The ability to generate customized partial monosomy cell lines on an isogenic, karyotypically normal background should facilitate the gain of novel insights into the impact of chromosome loss on cellular fitness.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems
*Chromosomes, Human, Pair 21/genetics
*Chromosome Deletion
Monosomy/genetics
Cell Line

@article {pmid39569987,
year = {2024},
author = {Yamamoto, S and Afifi, OA and Lam, LPY and Takeda-Kimura, Y and Osakabe, Y and Osakabe, K and Bartley, LE and Umezawa, T and Tobimatsu, Y},
title = {Disruption of aldehyde dehydrogenase decreases cell wall-bound p-hydroxycinnamates and improves cell wall digestibility in rice.},
journal = {The Plant journal : for cell and molecular biology},
volume = {120},
number = {6},
pages = {2828-2845},
doi = {10.1111/tpj.17148},
pmid = {39569987},
issn = {1365-313X},
support = {Exploratory Research on Humanosphere Science 2021//Research Institute for Sustainable Humanosphere, Kyoto University/ ; Exploratory Research on Humanosphere Science 2023//Research Institute for Sustainable Humanosphere, Kyoto University/ ; DE-SC0021126//Office of Science/ ; #20H03044//Japan Society for the Promotion of Science/ ; #22J13457//Japan Society for the Promotion of Science/ ; #24K01827//Japan Society for the Promotion of Science/ ; #1015621//National Institute of Food and Agriculture/ ; },
mesh = {*Cell Wall/metabolism ; *Oryza/genetics/metabolism/enzymology ; *Coumaric Acids/metabolism ; *Lignin/metabolism ; *Plant Proteins/genetics/metabolism ; Aldehyde Dehydrogenase/metabolism/genetics ; Xylans/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {In grass cell walls, ferulic acid (FA) serves as an important cross-linker between cell wall polymers, such as arabinoxylan (AX) and lignin, affecting the physicochemical properties of the cell walls as well as the utilization properties of grass lignocellulose for biorefinering. Here, we demonstrate that hydroxycinnamaldehyde dehydrogenase (HCALDH) plays a crucial role in the biosynthesis of the FA used for cell wall feruloylation in rice (Oryza sativa). Bioinformatic and gene expression analyses of aldehyde dehydrogenases (ALDHs) identified two rice ALDH subfamily 2C members, OsHCALDH2 (OsALDH2C2) and OsHCALDH3 (OsALDH2C3), potentially involved in cell wall feruloylation in major vegetative tissues of rice. CRISPR-Cas9 genome editing of OsHCALDH2 and OsHCALDH3 revealed that the contents of AX-bound ferulate were reduced by up to ~45% in the cell walls of the HCALDH-edited mutants, demonstrating their roles in cell wall feruloylation. The abundance of hemicellulosic sugars including arabinosyl units on AX was notably reduced in the cell walls of the HCALDH-edited mutants, whereas cellulose and lignin contents remained unaffected. In addition to reducing cell wall-bound ferulate, the loss of OsHCALDH2 and/or OsHCALDH3 also partially reduced cell wall-bound p-coumarate and sinapate in the vegetative tissues of rice, whereas it did not cause detectable changes in the amount of γ-oryzanol (feruloyl sterols) in rice seeds. Furthermore, the HCALDH-edited mutants exhibited improved cell wall saccharification efficiency, both with and without alkaline pretreatment, plausibly due to the reduction in cell wall cross-linking FA. Overall, HCALDH appears to present a potent bioengineering target for enhancing utilization properties of grass lignocellulose.},
}

*Cell Wall/metabolism
*Oryza/genetics/metabolism/enzymology
*Coumaric Acids/metabolism
*Lignin/metabolism
*Plant Proteins/genetics/metabolism
Aldehyde Dehydrogenase/metabolism/genetics
Xylans/metabolism
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid39560384,
year = {2024},
author = {Steiner, S and Roy, CR},
title = {CRISPR-Cas9-based approaches for genetic analysis and epistatic interaction studies in Coxiella burnetii.},
journal = {mSphere},
volume = {9},
number = {12},
pages = {e0052324},
doi = {10.1128/msphere.00523-24},
pmid = {39560384},
issn = {2379-5042},
support = {R21AI171333,R56AI182383//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {*Coxiella burnetii/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epistasis, Genetic ; Bacterial Proteins/genetics ; Humans ; Animals ; Phenotype ; },
abstract = {UNLABELLED: Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates to high numbers in an acidified lysosome-derived vacuole. Intracellular replication requires the Dot/Icm type IVB secretion system, which translocates over 100 different effector proteins into the host cell. Screens employing random transposon mutagenesis have identified several C. burnetii effectors that play an important role in intracellular replication; however, the difficulty in conducting directed mutagenesis has been a barrier to the systematic analysis of effector mutants and to the construction of double mutants to assess epistatic interactions between effectors. Here, two CRISPR-Cas9 technology-based approaches were developed to study C. burnetii phenotypes resulting from targeted gene disruptions. CRISPRi was used to silence gene expression and demonstrated that silencing of effectors or Dot/Icm system components resulted in phenotypes similar to those of transposon insertion mutants. A CRISPR-Cas9-mediated cytosine base editing protocol was developed to generate targeted loss-of-function mutants through the introduction of premature stop codons into C. burnetii genes. Cytosine base editing successfully generated double mutants in a single step. A double mutant deficient in both cig57 and cig2 had a robust and additive intracellular replication defect when compared to either single mutant, which is consistent with Cig57 and Cig2 functioning in independent pathways that both contribute to a vacuole that supports C. burnetii replication. Thus, CRISPR-Cas9-based technologies expand the genetic toolbox for C. burnetii and will facilitate genetic studies aimed at investigating the mechanisms this pathogen uses to replicate inside host cells.

IMPORTANCE: Understanding the genetic mechanisms that enable C. burnetii to replicate in mammalian host cells has been hampered by the difficulty in making directed mutations. Here, a reliable and efficient system for generating targeted loss-of-function mutations in C. burnetii using a CRISPR-Cas9-assisted base editing approach is described. This technology was applied to make double mutants in C. burnetii that enabled the genetic analysis of two genes that play independent roles in promoting the formation of vacuoles that support intracellular replication. This advance will accelerate the discovery of mechanisms important for C. burnetii host infection and disease.},
}

*Coxiella burnetii/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Epistasis, Genetic
Bacterial Proteins/genetics
Humans
Animals
Phenotype

@article {pmid39546384,
year = {2024},
author = {Birchler, JA and Kelly, J and Singh, J and Liu, H and Zhang, Z and Char, SN and Sharma, M and Yang, H and Albert, PS and Yang, B},
title = {Synthetic minichromosomes in plants: past, present, and promise.},
journal = {The Plant journal : for cell and molecular biology},
volume = {120},
number = {6},
pages = {2356-2366},
doi = {10.1111/tpj.17142},
pmid = {39546384},
issn = {1365-313X},
support = {PGRP 2221891//National Science Foundation/ ; },
mesh = {*Chromosomes, Plant/genetics ; *Chromosomes, Artificial/genetics ; *Centromere/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Genetic Engineering/methods ; Plants/genetics ; Telomere/genetics ; Plants, Genetically Modified ; },
abstract = {The status of engineered mini-chromosomes/artificial chromosomes/synthetic chromosomes in plants is summarized. Their promise is that they provide a means to accumulate foreign genes on an independent entity other than the normal chromosomes, which would facilitate stacking of novel traits in a way that would not be linked to endogenous genes and that would facilitate transfer between lines. Centromeres in plants are epigenetic, and therefore the isolation of DNA underlying centromeres and reintroduction into plant cells will not establish a functional kinetochore, which obviates this approach for in vitro assembly of plant artificial chromosomes. This issue was bypassed by using telomere-mediated chromosomal truncation to produce mini-chromosomes with little more than an endogenous centromere that could in turn be used as a foundation to build synthetic chromosomes. Site-specific recombinases and various iterations of CRISPR-Cas9 editing provide many tools for the development and re-engineering of synthetic chromosomes.},
}

*Chromosomes, Plant/genetics
*Chromosomes, Artificial/genetics
*Centromere/genetics
*CRISPR-Cas Systems
Gene Editing/methods
Genetic Engineering/methods
Plants/genetics
Telomere/genetics
Plants, Genetically Modified

@article {pmid39521313,
year = {2025},
author = {Levis, H and Lewis, C and Fainor, M and Lawal, A and Stockham, E and Weston, J and Farhang, N and Gullbrand, SE and Bowles, RD},
title = {Targeted CRISPR regulation of ZNF865 enhances stem cell cartilage deposition, tissue maturation rates, and mechanical properties in engineered intervertebral discs.},
journal = {Acta biomaterialia},
volume = {191},
number = {},
pages = {276-291},
doi = {10.1016/j.actbio.2024.11.007},
pmid = {39521313},
issn = {1878-7568},
support = {R01 AR074998/AR/NIAMS NIH HHS/United States ; R01 AR083990/AR/NIAMS NIH HHS/United States ; },
mesh = {Humans ; *Intervertebral Disc/metabolism/cytology ; *Tissue Engineering/methods ; *Stem Cells/metabolism/cytology ; Cartilage/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Biomechanical Phenomena ; },
abstract = {Cell and tissue engineering based approaches have garnered significant interest for treating intervertebral disc degeneration and associated low back pain due to the substantial limitations of currently available clinical treatments. Herein we present a clustered regularly interspaced short palindromic repeats (CRISPR)-guided gene modulation strategy to improve the therapeutic potential of cell and tissue engineering therapies for treating intervertebral disc disease. Recently, we discovered a zinc finger (ZNF) protein, ZNF865 (BLST), which is associated with no in-depth publications and has not been functionally characterized. Utilizing CRISPR-guided gene modulation, we show that ZNF865 regulates cell cycle progression and protein processing. As a result, regulating this gene acts as a primary titratable regulator of cell activity. We also demonstrate that targeted ZNF865 regulation can enhance protein production and fibrocartilage-like matrix deposition in human adipose-derived stem cells (hASCs). Furthermore, we demonstrate CRISPR-engineered hASCs ability to increase GAG and collagen II matrix deposition in human-size tissue-engineered discs by 8.5-fold and 88.6-fold, respectively, while not increasing collagen X expression compared to naive hASCs dosed with growth factors. With this increased tissue deposition, we observe significant improvements in compressive mechanical properties, generating a stiffer and more robust tissue. Overall, we present novel biology on ZNF865 and display the power of CRISPR-cell engineering to enhance strategies treating musculoskeletal disease. STATEMENT OF SIGNIFICANCE: This work reports on a novel gene, ZNF865 (also known as BLST), that when regulated with CRISPRa, improves cartilagenous tissue deposition in human sized tissue engineering constructs. Producing tissue engineering constructs at human scale has proven difficult, and this strategy presents a broadly applicable tool to enhance a cells ability to produce tissue at these scales, as we saw an ∼8-88 fold increase in tissue deposition and significantly improved biomechanics in large tissue engineered intervertebral disc compared to traditional growth factor differentiation methods. Additionally, this work begins to elucidate the biology of this novel zinc finger protein, which appears to be critical in regulating cell function and activity.},
}

Humans
*Intervertebral Disc/metabolism/cytology
*Tissue Engineering/methods
*Stem Cells/metabolism/cytology
Cartilage/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Biomechanical Phenomena

@article {pmid39504234,
year = {2024},
author = {Xie, Y and Xu, Y and Jia, H and Wang, K and Chen, S and Ma, T and Deng, Y and Lang, Z and Niu, Q},
title = {Tomato MADS-RIN regulates GAME5 expression to promote non-bitter glycoalkaloid biosynthesis in fruit.},
journal = {The Plant journal : for cell and molecular biology},
volume = {120},
number = {6},
pages = {2500-2514},
doi = {10.1111/tpj.17125},
pmid = {39504234},
issn = {1365-313X},
support = {2022AH020061//the Science Fund for Distinguished Young Scholars of Anhui Province/ ; 32270367; 32400275//the National Natural Science Foundation of China/ ; NK2022010301//the National Key Research and Development Program of China/ ; jxsq2020101081//the Double Thousand Plan of Jiangxi Province/ ; jxsq2020101082//the Double Thousand Plan of Jiangxi Province/ ; 2022AH010056//the Innovative research team of Anhui Education/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism ; *Fruit/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; MADS Domain Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Alkaloids/metabolism/biosynthesis ; Gene Editing ; Sapogenins ; },
abstract = {A well-known defense-associated steroidal glycoalkaloid (SGA) metabolic shift eliminates the bitterness and toxicity of ripe tomato fruits. This study was conducted to clarify the effects of MADS-RIN (RIN) and its cofactors on SGA metabolism in tomato fruits. Using a CRISPR/Cas9-based gene-editing system, we mutated RIN and two cofactor genes (FUL1 and FUL2). The observed changes to fruit color and size in the mutants reflected the overlapping and distinct effects of RIN, FUL1, and FUL2 on fruit ripening. According to a UPLC-MS/MS analysis, the RIN and cofactor mutants had decreased levels of the relatively non-toxic metabolite esculeoside A, but they accumulated toxic SGA pathway intermediates, suggesting RIN and its cofactors are directly involved in esculeoside A biosynthesis. Transcriptome and qPCR analyses detected the downregulated expression of GAME5, which encodes a key enzyme mediating esculeoside A biosynthesis. ChIP-seq and ChIP-qPCR analyses confirmed GAME5 is targeted by RIN. RIN was observed to activate GAME5 transcription by binding to two non-canonical CArG-boxes in the GAME5 promoter. Additionally, RIN promotes SGA metabolism independently of ethylene. Collectively, these findings enhance our understanding of the molecular mechanism governing tomato fruit ripening and SGA biosynthesis. Furthermore, they may be useful for improving tomato fruit quality and safety.},
}

*Solanum lycopersicum/genetics/metabolism
*Fruit/genetics/metabolism
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
MADS Domain Proteins/genetics/metabolism
CRISPR-Cas Systems
Alkaloids/metabolism/biosynthesis
Gene Editing
Sapogenins

@article {pmid39697359,
year = {2022},
author = {Hegeman, CV and de Jong, OG and Lorenowicz, MJ},
title = {A kaleidoscopic view of extracellular vesicles in lysosomal storage disorders.},
journal = {Extracellular vesicles and circulating nucleic acids},
volume = {3},
number = {4},
pages = {393-421},
pmid = {39697359},
issn = {2767-6641},
abstract = {Extracellular vesicles (EVs) are a heterogeneous population of stable lipid membrane particles that play a critical role in the regulation of numerous physiological and pathological processes. EV cargo, which includes lipids, proteins, and RNAs including miRNAs, is affected by the metabolic status of the parental cell. Concordantly, abnormalities in the autophagic-endolysosomal pathway, as seen in lysosomal storage disorders (LSDs), can affect EV release as well as EV cargo. LSDs are a group of over 70 inheritable diseases, characterized by lysosomal dysfunction and gradual accumulation of undigested molecules. LSDs are caused by single gene mutations that lead to a deficiency of a lysosomal protein or lipid. Lysosomal dysfunction sets off a cascade of alterations in the endolysosomal pathway that can affect autophagy and alter calcium homeostasis, leading to energy imbalance, oxidative stress, and apoptosis. The pathophysiology of these diseases is very heterogenous, complex, and currently incompletely understood. LSDs lead to progressive multisystemic symptoms that often include neurological deficits. In this review, a kaleidoscopic overview will be given on the roles of EVs in LSDs, from their contribution to pathology and diagnostics to their role as drug delivery vehicles. Furthermore, EV cargo and surface engineering strategies will be discussed to show the potential of EVs in future LSD treatment, both in the context of enzyme replacement therapy, as well as future gene editing strategies like CRISPR/Cas. The use of engineered EVs as drug delivery vehicles may mask therapeutic cargo from the immune system and protect it from degradation, improving circulation time and targeted delivery.},
}

@article {pmid39693337,
year = {2024},
author = {Li, LL and Xiao, Y and Wang, B and Zhuang, Y and Chen, Y and Lu, J and Lou, Y and Li, R},
title = {A frameshift mutation in JAZ10 resolves the growth versus defense dilemma in rice.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {52},
pages = {e2413564121},
doi = {10.1073/pnas.2413564121},
pmid = {39693337},
issn = {1091-6490},
support = {32372551//MOST | National Natural Science Foundation of China (NSFC)/ ; 32402385//MOST | National Natural Science Foundation of China (NSFC)/ ; 2021YFD1401100//MOST | National Key Research and Development Program of China (NKPs)/ ; NA//Max Planck Partner Group Program/ ; 226-2024-00159//MOE | Fundamental Research Funds for the Central Universities (Fundamental Research Fund for the Central Universities)/ ; },
mesh = {*Oryza/genetics/growth & development/parasitology/metabolism ; *Frameshift Mutation ; *Cyclopentanes/metabolism ; *Plant Proteins/genetics/metabolism ; *Oxylipins/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Animals ; Plant Diseases/parasitology/genetics ; Hemiptera/genetics/growth & development/metabolism ; Gene Editing ; Signal Transduction ; Disease Resistance/genetics ; Plants, Genetically Modified/genetics ; Gibberellins/metabolism ; },
abstract = {CRISPR-Cas9 genome editing systems have revolutionized plant gene functional studies by enabling the targeted introduction of insertion-deletions (INDELs) via the nonhomologous end-joining (NHEJ) pathway. Frameshift-inducing INDELs can introduce a premature termination codon and, in other instances, can lead to the appearance of new proteins. Here, we found that mutations in the rice jasmonate (JA) signaling gene OsJAZ10 by CRISPR-Cas9-based genome editing did not affect canonical JA signaling. However, a type of mutant with an INDEL that yielded a novel frameshift protein named FJ10 (Frameshift mutation of JAZ10), exhibited enhanced rice growth and increased resistance to brown planthopper attacks. Overexpression of FJ10 in wild-type plants phenocopies OsJAZ10 frameshift mutants. Further characterization revealed that FJ10 interacts with Slender Rice 1 (OsSLR1) and F-box/Kelch 16 (OsFBK16). These interactions disrupt the function of OsSLR1 in suppressing gibberellin-mediated growth and the function of OsFBK16 in repressing lignin-mediated defense responses, respectively. Field experiments with FJ10-expressing plants demonstrate that this protein uncouples the growth-defense tradeoff, opening broad avenues to obtain cultivars with enhanced yield without compromised defenses.},
}

*Oryza/genetics/growth & development/parasitology/metabolism
*Frameshift Mutation
*Cyclopentanes/metabolism
*Plant Proteins/genetics/metabolism
*Oxylipins/metabolism
CRISPR-Cas Systems
Gene Expression Regulation, Plant
Animals
Plant Diseases/parasitology/genetics
Hemiptera/genetics/growth & development/metabolism
Gene Editing
Signal Transduction
Disease Resistance/genetics
Plants, Genetically Modified/genetics
Gibberellins/metabolism

@article {pmid39693330,
year = {2024},
author = {Perampalam, P and McDonald, JI and Dick, FA},
title = {GO-CRISPR: A highly controlled workflow to discover gene essentiality in loss-of-function screens.},
journal = {PloS one},
volume = {19},
number = {12},
pages = {e0315923},
doi = {10.1371/journal.pone.0315923},
pmid = {39693330},
issn = {1932-6203},
mesh = {Humans ; *Genes, Essential ; *CRISPR-Cas Systems ; *Workflow ; Ovarian Neoplasms/genetics/pathology ; Female ; Software ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; Loss of Function Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Genome-wide CRISPR screens are an effective discovery tool for genes that underlie diverse cellular mechanisms that can be scored through cell fitness. Loss-of-function screens are particularly challenging compared to gain-of-function because of the limited dynamic range of decreased sgRNA sequence detection. Here we describe Guide-Only control CRISPR (GO-CRISPR), an improved loss-of-function screening workflow, and its companion software package, Toolset for the Ranked Analysis of GO-CRISPR Screens (TRACS). We demonstrate a typical GO-CRISPR workflow in a non-proliferative 3D spheroid model of dormant high grade serous ovarian cancer and demonstrate superior performance to standard screening methods. The unique integration of the pooled sgRNA library quality and guide-only controls allows TRACS to identify novel molecular pathways that were previously unidentified in tumor dormancy and undetectable to analysis packages that lack the guide only controls. Together, GO-CRISPR and TRACS can robustly improve the discovery of essential genes in challenging biological scenarios such as growth arrested cells.},
}

Humans
*Genes, Essential
*CRISPR-Cas Systems
*Workflow
Ovarian Neoplasms/genetics/pathology
Female
Software
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics
Loss of Function Mutation
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39692063,
year = {2024},
author = {Yuan, P and Usman, M and Liu, W and Adhikari, A and Zhang, C and Njiti, V and Xia, Y},
title = {Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency.},
journal = {Biotechnology journal},
volume = {19},
number = {12},
pages = {e202400457},
doi = {10.1002/biot.202400457},
pmid = {39692063},
issn = {1860-7314},
support = {OHO01511//USDA Hatch Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; Transformation, Genetic ; Biotechnology/methods ; Plants/genetics ; },
abstract = {Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to nonmodel plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.},
}

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

@article {pmid39690340,
year = {2024},
author = {Dixit, Y and Yadav, P and Asnani, H and Sharma, AK},
title = {CRISPR/Cas9-Engineering for Increased Amylolytic Potential of Microbes for Sustainable Wastewater Treatment: A Review.},
journal = {Current microbiology},
volume = {82},
number = {1},
pages = {44},
pmid = {39690340},
issn = {1432-0991},
mesh = {*CRISPR-Cas Systems ; *Wastewater/microbiology ; *Amylases/genetics/metabolism ; Bacteria/genetics/metabolism/enzymology ; Gene Editing/methods ; Water Purification/methods ; Genetic Engineering/methods ; },
abstract = {Amylases are pivotal enzymes with extensive industrial applications, including food processing, textile manufacturing, pharmaceuticals, and biofuel production. Traditional methods for enhancing amylase production in microbial strains often lack precision and efficiency. The advent of CRISPR/Cas9 technology has revolutionized genetic engineering, offering precise and targeted modifications to microbial genomes. This review explores the potential of CRISPR/Cas9 for improving amylase production, highlighting its advantages over conventional methods. This review discusses the mechanism of CRISPR/Cas9, the identification and targeting of key genes involved in amylase synthesis and regulation, and the optimization of expression systems. Additionally, current review examines case studies demonstrating successful CRISPR/Cas9 applications in various microbial hosts. The review also delves into the integration of CRISPR/Cas9 in wastewater treatment, where genetically engineered amylolytic strains enhance the degradation of complex organic pollutants. Despite the promising prospects, challenges such as off-target effects and regulatory considerations remain. This review provides a comprehensive overview of the current advancements, challenges, and future directions in the application of CRISPR/Cas9 technology for amylase production and environmental biotechnology.},
}

*CRISPR-Cas Systems
*Wastewater/microbiology
*Amylases/genetics/metabolism
Bacteria/genetics/metabolism/enzymology
Gene Editing/methods
Water Purification/methods
Genetic Engineering/methods

@article {pmid39690326,
year = {2024},
author = {Barber, HM and Pater, AA and Gagnon, KT and Damha, MJ and O'Reilly, D},
title = {Chemical engineering of CRISPR-Cas systems for therapeutic application.},
journal = {Nature reviews. Drug discovery},
volume = {},
number = {},
pages = {},
pmid = {39690326},
issn = {1474-1784},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed molecular biology and the future of gene-targeted therapeutics. CRISPR systems comprise a CRISPR-associated (Cas) endonuclease and a guide RNA (gRNA) that can be programmed to guide sequence-specific binding, cleavage, or modification of complementary DNA or RNA. However, the application of CRISPR-based therapeutics is challenged by factors such as molecular size, prokaryotic or phage origins, and an essential gRNA cofactor requirement, which impact efficacy, delivery and safety. This Review focuses on chemical modification and engineering approaches for gRNAs to enhance or enable CRISPR-based therapeutics, emphasizing Cas9 and Cas12a as therapeutic paradigms. Issues that chemically modified gRNAs seek to address, including drug delivery, physiological stability, editing efficiency and off-target effects, as well as challenges that remain, are discussed.},
}

@article {pmid39688838,
year = {2024},
author = {Xu, X and Zhang, Y and Liu, J and Wei, S and Li, N and Yao, X and Wang, M and Su, X and Jing, G and Xu, J and Liu, Y and Lu, Y and Cheng, J and Xu, Y},
title = {Concurrent Detection of Protein and miRNA at the Single Extracellular Vesicle Level Using a Digital Dual CRISPR-Cas Assay.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.4c13557},
pmid = {39688838},
issn = {1936-086X},
abstract = {The simultaneous detection of proteins and microRNA (miRNA) at the single extracellular vesicle (EV) level shows great promise for precise disease profiling, owing to the heterogeneity and scarcity of tumor-derived EVs. However, a highly reliable method for multiple-target analysis of single EVs remains to be developed. In this study, a digital dual CRISPR-Cas-powered Single EV Evaluation (ddSEE) system was proposed to enable the concurrent detection of surface protein and inner miRNA of EVs at the single-molecule level. By optimizing simultaneous reaction conditions of CRISPR-Cas12a and CRISPR-Cas13a, the surface protein of EVs was detected by Cas12a using antibody-DNA conjugates to transfer the signal of the protein to DNA, while the inner miRNA was analyzed by Cas13a through EV-liposome fusion. A microfluidic chip containing 188,000 microwells was used to convert the CRISPR-Cas system into a digital assay format to enable the absolute quantification of miRNA/protein-positive EVs without bias through fluorescence imaging, which can detect as few as 214 EVs/μL. Finally, a total of 31 blood samples, 21 from breast cancer patients and 10 from healthy donors, were collected and tested, achieving a diagnostic accuracy of 92% in distinguishing patients with breast cancer from healthy donors. With its absolute quantification, ease of use, and multiplexed detection capability, the ddSEE system demonstrates its great potential for both EV research and clinical applications.},
}

@article {pmid39687096,
year = {2024},
author = {Karpenko, A and Shelenkov, A and Petrova, L and Gusarov, V and Zamyatin, M and Mikhaylova, Y and Akimkin, V},
title = {Two multidrug-resistant Proteus mirabilis clones carrying extended spectrum beta-lactamases revealed in a single hospital department by whole genome sequencing.},
journal = {Heliyon},
volume = {10},
number = {23},
pages = {e40821},
doi = {10.1016/j.heliyon.2024.e40821},
pmid = {39687096},
issn = {2405-8440},
abstract = {Proteus mirabilis bacteria is a component of normal intestinal microflora of humans and animals, but can also be found in hospital settings causing urinary tract infections and sepsis. The problem of treating such infections is complicated by multidrug-resistant isolates producing extended spectrum beta-lactamases (ESBL), and the number of ESBL-carrying P. mirabilis strains has significantly increased recently. This study presents a detailed analysis of 12 multidrug-resistant P. mirabilis isolates obtained from the wounds of different patients in one surgical department of a multidisciplinary hospital in Moscow, Russia, using the short- and long-read whole genome sequencing. The isolates under investigation divided into two clusters (clones) C1 and C2 based on their genomic profiles and carried antimicrobial resistance (AMR) genes corresponding well with phenotypic profiles, which was the first case of reporting two different P. mirabilis clones obtained simultaneously from the same specimens at one hospital, to the best of our knowledge. Some genes, including ESBL encoding ones, were specific for either C1 or C2 (aac(6')-Ib10, ant(2″)-Ia, qnrA1, bla VEB-6 and fosA3, bla CTX -M-65 , correspondingly). Additionally, the Salmonella genomic islands 1 were found that differed in composition of multiple antibiotic resistance regions between C1 and C2 groups. CRISPR-Cas system type I-E was revealed only in C2 isolates, while the same set of virulence factors was determined for both P. mirabilis clones. Diversity of all genetic factors found in case of simultaneous existence of two clones collected from the same source at one department indicates high pathogenic potential of P. mirabilis and poses a requirement of proper spreading monitoring. The data obtained will facilitate the understanding of AMR transfer and dynamics within clinical P. mirabilis isolates and contribute to epidemiological surveillance of this pathogen.},
}

@article {pmid39686519,
year = {2024},
author = {Hernandez, FJ},
title = {Nucleases: From Primitive Immune Defenders to Modern Biotechnology Tools.},
journal = {Immunology},
volume = {},
number = {},
pages = {},
doi = {10.1111/imm.13884},
pmid = {39686519},
issn = {1365-2567},
support = {2021-05641//Vetenskapsrådet/ ; //HORIZON-MSCA-2022-COFUND-101126600-SmartBRAIN3/ ; },
abstract = {The story of nucleases begins on the ancient battlefields of early Earth, where simple bacteria fought to survive against viral invaders. Nucleases are enzymes that degrade nucleic acids, with restriction endonucleases emerging as some of the earliest defenders, cutting foreign DNA to protect their bacteria hosts. However, bacteria sought more than just defence. They evolved the CRISPR-Cas system, an adaptive immune mechanism capable of remembering past invaders. The now-famous Cas9 nuclease, a key player in this system, has been harnessed for genome editing, revolutionising biotechnology. Over time, nucleases evolved from basic viral defence tools into complex regulators of immune function in higher organisms. In humans, DNases and RNases maintain immune balance by clearing cellular debris, preventing autoimmunity, and defending against pathogens. These enzymes have transformed from simple bacterial defenders to critical players in both human immunity and biotechnology. This review explores the evolutionary history of nucleases and their vital roles as protectors in the story of life's defence mechanisms.},
}

@article {pmid39684611,
year = {2024},
author = {Pavlova, SV and Shulgina, AE and Minina, JM and Zakian, SM and Dementyeva, EV},
title = {Generation of Isogenic iPSC Lines for Studying the Effect of the p.N515del (c.1543_1545delAAC) Variant on MYBPC3 Function and Hypertrophic Cardiomyopathy Pathogenesis.},
journal = {International journal of molecular sciences},
volume = {25},
number = {23},
pages = {},
pmid = {39684611},
issn = {1422-0067},
support = {22-15-00271//Russian Science Foundation/ ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Cardiomyopathy, Hypertrophic/genetics/pathology/metabolism ; Humans ; *Myocytes, Cardiac/metabolism/cytology/pathology ; *Carrier Proteins/genetics/metabolism ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing ; },
abstract = {The clinical significance of numerous cardiovascular gene variants remains to be determined. CRISPR/Cas9 allows for the introduction and/or correction of a certain variant in induced pluripotent stem cells (iPSCs). The resulting isogenic iPSC lines can be differentiated into cardiomyocytes and used as a platform to assess the pathogenicity of the variant. In this study, isogenic iPSC lines were generated for a variant of unknown significance found previously in a patient with hypertrophic cardiomyopathy (HCM), p.N515del (c.1543_1545delAAC) in MYBPC3. The deletion was corrected with CRISPR/Cas9 in the patient-specific iPSCs. The iPSC lines with the corrected deletion in MYBPC3 maintained pluripotency and a normal karyotype and showed no off-target CRISPR/Cas9 activity. The isogenic iPSC lines, together with isogenic iPSC lines generated earlier via introducing the p.N515del (c.1543_1545delAAC) variant in MYBPC3 of iPSCs of a healthy donor, were differentiated into cardiomyocytes. The cardiomyocytes derived from both panels of the isogenic iPSCs showed an increased size in the presence of the deletion in MYBPC3, which is one of the HCM traits at the cellular level. This finding indicates the effectiveness of these iPSC lines for studying the impact of the variant on HCM development.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
*Cardiomyopathy, Hypertrophic/genetics/pathology/metabolism
Humans
*Myocytes, Cardiac/metabolism/cytology/pathology
*Carrier Proteins/genetics/metabolism
*Cell Differentiation/genetics
*CRISPR-Cas Systems
Cell Line
Gene Editing

@article {pmid39637860,
year = {2024},
author = {Ursch, LT and Müschen, JS and Ritter, J and Klermund, J and Bernard, BE and Kolb, S and Warmuth, L and Andrieux, G and Miller, G and Jiménez-Muñoz, M and Theis, FJ and Boerries, M and Busch, DH and Cathomen, T and Schumann, K},
title = {Modulation of TCR stimulation and pifithrin-α improves the genomic safety profile of CRISPR-engineered human T cells.},
journal = {Cell reports. Medicine},
volume = {5},
number = {12},
pages = {101846},
doi = {10.1016/j.xcrm.2024.101846},
pmid = {39637860},
issn = {2666-3791},
mesh = {Humans ; *T-Lymphocytes/immunology/drug effects ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; *CRISPR-Cas Systems/genetics ; *Benzothiazoles/pharmacology ; *Lymphocyte Activation/drug effects/immunology ; *Toluene/analogs & derivatives/pharmacology ; *Gene Editing/methods ; Cell Proliferation/drug effects ; Receptors, Chimeric Antigen/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-engineered chimeric antigen receptor (CAR) T cells are at the forefront of novel cancer treatments. However, several reports describe the occurrence of CRISPR-induced chromosomal aberrations. So far, measures to increase the genomic safety of T cell products focused mainly on the components of the CRISPR-Cas9 system and less on T cell-intrinsic features, such as their massive expansion after T cell receptor (TCR) stimulation. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of large deletions with the downside of reduced knockout efficiencies. Alternatively, the addition of the small-molecule pifithrin-α limits large deletions, chromosomal translocations, and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells, including CAR T cells. Controlling T cell activation and pifithrin-α treatment are easily implementable strategies to improve the genomic integrity of CRISPR-engineered T cells.},
}

Humans
*T-Lymphocytes/immunology/drug effects
*Receptors, Antigen, T-Cell/genetics/immunology/metabolism
*CRISPR-Cas Systems/genetics
*Benzothiazoles/pharmacology
*Lymphocyte Activation/drug effects/immunology
*Toluene/analogs & derivatives/pharmacology
*Gene Editing/methods
Cell Proliferation/drug effects
Receptors, Chimeric Antigen/genetics/immunology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39627502,
year = {2025},
author = {Ngoi, NYL and Gallo, D and Torrado, C and Nardo, M and Durocher, D and Yap, TA},
title = {Synthetic lethal strategies for the development of cancer therapeutics.},
journal = {Nature reviews. Clinical oncology},
volume = {22},
number = {1},
pages = {46-64},
pmid = {39627502},
issn = {1759-4782},
mesh = {Humans ; *Neoplasms/drug therapy/genetics ; *Synthetic Lethal Mutations ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use ; CRISPR-Cas Systems ; Antineoplastic Agents/therapeutic use ; Drug Discovery/methods ; DNA Damage/drug effects ; },
abstract = {Synthetic lethality is a genetic phenomenon whereby the simultaneous presence of two different genetic alterations impairs cellular viability. Importantly, targeting synthetic lethal interactions offers potential therapeutic strategies for cancers with alterations in pathways that might otherwise be considered undruggable. High-throughput screening methods based on modern CRISPR-Cas9 technologies have emerged and become crucial for identifying novel synthetic lethal interactions with the potential for translation into biologically rational cancer therapeutic strategies as well as associated predictive biomarkers of response capable of guiding patient selection. Spurred by the clinical success of PARP inhibitors in patients with BRCA-mutant cancers, novel agents targeting multiple synthetic lethal interactions within DNA damage response pathways are in clinical development, and rational strategies targeting synthetic lethal interactions spanning alterations in epigenetic, metabolic and proliferative pathways have also emerged and are in late preclinical and/or early clinical testing. In this Review, we provide a comprehensive overview of established and emerging technologies for synthetic lethal drug discovery and development and discuss promising therapeutic strategies targeting such interactions.},
}

Humans
*Neoplasms/drug therapy/genetics
*Synthetic Lethal Mutations
Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use
CRISPR-Cas Systems
Antineoplastic Agents/therapeutic use
Drug Discovery/methods
DNA Damage/drug effects

@article {pmid39626673,
year = {2024},
author = {Awasthi, S and Dobrolecki, LE and Sallas, C and Zhang, X and Li, Y and Khazaei, S and Ghosh, S and Jeter, CR and Liu, J and Mills, GB and Westin, SN and Lewis, MT and Peng, W and Sood, AK and Yap, TA and Yi, SS and McGrail, DJ and Sahni, N},
title = {UBA1 inhibition sensitizes cancer cells to PARP inhibitors.},
journal = {Cell reports. Medicine},
volume = {5},
number = {12},
pages = {101834},
doi = {10.1016/j.xcrm.2024.101834},
pmid = {39626673},
issn = {2666-3791},
mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Ubiquitin-Activating Enzymes/antagonists & inhibitors/metabolism/genetics ; Animals ; Cell Line, Tumor ; Female ; Mice ; Xenograft Model Antitumor Assays ; Ovarian Neoplasms/drug therapy/pathology/genetics ; DNA Damage/drug effects ; CRISPR-Cas Systems/genetics ; Poly(ADP-ribose) Polymerases/metabolism/genetics ; },
abstract = {Therapeutic strategies targeting the DNA damage response, such as poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi), have revolutionized cancer treatment in tumors deficient in homologous recombination (HR). However, overcoming innate and acquired resistance to PARPi remains a significant challenge. Here, we employ a genome-wide CRISPR knockout screen and discover that the depletion of ubiquitin-activating enzyme E1 (UBA1) enhances sensitivity to PARPi in HR-proficient ovarian cancer cells. We show that silencing or pharmacological inhibition of UBA1 sensitizes multiple cell lines and organoid models to PARPi. Mechanistic studies uncover that UBA1 inhibition not only impedes HR repair to sensitize cells to PARP inhibition but also increases PARylation, which may subsequently be targeted by PARP inhibition. In vivo experiments using patient-derived xenografts demonstrate that combining PARP and UBA1 inhibition provided significant survival benefit compared to individual therapies with no detectable signs of toxicity, establishing this combination approach as a promising strategy to extend PARPi benefit.},
}

Humans
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
*Ubiquitin-Activating Enzymes/antagonists & inhibitors/metabolism/genetics
Animals
Cell Line, Tumor
Female
Mice
Xenograft Model Antitumor Assays
Ovarian Neoplasms/drug therapy/pathology/genetics
DNA Damage/drug effects
CRISPR-Cas Systems/genetics
Poly(ADP-ribose) Polymerases/metabolism/genetics

@article {pmid39505154,
year = {2024},
author = {Yu, X and Peng, J and Zhong, Q and Wu, A and Deng, X and Zhu, Y},
title = {Caspase-1 knockout disrupts pyroptosis and protects photoreceptor cells from photochemical damage.},
journal = {Molecular and cellular probes},
volume = {78},
number = {},
pages = {101991},
doi = {10.1016/j.mcp.2024.101991},
pmid = {39505154},
issn = {1096-1194},
mesh = {*Pyroptosis/genetics ; *Caspase 1/metabolism/genetics ; Animals ; *Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; Cell Line ; Mice ; Reactive Oxygen Species/metabolism ; Cell Proliferation ; Photoreceptor Cells/metabolism/radiation effects ; Light/adverse effects ; Cell Survival/radiation effects ; },
abstract = {AIM: Retinal photochemical damage (RPD) plays a significant role in the development of various ocular diseases, with Caspase-1 being a key contributor. This study investigates the protective effects of Caspase-1 gene-mediated pyroptosis against RPD.

METHODS: Differentially expressed genes (DEGs) associated with RPD were identified through the analysis of two expression profiles from the GEO database. Correlation analysis was used to pinpoint pyroptosis-related genes (PRGs) linked to RPD. A Caspase-1 knockout 661 W cell line was generated via CRISPR-Cas9 gene editing, and single-cell colonies were screened and purified. Validation of knockout cells was performed through RT-qPCR, gene sequencing, and Western blot analysis. Comparative assays on cell proliferation, intracellular reactive oxygen species (ROS), and cytotoxicity were conducted between wild-type and Caspase-1 knockout cells under light exposure. Further RT-qPCR and Western blot experiments examined changes in the mRNA and protein levels of key pyroptosis pathway components.

RESULTS: Significant alterations in Caspase-1 expression were observed among PRGs. Homozygous Caspase-1 knockout cell lines were confirmed through RT-qPCR, genomic PCR product sequencing, and Western blot analysis. Compared to wild-type 661 W cells, Caspase-1 knockout cells exhibited higher viability and proliferation rates after 24 h of light exposure, alongside reduced LDH release. The expression of downstream pyroptosis factors at both the mRNA and protein levels was markedly decreased in the knockout group.

CONCLUSION: CRISPR/Cas9-mediated Caspase-1 knockout enhanced the resistance of 661 W cells to photochemical damage, suggesting that Caspase-1 may serve as a potential therapeutic target for RPD-related diseases.},
}

*Pyroptosis/genetics
*Caspase 1/metabolism/genetics
Animals
*Gene Knockout Techniques
CRISPR-Cas Systems/genetics
Cell Line
Mice
Reactive Oxygen Species/metabolism
Cell Proliferation
Photoreceptor Cells/metabolism/radiation effects
Light/adverse effects
Cell Survival/radiation effects

@article {pmid39278404,
year = {2025},
author = {Liu, N and Zhang, J and Chen, W and Ma, W and Wu, T},
title = {RBM39 Enhances Cholangiocarcinoma Growth Through EZH2-mediated WNT7B/β-catenin Pathway.},
journal = {Cellular and molecular gastroenterology and hepatology},
volume = {19},
number = {1},
pages = {101404},
pmid = {39278404},
issn = {2352-345X},
mesh = {Humans ; *Cholangiocarcinoma/pathology/metabolism/genetics ; *Enhancer of Zeste Homolog 2 Protein/metabolism/genetics/antagonists & inhibitors ; Animals ; Mice ; *RNA-Binding Proteins/metabolism/genetics ; *Bile Duct Neoplasms/pathology/metabolism/genetics ; *Cell Proliferation ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Wnt Proteins/metabolism ; Wnt Signaling Pathway ; beta Catenin/metabolism ; Male ; Xenograft Model Antitumor Assays ; Female ; Mice, Nude ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND & AIMS: The RNA-binding motif protein 39 (RBM39) functions as both an RNA-binding protein and a splicing factor in a variety of cancer types. However, the function of RBM39 in cholangiocarcinoma (CCA) remains undefined. In this study, we aimed to investigate the role of RBM39 in CCA and explore its potential as a therapeutic target.

METHODS: The expression of RBM39 in CCA was investigated by analyzing human CCA tumor specimens. CRISPR/Cas9 or shRNA-mediated depletion of RBM39 was performed in vitro and in vivo to document the oncogenic role of RBM39 in CCA. The anti-tumor effect of the RBM39 inhibitor, Indisulam, in combination with the EZH2 degrader MS177 was assessed in vitro and in vivo.

RESULTS: RBM39 is significantly increased in human CCA tissues and associated with a poor prognosis in patients with CCA. Depletion of RBM39 by CRISPR/Cas9 or shRNA inhibited CCA cell proliferation in vitro and prevented CCA development and tumor growth in mice. Mechanistically, our results showed that depletion of RBM39 suppressed EZH2 expression via disrupting its mRNA splicing. RBM39-regulated EZH2 controls WNT7B/β-catenin activity. Pharmacological co-targeting of RBM39 (with Indisulam) and EZH2 (with MS177) resulted in a synergistic antitumor effect, both in vitro and in vivo.

CONCLUSION: This study discloses a novel RBM39-EZH2-β-catenin signaling axis that is crucial for CCA growth. Our findings suggest that simultaneous inhibition of RBM39 and EZH2 presents a promising therapeutic strategy for CCA treatment.},
}

Humans
*Cholangiocarcinoma/pathology/metabolism/genetics
*Enhancer of Zeste Homolog 2 Protein/metabolism/genetics/antagonists & inhibitors
Animals
Mice
*RNA-Binding Proteins/metabolism/genetics
*Bile Duct Neoplasms/pathology/metabolism/genetics
*Cell Proliferation
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
Wnt Proteins/metabolism
Wnt Signaling Pathway
beta Catenin/metabolism
Male
Xenograft Model Antitumor Assays
Female
Mice, Nude
CRISPR-Cas Systems

@article {pmid39684477,
year = {2024},
author = {Borovikova, SE and Shepelev, MV and Mazurov, DV and Kruglova, NA},
title = {Efficient Genome Editing Using 'NanoMEDIC' AsCas12a-VLPs Produced with Pol II-Transcribed crRNA.},
journal = {International journal of molecular sciences},
volume = {25},
number = {23},
pages = {},
doi = {10.3390/ijms252312768},
pmid = {39684477},
issn = {1422-0067},
support = {22-15-00381//Russian Science Foundation/ ; 075-15-2019-1661//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *RNA Polymerase II/metabolism/genetics ; Jurkat Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble in the cytoplasm, but U6-driven sgRNA is localized in the nucleus, which hinders the efficient formation and packaging of RNPs into VLPs. In this study, using the nuclease packaging mechanism of 'NanoMEDIC' VLPs, we produced VLPs with AsCas12a and exploited its ability to process pre-crRNA. This allowed us to direct crRNA in the cytoplasm as part of a Pol II-driven transcript where AsCas12a excised mature crRNA, thus boosting RNP incorporation into VLPs. CMV-driven crRNA increased Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50-90% and raised the level of endogenous CXCR4 knockout in Jurkat T cells from 1% to 20%. Changing a single crRNA to an array of three or six identical crRNAs improved CXCR4 knockout rates by up to 60-70%. Compared to SpCas9-VLPs, the editing efficiencies of AsCas12a-VLPs were higher, regardless of promoter usage. Thus, we showed that AsCas12a and CMV-driven crRNA could be efficiently packaged into VLPs and mediate high levels of gene editing. AsCas12a-VLPs are a new and promising tool for the delivery of RNPs into mammalian cells that will allow efficient target genome editing and may be useful for gene therapy applications.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
HEK293 Cells
*RNA Polymerase II/metabolism/genetics
Jurkat Cells
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid39684395,
year = {2024},
author = {Xuan, Q and Wang, J and Nie, Y and Fang, C and Liang, W},
title = {Research Progress and Application of Miniature CRISPR-Cas12 System in Gene Editing.},
journal = {International journal of molecular sciences},
volume = {25},
number = {23},
pages = {},
doi = {10.3390/ijms252312686},
pmid = {39684395},
issn = {1422-0067},
support = {32201248//National Natural Science Foundation of China/ ; 242102111164//Key R&D and Promotion Projects in Henan Province/ ; 222301420106//Natural Science Foundation of Henan/ ; 222300420202//Natural Science Foundation of Henan/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Plants/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; },
abstract = {CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used in many research fields such as synthetic biology, crop genetics and breeding, precision medicine, and so on. The miniature CRISPR-Cas12 system has been an emerging genome editing tool in recent years. Compared to the commonly used CRISPR-Cas9 and CRISPR-Cas12a, the miniature CRISPR-Cas12 system has unique advantages, such as rich PAM sites, higher specificity, smaller volume, and cytotoxicity. However, the application of miniature Cas12 proteins and the methods to improve its editing efficiency have not been systematically summarized. In this review, we introduce the classification of CRISPR-Cas system and summarize the structural characteristics of type V CRISPR-Cas system and the cleavage mechanism of five miniature Cas12 proteins. The application of a miniature CRISPR-Cas12 system in the gene editing of animals, plants, and microorganisms is summarized, and the strategies to improve the editing efficiency of the miniature CRISPR-Cas12 system are discussed, aiming to provide reference for further understanding the functional mechanism and engineering modification of the miniature CRISPR-Cas12 system.},
}

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

@article {pmid39684387,
year = {2024},
author = {Aksoy, MO and Bilinska, A and Stachowiak, M and Flisikowska, T and Szczerbal, I},
title = {Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing.},
journal = {International journal of molecular sciences},
volume = {25},
number = {23},
pages = {},
doi = {10.3390/ijms252312677},
pmid = {39684387},
issn = {1422-0067},
support = {2018/29/B/NZ2/00956//National Science Center/ ; },
mesh = {Animals ; *Adipogenesis/genetics ; *Sterol Regulatory Element Binding Protein 1/genetics/metabolism ; *CRISPR-Cas Systems ; Swine ; *Gene Editing/methods ; Adipocytes/metabolism/cytology ; Gene Expression Regulation ; Lipid Metabolism/genetics ; },
abstract = {Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5'-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.},
}

Animals
*Adipogenesis/genetics
*Sterol Regulatory Element Binding Protein 1/genetics/metabolism
*CRISPR-Cas Systems
Swine
*Gene Editing/methods
Adipocytes/metabolism/cytology
Gene Expression Regulation
Lipid Metabolism/genetics

@article {pmid39684256,
year = {2024},
author = {Yang, P and Zhang, S and Hu, D and Li, X and Guo, Y and Guo, H and Zhang, L and Ding, X},
title = {Research Progress on the Mechanism and Application of the Type I CRISPR-Cas System.},
journal = {International journal of molecular sciences},
volume = {25},
number = {23},
pages = {},
doi = {10.3390/ijms252312544},
pmid = {39684256},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Archaea/genetics ; Bacteria/genetics ; Genetic Engineering/methods ; },
abstract = {The CRISPR-Cas system functions as an adaptive immune mechanism in archaea and bacteria, providing defense against the invasion of foreign nucleic acids. Most CRISPR-Cas systems are classified into class 1 or class 2, with further subdivision into several subtypes. The primary distinction between class 1 and class 2 systems lies in the assembly of their effector modules. In class 1 systems, the effector complex consists of multiple proteins with distinct functions, whereas in class 2 systems, the effector is associated with a single protein. Class 1 systems account for approximately 90% of the CRISPR-Cas repertoire and are categorized into three types (type I, type IV, and type III) and 12 subtypes. To date, various CRISPR-Cas systems have been widely employed in the field of genetic engineering as essential tools and techniques for genome editing. Type I CRISPR-Cas systems remain a valuable resource for developing sophisticated application tools. This review provides a comprehensive review of the characteristics, mechanisms of action, and applications of class 1 type I CRISPR-Cas systems, as well as transposon-associated systems, offering effective approaches and insights for future research on the mechanisms of action, as well as the subsequent development and application of type I CRISPR-Cas systems.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Archaea/genetics
Bacteria/genetics
Genetic Engineering/methods

@article {pmid39684244,
year = {2024},
author = {Wattad, H and Molcho, J and Manor, R and Weil, S and Aflalo, ED and Chalifa-Caspi, V and Sagi, A},
title = {Roadmap and Considerations for Genome Editing in a Non-Model Organism: Genetic Variations and Off-Target Profiling.},
journal = {International journal of molecular sciences},
volume = {25},
number = {23},
pages = {},
doi = {10.3390/ijms252312530},
pmid = {39684244},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; Palaemonidae/genetics ; Genetic Variation ; Polymorphism, Single Nucleotide ; High-Throughput Nucleotide Sequencing ; Molecular Sequence Annotation ; Sex Chromosomes/genetics ; },
abstract = {The CRISPR/Cas genome editing approach in non-model organisms poses challenges that remain to be resolved. Here, we demonstrated a generalized roadmap for a de novo genome annotation approach applied to the non-model organism Macrobrachium rosenbergii. We also addressed the typical genome editing challenges arising from genetic variations, such as a high frequency of single nucleotide polymorphisms, differences in sex chromosomes, and repetitive sequences that can lead to off-target events. For the genome editing of M. rosenbergii, our laboratory recently adapted the CRISPR/Cas genome editing approach to embryos and the embryonic primary cell culture. In this continuation study, an annotation pipeline was trained to predict the gene models by leveraging the available genomic, transcriptomic, and proteomic data, and enabling accurate gene prediction and guide design for knock-outs. A next-generation sequencing analysis demonstrated a high frequency of genetic variations in genes on both autosomal and sex chromosomes, which have been shown to affect the accuracy of editing analyses. To enable future applications based on the CRISPR/Cas tool in non-model organisms, we also verified the reliability of editing efficiency and tracked off-target frequencies. Despite the lack of comprehensive information on non-model organisms, this study provides an example of the feasibility of selecting and editing specific genes with a high degree of certainty.},
}

*Gene Editing/methods
Animals
*CRISPR-Cas Systems
Palaemonidae/genetics
Genetic Variation
Polymorphism, Single Nucleotide
High-Throughput Nucleotide Sequencing
Molecular Sequence Annotation
Sex Chromosomes/genetics

@article {pmid39683106,
year = {2024},
author = {Zhan, X and Zhang, F and Li, N and Xu, K and Wang, X and Gao, S and Yin, Y and Yuan, W and Chen, W and Ren, Z and Yao, M and Wang, F},
title = {CRISPR/Cas: An Emerging Toolbox for Engineering Virus Resistance in Plants.},
journal = {Plants (Basel, Switzerland)},
volume = {13},
number = {23},
pages = {},
doi = {10.3390/plants13233313},
pmid = {39683106},
issn = {2223-7747},
support = {2023BBB044//Fei Wang/ ; 2022BBA0060//Fei Wang/ ; HBZY2023B004-3//Minghua Yao/ ; 2023HBSTX4-06//Ning Li/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas have been recognized as powerful genome-editing tools in diverse eukaryotic species, including plants, and thus hold great promise for engineering virus resistance in plants. Nevertheless, further attention is required regarding various issues associated with applying new powerful technologies in the field. This mini-review focuses on the recent advances in using CRISPR/Cas9 and CRISPR/Cas13 systems to combat DNA and RNA viruses in plants. We explored the utility of CRISPR/Cas for targeting the viral genome and editing host susceptibility genes in plants. We also provide insights into the limitations and challenges of using CRISPR/Cas for plant virus interference and propose individual combinatorial solutions. In conclusion, CRISPR/Cas technology has the potential to offer innovative and highly efficient approaches for controlling viruses in important crops in the near future.},
}

@article {pmid39682777,
year = {2024},
author = {Ryu, M and Yurube, T and Takeoka, Y and Kanda, Y and Tsujimoto, T and Miyazaki, K and Ohnishi, H and Matsuo, T and Kumagai, N and Kuroshima, K and Hiranaka, Y and Kuroda, R and Kakutani, K},
title = {Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An In Vitro Comparative Study Between RNA Interference and CRISPR-Cas9.},
journal = {Cells},
volume = {13},
number = {23},
pages = {},
doi = {10.3390/cells13232030},
pmid = {39682777},
issn = {2073-4409},
support = {JP21K09323//Japan Society for the Promotion of Science/ ; JP24K02565//Japan Society for the Promotion of Science/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *TOR Serine-Threonine Kinases/metabolism ; *Proto-Oncogene Proteins c-akt/metabolism ; *Signal Transduction ; *RNA Interference ; *Intervertebral Disc Degeneration/therapy/genetics/metabolism/pathology ; Gene Silencing ; Phosphatidylinositol 3-Kinases/metabolism ; Nucleus Pulposus/metabolism/pathology ; Intervertebral Disc/metabolism/pathology ; Autophagy/genetics ; Regulatory-Associated Protein of mTOR/metabolism/genetics ; RNA, Small Interfering/metabolism/genetics ; Male ; Female ; Middle Aged ; },
abstract = {The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR-Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8-60.3% for RNAi and 88.1-89.3% for CRISPR-Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR-Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR-Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR-Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease.},
}

Humans
*CRISPR-Cas Systems/genetics
*TOR Serine-Threonine Kinases/metabolism
*Proto-Oncogene Proteins c-akt/metabolism
*Signal Transduction
*RNA Interference
*Intervertebral Disc Degeneration/therapy/genetics/metabolism/pathology
Gene Silencing
Phosphatidylinositol 3-Kinases/metabolism
Nucleus Pulposus/metabolism/pathology
Intervertebral Disc/metabolism/pathology
Autophagy/genetics
Regulatory-Associated Protein of mTOR/metabolism/genetics
RNA, Small Interfering/metabolism/genetics
Male
Female
Middle Aged

@article {pmid39681230,
year = {2024},
author = {Nafari, NB and Zamani, M and Mosayyebi, B},
title = {Recent advances in lateral flow assays for MicroRNA detection.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {567},
number = {},
pages = {120096},
doi = {10.1016/j.cca.2024.120096},
pmid = {39681230},
issn = {1873-3492},
abstract = {Lateral flow assays (LFAs) have emerged as pivotal tools for the rapid and reliable detection of microRNAs (miRNAs). It is believed that these biomarkers are crucial for the diagnosis and prognosis of various diseases, particularly cancer. Traditional miRNA detection techniques, such as quantitative PCR, are highly sensitive but have limited efficacy due to their complexity, high cost, and technical requirements. LFAs are valuable due to their simplicity, affordability, and portability, making them ideal for point-of-care testing in low-resource environments. However, challenges remain in developing highly sensitive and accurate LFA devices for miRNA detection. This review explores recent advancements in LFAs to improve miRNA detection sensitivity and specificity. Key innovations include signal amplification using isothermal methods, the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems for direct targeting of miRNAs, and the incorporation of nanomaterials, such as gold nanoparticles and nanorods, to enhance signal intensity. Using artificial intelligence (AI) algorithms enables precise, automated, and rapid quantification of miRNAs. Moreover, this review examines the ability of LFA-based devices to detect multiple miRNAs simultaneously. One of the most significant advancements is the detection of miR-21 levels as low as 20 pM and let-7a levels as low as 40 pM within ten minutes. This highlights the potential of these devices for clinical diagnostics.},
}

@article {pmid39680738,
year = {2024},
author = {Yang, S and Hu, G and Wang, J and Song, J},
title = {CRISPR/Cas-Based Gene Editing Tools for Large DNA Fragment Integration.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00632},
pmid = {39680738},
issn = {2161-5063},
abstract = {In recent years, gene editing technologies have rapidly evolved to enable precise and efficient genomic modification. These strategies serve as a crucial instrument in advancing our comprehension of genetics and treating genetic disorders. Of particular interest is the manipulation of large DNA fragments, notably the insertion of large fragments, which has emerged as a focal point of research in recent years. Nevertheless, the techniques employed to integrate larger gene fragments are frequently confronted with inefficiencies, off-target effects, and elevated costs. It is therefore imperative to develop efficient tools capable of precisely inserting kilobase-sized DNA fragments into mammalian genomes to support genetic engineering, gene therapy, and synthetic biology applications. This review provides a comprehensive overview of methods developed in the past five years for integrating large DNA fragments with a particular focus on burgeoning CRISPR-related technologies. We discuss the opportunities associated with homology-directed repair (HDR) and emerging CRISPR-transposase and CRISPR-recombinase strategies, highlighting their potential to revolutionize gene therapies for complex diseases. Additionally, we explore the challenges confronting these methodologies and outline potential future directions for their improvement with the overarching goal of facilitating the utilization and advancement of tools for large fragment gene editing.},
}

@article {pmid39680522,
year = {2024},
author = {Zheng, J and Li, B and Jia, L and Zhang, J and Gong, Z and Le, Y and Nian, X and Li, X and Liu, B and Yu, D and Zhang, Z and Li, C},
title = {Tumorigenicity decrease in Bcl-xL deficient MDCK cells ensuring the safety for influenza vaccine production.},
journal = {PloS one},
volume = {19},
number = {12},
pages = {e0311069},
doi = {10.1371/journal.pone.0311069},
pmid = {39680522},
issn = {1932-6203},
mesh = {Animals ; Dogs ; *bcl-X Protein/genetics/metabolism ; Madin Darby Canine Kidney Cells ; *Influenza Vaccines ; Mice ; *Apoptosis ; Cell Proliferation ; CRISPR-Cas Systems ; Mice, Nude ; Carcinogenesis/genetics ; Cell Movement ; Gene Editing ; Mice, Inbred BALB C ; },
abstract = {Madin-Darby canine kidney (MDCK) cells are the recognized cell strain for influenza vaccine production. However, the tumorigenic potential of MDCK cells raises concerns about their use in biological product manufacturing. To reduce MDCK cells' tumorigenicity and ensure the safety of influenza vaccine production, a B-cell lymphoma extra-large (Bcl-xL) gene, which plays a pivotal role in apoptosis regulation, was knocked-out in original MDCK cells by CRISPR-Cas9 gene editing technology, so that a homozygous MDCK-Bcl-xL-/- cell strain was acquired and named as BY-02. Compared with original MDCK cells, the proliferation and migration ability of BY-02 were significantly reduced, while apoptosis level was significantly increased, the endogenous mitochondrial apoptotic pathway were also modulated after Bcl-xL knock-out in MDCK cells. For tumor formation assays in nude mouse tests, all ten mice injected with original MDCK cells presented tumors growth in the injection site, in contrast to only one mouse injected with BY-02 cells presented tumors growth. These findings suggest that Bcl-xL knock-down is an effective strategy to inhibit tumor formation in MDCK cells, making BY-02 a promising genetically engineered cell strain for influenza vaccine production.},
}

Animals
Dogs
*bcl-X Protein/genetics/metabolism
Madin Darby Canine Kidney Cells
*Influenza Vaccines
Mice
*Apoptosis
Cell Proliferation
CRISPR-Cas Systems
Mice, Nude
Carcinogenesis/genetics
Cell Movement
Gene Editing
Mice, Inbred BALB C

@article {pmid39678914,
year = {2024},
author = {Rostampour, M and Panahi, B and Masoumi Jahandizi, R},
title = {The CRISPR-Cas system in Lactiplantibacillus plantarum strains: identification and characterization using a genome mining approach.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1394756},
pmid = {39678914},
issn = {1664-302X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system against bacteriophages. In this study, 675 sequences of Lactiplantibacillus plantarum isolates deposited in GenBank were analyzed in terms of diversity, occurrence, and evolution of the CRISPR-Cas system. This study investigated the presence, structural variations, phylogenetic relationships, and diversity of CRISPR-Cas systems in 675 L. plantarum strains. The analysis revealed that 143 strains harbor confirmed CRISPR-Cas systems, with subtype II-A being predominant. Moreover, targeting phages and plasmid diversity between the predicted systems were dissected. The results indicated that approximately 22% of the isolates with verified and complete CRISPR systems exhibited the coexistence of both subtypes II-A and I-E within their genomes. The results further showed that in subtype II-A, the length of the repeat sequence was 36 nucleotides, on average. In addition, the number of spacers in subtypes II-A and I-E varied between 1-24 and 3-16 spacers, respectively. The results also indicated that subtype II-A has nine protospacer adjacent motifs, which are 5'-CC-3', 5'-GAA-3', 5'-TGG-3', 5'-CTT-3', 5'-GGG-3', 5'-CAT-3', 5'-CTC-3', 5'-CCT-3', and 5'-CGG-3'. In addition, the identified systems displayed a potential for targeting Lactobacillus phages. The investigation of the relationship between the targeting of Lactobacillus phages by the antiphage system in L. plantarum species showed that subtype II-A had the highest diversity in targeting Lactobacillus phages than subtype I-E. In conclusion, current findings offer a perspective on the prevalence and evolution of the CRISPR-Cas system in L. plantarum, contributing novel insights to the expanding field of CRISPR-Cas systems within lactobacillus strains. This knowledge establishes a foundation for future applied studies focused on enhancing phage resistance in industrial fermentation, reducing contamination risks, and improving product quality. The identified targeting diversity may also foster advancements in phage therapy through the development of CRISPR-based antimicrobials.},
}

@article {pmid39677770,
year = {2024},
author = {Hoelting, K and Madlmayr, A and Hoeger, B and Lewitz, D and Weng, M and Haider, T and Duggan, M and Ross, R and Horgen, FD and Sperandio, M and Dietrich, A and Gudermann, T and Zierler, S},
title = {TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.12.04.626765},
pmid = {39677770},
issn = {2692-8205},
abstract = {UNLABELLED: T lymphocyte activation is a crucial process in the regulation of innate and adaptive immune responses. The ion channel-kinase TRPM7 has previously been implicated in cellular Mg [2+] homeostasis, proliferation, and immune cell modulation. Here, we show that pharmacological and genetic silencing of TRPM7 leads to diminished human CD4 T-cell activation and proliferation following TCR mediated stimulation. In both primary human CD4 T cells and CRISPR/Cas-9 engineered Jurkat T cells, loss of TRPM7 led to altered Mg [2+] homeostasis, Ca [2+] signaling, reduced NFAT translocation, decreased IL-2 secretion and ultimately diminished proliferation and differentiation. While the activation of primary human CD4 T cells was dependent on TRPM7, polarization of naïve CD4 T cells into regulatory T cells (T reg) was not. Taken together, these results highlight TRPM7 as a key protein of cellular Mg [2+] homeostasis and CD4 T-cell activation. Its role in lymphocyte activation suggests therapeutic potential for TRPM7 in numerous T-cell mediated diseases.

SUMMARY: TRPM7 is crucial to maintaining cellular Mg [2+] homeostasis and regulates human CD4 T-cell activation by modulating early Ca [2+] signaling events in response to TCR-mediated stimulation subsequently, influencing T-cell differentiation in a Mg [2+] dependent manner.},
}

@article {pmid39677748,
year = {2024},
author = {Shamloo, S and Schloßhauer, JL and Tiwari, S and Fischer, KD and Ghebrechristos, Y and Kratzenberg, L and Bejoy, AM and Aifantis, I and Wang, E and Imig, J},
title = {RNA Binding of GAPDH Controls Transcript Stability and Protein Translation in Acute Myeloid Leukemia.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.12.02.626357},
pmid = {39677748},
issn = {2692-8205},
abstract = {Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukemia (AML) RBPs are key regulators of tumor proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5'UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.},
}

@article {pmid39677651,
year = {2024},
author = {Zheng, Y and Mayourian, J and King, JS and Li, Y and Bezzerides, VJ and Pu, WT and VanDusen, NJ},
title = {Cardiac Applications of CRISPR/AAV-Mediated Precise Genome Editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.12.03.626493},
pmid = {39677651},
issn = {2692-8205},
abstract = {The ability to efficiently make precise genome edits in somatic tissues will have profound implications for gene therapy and basic science. CRISPR/Cas9 mediated homology-directed repair (HDR) is one approach that is commonly used to achieve precise and efficient editing in cultured cells. Previously, we developed a platform capable of delivering CRISPR/ Cas 9 gRNAs and donor templates via a deno- a ssociated v irus to induce HDR (CASAAV-HDR). We demonstrated that CASAAV-HDR is capable of creating precise genome edits in vivo within mouse cardiomyocytes at the neonatal and adult stages. Here, we report several applications of CASAAV-HDR in cardiomyocytes. First, we show the utility of CASAAV-HDR for disease modeling applications by using CASAAV-HDR to create and precisely tag two pathological variants of the titin gene observed in cardiomyopathy patients. We used this approach to monitor the cellular localization of the variants, resulting in mechanistic insights into their pathological functions. Next, we utilized CASAAV-HDR to create another mutation associated with human cardiomyopathy, arginine 14 deletion (R14Del) within the N-terminus of Phospholamban (PLN). We assessed the localization of PLN-R14Del and quantified cardiomyocyte phenotypes associated with cardiomyopathy, including cell morphology, activation of PLN via phosphorylation, and calcium handling. After demonstrating CASAAV-HDR utility for disease modeling we next tested its utility for functional genomics, by targeted genomic insertion of a library of enhancers for a massively parallel reporter assay (MPRA). We show that MPRAs with genomically integrated enhancers are feasible, and can yield superior assay sensitivity compared to tests of the same enhancers in an AAV/episomal context. Collectively, our study showcases multiple applications for in vivo precise editing of cardiomyocyte genomes via CASAAV-HDR.},
}

@article {pmid39644903,
year = {2024},
author = {Kohabir, KAV and Linthorst, J and Nooi, LO and Brouwer, R and Wolthuis, RMF and Sistermans, EA},
title = {Synthetic mismatches enable specific CRISPR-Cas12a-based detection of genome-wide SNVs tracked by ARTEMIS.},
journal = {Cell reports methods},
volume = {4},
number = {12},
pages = {100912},
doi = {10.1016/j.crmeth.2024.100912},
pmid = {39644903},
issn = {2667-2375},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Proto-Oncogene Proteins B-raf/genetics ; Aldehyde Dehydrogenase, Mitochondrial/genetics ; Genome, Human/genetics ; Polymorphism, Single Nucleotide/genetics ; Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; BRCA2 Protein/genetics ; Neoplasms/genetics/diagnosis ; Tumor Suppressor Protein p53/genetics ; Base Pair Mismatch ; Bacterial Proteins ; },
abstract = {Detection of pathogenic DNA variants is vital in cancer diagnostics and treatment monitoring. While CRISPR-based diagnostics (CRISPRdx) offer promising avenues for cost-effective, rapid, and point-of-care testing, achieving single-nucleotide detection fidelity remains challenging. We present an in silico pipeline that scans the human genome for targeting pathogenic mutations in the seed region (ARTEMIS), the most stringent crRNA domain. ARTEMIS identified 12% of pathogenic SNVs as Cas12a recognizable, including 928 cancer-associated variants such as BRAF[V600E], BRCA2[E1953∗], TP53[V272M], and ALDH2[E504K]. Cas12a exhibited remarkable tolerance to single mismatches within the seed region. Introducing deliberate synthetic mismatches within the seed region yielded on-target activity with single-nucleotide fidelity. Both positioning and nucleobase types of mismatches influenced detection accuracy. With improved specificity, Cas12a could accurately detect and semi-quantify BRAF[V600E] in cfDNA from cell lines and patient liquid biopsies. These results provide insights toward rationalized crRNA design for high-fidelity CRISPRdx, supporting personalized and cost-efficient healthcare solutions in oncologic diagnostics.},
}

Humans
*CRISPR-Cas Systems/genetics
Proto-Oncogene Proteins B-raf/genetics
Aldehyde Dehydrogenase, Mitochondrial/genetics
Genome, Human/genetics
Polymorphism, Single Nucleotide/genetics
Endodeoxyribonucleases/genetics/metabolism
CRISPR-Associated Proteins/genetics/metabolism
BRCA2 Protein/genetics
Neoplasms/genetics/diagnosis
Tumor Suppressor Protein p53/genetics
Base Pair Mismatch
Bacterial Proteins

@article {pmid39641617,
year = {2024},
author = {Jiang, H and Qian, C and Deng, Y and Lv, X and Liu, Y and Li, A and Li, X},
title = {Novel Multimode Assay Based on Asymmetrically Competitive CRISPR and Raman Barcode Spectra for Multiple Hepatocellular Carcinoma Biomarkers Detection.},
journal = {Analytical chemistry},
volume = {96},
number = {50},
pages = {20004-20014},
doi = {10.1021/acs.analchem.4c04593},
pmid = {39641617},
issn = {1520-6882},
mesh = {*Spectrum Analysis, Raman/methods ; *Carcinoma, Hepatocellular/diagnosis/genetics ; Humans ; *Liver Neoplasms/diagnosis/genetics ; *Biomarkers, Tumor/genetics ; *MicroRNAs/analysis ; Gold/chemistry ; Metal Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; },
abstract = {Commercial pregnancy test strips (PTS) possess the advantages of lower price, higher stability, and better repeatability and have been popularized to integrate with novel sensing strategies to detect other disease biomarkers, which accelerates the commercialization process of those novel sensing strategies. However, the current integration of novel sensing strategies into commercial PTS still faced the problems of insufficient quantification, low sensitivity, and lack of multiple detection capabilities. Hence, we proposed the concept of "visual classification recognition, spectral signal subdivision" for multiple hepatocellular carcinoma biomarkers (miRNA122 and miRNA233) detection with dual signals based on asymmetric competitive CRISPR (acCRISPR) and surface-enhanced Raman spectroscopy coupling with PTS, named the acCRISPR-PTS-SERS assay. In this assay, acCRISPR was used as a nonamplified cascaded signal amplification method to improve the sensitivity of detection. Two AuNPs-based core-shell Raman tags, each corresponding to different miRNA biomarkers, were used to achieve both visual recognition and spectral segmentation to enhance the quantification of PTS detection and the capability for multiple detection. Under the optimal conditions, the LOD for miRNA122 and miRNA223 were 10.36 and 4.65 fM, respectively. The sensitivity was enhanced by nearly 2 orders of magnitude. In the future, simultaneous hand-held detection for fingerprint barcodes of different cancers can be achieved with the assistance of a microfluidic chip and smartphone.},
}

*Spectrum Analysis, Raman/methods
*Carcinoma, Hepatocellular/diagnosis/genetics
Humans
*Liver Neoplasms/diagnosis/genetics
*Biomarkers, Tumor/genetics
*MicroRNAs/analysis
Gold/chemistry
Metal Nanoparticles/chemistry
CRISPR-Cas Systems/genetics

@article {pmid39639565,
year = {2024},
author = {Yuan, A and Sun, T and Chen, L and Zhang, D and Xie, W and Peng, H},
title = {CRISPR/Cas12a Corona Nanomachine for Detecting Circulating Tumor Nucleic Acids in Serum.},
journal = {Analytical chemistry},
volume = {96},
number = {50},
pages = {20074-20081},
doi = {10.1021/acs.analchem.4c04993},
pmid = {39639565},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gold/chemistry ; Circulating Tumor DNA/blood/genetics ; Metal Nanoparticles/chemistry ; Endodeoxyribonucleases/chemistry/metabolism ; CRISPR-Associated Proteins/chemistry/metabolism ; Biosensing Techniques ; Limit of Detection ; Bacterial Proteins ; },
abstract = {Circulating tumor nucleic acids (CTNAs), which consist of cell-free DNA or RNA released from tumor cells, are utilized as potential biomarkers for diagnosing and managing tumor prognosis. There is a significant demand for developing a highly sensitive and reliable assay for CTNAs detection. In this study, we engineered a CRISPR/Cas12a corona nanomachine capable of detecting circulating tumor DNA and RNA in serum. This nanomachine consists of a protein shell incorporating Cas12a/crRNA ribonucleoprotein complexes and a scaffold AuNP core decorated with substrate ssDNA strands. The protective CRISPR corona shields the nucleic acid core from degradation by nuclease DNase/RNase, thereby enhancing the stability of the CRISPR/Cas12a corona nanomachine in biological fluids, even tolerating up to undiluted human serum and FBS. Upon encountering target CTNAs, the CRISPR/Cas12a is activated through the sequence-specific hybridization between crRNA and CTNAs. Subsequently, the activated CRISPR/Cas12a autonomously cleaves the collateral ssDNA substrates on AuNPs, releasing the fluorophore-labeled fragment and generating an increasing fluorescent signal. The CRISPR corona nanomachine was successfully employed to detect various CTNAs, including circulating tumor (ct)DNA/RNA (EGFR L858R) and microRNA-21, achieving a limit of detection of 0.14 pM for ctDNA and 1.0 pM for RNA. This CRISPR corona nanomachine enables simultaneous detection of both DNA and RNA in complex biological samples, offering a promising tool for early diagnosis.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gold/chemistry
Circulating Tumor DNA/blood/genetics
Metal Nanoparticles/chemistry
Endodeoxyribonucleases/chemistry/metabolism
CRISPR-Associated Proteins/chemistry/metabolism
Biosensing Techniques
Limit of Detection
Bacterial Proteins

@article {pmid39603242,
year = {2024},
author = {Pahl, V and Lubrano, P and Troßmann, F and Petras, D and Link, H},
title = {Intact protein barcoding enables one-shot identification of CRISPRi strains and their metabolic state.},
journal = {Cell reports methods},
volume = {4},
number = {12},
pages = {100908},
doi = {10.1016/j.crmeth.2024.100908},
pmid = {39603242},
issn = {2667-2375},
mesh = {*Mass Spectrometry/methods ; Ubiquitin/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Metabolome ; },
abstract = {Detecting strain-specific barcodes with mass spectrometry can facilitate the screening of genetically engineered bacterial libraries. Here, we introduce intact protein barcoding, a method to measure protein-based library barcodes and metabolites using flow injection mass spectrometry (FI-MS). Protein barcodes are based on ubiquitin with N-terminal tags of six amino acids. We demonstrate that FI-MS detects intact ubiquitin proteins and identifies the mass of N-terminal barcodes. In the same analysis, we measured relative concentrations of primary metabolites. We constructed six ubiquitin-barcoded CRISPR interference (CRISPRi) strains targeting metabolic enzymes and analyzed their metabolic profiles and ubiquitin barcodes. FI-MS detected barcodes and distinct metabolome changes in CRISPRi-targeted pathways. We demonstrate the scalability of intact protein barcoding by measuring 132 ubiquitin barcodes in microtiter plates. These results show that intact protein barcoding enables fast and simultaneous detection of library barcodes and intracellular metabolites, opening up new possibilities for mass spectrometry-based barcoding.},
}

*Mass Spectrometry/methods
Ubiquitin/metabolism/genetics
CRISPR-Cas Systems/genetics
Escherichia coli/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Metabolome

@article {pmid39588656,
year = {2024},
author = {Jiang, Y and Wang, Y and Luo, W and Luan, X and Zhang, Z and Pan, Y and He, B and Gao, Y and Song, Y},
title = {Detecting telomerase activity at the single-cell level using a CRISPR-Cas12a-based chip.},
journal = {Lab on a chip},
volume = {25},
number = {1},
pages = {49-56},
doi = {10.1039/d4lc00619d},
pmid = {39588656},
issn = {1473-0189},
mesh = {*Telomerase/metabolism ; *CRISPR-Cas Systems ; Humans ; *Single-Cell Analysis/instrumentation ; Lab-On-A-Chip Devices ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The intimate association between telomerase activity and cancer has driven the exploration of diverse methodologies for its precise detection. However, detecting telomerase activity at the single-cell level remains a significant challenge. Herein, we present a MOF-DNA barcode-amplified CRISPR-Cas12a strategy integrated with a single-cell microfluidic chip for ultrasensitive detection of telomerase activity. DNA-functionalized UiO-66 nanoparticles act as signal transducers, effectively converting telomerase activity into DNA activation strands, which subsequently trigger the trans-cleavage activity of CRISPR-Cas12a. This amplification-based assay could be integrated with a microfluidic chip to enable highly sensitive detection of telomerase activity at the single-cell level, offering promising advancements in early cancer diagnosis.},
}

*Telomerase/metabolism
*CRISPR-Cas Systems
Humans
*Single-Cell Analysis/instrumentation
Lab-On-A-Chip Devices
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39571855,
year = {2024},
author = {Wan, Y and Huang, C and Feng, D and Wang, L and Lin, X and Zhao, X and Han, L and Zhu, Y and Hao, L and Du, H and Huang, L},
title = {Characterizing the collateral activity of CRISPR/Cas13 in mammalian cells: Implications for RNA editing and therapeutic applications.},
journal = {International journal of biological macromolecules},
volume = {283},
number = {Pt 4},
pages = {137861},
doi = {10.1016/j.ijbiomac.2024.137861},
pmid = {39571855},
issn = {1879-0003},
mesh = {Humans ; *RNA Editing/genetics ; *CRISPR-Cas Systems ; Animals ; Swine ; Cell Proliferation ; HEK293 Cells ; Cell Survival ; Embryonic Development/genetics ; },
abstract = {The CRISPR/Cas13 system has garnered attention as a potential tool for RNA editing. However, the degree of collateral activity among various Cas13 orthologs and their cytotoxic effects in mammalian cells remain contentious, potentially impacting their applications. In this study, we observed differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter, with LwaCas13a displaying notable activity contrary to previous reports. However, significant collateral RNA cleavage exerted only a modest impact on cell viability. Furthermore, collateral activity of LwaCas13a mildly impeded, but did not arrest, porcine embryo development. Our findings reveal that distinct collateral RNA cleavage by Cas13 slightly suppresses mammalian cell proliferation and embryo development. This could account for the lack of reported collateral effects in numerous prior studies and offers new insights into the implications of the collateral activity of Cas13 for clinical application.},
}

Humans
*RNA Editing/genetics
*CRISPR-Cas Systems
Animals
Swine
Cell Proliferation
HEK293 Cells
Cell Survival
Embryonic Development/genetics

@article {pmid39566771,
year = {2024},
author = {Panda, G and Ray, A},
title = {Deciphering Cas9 specificity: Role of domain dynamics and RNA:DNA hybrid interactions revealed through machine learning and accelerated molecular simulations.},
journal = {International journal of biological macromolecules},
volume = {283},
number = {Pt 4},
pages = {137835},
doi = {10.1016/j.ijbiomac.2024.137835},
pmid = {39566771},
issn = {1879-0003},
mesh = {*Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; *Machine Learning ; *DNA/chemistry/metabolism ; *RNA/chemistry/metabolism ; Francisella/enzymology ; Protein Domains ; CRISPR-Cas Systems ; Substrate Specificity ; Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {CRISPR/Cas9 technology is widely used for gene editing, but off-targeting still remains a major concern in therapeutic applications. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. This study compares the dynamics of the highly specific Cas9 from Francisella novicida (FnCas9) to the commonly used SpCas9. Using long-scale atomistic Gaussian accelerated molecular dynamic simulations and machine learning techniques, we deciphered the structural factors behind FnCas9's higher specificity in native and off-target forms. Our analysis revealed that Cas9's cleavage specificity relies more on its domain rearrangement than on RNA:DNA heteroduplex shape, with significant conformational variations in Cas9 domains among off-target forms, while the RNA:DNA hybrid showed minimal changes, especially in FnCas9 compared to SpCas9. REC1-REC3 domains contacts with the RNA:DNA hybrid in FnCas9 acted as critical discriminator of off-target effects playing a pivotal role in influencing specificity. In FnCas9, allosteric signal transmission involves the REC3 and HNH domain, bypassing REC2, leading to a superior efficiency in information transmission. This study offers a quantitative framework for understanding the structural basis of elevated specificity, paving the way for the rational design of Cas9 variants with improved precision and specificity in genome editing applications.},
}

*Molecular Dynamics Simulation
*CRISPR-Associated Protein 9/chemistry/metabolism/genetics
*Machine Learning
*DNA/chemistry/metabolism
*RNA/chemistry/metabolism
Francisella/enzymology
Protein Domains
CRISPR-Cas Systems
Substrate Specificity
Bacterial Proteins/chemistry/metabolism/genetics

@article {pmid39542328,
year = {2024},
author = {Li, S and Wang, F and Hao, L and Zhang, P and Song, G and Zhang, Y and Wang, C and Wang, Z and Wu, Q},
title = {Enhancing peroxidase activity of NiCo2O4 nanoenzyme by Mn doping for catalysis of CRISPR/Cas13a-mediated non-coding RNA detection.},
journal = {International journal of biological macromolecules},
volume = {283},
number = {Pt 1},
pages = {137594},
doi = {10.1016/j.ijbiomac.2024.137594},
pmid = {39542328},
issn = {1879-0003},
mesh = {*Manganese/chemistry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Peroxidase/metabolism/chemistry ; Catalysis ; Nickel/chemistry ; Humans ; Colorimetry/methods ; Cobalt/chemistry ; },
abstract = {CRISPR/Cas13a with precise and controllable programming of endonuclease activity has been served as powerful tool for RNA sensing. Although with high sensitivity, existing CRISPR/Cas13a-based biosensors need complex amplification procedure or special equipment that limited quantification capability. Here, Mn-doped NiCo2O4 (Mn/NiCo2O4) nanozyme with enhanced peroxidase activity was synthesized and combined with CRISPR/Cas13a-based reaction to develop a simple, sensitive and universal biosensor for RNA detection, which is achieved through target recognition that activates Cas enzymes to cleave RNA reporter for inhibiting Mn/NiCo2O4 nanozyme to assemble on microplate. The Mn/NiCo2O4 nanozyme assembled on microplate can be monitored through colorimetric and fluorometric approaches. On one hand, Mn/NiCo2O4 nanozyme offers ideal peroxidase activity to catalyze colorimetric reaction, and as low as dozens of amol level of RNA target can be sensitively detected by naked eyes without any amplification procedures. On the other hand, Mn/NiCo2O4 can be also served as a signal amplifier to produce large amount of Co[2+], Mn[2+]and Ni[2+] to quench the fluorescence of calcein. The fluorescent approach can achieve higher sensitivity (about 40-fold) than colorimetric method. More importantly, the proposed biosensor can work well for multiple RNA detection in real biological samples, showing a great potential for monitoring non-coding RNA-related diseases.},
}

*Manganese/chemistry
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Peroxidase/metabolism/chemistry
Catalysis
Nickel/chemistry
Humans
Colorimetry/methods
Cobalt/chemistry

@article {pmid39528193,
year = {2024},
author = {Zhang, K and Luo, H and Zhu, X and Liu, W and Yu, X and Tao, W and Lin, H and Hou, M and Wu, J},
title = {Construction of Bacillus subtilis chassis strain with enhanced α-amylase expression capability based on CRISPRi screening.},
journal = {International journal of biological macromolecules},
volume = {283},
number = {Pt 1},
pages = {137497},
doi = {10.1016/j.ijbiomac.2024.137497},
pmid = {39528193},
issn = {1879-0003},
mesh = {*alpha-Amylases/genetics/biosynthesis/metabolism ; *Bacillus subtilis/genetics/enzymology ; Recombinant Proteins/genetics/metabolism/biosynthesis ; CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics/metabolism/biosynthesis ; Fermentation ; Gene Expression Regulation, Bacterial ; },
abstract = {Bacillus subtilis has been widely used in the expression of recombinant proteins due to its food safe and powerful secretion characteristic, but the current production level cannot meet the increasing industrial needs. To enhance the production of recombinant protein, we first screened target key genes that are directly or indirectly involved in protein synthesis, using CRISPRi technology targeting the whole genome, with industrial valuable Bacillus stearothermophilus α-amylase as the model protein. Then the screened key genes were combined, yielding a chassis strain that owning enhanced protein expression capability. Following overlaying molecular chaperone GroES/L and peptidoglycan glycosyltransferase PonA, α-amylase activity reached 102,893 U/mL in a 3-L fermenter, the highest level reported till now. Finally, transcriptome analysis showed that the enhanced recombinant expression may be due to more rational allocation of energy and resources. These strategies can be well implicated in engineering other microbial cell factories for higher industrial production.},
}

*alpha-Amylases/genetics/biosynthesis/metabolism
*Bacillus subtilis/genetics/enzymology
Recombinant Proteins/genetics/metabolism/biosynthesis
CRISPR-Cas Systems/genetics
Bacterial Proteins/genetics/metabolism/biosynthesis
Fermentation
Gene Expression Regulation, Bacterial

@article {pmid39522648,
year = {2024},
author = {Lee, SH and Park, J and Hwang, B},
title = {Multiplexed multimodal single-cell technologies: From observation to perturbation analysis.},
journal = {Molecules and cells},
volume = {47},
number = {12},
pages = {100147},
pmid = {39522648},
issn = {0219-1032},
mesh = {*Single-Cell Analysis/methods ; Humans ; CRISPR-Cas Systems ; Animals ; Genomics/methods ; },
abstract = {Single-cell technologies have undergone a significant transformation, expanding from their initial focus on transcriptomics to encompass a diverse range of modalities. Recent advancements have markedly improved scalability and reduced costs, facilitating the processing of larger cell populations and broadening the scope of single-cell research. The incorporation of clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations has revolutionized the field by enabling precise functional genomics and detailed studies of gene regulation at the single-cell level. Despite these advancements, challenges persist, particularly in achieving genome-wide perturbations and managing the complexity of high-throughput data. This review discusses the technological milestones that have driven these changes, the current limitations of single-cell CRISPR technologies, and the future directions needed to address these challenges and advance our understanding of cellular biology.},
}

*Single-Cell Analysis/methods
Humans
CRISPR-Cas Systems
Animals
Genomics/methods

@article {pmid39515687,
year = {2024},
author = {Yang, H and Guo, Y and Wang, J and Tao, C and Cao, J and Cheng, T and Liu, C},
title = {Bmgsb is involved in the determination of cell fate by affecting the cell cycle genes in the silk gland of Bombyx mori.},
journal = {International journal of biological macromolecules},
volume = {283},
number = {Pt 2},
pages = {136914},
doi = {10.1016/j.ijbiomac.2024.136914},
pmid = {39515687},
issn = {1879-0003},
mesh = {*Bombyx/genetics/metabolism ; Animals ; *Silk/genetics ; *Insect Proteins/genetics/metabolism ; Fibroins/genetics ; Cell Cycle/genetics ; Transcription Factors/genetics/metabolism ; Cell Differentiation/genetics ; CRISPR-Cas Systems ; },
abstract = {Silk gland is the only organ of silkworm that can produce silk protein, which is a natural macromolecular protein complex and widely utilized in various fields such as biomaterials and biomedicine. The development of silk gland and the expression patterns of silk protein crucial for the silk industry. In this study, the function of a transcription factor Bmgsb was investigated with CRISPR/Cas9 and transgenic system. It was found that the homozygous individuals in the Bmgsb KO line experienced spinning failure and pupae death, the AMSG exhibited defects, and the ASG displayed abnormal curvature. These phenotypes were accompanied by increased DNA endoreplication and significantly upregulated expression of fibroin genes in the ASG. RT-qPCR results confirmed significant upregulation of cell cycle-related genes, including cyclin G and cyclin T in the Bmgsb KO line. Furthermore, ectopic expression of Bmgsb in the PSG weakened PSG curvature, inhibited DNA endoreplication, and downregulated the expression of fibroin genes. These findings strongly suggest that Bmgsb plays a crucial role in determining cell fate in the silk gland and regulating the expression of silk protein through the cyclin pathway. Our research provides a theoretical foundation for further studies on organ differentiation and have implications for the silk industry.},
}

*Bombyx/genetics/metabolism
Animals
*Silk/genetics
*Insect Proteins/genetics/metabolism
Fibroins/genetics
Cell Cycle/genetics
Transcription Factors/genetics/metabolism
Cell Differentiation/genetics
CRISPR-Cas Systems

@article {pmid39494910,
year = {2024},
author = {Stevens, CS and Carmichael, JC and Watkinson, R and Kowdle, S and Reis, RA and Hamane, K and Jang, J and Park, A and Pernet, O and Khamaikawin, W and Hong, P and Thibault, P and Gowlikar, A and An, DS and Lee, B},
title = {A temperature-sensitive and less immunogenic Sendai virus for efficient gene editing.},
journal = {Journal of virology},
volume = {98},
number = {12},
pages = {e0083224},
doi = {10.1128/jvi.00832-24},
pmid = {39494910},
issn = {1098-5514},
support = {R01 AI138921/AI/NIAID NIH HHS/United States ; T32 AI07647//HHS | National Institutes of Health (NIH)/ ; F32 HL158173/HL/NHLBI NIH HHS/United States ; ALTF 628-2015//European Molecular Biology Organization (EMBO)/ ; CIHR 359156//Canadian Government | Canadian Institutes of Health Research (CIHR)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Sendai virus/genetics/immunology ; *Receptors, CCR5/genetics ; *Hematopoietic Stem Cells/virology/immunology ; *Genetic Vectors/genetics ; CRISPR-Cas Systems ; Temperature ; HIV-1/genetics/immunology ; Transduction, Genetic ; HIV Infections/immunology/therapy/virology/genetics ; Monocytes/immunology ; },
abstract = {UNLABELLED: The therapeutic potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and less immunogenic Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types with limited induction of an innate immune response. ts SeV demonstrates high transduction efficiency in human CD34[+] hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34[+]/CD38[-]/CD45RA[-]/CD90[+](Thy1[+])/CD49f[high] stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14[+] monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help further expand the possibilities in personalized medicine and the treatment of genetic disorders.

IMPORTANCE: Gene editing has the potential to be a powerful tool for the treatment of human diseases including HIV, β-thalassemias, and sickle cell disease. Recent advances have begun to overcome one of the major limiting factors of this technology, namely delivery of the CRISPR-Cas9 gene editing machinery, by utilizing viral vectors. However, gene editing therapies have yet to be implemented due to inherent risks associated with the DNA viral vectors typically used for delivery. As an alternative strategy, we have developed an RNA-based Sendai virus CRISPR-Cas9 delivery vector that does not integrate into the genome, is temperature sensitive, and does not induce a significant host interferon response. This recombinant SeV successfully delivered CRISPR-Cas9 in primary human CD14+ monocytes ex vivo resulting in a high level of CCR5 editing and inhibition of HIV infection.},
}

*Gene Editing/methods
Humans
*Sendai virus/genetics/immunology
*Receptors, CCR5/genetics
*Hematopoietic Stem Cells/virology/immunology
*Genetic Vectors/genetics
CRISPR-Cas Systems
Temperature
HIV-1/genetics/immunology
Transduction, Genetic
HIV Infections/immunology/therapy/virology/genetics
Monocytes/immunology

@article {pmid39419543,
year = {2024},
author = {Lamb, CH and Pitré, EM and Ajufo, S and Rigby, CV and Bisht, K and Oade, MS and Jalal, H and Myhrvold, C and Te Velthuis, AJW},
title = {Quantification of influenza virus mini viral RNAs using Cas13.},
journal = {RNA (New York, N.Y.)},
volume = {31},
number = {1},
pages = {126-138},
doi = {10.1261/rna.080174.124},
pmid = {39419543},
issn = {1469-9001},
mesh = {*RNA, Viral/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Influenza A virus/genetics ; Animals ; Genome, Viral ; Influenza, Human/virology/genetics ; Dogs ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Influenza A virus (IAV) RNA synthesis produces full-length and deletion-containing RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that DVG and mvRNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host-pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Measuring the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we explored if IAV mvRNA molecules can be detected and quantified using amplification-free, CRISPR-LbuCas13a-based detection. We show that CRISPR-LbuCas13a can be used to measure the copy numbers of specific mvRNAs in samples from infected tissue culture cells. However, to efficiently detect mvRNAs in other samples, promiscuous CRISPR guide RNAs are required that activate LbuCas13a in the presence of multiple mvRNA sequences. One crRNA was able to detect full-length IAV segment 5 without amplification, allowing it to be used for general IAV infection detection nasopharyngeal swabs. Using CRISPR-LbuCas13a, we confirm that mvRNAs are present in ferret upper and lower respiratory tract tissue, as well as clinical nasopharyngeal swab extracts of hospitalized patients. Overall, CRISPR-LbuCas13a-based RNA detection is a useful tool for studying deletion-containing viral RNAs, and it complements existing amplification-based approaches.},
}

*RNA, Viral/genetics/metabolism
Humans
*CRISPR-Cas Systems
*Influenza A virus/genetics
Animals
Genome, Viral
Influenza, Human/virology/genetics
Dogs
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39212195,
year = {2024},
author = {Sarkar, S and Moitra, P and Duan, W and Bhattacharya, S},
title = {A Multifunctional Aptamer Decorated Lipid Nanoparticles for the Delivery of EpCAM-targeted CRISPR/Cas9 Plasmid for Efficacious In Vivo Tumor Regression.},
journal = {Advanced healthcare materials},
volume = {13},
number = {31},
pages = {e2402259},
doi = {10.1002/adhm.202402259},
pmid = {39212195},
issn = {2192-2659},
mesh = {*Epithelial Cell Adhesion Molecule/metabolism/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; Humans ; *Aptamers, Nucleotide/chemistry ; *Plasmids/genetics/chemistry ; Mice ; Cell Line, Tumor ; Lipids/chemistry ; Neoplasms/therapy/genetics/metabolism/pathology ; Liposomes ; },
abstract = {Epithelial cell adhesion molecule (EpCAM) gene encodes a type-I trans-membrane glycoprotein that is overexpressed in many cancerous epithelial cells and promotes tumor progression by regulating the expression of several oncogenes like c-myc and other cyclins. Because of this tumorigenic association, the EpCAM gene has been a potential target for anti-cancer therapy in recent days. Herein, it is attempted to knockout the proto-oncogenic EpCAM expression by efficiently delivering an all-in-one Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) plasmid via a lipid nanoparticle system made out of synthetic stimuli-sensitive lipids. The plasmid possesses the necessary information in the form of a guide RNA targeted to the EpCAM gene. The aptamer decorated system selectively targets EpCAM overexpressed cells and efficiently inhibits the genetic expression. It has explored the pH-responsive property of the developed lipid nanoparticles and monitored their efficacy in various cancer cell lines of different origins with elevated EpCAM levels. The phenomenon has further been validated in vivo in non-immunocompromised mouse tumor models. Overall, the newly developed aptamer decorated lipid nanoparticle system has been proven to be efficacious for the delivery of EpCAM-targeted CRISPR/Cas9 plasmid.},
}

*Epithelial Cell Adhesion Molecule/metabolism/genetics
Animals
*CRISPR-Cas Systems/genetics
*Nanoparticles/chemistry
Humans
*Aptamers, Nucleotide/chemistry
*Plasmids/genetics/chemistry
Mice
Cell Line, Tumor
Lipids/chemistry
Neoplasms/therapy/genetics/metabolism/pathology
Liposomes

@article {pmid39172347,
year = {2024},
author = {Zhou, Z and Wang, YQ and Zheng, XN and Zhang, XH and Ji, LY and Han, JY and Zuo, ZC and Mo, WL and Zhang, L},
title = {Optimizing ABA-based chemically induced proximity for enhanced intracellular transcriptional activation and modification response to ABA.},
journal = {Science China. Life sciences},
volume = {67},
number = {12},
pages = {2650-2663},
pmid = {39172347},
issn = {1869-1889},
mesh = {*Abscisic Acid/metabolism/pharmacology ; Humans ; HEK293 Cells ; *Transcriptional Activation/drug effects ; *Arabidopsis Proteins/genetics/metabolism ; Arabidopsis/genetics/metabolism/drug effects ; CRISPR-Cas Systems ; Cell Proliferation/drug effects ; Signal Transduction/drug effects ; Membrane Transport Proteins ; },
abstract = {Abscisic acid (ABA)-based chemically induced proximity (CIP) is primarily mediated by the interaction of the ABA receptor pyrabactin resistance 1-like 1 (PYL1) and the 2C-type protein phosphatase ABI1, which confers ABA-induced proximity to their fusion proteins, and offers precise temporal control of a wide array of biological processes. However, broad application of ABA-based CIP has been limited by ABA response intensity. In this study, we demonstrated that ABA-induced interaction between another ABA receptor pyrabactin resistance 1 (PYR1) and ABI1 exhibited higher ABA response intensity than that between PYL1 and ABI1 in HEK293T cells. We engineered PYR1-ABI1 and PYL1-ABI1 into ABA-induced transcriptional activation tools in mammalian cells by integration with CRISPR/dCas9 and found that the tool based on PYR1-ABI1 demonstrated better ABA response intensity than that based on PYL1-ABI1 for both exogenous and endogenous genes in mammalian cells. We further achieved ABA-induced RNA m[6]A modification installation and erasure by combining ABA-induced PYR1-ABI1 interaction with CRISPR/dCas13, successfully inhibiting tumor cell proliferation. We subsequently improved the interaction of PYR1-ABI1 through phage-assisted continuous evolution (PACE), successfully generating a PYR1 mutant (PYR1m) whose interaction with ABI1 exhibited a higher ABA response intensity than that of the wild-type. In addition, we tested the transcriptional activation tool based on PYRm-ABI1 and found that it also showed a higher ABA response intensity than that of the wild type. These results demonstrate that we have developed a novel ABA-based CIP and further improved upon it using PACE, providing a new approach for the modification of other CIP systems.},
}

*Abscisic Acid/metabolism/pharmacology
Humans
HEK293 Cells
*Transcriptional Activation/drug effects
*Arabidopsis Proteins/genetics/metabolism
Arabidopsis/genetics/metabolism/drug effects
CRISPR-Cas Systems
Cell Proliferation/drug effects
Signal Transduction/drug effects
Membrane Transport Proteins

@article {pmid39158766,
year = {2024},
author = {He, Y and Liu, S and Chen, L and Pu, D and Zhong, Z and Xu, T and Ren, Q and Dong, C and Wang, Y and Wang, D and Zheng, X and Guo, F and Zhang, T and Qi, Y and Zhang, Y},
title = {Versatile plant genome engineering using anti-CRISPR-Cas12a systems.},
journal = {Science China. Life sciences},
volume = {67},
number = {12},
pages = {2730-2745},
pmid = {39158766},
issn = {1869-1889},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Plant ; *Plants, Genetically Modified/genetics ; *Oryza/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Protoplasts/metabolism ; Escherichia coli/genetics/metabolism ; Genetic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endodeoxyribonucleases ; },
abstract = {CRISPR-Cas12a genome engineering systems have been widely used in plant research and crop breeding. To date, the performance and use of anti-CRISPR-Cas12a systems have not been fully established in plants. Here, we conduct in silico analysis to identify putative anti-CRISPR systems for Cas12a. These putative anti-CRISPR proteins, along with known anti-CRISPR proteins, are assessed for their ability to inhibit Cas12a cleavage activity in vivo and in planta. Among all anti-CRISPR proteins tested, AcrVA1 shows robust inhibition of Mb2Cas12a and LbCas12a in E. coli. Further tests show that AcrVA1 inhibits LbCas12a mediated genome editing in rice protoplasts and stable transgenic lines. Impressively, co-expression of AcrVA1 mitigates off-target effects by CRISPR-LbCas12a, as revealed by whole genome sequencing. In addition, transgenic plants expressing AcrVA1 exhibit different levels of inhibition to LbCas12a mediated genome editing, representing a novel way of fine-tuning genome editing efficiency. By controlling temporal and spatial expression of AcrVA1, we show that inducible and tissue specific genome editing can be achieved in plants. Furthermore, we demonstrate that AcrVA1 also inhibits LbCas12a-based CRISPR activation (CRISPRa) and based on this principle we build logic gates to turn on and off target genes in plant cells. Together, we have established an efficient anti-CRISPR-Cas12a system in plants and demonstrate its versatile applications in mitigating off-target effects, fine-tuning genome editing efficiency, achieving spatial-temporal control of genome editing, and generating synthetic logic gates for controlling target gene expression in plant cells.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Genome, Plant
*Plants, Genetically Modified/genetics
*Oryza/genetics
*CRISPR-Associated Proteins/genetics/metabolism
Protoplasts/metabolism
Escherichia coli/genetics/metabolism
Genetic Engineering/methods
Bacterial Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Endodeoxyribonucleases

@article {pmid39676682,
year = {2024},
author = {Chen, J and Lin, X and Xiang, W and Chen, Y and Zhao, Y and Huang, L and Liu, L},
title = {DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae1241},
pmid = {39676682},
issn = {1362-4962},
support = {32022047//National Natural Science Foundation of China/ ; 3502Z20227020//Natural Science Foundation of Xiamen, China/ ; 2023J01023//Natural Science Foundation of Fujian Province/ ; },
abstract = {Precursor (pre)-CRISPR RNA (crRNA) processing can occur in both the repeat and spacer regions, leading to the removal of specific segments from the repeat and spacer sequences, thereby facilitating crRNA maturation. The processing of pre-crRNA repeat by Cas effector and ribonuclease has been observed in CRISPR-Cas9 and CRISPR-Cas12a systems. However, no evidence of pre-crRNA spacer cleavage by any enzyme has been reported in these systems. In this study, we demonstrate that DNA target binding triggers efficient cleavage of pre-crRNA spacers by type II and V Cas effectors such as Cas12a, Cas12b, Cas12i, Cas12j and Cas9. We show that the pre-crRNA spacer cleavage catalyzed by Cas12a and Cas9 has distinct characteristics. Activation of the cleavage activity in Cas12a is induced by both single-stranded DNA (ssDNA) and double-stranded DNA target binding, whereas only ssDNA target binding triggers cleavage in Cas9 toward the pre-crRNA spacer. We present a series of structures elucidating the underlying mechanisms governing conformational activation in both Cas12a and Cas9. Furthermore, leveraging the trans-cutting activity of the pre-crRNA spacer, we develop a one-step DNA detection method characterized by its simplicity, high sensitivity, and excellent specificity.},
}

@article {pmid39676076,
year = {2024},
author = {Gao, Z and Bak, RO},
title = {Integration of large genetic payloads using prime editing and site-specific integrases.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
pmid = {39676076},
issn = {1750-2799},
support = {101041231//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; R238-2016-3349//Lundbeckfonden (Lundbeck Foundation)/ ; 8056-00010B//Innovationsfonden (Innovation Fund Denmark)/ ; NNF21OC0071259//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; },
}

@article {pmid39675005,
year = {2024},
author = {Rubio, A and Garzón, A and Moreno-Rodríguez, A and Pérez-Pulido, AJ},
title = {Biological warfare between two bacterial viruses in a defense archipelago sheds light on the spread of CRISPR-Cas systems.},
journal = {Cell reports},
volume = {43},
number = {12},
pages = {115085},
doi = {10.1016/j.celrep.2024.115085},
pmid = {39675005},
issn = {2211-1247},
abstract = {CRISPR-Cas systems are adaptive immunity systems of bacteria and archaea that prevent infection by viruses and other external mobile genetic elements. It is currently known that these defense systems can be co-opted by the same viruses. We have found one of these viruses in the opportunistic pathogen Acinetobacter baumannii, and the same system has been also found in an integration hotspot of the bacterial genome that harbors other multiple defense systems. The CRISPR-Cas system appears to especially target another virus that could compete with the system itself for the same integration site. This virus is prevalent in strains of the species belonging to the so-called Global Clone 2, which causes the most frequent outbreaks worldwide. Knowledge of this viral warfare involving antiviral systems could be useful in the fight against infections caused by bacteria, and it would also shed light on how CRISPR-Cas systems expand in bacteria.},
}

@article {pmid39673405,
year = {2024},
author = {Moazed, N and Tahmasebi Nezhad, N and Karami Nezhad, Z and Khosravi, A},
title = {Application of CRISPR/Cas9 in CAR-T Cell Therapy: A Systematic Review Study.},
journal = {Iranian biomedical journal},
volume = {28},
number = {7},
pages = {40},
pmid = {39673405},
issn = {2008-823X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; *Gene Editing/methods ; Neoplasms/therapy/genetics ; Receptors, Chimeric Antigen/genetics/immunology ; T-Lymphocytes/immunology ; Animals ; },
abstract = {INTRODUCTION: Currently, several attempts have been made to find effective treatments for cancer, with various options suggested, including chimeric antigen receptor-engineered T-cell (CAR-T) therapy. CAR-T cell therapy is a type of cell therapy that targets cancerous cells and is used in adoptive cell immunotherapy. Recent studies indicate that certain genetic therapeutic methods, such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing technology, can enhance the efficacy of this treatment and have garnered significant attention.

SEARCH STRATEGY: This article presents a review study that involved searching reputable databases, including Google Scholar, PubMed, Scopus, Cochrane Library, Web of Science, using keywords such as "CRISPR/Cas9", "CAR-T cell therapy", "Cancer", "Gene editing", and "immunotherapy" to ensure the comprehensive search results. The sources of the articles were thoroughly reviewed. After removing duplicate titles using EndNote software and examining the titles and abstracts, the articles relavant to the JBI tools were selected for further review. The appropriate checklist was then applied.

RESULTS: Studies showed that despite significant success and promising result in CAR-T cell therapy, there were several challenges, including cell malfunction, toxicity, in adequate persistence of CAR-T cells, and T-cell exhaustion. These issues can adversely affect the efficacy and safety of this treatment. CRISPR/Cas9 technology has the potential to enhance CAR-T cell therapy by increasing resistance to immune cell-suppressive molecules. By enabling genome-targeted manipulation, this method can improve the effectiveness of CAR-T cell therapy through precise editing of these cells. CRISPR/Cas9 technology is notable for its remarkable efficacy in genome editing, flexibility, and ease of use. One of its most significant advantages is the utilization of a single nuclease combined with two short RNA molecules, which facilitates efficient genome editing.

CONCLUSION AND DISCUSSION: Despite the substantial advantages demonstrated by CRISPR/Cas9 technology, this method represents a novel approach to genome editing and offers rapid improvements in gene therapy. However, our understanding of all aspects of this technique, particularly its therapeutic application in cancer treatment, remains incomplete. Therefore, we recommend further studies to enhance our knowledge in this area.},
}

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

@article {pmid39673266,
year = {2024},
author = {Molina-Sánchez, MD and Martínez-Abarca, F and Millán, V and Mestre, MR and Stehantsev, P and Stetsenko, A and Guskov, A and Toro, N},
title = {Adaptive immunity of type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae1154},
pmid = {39673266},
issn = {1362-4962},
support = {P20_00047//Consejeria de Transformacion Economica, industria, Conocimiento y Universidades/ ; //ERDF A way of making Europe/ ; },
abstract = {Cas13-containing type VI CRISPR-Cas systems specifically target RNA; however, the mechanism of spacer acquisition remains unclear. We have previously reported the association of reverse transcriptase-Cas1 (RT-Cas1) fusion proteins with certain types of VI-A systems. Here, we show that RT-Cas1 fusion proteins are also recruited by type VI-B systems in bacteria from gut microbiomes, constituting a VI-B1 variant system that includes a CorA-encoding locus in addition to the CRISPR array and the RT-Cas1/Cas2 adaptation module. We found that type VI RT-CRISPR systems were functional for spacer acquisition, CRISPR array processing and interference activity, demonstrating that adaptive immunity mediated by these systems can function independently of other in trans systems. We provide evidence that the RT associated with these systems enables spacer acquisition from RNA molecules. We also found that CorA encoded by type VI-B1 RT-associated systems can transport divalent metal ions and downregulate Cas13b-mediated RNA interference. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, potentially enabling the integration of RNA-derived spacers into CRISPR arrays as a mechanism against RNA-based invaders in specific environments.},
}

@article {pmid39671335,
year = {2024},
author = {Kolodziejczyk, A and Hoffmann, J and Cubillos, P and Albert, M},
title = {Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {213},
pages = {},
doi = {10.3791/67598},
pmid = {39671335},
issn = {1940-087X},
mesh = {Humans ; *Electroporation/methods ; *Organoids/cytology/metabolism ; *CRISPR-Cas Systems ; Cerebral Cortex/cytology ; Gene Knockout Techniques/methods ; Neocortex/cytology ; },
abstract = {Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.},
}

Humans
*Electroporation/methods
*Organoids/cytology/metabolism
*CRISPR-Cas Systems
Cerebral Cortex/cytology
Gene Knockout Techniques/methods
Neocortex/cytology

@article {pmid39671009,
year = {2024},
author = {Yu, Y and Li, Q and Yu, H and Li, Q},
title = {Comparative Analysis of Promoter Activity in Crassostrea gigas Embryos: Implications for Bivalve Gene Editing.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {27},
number = {1},
pages = {20},
pmid = {39671009},
issn = {1436-2236},
support = {42276111//National Natural Science Foundation of China/ ; ZR2022MC171//Natural Science Foundation of Shandong Province/ ; },
mesh = {Animals ; *Crassostrea/genetics/embryology ; *Promoter Regions, Genetic ; *Green Fluorescent Proteins/genetics/metabolism ; *Gene Editing/methods ; *Histones/metabolism/genetics ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Peptide Elongation Factor 1/genetics/metabolism ; Aquaculture ; },
abstract = {In recent years, CRISPR/Cas9 gene editing technology has emerged as a powerful genetic tool with potential application in aquaculture. Crassostrea gigas, as a valuable species in aquaculture, holds promising potential for genetic enhancement and breeding through gene editing. However, the lack of efficient promoters for driving exogenous gene expression poses a major obstacle in bivalve gene editing. In this study, we isolated the promoter sequences of the β-tub and histone H3.3A genes from C. gigas. DNA expression constructs were generated by linking the promoters with the enhanced green fluorescent protein (EGFP) reporter and compared with the promoter activity of the endogenous EF-1α gene and an exogenous OsHV-1 promoter in C. gigas embryos. All four promoters effectively drive the expression of EGFP during early embryonic development in C. gigas. Among these four promoters, the β-tub promoter is the most potent promoter in driving EGFP expression in C. gigas embryos as early as 4.5 h after fertilization. The OsHV-1 promoter showed similar activity as β-tub promoter and appeared to be more active than the EF-1α and histone H3.3A promoters in C. gigas embryos. Furthermore, we assessed their performance in other three C. gigas relatives (Crassostrea ariakensis, Crassostrea nippona, and Crassostrea sikamea) and similar results were found. Collectively, these data suggest that the β-tub promoter is an effective promoter in directing gene expression in directing gene expression in oyster embryos, thus offering a potential application for gene editing in bivalves.},
}

Animals
*Crassostrea/genetics/embryology
*Promoter Regions, Genetic
*Green Fluorescent Proteins/genetics/metabolism
*Gene Editing/methods
*Histones/metabolism/genetics
Embryo, Nonmammalian/metabolism
CRISPR-Cas Systems
Peptide Elongation Factor 1/genetics/metabolism
Aquaculture

@article {pmid39670656,
year = {2024},
author = {Jung, JK and Dreyer, KS and Dray, KE and Muldoon, JJ and George, J and Shirman, S and Cabezas, MD and d'Aquino, AE and Verosloff, MS and Seki, K and Rybnicky, GA and Alam, KK and Bagheri, N and Jewett, MC and Leonard, JN and Mangan, NM and Lucks, JB},
title = {Developing, Characterizing, and Modeling CRISPR-Based Point-of-Use Pathogen Diagnostics.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00469},
pmid = {39670656},
issn = {2161-5063},
abstract = {Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.},
}

@article {pmid39670632,
year = {2024},
author = {Chen, W and Liu, L and Cheng, L},
title = {Conditionally Activated Cross-Linked crRNAs for CRISPR/Cas12a Based Nucleic Acid Detection.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00695},
pmid = {39670632},
issn = {2161-5063},
abstract = {CRISPR/Cas systems, particularly CRISPR/Cas12a, have revolutionized nucleic acid detection due to their exceptional specificity and sensitivity. However, CRISPR/Cas12a's cleavage activity can interfere with amplification processes, such as reverse transcription (RT) and isothermal amplification (e.g., RPA), potentially compromising detection sensitivity and accuracy. While modified CRISPR/Cas12a systems employing caging and decaging strategies have been developed to address this, these approaches typically require extensive optimization of photolabile groups and complex assay configurations. Here, we present a universal, photochemically controlled strategy for CRISPR/Cas12a-based detection that overcomes these challenges. Our approach involves cross-linking a polymeric crRNA with a photoresponsive cross-linker, effectively inactivating it during amplification and enabling rapid activation through brief light exposure to cleave the cross-linker and release active crRNA. This method obviates the need for labor-intensive optimizations and modifications, making it highly versatile and suitable for rapid, on-site detection applications. Our strategy demonstrates enhanced versatility and applicability, particularly for the immediate detection of newly emerging or unexpected nucleic acid sequences, supporting applications in pathogen detection, genetic screening, and point-of-care diagnostics.},
}

@article {pmid39601618,
year = {2024},
author = {Liu, X and Peng, H and Gong, L and Zhang, H and Zhao, C and Lai, W and An, G and Zhao, X},
title = {Reliable and precise lipoprotein detection based on a self-priming hairpin-triggered Cas12a/crRNA based signaling strategy.},
journal = {The Analyst},
volume = {150},
number = {1},
pages = {46-54},
doi = {10.1039/d4an01167h},
pmid = {39601618},
issn = {1364-5528},
mesh = {Humans ; *Limit of Detection ; *Lipoproteins, LDL/blood/analysis ; Lipoproteins, HDL/blood/analysis ; Antibodies, Immobilized/immunology ; CRISPR-Cas Systems ; CRISPR-Associated Proteins/metabolism ; Lipoproteins/chemistry/blood ; },
abstract = {Cardiovascular disease, intimately linked to dyslipidemia, is one of the leading global causes of mortality. Dyslipidaemia often presents as an elevated concentration of low-density lipoprotein (LDL) and a decreased concentration of high-density lipoprotein (HDL). Therefore, accurately measuring the levels of LDL and HDL particles is crucial for assessing the risk of developing cardiovascular diseases. However, conventional approaches can commonly quantify HDL/LDL particles by detecting cholesterol or protein molecules within them, which may fail to reflect the number of intact particles. In addition, these approaches are sometimes tedious and time-consuming, highlighting the need for a novel method for precise and effective identification of intact HDL and LDL particles. We have devised a technique that allows accurately and sensitively determining the levels of intact HDL and LDL in a sample without the need for isolation. This method relies on antibody-based immobilization and a self-priming hairpin-triggered Cas12a/crRNA signaling strategy. Based on the elegant design, this technique can be employed to directly and precisely measure the concentration of "actual" HDL and LDL particles, rather than the cholesterol content inside HDL and LDL. The approach has detection limits of 12.3 mg dL[-1] and 5.4 mg dL[-1] for HDL and LDL, respectively, and is also suitable for analyzing lipoproteins in clinical samples. Hence, this platform exhibits immense potential for clinical applications and health management.},
}

Humans
*Limit of Detection
*Lipoproteins, LDL/blood/analysis
Lipoproteins, HDL/blood/analysis
Antibodies, Immobilized/immunology
CRISPR-Cas Systems
CRISPR-Associated Proteins/metabolism
Lipoproteins/chemistry/blood

@article {pmid39556313,
year = {2024},
author = {Liu, S and Zhao, Y and Mo, Q and Sun, Y and Ma, H},
title = {Engineering CjCas9 for Efficient Base Editing and Prime Editing.},
journal = {The CRISPR journal},
volume = {7},
number = {6},
pages = {395-405},
doi = {10.1089/crispr.2024.0018},
pmid = {39556313},
issn = {2573-1602},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; Campylobacter jejuni/genetics ; Cytosine/metabolism ; Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Adenine/metabolism ; Bacterial Proteins/genetics ; Streptococcus pyogenes/genetics/enzymology ; },
abstract = {The CRISPR-Cas9 system has been applied for clinical applications of gene therapy. Most CRISPR-based gene therapies are derived from Streptococcus pyogenes Cas9, which is challenging to package into a single adeno-associated virus vector and limits its clinical applications. Campylobacter jejuni Cas9 (CjCas9) is one of the smallest Cas9 proteins. CjCas9-mediated base editing (CjBE) efficiency varies across genomic sites, while CjCas9-mediated prime editing (CjPE) efficiency is less than 5% on average. Here we developed enhanced cytosine base editors (enCjCBEs) and adenine base editors (enCjABEs) by engineered CjCas9[P47K]. We demonstrated the robust C-to-T conversion (70% on average) by enCjCBE or A-to-G conversion (76% on average) by enCjABE. Meanwhile, we applied the CjCas9[P47K] variant to generate enhanced CjPE (enCjPE), which increases the editing efficiency 17-fold at the PRNP site over wild-type CjPE. Fusing nonspecific DNA binding protein Sso7d to enCjCas9 and MS2 stem-loop RNA aptamer to the 3-terminal of cognate pegRNA resulted in 12% editing efficiency on average with a 24-fold increase over wild-type CjPE, and we termed it SsenCjPE. The SsenCjPE can also be combined with hMLH1dn to further increase the editing efficiency and MMLV RTaseΔRnH to reduce size. Finally, we introduced an additional mutation D829R into SsenCjPE and generated SsenCjPE-M2 with a 61-fold increase of PE efficiency over wild-type at the PRNP site. In summary, enCjBEs, SsenCjPEs, or SsenCjPE-M2 are compact Cas9-derived BE or prime editors in biological research or biomedical applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
Humans
Campylobacter jejuni/genetics
Cytosine/metabolism
Genetic Therapy/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Adenine/metabolism
Bacterial Proteins/genetics
Streptococcus pyogenes/genetics/enzymology

@article {pmid39239833,
year = {2024},
author = {Ma, R and Liang, S and Zeng, W and Li, J and Lai, Y and Yang, X and Diao, F},
title = {Single-cell RNA sequencing reveals the important role of Dcaf17 in spermatogenesis of golden hamsters†.},
journal = {Biology of reproduction},
volume = {111},
number = {6},
pages = {1326-1340},
doi = {10.1093/biolre/ioae132},
pmid = {39239833},
issn = {1529-7268},
support = {2021YFC2700605//National Key Research and Development Program of China/ ; 82271638//National Natural Science Foundation of China/ ; 2022YFC2702505//National Key Research and Development Program of China/ ; },
mesh = {Animals ; Male ; *Spermatogenesis/genetics ; *Mesocricetus ; Single-Cell Analysis ; Cricetinae ; Sequence Analysis, RNA ; Infertility, Male/genetics ; CRISPR-Cas Systems ; },
abstract = {Dcaf17, also known as DDB1- and CUL4-associated factor 17, is a member of the DCAF family and acts as the receptor for the CRL4 ubiquitin E3 ligase complex. Several previous studies have reported that mutations in Dcaf17 cause Woodhouse-Sakati syndrome, which results in oligoasthenoteratozoospermia and male infertility. As a model to explore the role of Dcaf17 in the male reproductive system, we created Dcaf17-deficient male golden hamsters using CRISPR-Cas9 technology; the results of which demonstrate that deletion of Dcaf17 led to abnormal spermatogenesis and infertility. To uncover the underlying molecular mechanisms involved, we conducted single cell Ribonucleic Acid sequencing analysis to evaluate the effect of Dcaf17 deficiency on transcriptional levels in spermatogenic cells during various stages of spermatogenesis. These data emphasize the significant regulatory role played by Dcaf17 in early spermatogenic cells, with many biological processes being affected, including spermatogenesis and protein degradation. Dysregulation of genes associated with these functions ultimately leads to abnormalities. In summary, our findings highlight the critical function of Dcaf17 in spermatogenesis and clarify the specific stage at which Dcaf17 exerts its effects, while simultaneously providing a novel animal model for the study of Dcaf17.},
}

Animals
Male
*Spermatogenesis/genetics
*Mesocricetus
Single-Cell Analysis
Cricetinae
Sequence Analysis, RNA
Infertility, Male/genetics
CRISPR-Cas Systems

@article {pmid39218670,
year = {2024},
author = {Lin, Q and Takebayashi, K and Torigoe, N and Liu, B and Namula, Z and Hirata, M and Tanihara, F and Nagahara, M and Otoi, T},
title = {Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.},
journal = {The Journal of reproduction and development},
volume = {70},
number = {6},
pages = {356-361},
doi = {10.1262/jrd.2024-054},
pmid = {39218670},
issn = {1348-4400},
mesh = {Animals ; *Gene Editing/methods ; *Zygote/metabolism ; *CRISPR-Cas Systems ; Swine ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Galactosyltransferases/genetics/metabolism ; Fertilization in Vitro ; Receptors, Somatotropin/genetics/metabolism ; Female ; Mutation ; Transfection ; Lipids/chemistry ; },
abstract = {CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneously double-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.},
}

Animals
*Gene Editing/methods
*Zygote/metabolism
*CRISPR-Cas Systems
Swine
*RNA, Guide, CRISPR-Cas Systems/genetics
Galactosyltransferases/genetics/metabolism
Fertilization in Vitro
Receptors, Somatotropin/genetics/metabolism
Female
Mutation
Transfection
Lipids/chemistry

@article {pmid38165445,
year = {2024},
author = {Iannuzzi, RM and Manipur, I and Pacini, C and Behan, FM and Guarracino, MR and Garnett, MJ and Savino, A and Iorio, F},
title = {Benchmark Software and Data for Evaluating CRISPR-Cas9 Experimental Pipelines Through the Assessment of a Calibration Screen.},
journal = {The CRISPR journal},
volume = {7},
number = {6},
pages = {355-365},
doi = {10.1089/crispr.2023.0040},
pmid = {38165445},
issn = {2573-1602},
mesh = {Humans ; *Software ; *CRISPR-Cas Systems ; *Benchmarking ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Calibration ; HT29 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; Colorectal Neoplasms/genetics ; Gene Editing/methods ; Cell Line, Tumor ; },
abstract = {Genome-wide genetic screens using CRISPR-guide RNA libraries are widely performed in mammalian cells to functionally characterize individual genes and for the discovery of new anticancer therapeutic targets. As the effectiveness of such powerful and precise tools for cancer pharmacogenomics is emerging, tools and methods for their quality assessment are becoming increasingly necessary. Here, we provide an R package and a high-quality reference data set for the assessment of novel experimental pipelines through which a single calibration experiment has been executed: a screen of the HT-29 human colorectal cancer cell line with a commercially available genome-wide library of single-guide RNAs. This package and data allow experimental researchers to benchmark their screens and produce a quality-control report, encompassing several quality and validation metrics. The R code used for processing the reference data set, for its quality assessment, as well as to evaluate the quality of a user-provided screen, and to reproduce the figures presented in this article is available at https://github.com/DepMap-Analytics/HT29benchmark. The reference data is publicly available on FigShare.},
}

Humans
*Software
*CRISPR-Cas Systems
*Benchmarking
*RNA, Guide, CRISPR-Cas Systems/genetics
Calibration
HT29 Cells
Clustered Regularly Interspaced Short Palindromic Repeats
Colorectal Neoplasms/genetics
Gene Editing/methods
Cell Line, Tumor

@article {pmid39656986,
year = {2024},
author = {Jordan, B},
title = {[New-generation editing].},
journal = {Medecine sciences : M/S},
volume = {40},
number = {11},
pages = {869-871},
doi = {10.1051/medsci/2024149},
pmid = {39656986},
issn = {1958-5381},
mesh = {*Gene Editing/methods/trends ; Humans ; CRISPR-Cas Systems ; Animals ; Bacteria/genetics ; },
abstract = {Recent work on bacterial insertion sequences reveals that some of them use an RNA sequence (called Bridge RNA or Seek RNA) to define both donor and target DNA specificity. This opens the way to easy insertion of kilobase DNA sequences at pre-defined sites in the genome, announcing a host of new possibilities. The system still needs a lot of tweaking, as it has only been demonstrated in bacteria, but it holds great promise for genome editing and engineering.},
}

*Gene Editing/methods/trends
Humans
CRISPR-Cas Systems
Animals
Bacteria/genetics

@article {pmid39670384,
year = {2025},
author = {Blennemann, M and Verma, A and Bachl, S and Carnevale, J and Engelhardt, BE},
title = {Understanding TCR T cell knockout behavior using interpretable machine learning.},
journal = {Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing},
volume = {30},
number = {},
pages = {382-393},
pmid = {39670384},
issn = {2335-6936},
mesh = {Humans ; *Receptors, Antigen, T-Cell/immunology/genetics ; *Machine Learning ; *Computational Biology ; *T-Lymphocytes/immunology ; *Neoplasms/immunology/genetics ; Gene Knockout Techniques ; Neural Networks, Computer ; CRISPR-Cas Systems ; },
abstract = {Genetic perturbation of T cell receptor (TCR) T cells is a promising method to unlock better TCR T cell performance to create more powerful cancer immunotherapies, but understanding the changes to T cell behavior induced by genetic perturbations remains a challenge. Prior studies have evaluated the effect of different genetic modifications with cytokine production and metabolic activity assays. Live-cell imaging is an inexpensive and robust approach to capture TCR T cell responses to cancer. Most methods to quantify T cell responses in live-cell imaging data use simple approaches to count T cells and cancer cells across time, effectively quantifying how much space in the 2D well each cell type covers, leaving actionable information unexplored. In this study, we characterize changes in TCR T cell's interactions with cancer cells from live-cell imaging data using explainable artificial intelligence (AI). We train convolutional neural networks to distinguish behaviors in TCR T cell with CRISPR knock outs of CUL5, RASA2, and a safe harbor control knockout. We use explainable AI to identify specific interaction types that define different knock-out conditions. We find that T cell and cancer cell coverage is a strong marker of TCR T cell modification when comparing similar experimental time points, but differences in cell aggregation characterize CUL5KO and RASA2KO behavior across all time points. Our pipeline for discovery in live-cell imaging data can be used for characterizing complex behaviors in arbitrary live-cell imaging datasets, and we describe best practices for this goal.},
}

Humans
*Receptors, Antigen, T-Cell/immunology/genetics
*Machine Learning
*Computational Biology
*T-Lymphocytes/immunology
*Neoplasms/immunology/genetics
Gene Knockout Techniques
Neural Networks, Computer
CRISPR-Cas Systems

@article {pmid39667861,
year = {2025},
author = {Wu, P and Zhang, Y and Shan, Q and Wang, Z and Cheng, S and Wang, L and Liu, B and Li, W and Chen, Z and Luo, J and Liang, Y},
title = {The investigation of the mechanism underlying variations in oxidative stress tolerance of Lacticaseibacillus paracasei resulting from fermentation methods through endogenous CRISPR-Cas9 editing methodology.},
journal = {Food microbiology},
volume = {127},
number = {},
pages = {104697},
doi = {10.1016/j.fm.2024.104697},
pmid = {39667861},
issn = {1095-9998},
mesh = {*Oxidative Stress ; *Fermentation ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Lacticaseibacillus paracasei/genetics/metabolism/physiology/growth & development ; Probiotics ; Bacterial Proteins/genetics/metabolism ; },
abstract = {The probiotic effects of lactic acid bacteria make them widely used in human and animal breeding industry. However, the presence of oxidative stress during the production and application process can cause bacterial damage or even death, significantly compromising the functionality of probiotics. Despite its potential for broader application scenarios that could provide a more comprehensive understanding of bacteria's internal adaptation strategies, there is a lack of research investigating oxidative stress from the perspective of culture methods. In this study, the tolerance to oxidative stress was compared between bacteria cultivated through solid-state fermentation (SSF) and liquid-state fermentation (LSF), and the physiological and transcriptional disparities between these two bacterial strains were investigated. Additionally, a novel and efficient gene editing method was developed to elucidate the genetic basis underlying these differences in tolerance. The results demonstrated a significantly higher tolerance to oxidative stress in SSF bacteria compared to LSF bacteria, along with a stronger capacity for maintaining intracellular microenvironment stability and the activity of key metabolic enzymes. It is noteworthy that the bacteria from SSF significantly enhance the transport of carbohydrate substances and facilitate intracellular metabolic flow. Gene editing experiments have confirmed the crucial role of genes glpF and glpO in regulating the glycerol metabolism pathway, which is essential for enhancing the tolerance of bacteria from SSF to oxidative stress. Based on these findings, the mechanism underlying the disparity in oxidative stress tolerance resulting from different culture methods has been summarized. Furthermore, investigation into different culture modes has revealed that moderate oxygen levels during cultivation significantly influence variation in bacterial tolerance to oxidative stress. Importantly, these variations are species-specific and depend on the ecological niche distribution of Lactobacilli. These findings elucidate a novel mechanism by which Lacticaseibacillus paracasei Zhang tolerates oxidative stress, and also suggest that distinct cultivation and processing methods should be tailored based on the specific Lactobacilli groups to achieve optimal application effects in production.},
}

*Oxidative Stress
*Fermentation
*Gene Editing/methods
*CRISPR-Cas Systems
*Lacticaseibacillus paracasei/genetics/metabolism/physiology/growth & development
Probiotics
Bacterial Proteins/genetics/metabolism

@article {pmid39667860,
year = {2025},
author = {Zhang, X and Chen, W and Wang, C},
title = {Regulation of citrinin biosynthesis in Monascus purpureus: Impacts on growth, morphology, and pigments production.},
journal = {Food microbiology},
volume = {127},
number = {},
pages = {104698},
doi = {10.1016/j.fm.2024.104698},
pmid = {39667860},
issn = {1095-9998},
mesh = {*Monascus/metabolism/growth & development/genetics ; *Citrinin/biosynthesis/metabolism ; *Fungal Proteins/genetics/metabolism ; *Pigments, Biological/biosynthesis/metabolism ; Gene Expression Regulation, Fungal ; Mycelium/growth & development/metabolism/genetics ; CRISPR-Cas Systems ; Hyphae/growth & development/metabolism ; Spores, Fungal/growth & development/metabolism/genetics ; Gene Knockout Techniques ; },
abstract = {Fungal hyphae self-assemble a variety of cellular macrostates, ranging from suspended mycelium to dense pellets, all inextricably linked to their productivity. In this study, using CRISPR/Cas technology, we constructed a ctnA knockout strain (ΔctnA) and an overexpression strain (A2) so as to investigate the effects of interfering with citrinin biosynthesis on the growth, morphology and pigmentation of M.purpureus. Results indicated that deletion of ctnA in M. purpureus RP2 led to increased mycelium length, delayed conidium formation, and a citrinin content of 22% of the wild-type strain. Conversely, ctnA overexpression in strain A2 resulted in delayed mycelial growth, normal conidium formation, and a citrinin content of 120% compared to the wild-type strain, with minimal effects on pigments content. Notably, the ΔctnA strain formed small, tightly structured pellets (mean diameter 1.2 ± 0.06 mm) and exhibited low citrinin content, promoting pigments production. Our findings suggest a complex interplay between citrinin biosynthesis and morphological development, providing insights for optimizing metabolite production in industrial applications.},
}

*Monascus/metabolism/growth & development/genetics
*Citrinin/biosynthesis/metabolism
*Fungal Proteins/genetics/metabolism
*Pigments, Biological/biosynthesis/metabolism
Gene Expression Regulation, Fungal
Mycelium/growth & development/metabolism/genetics
CRISPR-Cas Systems
Hyphae/growth & development/metabolism
Spores, Fungal/growth & development/metabolism/genetics
Gene Knockout Techniques

@article {pmid39667702,
year = {2024},
author = {Chaudhary, N and Sharma, K and Harpreet Kaur, and Prajapati, S and Mohan, B and Taneja, N},
title = {CRISPR-Cas-Assisted Phage Engineering for Personalized Antibacterial Treatments.},
journal = {Indian journal of medical microbiology},
volume = {},
number = {},
pages = {100771},
doi = {10.1016/j.ijmmb.2024.100771},
pmid = {39667702},
issn = {1998-3646},
abstract = {BACKGROUND: In the age of modern medicine, CRISPR-Cas system-aided phage engineering has emerged as a major game changer for developing personalised antibacterial treatments. Modifying genomic DNA at a specific location leads to the inactivation of target genes, the acquisition of novel genetic features, and the correction of lethal gene mutations. Phages can be modified to precisely detect and control bacteria because of the vast possibilities of CRISPR-Cas-based genetic engineering.

OBJECTIVES: The primary objective of this review is to explore the basic principles, mechanisms, limitations, and perspectives of CRISPR-Cas system-aided phage engineering in producing tailored antibacterial therapeutics. Furthermore, this study will address how editing phage genomes using CRISPR-Cas technology allows for precise bacteria targeting, broadening phage host range, and improving infection control tactics.

CONTENT: The arrival of the CRISPR-Cas system has transformed the field of phage engineering and aided in the precise modification of phagе genomes to broaden the phage host range. This novel strategy uses the accuracy of the CRISPR-Cas system to design engineered bacteriophages, giving targeted options for infection control. These recent advancements have the potential to alter the era of modern medicine.},
}

@article {pmid39666857,
year = {2024},
author = {Xie, F and Zhao, H and Liu, J and Yang, X and Neuber, M and Agrawal, AA and Kaur, A and Herrmann, J and Kalinina, OV and Wei, X and Müller, R and Fu, C},
title = {Autologous DNA mobilization and multiplication expedite natural products discovery from bacteria.},
journal = {Science (New York, N.Y.)},
volume = {386},
number = {6727},
pages = {eabq7333},
doi = {10.1126/science.abq7333},
pmid = {39666857},
issn = {1095-9203},
mesh = {*Biological Products/metabolism ; *Streptomyces/genetics/metabolism ; *CRISPR-Cas Systems ; *Multigene Family ; *Plasmids/genetics ; DNA, Bacterial/genetics ; Genome, Bacterial ; Drug Discovery ; Biosynthetic Pathways ; DNA Replication ; Drug Resistance, Bacterial/genetics ; },
abstract = {The transmission of antibiotic-resistance genes, comprising mobilization and relocation events, orchestrates the dissemination of antimicrobial resistance. Inspired by this evolutionarily successful paradigm, we developed ACTIMOT, a CRISPR-Cas9-based approach to unlock the vast chemical diversity concealed within bacterial genomes. ACTIMOT enables the efficient mobilization and relocation of large DNA fragments from the chromosome to replicative plasmids within the same bacterial cell. ACTIMOT circumvents the limitations of traditional molecular cloning methods involving handling and replicating large pieces of genomic DNA. Using ACTIMOT, we mobilized and activated four cryptic biosynthetic gene clusters from Streptomyces, leading to the discovery of 39 compounds across four distinct classes. This work highlights the potential of ACTIMOT for accelerating the exploration of biosynthetic pathways and the discovery of natural products.},
}

*Biological Products/metabolism
*Streptomyces/genetics/metabolism
*CRISPR-Cas Systems
*Multigene Family
*Plasmids/genetics
DNA, Bacterial/genetics
Genome, Bacterial
Drug Discovery
Biosynthetic Pathways
DNA Replication
Drug Resistance, Bacterial/genetics

@article {pmid39666823,
year = {2024},
author = {Li, Y and Li, Z and Yan, P and Hua, C and Kong, J and Wu, W and Cui, Y and Duan, Y and Li, S and Li, G and Ji, S and Chen, Y and Zhao, Y and Yang, P and Hu, C and Lu, M and Chen, M and Xiao, Y},
title = {Antiviral signaling of a type III CRISPR-associated deaminase.},
journal = {Science (New York, N.Y.)},
volume = {},
number = {},
pages = {eadr0393},
doi = {10.1126/science.adr0393},
pmid = {39666823},
issn = {1095-9203},
abstract = {Prokaryotes have evolved diverse defense strategies against viral infection, such as foreign nucleic acid degradation by CRISPR-Cas systems and DNA/RNA synthesis inhibition via nucleotide pool depletion. Here, we report an antiviral mechanism of type III CRISPR-Cas-regulated ATP depletion, where ATP is converted into ITP by CRISPR-Cas-associated adenosine deaminase (CAAD) upon activation by either cA4 or cA6, followed by hydrolysis into IMP by Nudix hydrolase, ultimately resulting in cell growth arrest. The cryo-electron microscopy structures of CAAD in its apo and activated forms, together with biochemical evidence, revealed how cA4/cA6 binds to the CARF domain and abrogates CAAD autoinhibition, inducing substantial conformational changes that reshape the structure of CAAD and induce its deaminase activity. Our results reveal the mechanism of a CRISPR-Cas-regulated ATP depletion antiviral strategy.},
}

@article {pmid39666677,
year = {2024},
author = {Blokhina, Y and Buchwalter, A},
title = {Modeling the consequences of age-linked rDNA hypermethylation with dCas9-directed DNA methylation in human cells.},
journal = {PloS one},
volume = {19},
number = {12},
pages = {e0310626},
pmid = {39666677},
issn = {1932-6203},
mesh = {Humans ; *DNA Methylation ; *DNA, Ribosomal/genetics/metabolism ; *Promoter Regions, Genetic ; *Aging/genetics ; Pol1 Transcription Initiation Complex Proteins/metabolism/genetics ; RNA Polymerase I/metabolism/genetics ; CRISPR-Cas Systems ; Transcription, Genetic ; CRISPR-Associated Protein 9/metabolism/genetics ; Animals ; },
abstract = {Ribosomal DNA (rDNA) genes encode the structural RNAs of the ribosome and are present in hundreds of copies in mammalian genomes. Age-linked DNA hypermethylation throughout the rDNA constitutes a robust "methylation clock" that accurately reports age, yet the consequences of hypermethylation on rDNA function are unknown. We confirmed that pervasive hypermethylation of rDNA occurs during mammalian aging and senescence while rDNA copy number remains stable. We found that DNA methylation is exclusively found on the promoters and gene bodies of inactive rDNA. To model the effects of age-linked methylation on rDNA function, we directed de novo DNA methylation to the rDNA promoter or gene body with a nuclease-dead Cas9 (dCas9)-DNA methyltransferase fusion enzyme in human cells. Hypermethylation at each target site had no detectable effect on rRNA transcription, nucleolar morphology, or cellular growth rate. Instead, human UBF and Pol I remain bound to rDNA promoters in the presence of increased DNA methylation. These data suggest that promoter methylation is not sufficient to impair transcription of the human rDNA and imply that the human rDNA transcription machinery may be resilient to age-linked rDNA hypermethylation.},
}

Humans
*DNA Methylation
*DNA, Ribosomal/genetics/metabolism
*Promoter Regions, Genetic
*Aging/genetics
Pol1 Transcription Initiation Complex Proteins/metabolism/genetics
RNA Polymerase I/metabolism/genetics
CRISPR-Cas Systems
Transcription, Genetic
CRISPR-Associated Protein 9/metabolism/genetics
Animals

@article {pmid39615461,
year = {2024},
author = {Li, CY and Chen, LW and Tsai, MC and Chou, YY and Lin, PX and Chang, YM and Hwu, WL and Chien, YH and Lin, JL and Chen, HA and Lee, NC and Su, PH and Hsieh, TC and Klinkhammer, H and Wang, YC and Huang, YT and Krawitz, PM and Lin, SH and Huang, LLH and Chiang, PM and Shih, MH and Chen, PC},
title = {Homozygous variant in translocase of outer mitochondrial membrane 7 leads to metabolic reprogramming and microcephalic osteodysplastic dwarfism with moyamoya disease.},
journal = {EBioMedicine},
volume = {110},
number = {},
pages = {105476},
doi = {10.1016/j.ebiom.2024.105476},
pmid = {39615461},
issn = {2352-3964},
mesh = {Humans ; Animals ; *Zebrafish ; *Moyamoya Disease/genetics/metabolism/pathology ; *Microcephaly/genetics/metabolism ; Male ; *Homozygote ; Female ; Mitochondrial Precursor Protein Import Complex Proteins ; Proteomics/methods ; Mitochondria/metabolism/genetics ; Induced Pluripotent Stem Cells/metabolism ; Endothelial Cells/metabolism ; Dwarfism/genetics/metabolism/pathology ; Child ; Pedigree ; CRISPR-Cas Systems ; Child, Preschool ; Phenotype ; Disease Models, Animal ; Metabolic Reprogramming ; },
abstract = {BACKGROUND: Impaired mitochondrial protein import machinery leads to phenotypically heterogeneous diseases. Here, we report a recurrent homozygous missense variant in the gene that encodes the translocase of outer mitochondrial membrane 7 (TOMM7) in nine patients with microcephaly, short stature, facial dysmorphia, atrophic macular scarring, and moyamoya disease from seven unrelated families.

METHODS: To prove the causality of the TOMM7 variant, mitochondrial morphology, proteomics, and respiration were investigated in CRISPR/Cas9-edited iPSCs-derived endothelial cells. Cerebrovascular defects and mitochondrial respiration were also examined in CRISPR/Cas9-edited zebrafish.

FINDINGS: iPSC-derived endothelial cells with homozygous TOMM7 p.P29L showed increased TOM7 stability, enlarged mitochondria, increased senescence, and defective tube formation. In addition, proteomic analysis revealed a reduced abundance of mitochondrial proteins involved in ATP synthesis or coordinating TCA cycle and gluconeogenesis. Moreover, mitochondrial respiration was slightly decreased while ATP production from glycolysis was significantly increased. Furthermore, deletion of tomm7 in zebrafish caused craniofacial and cerebrovascular defects that recapitulated human phenotypes. Notably, homozygous iPSCs differentially expressed genes involved in glycolysis and response to hypoxia. Finally, the metabolic imbalance was evidenced by decreased oxygen consumption, increased level of hexokinase 2, and enhanced glycolysis in endothelial cells derived from the patient's iPSCs.

INTERPRETATION: These results revealed the essential role of TOMM7 in balancing cellular sources of energy production at both proteomic and transcriptomic levels and provided the molecular mechanisms through which TOMM7 p.P29L variant leads to an autosomal recessive microcephalic osteodysplastic dwarfism with moyamoya disease.

FUNDING: This work is supported by National Science and Technology Council grants and National Cheng Kung University Hospital.},
}

Humans
Animals
*Zebrafish
*Moyamoya Disease/genetics/metabolism/pathology
*Microcephaly/genetics/metabolism
Male
*Homozygote
Female
Mitochondrial Precursor Protein Import Complex Proteins
Proteomics/methods
Mitochondria/metabolism/genetics
Induced Pluripotent Stem Cells/metabolism
Endothelial Cells/metabolism
Dwarfism/genetics/metabolism/pathology
Child
Pedigree
CRISPR-Cas Systems
Child, Preschool
Phenotype
Disease Models, Animal
Metabolic Reprogramming

@article {pmid39603092,
year = {2024},
author = {Zhang, S and Li, J and Pan, W and Ren, Q and Zhou, Y and Chen, M and Qu, J and Li, S},
title = {Establishment of a CPAMD8-GFP reporter human embryonic stem cell line, IBBDe001-B, using CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {81},
number = {},
pages = {103615},
doi = {10.1016/j.scr.2024.103615},
pmid = {39603092},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Genes, Reporter ; },
abstract = {The human CPAMD8 gene encodes proteins in the A2M/C3 (alpha-2-macroglobulin/complement 3) family, predominantly expressed in the distal tips of the retinal neuroepithelium that form the iris and ciliary body. Mutations in CPAMD8 have been linked to anterior segment dysplasia and congenital glaucoma. Using CRISPR/Cas9 editing, we inserted a 3*EAAAK-EGFP fluorescent tag into the CPAMD8 gene, enabling real-time observation of its expression and providing insights into its biological functions. The resulting gene-edited cell line retained normal stem cell morphology and karyotype, expressed essential pluripotency markers, and exhibited differentiation potential.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Editing/methods
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
Cell Differentiation
Genes, Reporter

@article {pmid39577309,
year = {2024},
author = {Tran, HD and Denman, CR and Shin, MK and Jeon, D and Kuhn, B and Jo, J},
title = {Establishment of TH-EGFP human embryonic stem cell line for specific labeling of dopaminergic neurons.},
journal = {Stem cell research},
volume = {81},
number = {},
pages = {103613},
doi = {10.1016/j.scr.2024.103613},
pmid = {39577309},
issn = {1876-7753},
mesh = {Humans ; *Dopaminergic Neurons/metabolism/cytology ; *Human Embryonic Stem Cells/metabolism/cytology ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Differentiation ; Cell Line ; Tyrosine 3-Monooxygenase/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Dopaminergic (DA) neurons play critical roles in various neurological processes and disorders, particularly Parkinson's disease. To enable precise visualization and tracking of DA neurons, we generated TH-EGFP, a tyrosine hydroxylase (TH)-driven enhanced green fluorescent protein (EGFP)-expressing knock-in cell line, by employing CRISPR/Cas9 technology. We introduced EGFP into the targeted genomic region of human embryonic stem cells (hESCs) and successfully established a TH-EGFP hESC line. Differentiation of TH-EGFP hESCs into human midbrain organoids confirmed the accurate integration of EGFP into TH-positive cells. The TH-EGFP hESC line serves as a valuable reporter for studying the development, maturation, and function of DA neurons.},
}

Humans
*Dopaminergic Neurons/metabolism/cytology
*Human Embryonic Stem Cells/metabolism/cytology
*Green Fluorescent Proteins/metabolism/genetics
Cell Differentiation
Cell Line
Tyrosine 3-Monooxygenase/metabolism/genetics
CRISPR-Cas Systems

@article {pmid39557225,
year = {2025},
author = {Zuo, M and Du, J and Liu, Y and Chen, M and Liu, B and Li, G and Li, M and Huang, S and Yu, G},
title = {Deletion of the gsk-3β (Glycogen synthase kinase-3β) in zebrafish results in decreased susceptibility to Aeromonas hydrophila.},
journal = {Microbial pathogenesis},
volume = {198},
number = {},
pages = {107129},
doi = {10.1016/j.micpath.2024.107129},
pmid = {39557225},
issn = {1096-1208},
mesh = {Animals ; *Aeromonas hydrophila/pathogenicity ; *Zebrafish/microbiology/immunology ; *Glycogen Synthase Kinase 3 beta/genetics/metabolism ; *Gram-Negative Bacterial Infections/immunology/microbiology ; *Fish Diseases/microbiology/immunology ; *Immunity, Innate/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; Autophagy/genetics ; Disease Models, Animal ; Gene Deletion ; Aquaculture ; Zebrafish Proteins/genetics/metabolism ; Disease Susceptibility ; },
abstract = {Aeromonas hydrophila is a significant pathogen in the field of fish farming, resulting in substantial financial losses for the aquaculture industry. As the pathogen's resistance to commercially available antibiotics continues to rise, the identification of novel antimicrobial strategies becomes increasingly crucial. This study aims to explore the modulatory impact of gsk-3β (Glycogen synthase kinase-3β) on the intrinsic immunity against Aeromonas hydrophila in zebrafish, with the objective of uncovering a new avenue for enhancing fish antimicrobial activity through gene editing. Our investigation involved an analysis of the evolutionary patterns and protein sequence of gsk-3β, elucidating its conserved characteristics in zebrafish and fish species of economic importance. In this research, CRISPR-Cas9 technology was employed to generate a zebrafish model with a knockout of gsk-3β, resulting in a decreased resistance of zebrafish to Aeromonas hydrophila (ATCC 7966) infection. Furthermore, we conducted preliminary investigations into the potential mechanisms through which gsk-3β governs antimicrobial immunity. Our findings revealed that knockout of gsk-3β resulted in diminished activation of innate immunity, antioxidant capacity, and autophagy. Hence, the findings of this study are highly significant in improving the economic benefits of aquaculture and in effectively preventing and controlling infection caused by Aeromonas hydrophila.},
}

Animals
*Aeromonas hydrophila/pathogenicity
*Zebrafish/microbiology/immunology
*Glycogen Synthase Kinase 3 beta/genetics/metabolism
*Gram-Negative Bacterial Infections/immunology/microbiology
*Fish Diseases/microbiology/immunology
*Immunity, Innate/genetics
*CRISPR-Cas Systems
*Gene Editing
Gene Knockout Techniques
Autophagy/genetics
Disease Models, Animal
Gene Deletion
Aquaculture
Zebrafish Proteins/genetics/metabolism
Disease Susceptibility

@article {pmid39546944,
year = {2024},
author = {Haferkamp, U and Telugu, N and Krieg, K and Schaefer, W and Lam, D and Binkle-Ladisch, L and Friese, MA and Diecke, S and Pless, O},
title = {Generation of two isogenic human iPSC lines (ZIPi013-B-1, ZIPi013-B-2) carrying a CRISPR/Cas9-mediated deletion of TRPM4.},
journal = {Stem cell research},
volume = {81},
number = {},
pages = {103609},
doi = {10.1016/j.scr.2024.103609},
pmid = {39546944},
issn = {1876-7753},
mesh = {Humans ; *TRPM Cation Channels/metabolism/genetics ; *CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells/metabolism ; Cell Line ; Gene Deletion ; },
abstract = {Two isogenic hiPSC lines, ZIPi013-B-1 and ZIPi013-B-2, were generated by CRISPR/Cas9-mediated indels in the TRPM4 gene of the previously published ZIPi013-B. TRPM4 belongs to the evolutionarily conserved family of transient receptor potential (TRP) channels. It is expressed ubiquitously and its activity is regulated by intracellular calcium binding, changes in membrane potential, phosphoinositide lipids in the plasma membrane and the local concentration of cytoplasmic ATP and ADP. TRPM4 has been implicated in various diseases, including neurological and immune system disorders, cardiac diseases and cancer. Both new cell lines offer the opportunity to model human diseases and test therapeutic modalities addressing these.},
}

Humans
*TRPM Cation Channels/metabolism/genetics
*CRISPR-Cas Systems
*Induced Pluripotent Stem Cells/metabolism
Cell Line
Gene Deletion

@article {pmid39541768,
year = {2024},
author = {Jiang, X and Liu, Q and Yang, L and Zhang, X and Gao, J and Jiang, Y},
title = {Generation of a MYH6 (c.4034T > C) mutant human embryonic stem cell line via CRISPR base editing.},
journal = {Stem cell research},
volume = {81},
number = {},
pages = {103610},
doi = {10.1016/j.scr.2024.103610},
pmid = {39541768},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Myosin Heavy Chains/genetics/metabolism ; *Gene Editing ; *Cardiac Myosins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Mutation ; Cell Differentiation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The MYH6 gene encodes α-myosin heavy chain in the adult human heart. MYH6 c.4034T > C (p.Leu1345Pro) mutation in MYH6 gene have been reported in patients with hypertrophic cardiomyopathy (HCM), but its causal role in HCM is less certain and has not been established unambiguously. Here, we generated a MYH6 (c.4034T > C) mutant human embryonic stem cell line (WAe009-A-1D) based on the CRISPR adenine base editing system that converts base A/T to G/C. The WAe009-A-1D cell maintains the morphology, pluripotency, and normal karyotype of the stem cells and is capable of differentiating into all three germ layers in vivo.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*Myosin Heavy Chains/genetics/metabolism
*Gene Editing
*Cardiac Myosins/genetics/metabolism
CRISPR-Cas Systems
Cell Line
Mutation
Cell Differentiation
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39490211,
year = {2024},
author = {Tanzer, K and Meier, B and Vulinovic, F and Pawlack, H and Klein, C and Seibler, P and Rakovic, A},
title = {Generation of four human-derived iPSC TorsinA-3xFLAG reporter lines from a DYT-TOR1A patient.},
journal = {Stem cell research},
volume = {81},
number = {},
pages = {103595},
doi = {10.1016/j.scr.2024.103595},
pmid = {39490211},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Molecular Chaperones/metabolism/genetics ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; Dystonia/genetics/metabolism ; },
abstract = {A 3-bp deletion (ΔGAG) in TOR1A is a common cause of early-onset isolated dystonia DYT-TOR1A. The exact disease mechanism remains unknown. Here we describe the generation and characterization of four TorsinA-3xFLAG reporter induced pluripotent cell (iPSC) lines derived from a DYT-TOR1A patient. The cell lines carry either a ΔGAG variant or a corrected allele and a mono- or biallelic 3xFLAG-Tag introduced using CRISPR/Cas9 technology. These cells provide an opportunity to study differential protein stability, subcellular localization, and interactors of endogenous WT or ΔE variants of TorsinA in iPSCs, neural progenitor cells (smNPC), and neurons.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Molecular Chaperones/metabolism/genetics
Cell Line
CRISPR-Cas Systems
Cell Differentiation
Dystonia/genetics/metabolism

@article {pmid39490210,
year = {2024},
author = {Shan, W and Cui, X and Wang, D and Wang, B and Guo, X and Wang, X and Wang, J and Li, Y and An, G and Ren, Q},
title = {Generation of heterozygous KCNA2 knockout induced pluripotent stem cell line.},
journal = {Stem cell research},
volume = {81},
number = {},
pages = {103596},
doi = {10.1016/j.scr.2024.103596},
pmid = {39490210},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism ; Humans ; Kv1.2 Potassium Channel/metabolism/genetics ; Heterozygote ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {The KCNA2 gene is the voltage-gated ion channel from both functional and structural perspectives. KCNA2 is involved in diverse functions including the regulation of neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. To investigate the relevant pathophysiological mechanisms, we generated heterozygous KCNA2 knockout human induced pluripotent stem cell (iPSC) line via CRISPR/Cas9 gene editing. The generated iPSCs had a normal karyotype, were free of genetically integrated epitaxial plasmids, expressed pluripotency markers, and maintained trilineage differentiation potential.},
}

*Induced Pluripotent Stem Cells/metabolism
Humans
Kv1.2 Potassium Channel/metabolism/genetics
Heterozygote
Cell Line
Cell Differentiation
CRISPR-Cas Systems
Gene Knockout Techniques
Gene Editing

@article {pmid39476617,
year = {2024},
author = {Sun, H and Li, Q and Xu, T and Zhang, W and Sun, J and Liu, H},
title = {Generation of SFTPC-mCherry knock-in reporter human embryonic stem cell line, WAe001-A-2H, using CRISPR/Cas9-based gene targeting.},
journal = {Stem cell research},
volume = {81},
number = {},
pages = {103597},
doi = {10.1016/j.scr.2024.103597},
pmid = {39476617},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Pulmonary Surfactant-Associated Protein C/metabolism/genetics ; *Gene Knock-In Techniques/methods ; Cell Line ; Gene Targeting/methods ; Genes, Reporter ; Gene Editing/methods ; },
abstract = {The SFTPC gene is responsible for the production of the pulmonary surfactant protein C (SPC), a highly hydrophobic molecule that plays a crucial role in maintaining lung integrity through its influence on the synthesis of alveolar surfactant proteins. In this study, we harnessed the CRISPR/Cas9 system for precise genome editing to create a modified H1 human embryonic stem cell (hESC) line, incorporating the SFTPC-mCherry reporter construct. Therefore, the engineered SFTPC-mCherry knock-in (KI) hESC line can serve as an effective tool for tracking the expression patterns of the SFTPC gene as alveolar type 2 cells differentiate from hESCs.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
*Pulmonary Surfactant-Associated Protein C/metabolism/genetics
*Gene Knock-In Techniques/methods
Cell Line
Gene Targeting/methods
Genes, Reporter
Gene Editing/methods

@article {pmid39476616,
year = {2024},
author = {Srisantitham, J and Suwanpitak, S and Thongsin, N and Wattanapanitch, M},
title = {Generation of a homozygous TIGIT gene knockout (TIGIT[-/-]) human iPSC line (MUSIi001-A-3) using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {81},
number = {},
pages = {103601},
doi = {10.1016/j.scr.2024.103601},
pmid = {39476616},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *CRISPR-Cas Systems ; *Receptors, Immunologic/genetics/metabolism ; *Gene Knockout Techniques/methods ; Homozygote ; Cell Line ; },
abstract = {Adoptive cell therapy for solid cancers involves enhancing and reinfusing immune cells to target tumor cells. The advancement of induced pluripotent stem cell technology enables the generation of immune cell products like T and NK cells for ACT. However, the expression of inhibitory receptors, such as TIGIT, may limit the functionality of these immune effector cells. In this study, we generated a homozygousTIGITgene knockout iPSC line to potentially prevent inhibitory signaling and exhaustion, thereby creating potent "off-the-shelf" immune cell products for cellular immunotherapy applications. This approach could offer a new frontier in the fight against solid tumors.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*CRISPR-Cas Systems
*Receptors, Immunologic/genetics/metabolism
*Gene Knockout Techniques/methods
Homozygote
Cell Line

@article {pmid39476461,
year = {2024},
author = {Zhu, H and Zhu, D and Li, Y and Li, Y and Song, X and Mo, J and Liu, L and Liu, Z and Wang, S and Yao, Y and Yan, H and Wu, K and Wang, W and Yin, J and Lin, M and Li, J},
title = {Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {26},
number = {},
pages = {100568},
pmid = {39476461},
issn = {2211-3207},
mesh = {*Plasmodium falciparum/genetics/drug effects ; *Drug Resistance/genetics ; *Antimalarials/pharmacology ; *CRISPR-Cas Systems ; *Quinolines/pharmacology ; *Polymorphism, Single Nucleotide ; *Malaria, Falciparum/parasitology/diagnosis ; *Polymerase Chain Reaction/methods ; Humans ; Mutation ; Exonucleases/genetics/metabolism ; Alleles ; Genotype ; Piperazines ; },
abstract = {Malaria remains a major public health concern. The rapid spread of resistance to antimalarial drugs is a major challenge for malaria eradication. Timely and accurate molecular monitoring based on practical detection methods is a critical step toward malaria control and elimination. In this study, two rapid detection techniques, allele-specific PCR (AS‒PCR) and recombinase-aided amplification (RAA) combined with CRISPR/Cas12a, were established, optimized and assessed to detect single nucleotide polymorphisms in the Plasmodium falciparum exonuclease (Pfexo) gene related to suspected piperaquine resistance. Moreover, phosphorothioate and artificial mismatches were introduced into the allele-specific primers for AS‒PCR, and crRNA-mismatched bases were introduced into the RAA‒CRISPR/Cas12a assay because crRNAs designed according to conventional rules fail to discriminate genotypes. As a result, the detection limits of the AS‒PCR and RAA‒CRISPR/Cas12a assays were 10[4] copies/μL and 10[3] copies/μL, respectively. The detection threshold for dried blood spots was 100‒150 parasites/μL, with no cross-reactivity against other genotypes. The average cost of AS‒PCR is approximately $1 per test and takes 2-3 h, whereas that of the RAA‒CRISPR/Cas12a system is approximately $7 per test and takes 1 h or less. Therefore, we provide more options for testing single nucleotide polymorphisms in the Pfexo gene, considering economic conditions and the availability of instruments, equipment, and reagents, which can contribute to the molecular monitoring of antimalarial resistance.},
}

*Plasmodium falciparum/genetics/drug effects
*Drug Resistance/genetics
*Antimalarials/pharmacology
*CRISPR-Cas Systems
*Quinolines/pharmacology
*Polymorphism, Single Nucleotide
*Malaria, Falciparum/parasitology/diagnosis
*Polymerase Chain Reaction/methods
Humans
Mutation
Exonucleases/genetics/metabolism
Alleles
Genotype
Piperazines

@article {pmid39437564,
year = {2024},
author = {Zhang, C and Li, J and Sai, Y and Su, H and Jiang, Y and Zhang, L and Jian, L and Zhang, H and Guo, G and Li, E and Li, X and Sun, L},
title = {Establishment of heterozygous LMOD2 knockout human embryonic stem cell line (ZZUNEUi022-A-1) using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {81},
number = {},
pages = {103586},
doi = {10.1016/j.scr.2024.103586},
pmid = {39437564},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; Cardiomyopathy, Dilated/genetics/pathology ; Membrane Proteins/genetics/metabolism ; Cell Differentiation ; Gene Knockout Techniques ; Heterozygote ; Muscle Proteins/genetics/metabolism ; },
abstract = {Dilated Cardiomyopathy (DCM), a prevalent form of cardiomyopathy, is characterized by ventricular dilation and systolic dysfunction. Its etiology is intricate, encompassing multiple genetic and environmental elements. The LMOD2 (Leiomodin 2) gene has been demonstrated to be closely associated with the pathogenesis of DCM. In this study, a pure cell line was generated by knocking out the LMOD2 gene, and a DCM cell model was established through induced differentiation, thus providing a powerful experimental approach for further understanding the pathogenesis of DCM. It also provides a potential research orientation for the early diagnosis and individualized treatment of DCM.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
Cell Line
Cardiomyopathy, Dilated/genetics/pathology
Membrane Proteins/genetics/metabolism
Cell Differentiation
Gene Knockout Techniques
Heterozygote
Muscle Proteins/genetics/metabolism