@article {pmid39056796,
year = {2024},
author = {Choi, W and Cha, S and Kim, K},
title = {Navigating the CRISPR/Cas Landscape for Enhanced Diagnosis and Treatment of Wilson's Disease.},
journal = {Cells},
volume = {13},
number = {14},
pages = {},
doi = {10.3390/cells13141214},
pmid = {39056796},
issn = {2073-4409},
support = {NRF- 2023R1A2C2004222, RS-2023-00220894, and RS-2023-00261905//The Bio & Medical Technology Development Program of the National Research Foundation (NRF) of Korea/ ; },
mesh = {*Hepatolenticular Degeneration/genetics/therapy/diagnosis ; Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Gene Editing/methods ; Copper-Transporting ATPases/genetics/metabolism ; Animals ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system continues to evolve, thereby enabling more precise detection and repair of mutagenesis. The development of CRISPR/Cas-based diagnosis holds promise for high-throughput, cost-effective, and portable nucleic acid screening and genetic disease diagnosis. In addition, advancements in transportation strategies such as adeno-associated virus (AAV), lentiviral vectors, nanoparticles, and virus-like vectors (VLPs) offer synergistic insights for gene therapeutics in vivo. Wilson's disease (WD), a copper metabolism disorder, is primarily caused by mutations in the ATPase copper transporting beta (ATP7B) gene. The condition is associated with the accumulation of copper in the body, leading to irreversible damage to various organs, including the liver, nervous system, kidneys, and eyes. However, the heterogeneous nature and individualized presentation of physical and neurological symptoms in WD patients pose significant challenges to accurate diagnosis. Furthermore, patients must consume copper-chelating medication throughout their lifetime. Herein, we provide a detailed description of WD and review the application of novel CRISPR-based strategies for its diagnosis and treatment, along with the challenges that need to be overcome.},
}

*Hepatolenticular Degeneration/genetics/therapy/diagnosis
Humans
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
Gene Editing/methods
Copper-Transporting ATPases/genetics/metabolism
Animals

@article {pmid39056772,
year = {2024},
author = {Bailey, JK and Ma, D and Clegg, DO},
title = {Initial Characterization of WDR5B Reveals a Role in the Proliferation of Retinal Pigment Epithelial Cells.},
journal = {Cells},
volume = {13},
number = {14},
pages = {},
doi = {10.3390/cells13141189},
pmid = {39056772},
issn = {2073-4409},
support = {N/A//Garland Initiative for Vision/ ; DR1-01444//California Institute for Regenerative Medicine/ ; FA1-00616//California Institute for Regenerative Medicine/ ; TG2-01151//California Institute for Regenerative Medicine/ ; CL1-00521//California Institute for Regenerative Medicine/ ; N/A//Breaux Foundation/ ; Wynn-Gund Translational Research Acceleration Program/FFB/Foundation Fighting Blindness/United States ; },
mesh = {Humans ; *Retinal Pigment Epithelium/metabolism/cytology ; *Cell Proliferation ; CRISPR-Cas Systems/genetics ; Epithelial Cells/metabolism ; Cell Differentiation ; Intracellular Signaling Peptides and Proteins/metabolism/genetics ; Cell Line ; Animals ; Gene Editing ; },
abstract = {The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer. In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored. Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization. In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation. Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures. CUT&RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites. These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.},
}

Humans
*Retinal Pigment Epithelium/metabolism/cytology
*Cell Proliferation
CRISPR-Cas Systems/genetics
Epithelial Cells/metabolism
Cell Differentiation
Intracellular Signaling Peptides and Proteins/metabolism/genetics
Cell Line
Animals
Gene Editing

@article {pmid39056771,
year = {2024},
author = {Na, DH and Cui, S and Fang, X and Lee, H and Eum, SH and Shin, YJ and Lim, SW and Yang, CW and Chung, BH},
title = {Advancements in Research on Genetic Kidney Diseases Using Human-Induced Pluripotent Stem Cell-Derived Kidney Organoids.},
journal = {Cells},
volume = {13},
number = {14},
pages = {},
doi = {10.3390/cells13141190},
pmid = {39056771},
issn = {2073-4409},
mesh = {Humans ; *Organoids/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; *Kidney Diseases/genetics/pathology ; *Kidney/pathology ; Animals ; CRISPR-Cas Systems/genetics ; },
abstract = {Genetic or hereditary kidney disease stands as a pivotal cause of chronic kidney disease (CKD). The proliferation and widespread utilization of DNA testing in clinical settings have notably eased the diagnosis of genetic kidney diseases, which were once elusive but are now increasingly identified in cases previously deemed CKD of unknown etiology. However, despite these diagnostic strides, research into disease pathogenesis and novel drug development faces significant hurdles, chiefly due to the dearth of appropriate animal models and the challenges posed by limited patient cohorts in clinical studies. Conversely, the advent and utilization of human-induced pluripotent stem cells (hiPSCs) offer a promising avenue for genetic kidney disease research. Particularly, the development of hiPSC-derived kidney organoid systems presents a novel platform for investigating various forms of genetic kidney diseases. Moreover, the integration of the CRISPR/Cas9 technique into this system holds immense potential for efficient research on genetic kidney diseases. This review aims to explore the applications of in vitro kidney organoids generated from hiPSCs in the study of diverse genetic kidney diseases. Additionally, it will delve into the limitations of this research platform and outline future perspectives for advancing research in this crucial area.},
}

Humans
*Organoids/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism
*Kidney Diseases/genetics/pathology
*Kidney/pathology
Animals
CRISPR-Cas Systems/genetics

@article {pmid39056004,
year = {2024},
author = {Ghaffarian, S and Panahi, B},
title = {Occurrence and diversity pattern of CRISPR-Cas systems in Acetobacter genus provides insights on adaptive defense mechanisms against to invasive DNAs.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1357156},
doi = {10.3389/fmicb.2024.1357156},
pmid = {39056004},
issn = {1664-302X},
abstract = {The Acetobacter genus is primarily known for its significance in acetic acid production and its application in various industrial processes. This study aimed to shed light on the prevalence, diversity, and functional implications of CRISPR-Cas systems in the Acetobacter genus using a genome mining approach. The investigation analyzed the CRISPR-Cas architectures and components of 34 Acetobacter species, as well as the evolutionary strategies employed by these bacteria in response to phage invasion and foreign DNA. Furthermore, phylogenetic analysis based on CAS1 protein sequences was performed to gain insights into the evolutionary relationships among Acetobacter strains, with an emphasis on the potential of this protein for genotyping purposes. The results showed that 15 species had orphan, while20 species had complete CRISPR-Cas systems, resulting in an occurrence rate of 38% for complete systems in Acetobacter strains. The predicted complete CRISPR-Cas systems were categorized into I-C, I-F, I-E, and II-C subtypes, with subtype I-E being the most prevalent in Acetobacter. Additionally, spacer homology analysis revealed against such the dynamic interaction between Acetobacter strains and foreign invasive DNAs, emphasizing the pivotal role of CRISPR-Cas systems in defending against such invasions. Furthermore, the investigation of the secondary structures of CRISPR arrays revealed the conserved patterns within subtypes despite variations in repeat sequences. The exploration of protospacer adjacent motifs (PAMs) identified distinct recognition motifs in the flanking regions of protospacers. In conclusion, this research not only contributes to the growing body of knowledge on CRISPR-Cas systems but also establishes a foundation for future studies on the adaptive defense mechanisms of Acetobacter. The findings provide valuable insights into the intricate interplay between bacteria and phages, with implications for industrial applications and potential biotechnological advancements.},
}

@article {pmid39055984,
year = {2024},
author = {Kumar Sachan, RS and Choudhary, A and Devgon, I and Karnwal, A and Al-Tawaha, ARMS and Malik, T},
title = {Bibliometric analysis on CRISPR/Cas: a potential Sherlock Holmes for disease detection.},
journal = {Frontiers in molecular biosciences},
volume = {11},
number = {},
pages = {1383268},
doi = {10.3389/fmolb.2024.1383268},
pmid = {39055984},
issn = {2296-889X},
abstract = {CRISPR has revolutionized illness detection by using precision gene editing to identify specific sequences in recent years. Using the Scopus database, this study performs a comprehensive bibliometric analysis, looking at academic papers on CRISPR that were published between 1992 and 2023. After screening a dataset of 1407 articles using Zotero, trends in annual publishing, citation patterns, author affiliations, and keyword co-occurrence are revealed using analysis tools such as VOSviewer, RStudio, and MS Excel. According to the report, there was only one CRISPR publication in 1992. By 2017, there were a meager 64 papers. Nonetheless, there is a notable upsurge between 2018 and 2023. Leading nations involved in CRISPR-based illness detection research include Germany, the United States, China, India, and the United Kingdom. Chongqing University Three Gorges Hospital, Chongqing University Medical University, and Chongqing University Bioengineering College are a few of the top institutions. With the greatest publication numbers (1688 and 1616) and strong total link strengths (TLS) of 42 and 77, respectively, authors Liu, C., and Li, Y., stand out. The field with the greatest citation counts as of 2023 is Broughton's 2020 study on CRISPR-based SARS-CoV-2 detection in Nature Biotechnology, with 1598 citations. Biosensors and Bioelectronics comprise 14.99% of papers. Researchers, decision-makers, and interested parties can use this thorough summary to help them make well-informed decisions about future CRISPR-based disease detection studies.},
}

@article {pmid39054353,
year = {2024},
author = {Lee, CY and Kim, H and Degani, I and Lee, H and Sandoval, A and Nam, Y and Pascavis, M and Park, HG and Randall, T and Ly, A and Castro, CM and Lee, H},
title = {Empowering the on-site detection of nucleic acids by integrating CRISPR and digital signal processing.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {6271},
pmid = {39054353},
issn = {2041-1723},
support = {5R25CA174650//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Uterine Cervical Neoplasms/diagnosis/virology/genetics ; Female ; *Papillomavirus Infections/diagnosis/virology ; CRISPR-Cas Systems/genetics ; DNA, Viral/genetics ; Papillomaviridae/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Signal Processing, Computer-Assisted ; Cervix Uteri/virology ; },
abstract = {Addressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer, where molecular detection of human papillomavirus (HPV) has emerged as an accurate screening method. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests. Here, we present CreDiT (CRISPR Enhanced Digital Testing) for on-site NA detection. The CreDiT platform integrates i) a one-pot CRISPR strategy that simultaneously amplifies both target NAs and analytical signals and ii) a robust fluorescent detection based on digital communication (encoding/decoding) technology. These features enable a rapid assay (<35 minutes) in a single streamlined workflow. We demonstrate the sensitive detection of cell-derived HPV DNA targets down to single copies and accurate identification of HPV types in clinical cervical brushing specimens (n = 121).},
}

Humans
*Uterine Cervical Neoplasms/diagnosis/virology/genetics
Female
*Papillomavirus Infections/diagnosis/virology
CRISPR-Cas Systems/genetics
DNA, Viral/genetics
Papillomaviridae/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Signal Processing, Computer-Assisted
Cervix Uteri/virology

@article {pmid39052485,
year = {2024},
author = {Choi, H and Shin, H and Kim, CY and Park, J and Kim, H},
title = {Highly efficient CRISPR/Cas9-RNP mediated CaPAD1 editing in protoplasts of three pepper (Capsicum annuum L.) cultivars.},
journal = {Plant signaling & behavior},
volume = {19},
number = {1},
pages = {2383822},
doi = {10.1080/15592324.2024.2383822},
pmid = {39052485},
issn = {1559-2324},
mesh = {*Capsicum/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Protoplasts/metabolism ; *Ribonucleoproteins/metabolism/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Parthenocarpy, characterized by seedless fruit development without pollination or fertilization, offers the advantage of consistent fruit formation, even under challenging conditions such as high temperatures. It can be induced by regulating auxin homeostasis; PAD1 (PARENTAL ADVICE-1) is an inducer of parthenocarpy in Solanaceae plants. However, precise editing of PAD1 is not well studied in peppers. Here, we report a highly efficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) for CaPAD1 editing in three valuable cultivars of pepper (Capsicum annuum L.): Dempsey, a gene-editable bell pepper; C15, a transformable commercial inbred line; and Younggo 4, a Korean landrace. To achieve the seedless pepper trait under high temperatures caused by unstable climate change, we designed five single guide RNAs (sgRNAs) targeting the CaPAD1 gene. We evaluated the in vitro on-target activity of the RNP complexes in three cultivars. Subsequently, we introduced five CRISPR/Cas9-RNP complexes into protoplasts isolated from three pepper leaves and compared indel frequencies and patterns through targeted deep sequencing analyses. We selected two sgRNAs, sgRNA2 and sgRNA5, which had high in vivo target efficiencies for the CaPAD1 gene across the three cultivars and were validated as potential off-targets in their genomes. These findings are expected to be valuable tools for developing new seedless pepper cultivars through precise molecular breeding of recalcitrant crops in response to climate change.},
}

*Capsicum/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Protoplasts/metabolism
*Ribonucleoproteins/metabolism/genetics
Plant Proteins/genetics/metabolism

@article {pmid39051216,
year = {2024},
author = {Krishna, L and Prashant, A and Kumar, YH and Paneyala, S and Patil, SJ and Ramachandra, SC and Vishwanath, P},
title = {Molecular and Biochemical Therapeutic Strategies for Duchenne Muscular Dystrophy.},
journal = {Neurology international},
volume = {16},
number = {4},
pages = {731-760},
doi = {10.3390/neurolint16040055},
pmid = {39051216},
issn = {2035-8385},
abstract = {Significant progress has been achieved in understanding Duchenne muscular dystrophy (DMD) mechanisms and developing treatments to slow disease progression. This review article thoroughly assesses primary and secondary DMD therapies, focusing on innovative modalities. The primary therapy addresses the genetic abnormality causing DMD, specifically the absence or reduced expression of dystrophin. Gene replacement therapies, such as exon skipping, readthrough, and gene editing technologies, show promise in restoring dystrophin expression. Adeno-associated viruses (AAVs), a recent advancement in viral vector-based gene therapies, have shown encouraging results in preclinical and clinical studies. Secondary therapies aim to maintain muscle function and improve quality of life by mitigating DMD symptoms and complications. Glucocorticoid drugs like prednisone and deflazacort have proven effective in slowing disease progression and delaying loss of ambulation. Supportive treatments targeting calcium dysregulation, histone deacetylase, and redox imbalance are also crucial for preserving overall health and function. Additionally, the review includes a detailed table of ongoing and approved clinical trials for DMD, exploring various therapeutic approaches such as gene therapies, exon skipping drugs, utrophin modulators, anti-inflammatory agents, and novel compounds. This highlights the dynamic research field and ongoing efforts to develop effective DMD treatments.},
}

@article {pmid39051081,
year = {2024},
author = {Zheng, M and Liu, Y and Liu, J and Kang, Q and Wang, T},
title = {[Effect of deletion of protein 4.1R on proliferation, apoptosis and glycolysis of hepatocyte HL-7702 cells].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {44},
number = {7},
pages = {1355-1360},
doi = {10.12122/j.issn.1673-4254.2024.07.15},
pmid = {39051081},
issn = {1673-4254},
mesh = {*Apoptosis ; *Glycolysis ; *Cell Proliferation ; Humans ; *Hepatocytes/metabolism/cytology ; Cell Line ; AMP-Activated Protein Kinases/metabolism/genetics ; CRISPR-Cas Systems ; Glucose/metabolism ; Signal Transduction ; },
abstract = {OBJECTIVE: To explore the effects of deletion of protein 4.1R on hepatocyte proliferation, apoptosis, and glycolysis and the molecular mechanisms.

METHODS: A 4.1R[-/-] HL-7702 cell line was constructed using CRISPR/Cas9 technique, and with 4.1R[+/+]HL-7702 cells as the control, its proliferative capacity and cell apoptosis were assessed using CCK-8 assay, EdU-488 staining, flow cytometry and Annexin V-FITC/PI staining at 24, 48, 72 h of cell culture. The changes in glucose uptake, lactate secretion, ATP production and pH value of the culture supernatant of 4.1R[-/-] HL-7702 cells were determined. The mRNA expressions of the key regulatory enzymes HK2, PFKL, PKM2 and LDHA in glycolysis were detected with qRT-PCR, and the protein expressions of AMPK, p-AMPK, Raptor and p-Raptor were determined using Western blotting.

RESULTS: Western blotting and sequencing analysis both confirmed the successful construction of 4.1R[-/-] HL-7702 cell line. Compared with the wild-type cells, 4.1R[-/-] HL-7702 cells exhibited a lowered proliferative activity with increased cell apoptosis. The deletion of protein 4.1R also resulted in significantly decreased glucose uptake, lactate secretion and ATP production of the cells and increased pH value of the cell culture supernatant. qRT-PCR showed significantly decreased mRNA expressions of the key regulatory enzymes in glycolysis in 4.1R[-/-] HL-7702 cells. Compared with those in HL-7702 cells, the expression levels of AMPK and Raptor proteins were decreased while the expression levels of p-AMPK and p-Raptor proteins increased significantly in 4.1R[-/-] HL-7702 cells.

CONCLUSION: Deletion of protein 4.1R in HL-7702 cells results in reduced proliferative capacity, increased apoptosis and suppression of glycolysis, and this regulatory mechanism is closely related with the activation of the downstream AMPK-mTORC1 signaling pathway.},
}

*Apoptosis
*Glycolysis
*Cell Proliferation
Humans
*Hepatocytes/metabolism/cytology
Cell Line
AMP-Activated Protein Kinases/metabolism/genetics
CRISPR-Cas Systems
Glucose/metabolism
Signal Transduction

@article {pmid39049493,
year = {2024},
author = {Yao, Q and Shen, R and Shao, Y and Tian, Y and Han, P and Zhang, X and Zhu, JK and Lu, Y},
title = {Efficient and multiplex gene upregulation in plants through CRISPR/Cas-mediated knock-in of enhancers.},
journal = {Molecular plant},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molp.2024.07.009},
pmid = {39049493},
issn = {1752-9867},
abstract = {Gene upregulation through genome editing is important for plant research and breeding. Targeted insertion of short transcriptional enhancers (STEs) into gene promoters may offer a universal solution akin to transgene-mediated overexpression, while avoiding the drawbacks associated with transgenesis. Here, we introduce an "in-locus activation" technique in rice that leverages specifically screened STEs for refined, heritable, and multiplexed gene upregulation. To address the scarcity of potent enhancers, we developed a large-scale mining approach and discovered a suite of STEs capable of enhancing gene expression in rice protoplasts. The in-locus integration of these STEs into eight rice genes resulted in substantial transcriptional enhancements, with up to 869.1-fold increases in the edited plants. Employing a variety of STEs, we achieved delicate control of gene expression, enabling the fine-tuning of key phenotypic traits such as plant height. Our approach also enabled efficient multiplexed gene upregulation, with up to four genes simultaneously activated, significantly enhancing the nicotinamide mononucleotide (NMN) metabolic pathway. Importantly, heritability studies from the T0 to T3 generations confirmed the stable and heritable nature of STE-driven gene activation. Coupled with our STE-mining technique, in-locus activation holds great promise to make gene upregulation a major application of genome editing in plant research and breeding.},
}

@article {pmid39048937,
year = {2024},
author = {Aghaali, Z and Naghavi, MR},
title = {Developing benzylisoquinoline alkaloid-enriched opium poppy via CRISPR-directed genome editing: A review.},
journal = {BMC plant biology},
volume = {24},
number = {1},
pages = {700},
pmid = {39048937},
issn = {1471-2229},
mesh = {*Papaver/genetics/metabolism ; *Benzylisoquinolines/metabolism ; *Gene Editing ; *CRISPR-Cas Systems ; Metabolic Engineering/methods ; Genome, Plant ; },
abstract = {Among plant-derived secondary metabolites are benzylisoquinoline alkaloids (BIAs) that play a vital role in medicine. The most conspicuous BIAs frequently found in opium poppy are morphine, codeine, thebaine, papaverine, sanguinarine, and noscapine. BIAs have provided abundant clinically useful drugs used in the treatment of various diseases and ailments With an increasing demand for these herbal remedies, genetic improvement of poppy plants appears to be essential to live up to the expectations of the pharmaceutical industry. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9), the field of metabolic engineering has undergone a paradigm shift in its approach due to its appealing attributes, such as the transgene-free editing capability, precision, selectivity, robustness, and versatility. The potentiality of the CRISPR system for manipulating metabolic pathways in opium poppy was demonstrated, but further investigations regarding the use of CRISPR in BIA pathway engineering should be undertaken to develop opium poppy into a bioreactor synthesizing BIAs at the industrial-scale levels. In this regard, the recruitment of RNA-guided genome editing for knocking out miRNAs, flower responsible genes, genes involved in competitive pathways, and base editing are described. The approaches presented here have never been suggested or applied in opium poppy so far.},
}

*Papaver/genetics/metabolism
*Benzylisoquinolines/metabolism
*Gene Editing
*CRISPR-Cas Systems
Metabolic Engineering/methods
Genome, Plant

@article {pmid39048715,
year = {2024},
author = {Cao, Y and Li, X and Pan, Y and Wang, H and Yang, S and Hong, L and Ye, L},
title = {CRISPR-based genetic screens advance cancer immunology.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {39048715},
issn = {1869-1889},
abstract = {CRISPR technologies have revolutionized research areas ranging from fundamental science to translational medicine. CRISPR-based genetic screens offer a powerful platform for unbiased screening in various fields, such as cancer immunology. Immune checkpoint blockade (ICB) therapy has been shown to strongly affect cancer treatment. However, the currently available ICBs are limited and do not work in all cancer patients. Pooled CRISPR screens enable the identification of previously unknown immune regulators that can regulate T-cell activation, cytotoxicity, persistence, infiltration into tumors, cytokine secretion, memory formation, T-cell metabolism, and CD4[+] T-cell differentiation. These novel targets can be developed as new immunotherapies or used with the current ICBs as new combination therapies that may yield synergistic efficacy. Here, we review the progress made in the development of CRISPR technologies, particularly technological advances in CRISPR screens and their application in novel target identification for immunotherapy.},
}

@article {pmid39048567,
year = {2024},
author = {Hatanaka, F and Suzuki, K and Shojima, K and Yu, J and Takahashi, Y and Sakamoto, A and Prieto, J and Shokhirev, M and Nuñez Delicado, E and Rodriguez Esteban, C and Izpisua Belmonte, JC},
title = {Therapeutic strategy for spinal muscular atrophy by combining gene supplementation and genome editing.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {6191},
pmid = {39048567},
issn = {2041-1723},
mesh = {*Muscular Atrophy, Spinal/therapy/genetics ; Animals ; *Gene Editing/methods ; *Survival of Motor Neuron 1 Protein/genetics ; *CRISPR-Cas Systems ; Mice ; *Genetic Therapy/methods ; Disease Models, Animal ; Humans ; Motor Neurons/metabolism/pathology ; Mutation ; Male ; Female ; },
abstract = {Defect in the SMN1 gene causes spinal muscular atrophy (SMA), which shows loss of motor neurons, muscle weakness and atrophy. While current treatment strategies, including small molecules or viral vectors, have shown promise in improving motor function and survival, achieving a definitive and long-term correction of SMA's endogenous mutations and phenotypes remains highly challenging. We have previously developed a CRISPR-Cas9 based homology-independent targeted integration (HITI) strategy, enabling unidirectional DNA knock-in in both dividing and non-dividing cells in vivo. In this study, we demonstrated its utility by correcting an SMA mutation in mice. When combined with Smn1 cDNA supplementation, it exhibited long-term therapeutic benefits in SMA mice. Our observations may provide new avenues for the long-term and efficient treatment of inherited diseases.},
}

*Muscular Atrophy, Spinal/therapy/genetics
Animals
*Gene Editing/methods
*Survival of Motor Neuron 1 Protein/genetics
*CRISPR-Cas Systems
Mice
*Genetic Therapy/methods
Disease Models, Animal
Humans
Motor Neurons/metabolism/pathology
Mutation
Male
Female

@article {pmid39047725,
year = {2024},
author = {Zhang, C and Chen, F and Wang, F and Xu, H and Xue, J and Li, Z},
title = {Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2024.06.033},
pmid = {39047725},
issn = {1097-4164},
abstract = {The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-F[HNH] and I-E[HNH] Cascade complexes at different states are reported. In type I-F[HNH] Cascade, Cas8f[HNH] loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8f[HNH] to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-E[HNH] Cascade, Cas5e[HNH] domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.},
}

@article {pmid39047109,
year = {2024},
author = {Liu, H and Dong, J and Duan, Z and Xia, F and Willner, I and Huang, F},
title = {Light-activated CRISPR-Cas12a for amplified imaging of microRNA in cell cycle phases at single-cell levels.},
journal = {Science advances},
volume = {10},
number = {30},
pages = {eadp6166},
doi = {10.1126/sciadv.adp6166},
pmid = {39047109},
issn = {2375-2548},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Cell Cycle/genetics ; *Single-Cell Analysis/methods ; Light ; MCF-7 Cells ; Microscopy, Confocal/methods ; },
abstract = {An ortho-nitrobenzyl phosphate ester-caged nucleic acid hairpin structure coupled to the CRISPR-Cas12a complex is introduced as a functional reaction module for the light-induced activation of the CRISPR-Cas12a (LAC12a) machinery toward the amplified fluorescence detection of microRNA-21 (miRNA-21). The LAC12a machinery is applied for the selective, in vitro sensing of miRNA-21 and for the intracellular imaging of miRNA-21 in different cell lines. The LAC12a system is used to image miRNA-21 in different cell cycle phases of MCF-7 cells. Moreover, the LAC12a machinery integrated in cells enables the two-photon laser confocal microscopy-assisted, light-stimulated spatiotemporal, selective activation of the CRISPR-Cas12a miRNA-21 imaging machinery at the single-cell level and the evaluation of relative expression levels of miRNA-21 at distinct cell cycle phases. The method is implemented to map the distribution of cell cycle phases in an array of single cells.},
}

*MicroRNAs/genetics/metabolism
Humans
*CRISPR-Cas Systems
*Cell Cycle/genetics
*Single-Cell Analysis/methods
Light
MCF-7 Cells
Microscopy, Confocal/methods

@article {pmid39046587,
year = {2024},
author = {Zhou, J and Chen, X and Li, SM},
title = {Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum.},
journal = {Applied microbiology and biotechnology},
volume = {108},
number = {1},
pages = {427},
pmid = {39046587},
issn = {1432-0614},
support = {Li844/11-1//Deutsche Forschungsgemeinschaft/ ; INST 160/620-1//Deutsche Forschungsgemeinschaft/ ; 202206170029//Chinese Government Scholarship/ ; },
mesh = {*Penicillium/genetics/metabolism ; *Secondary Metabolism/genetics ; *CRISPR-Cas Systems ; Aspergillus nidulans/genetics/metabolism ; Polyketide Synthases/genetics/metabolism ; Multigene Family ; Gene Targeting/methods ; Gene Expression Regulation, Fungal ; Fungal Proteins/genetics/metabolism ; Biosynthetic Pathways/genetics ; Metabolic Engineering/methods ; Gene Expression ; },
abstract = {Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. KEY POINTS: Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans.},
}

*Penicillium/genetics/metabolism
*Secondary Metabolism/genetics
*CRISPR-Cas Systems
Aspergillus nidulans/genetics/metabolism
Polyketide Synthases/genetics/metabolism
Multigene Family
Gene Targeting/methods
Gene Expression Regulation, Fungal
Fungal Proteins/genetics/metabolism
Biosynthetic Pathways/genetics
Metabolic Engineering/methods
Gene Expression

@article {pmid38969245,
year = {2024},
author = {Li, Q and Yu, H and Li, Q},
title = {Dual sgRNA-directed tyrosinases knockout using CRISPR/Cas9 technology in Pacific oyster (Crassostrea gigas) reveals their roles in early shell calcification.},
journal = {Gene},
volume = {927},
number = {},
pages = {148748},
doi = {10.1016/j.gene.2024.148748},
pmid = {38969245},
issn = {1879-0038},
mesh = {Animals ; *CRISPR-Cas Systems ; *Crassostrea/genetics/growth & development/metabolism ; *Animal Shells/metabolism/growth & development ; *Gene Knockout Techniques/methods ; *Calcification, Physiologic/genetics ; *Calcium Carbonate/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Biomineralization/genetics ; },
abstract = {Biomineralization processes in bivalves, particularly the initial production of molecular components (such as matrix deposition and calcification) in the early stages of shell development are highly complex and well-organized. This study investigated the temporal dynamics of organic matrix and calcium carbonate (CaCO3) deposition in Pacific oysters (Crassostrea gigas) across various development stages. The shell-field initiated matrix secretion during the gastrula stage. Subsequent larval development triggered central shell-field calcification, accompanied by expansion of the calcium ring from its interior to the periphery. Notably, the expression patterns of CgTyrp-2 and CgTyr closely correlated with matrix deposition and calcification during early developmental stages, with peak expression occurring in oyster's gastrula and D-veliger stages. Subsequently, the CRISPR/Cas9 system was utilized to knock out CgTyrp-2 and CgTyr with more distinct phenotypic alterations observed when both genes were concurrently knocked out. The relative gene expression was analyzed post-knockout, indicating that the knockout of CgTyr or CgTyrp-2 led to reduced expression of CgChs1, along with increased expression of CgChit4. Furthermore, when dual-sgRNAs were employed to knockout CgTyrp-2, a large deletion (2 kb) within the CgTyrp-2 gene was identified. In summary, early shell formation in C. gigas is the result of a complex interplay of multiple molecular components with CgTyrp-2 and CgTyr playing key roles in regulating CaCO3 deposition.},
}

Animals
*CRISPR-Cas Systems
*Crassostrea/genetics/growth & development/metabolism
*Animal Shells/metabolism/growth & development
*Gene Knockout Techniques/methods
*Calcification, Physiologic/genetics
*Calcium Carbonate/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Biomineralization/genetics

@article {pmid38945310,
year = {2024},
author = {Wang, Y and Jiang, H and Li, M and Xu, Z and Xu, H and Chen, Y and Chen, K and Zheng, W and Lin, W and Liu, Z and Lin, Z and Zhang, M},
title = {Delivery of CRISPR/Cas9 system by AAV as vectors for gene therapy.},
journal = {Gene},
volume = {927},
number = {},
pages = {148733},
doi = {10.1016/j.gene.2024.148733},
pmid = {38945310},
issn = {1879-0038},
mesh = {*Dependovirus/genetics ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; Humans ; *Genetic Vectors/genetics ; Animals ; *Gene Editing/methods ; },
abstract = {The adeno-associated virus (AAV) is a defective single-stranded DNA virus with the simplest structure reported to date. It constitutes a capsid protein and single-stranded DNA. With its high transduction efficiency, low immunogenicity, and tissue specificity, it is the most widely used and promising gene therapy vector. The clustered regularly interspaced short palindromic sequence (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system is an emerging technology that utilizes cas9 nuclease to specifically recognize and cleave target genes under the guidance of small guide RNA and realizes gene editing through homologous directional repair and non-homologous recombination repair. In recent years, an increasing number of animal experiments and clinical studies have revealed the great potential of AAV as a vector to deliver the CRISPR/cas9 system for treating genetic diseases and viral infections. However, the immunogenicity, toxicity, low transmission efficiency in brain and ear tissues, packaging size limitations of AAV, and immunogenicity and off-target effects of Cas9 protein pose several clinical challenges. This research reviews the role, challenges, and countermeasures of the AAV-CRISPR/cas9 system in gene therapy.},
}

*Dependovirus/genetics
*Genetic Therapy/methods
*CRISPR-Cas Systems
Humans
*Genetic Vectors/genetics
Animals
*Gene Editing/methods

@article {pmid38897560,
year = {2024},
author = {Li, J and Wu, T and Wang, J and Chen, Y and Zhang, W and Cai, L and Lai, S and Hu, K and Jin, W},
title = {Dual auxotrophy coupled red labeling strategy for efficient genome editing in Saccharomyces cerevisiae.},
journal = {Fungal genetics and biology : FG & B},
volume = {173},
number = {},
pages = {103910},
doi = {10.1016/j.fgb.2024.103910},
pmid = {38897560},
issn = {1096-0937},
mesh = {*Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Homologous Recombination/genetics ; Genome, Fungal/genetics ; Gene Knockout Techniques/methods ; Saccharomyces cerevisiae Proteins/genetics ; },
abstract = {The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target genes in S. cerevisiae has also received a lot of attention in recent years. All findings seem to indicate that editing relevant target genes in S. cerevisiae is an extremely easy event. In this study, we systematically analyzed the advantages and disadvantages of homologous recombination (HR) strategy, CRISPR/Cas9 strategy, and CRISPR/Cas9 combined homology-mediated repair (CRISPR/Case9-HDR) strategy in knocking out BY4742 ade2. Our data showed that when the ade2 was knocked out by HR strategy, a large number of clones appeared to be off-target, and 10 %-80 % of the so-called knockout clones obtained were heteroclones. When the CRISPR/Cas9 strategy was applied, 60% of clones were off-target and the rest were all heteroclones. Interestingly, most of the cells were edited successfully, but at least 60 % of the clones were heteroclones, when the CRISPR/Cas9-HDR strategy was employed. Our results clearly showed that the emergence of heteroclone seems inevitable regardless of the strategies used for editing BY4742 ade2. Given the characteristics of BY4742 defective in ade2 showing red on the YPD plate, we attempted to build an efficient yeast gene editing strategy, in which the CRISPR/Cas9 combines homology-mediated repair template carrying an ade2 expression cassette, BY4742(ade2Δ0) as the start strain. We used this strategy to successfully achieve 100 % knockout efficiency of trp1, indicating that technical challenges of how to easily screen out pure knockout clones without color phenotype have been solved. Our data showed in this study not only establishes an efficient yeast gene knockout strategy with dual auxotrophy coupled red labeling but also provides new ideas and references for the knockout of target genes in the monokaryotic mycelium of macrofungi.},
}

*Saccharomyces cerevisiae/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Homologous Recombination/genetics
Genome, Fungal/genetics
Gene Knockout Techniques/methods
Saccharomyces cerevisiae Proteins/genetics

@article {pmid38878988,
year = {2024},
author = {Xiao, Z and Yang, W and Yang, A and Deng, L and Geng, R and Xiang, H and Kong, W and Jiang, C and Li, X and Chen, Z and Gao, Q},
title = {CRISPR/Cas9-mediated knockout of NtMYC2a gene involved in resistance to bacterial wilt in tobacco.},
journal = {Gene},
volume = {927},
number = {},
pages = {148622},
doi = {10.1016/j.gene.2024.148622},
pmid = {38878988},
issn = {1879-0038},
mesh = {*Nicotiana/genetics/microbiology ; *Disease Resistance/genetics ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology/genetics ; *Ralstonia solanacearum/pathogenicity ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Cyclopentanes/metabolism ; Signal Transduction ; Oxylipins/metabolism ; Plants, Genetically Modified/genetics ; },
abstract = {MYC2 is a class of bHLH family transcription factors and a major regulatory factor in the JA signaling pathway, and its molecular function in tobacco has not been reported. In this study, CRISPR/Cas9-mediated MYC2 gene NtMYC2a knockout mutants at tobacco was obtained and its agronomic traits, disease resistance, and chemical composition were identified. Comparing with the WT, the leaf width of the KO-NtMYC2a was narrowed, the nornicotine content and mecamylamine content increased significantly and the resistance to Ralstonia solanacearum significantly decreased. The transcriptome sequencing results showed that DEGs related to immunity, signal transduction and growth and development were enriched between KO-NtMYC2a and WT. NtJAR1 and NtCOI1 in KO-NtMYC2a were down-regulated to regulating the JA signaling pathway, result in a significant decrease in tobacco's resistance to R. solanacearum. Our research provides theoretical support for the functional research of MYC2 and the study of the mechanism of tobacco bacterial wilt resistance.},
}

*Nicotiana/genetics/microbiology
*Disease Resistance/genetics
*CRISPR-Cas Systems
*Plant Diseases/microbiology/genetics
*Ralstonia solanacearum/pathogenicity
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
Gene Knockout Techniques
Cyclopentanes/metabolism
Signal Transduction
Oxylipins/metabolism
Plants, Genetically Modified/genetics

@article {pmid38842342,
year = {2024},
author = {McGinn, J and Wen, A and Edwards, DL and Brinkley, DM and Lamason, RL},
title = {An expanded genetic toolkit for inducible expression and targeted gene silencing in Rickettsia parkeri.},
journal = {Journal of bacteriology},
volume = {206},
number = {7},
pages = {e0009124},
pmid = {38842342},
issn = {1098-5530},
support = {R01 AI155489/AI/NIAID NIH HHS/United States ; TB200032//U.S. Department of Defense (DOD)/ ; DRG-2396-20//Damon Runyon Cancer Research Foundation (DRCRF)/ ; },
mesh = {*Rickettsia/genetics ; *CRISPR-Cas Systems ; *Gene Silencing ; *Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; Promoter Regions, Genetic ; Humans ; },
abstract = {Pathogenic species within the Rickettsia genus are transmitted to humans through arthropod vectors and cause a spectrum of diseases ranging from mild to life-threatening. Despite rickettsiae posing an emerging global health risk, the genetic requirements of their infectious life cycles remain poorly understood. A major hurdle toward building this understanding has been the lack of efficient tools for genetic manipulation, owing to the technical difficulties associated with their obligate intracellular nature. To this end, we implemented the Tet-On system to enable conditional gene expression in Rickettsia parkeri. Using Tet-On, we show inducible expression of antibiotic resistance and a fluorescent reporter. We further used this inducible promoter to screen the ability of R. parkeri to express four variants of the catalytically dead Cas9 (dCas9). We demonstrate that all four dCas9 variants can be expressed in R. parkeri and used for CRISPR interference (CRISPRi)-mediated targeted gene knockdown. We show targeted knockdown of an antibiotic resistance gene as well as the endogenous virulence factor sca2. Altogether, we have developed systems for inducible gene expression and CRISPRi-mediated gene knockdown for the first time in rickettsiae, laying the groundwork for more scalable, targeted mechanistic investigations into their infectious life cycles.IMPORTANCEThe spotted fever group of Rickettsia contains vector-borne pathogenic bacteria that are neglected and emerging threats to public health. Due to the obligate intracellular nature of rickettsiae, the development of tools for genetic manipulation has been stunted, and the molecular and genetic underpinnings of their infectious lifecycle remain poorly understood. Here, we expand the genetic toolkit by introducing systems for conditional gene expression and CRISPR interference (CRISPRi)-mediated gene knockdown. These systems allow for relatively easy manipulation of rickettsial gene expression. We demonstrate the effectiveness of these tools by disrupting the intracellular life cycle using CRISPRi to deplete the sca2 virulence factor. These tools will be crucial for building a more comprehensive and detailed understanding of rickettsial biology and pathogenesis.},
}

*Rickettsia/genetics
*CRISPR-Cas Systems
*Gene Silencing
*Gene Expression Regulation, Bacterial
Bacterial Proteins/genetics/metabolism
Promoter Regions, Genetic
Humans

@article {pmid38838774,
year = {2024},
author = {Dey, D and Chakravarti, R and Bhattacharjee, O and Majumder, S and Chaudhuri, D and Ahmed, KT and Roy, D and Bhattacharya, B and Arya, M and Gautam, A and Singh, R and Gupta, R and Ravichandiran, V and Chattopadhyay, D and Ghosh, A and Giri, K and Roy, S and Ghosh, D},
title = {A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.},
journal = {The Journal of biological chemistry},
volume = {300},
number = {7},
pages = {107439},
pmid = {38838774},
issn = {1083-351X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Base Pair Mismatch ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; DNA/chemistry/metabolism ; Molecular Dynamics Simulation ; RNA/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry ; HEK293 Cells ; Gene Editing ; },
abstract = {The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.},
}

Humans
*CRISPR-Cas Systems
*Base Pair Mismatch
*CRISPR-Associated Protein 9/metabolism/genetics/chemistry
DNA/chemistry/metabolism
Molecular Dynamics Simulation
RNA/chemistry/metabolism
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry
HEK293 Cells
Gene Editing

@article {pmid38838231,
year = {2024},
author = {Mo, J and Wermeling, F and Nilsson, G and Dahlin, JS},
title = {CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation.},
journal = {Blood advances},
volume = {8},
number = {15},
pages = {3941-3945},
doi = {10.1182/bloodadvances.2023012279},
pmid = {38838231},
issn = {2473-9537},
mesh = {Humans ; *Microphthalmia-Associated Transcription Factor/genetics/metabolism ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; *Mast Cells/metabolism ; Trans-Activators/genetics/metabolism ; },
}

Humans
*Microphthalmia-Associated Transcription Factor/genetics/metabolism
*Cell Differentiation/genetics
*CRISPR-Cas Systems
*Mast Cells/metabolism
Trans-Activators/genetics/metabolism

@article {pmid38762829,
year = {2024},
author = {Yousefi, Y and Nejati, R and Eslahi, A and Alizadeh, F and Farrokhi, S and Asoodeh, A and Mojarrad, M},
title = {Enhancing Temozolomide (TMZ) chemosensitivity using CRISPR-dCas9-mediated downregulation of O[6]-methylguanine DNA methyltransferase (MGMT).},
journal = {Journal of neuro-oncology},
volume = {169},
number = {1},
pages = {129-135},
pmid = {38762829},
issn = {1573-7373},
mesh = {Humans ; *Temozolomide/pharmacology ; *Antineoplastic Agents, Alkylating/pharmacology/therapeutic use ; *CRISPR-Cas Systems ; *Down-Regulation ; *Glioblastoma/genetics/drug therapy ; DNA Repair Enzymes/genetics/metabolism ; Brain Neoplasms/genetics/drug therapy/pathology ; DNA Modification Methylases/genetics/metabolism ; Tumor Suppressor Proteins/genetics/metabolism ; HEK293 Cells ; Drug Resistance, Neoplasm/genetics ; DNA Methylation/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Line, Tumor ; Promoter Regions, Genetic ; },
abstract = {PURPOSE: Glioblastoma (GBM) stands out as the most prevalent and aggressive intracranial tumor, notorious for its poor prognosis. The current standard-of-care for GBM patients involves surgical resection followed by radiotherapy, combined with concurrent and adjuvant chemotherapy using Temozolomide (TMZ). The effectiveness of TMZ primarily relies on the activity of O[6]-methylguanine DNA methyltransferase (MGMT), which removes alkyl adducts from the O[6] position of guanine at the DNA level, thereby counteracting the toxic effects of TMZ.

METHOD: In this study, we employed fusions of catalytically-inactive Cas9 (dCas9) to DNA methyltransferases (dCas9-DNMT3A) to selectively downregulation MGMT transcription by inducing methylation at MGMT promoter and K-M enhancer.

RESULT: Our findings demonstrate a significant reduction in MGMT expression, leading to intensified TMZ sensitivity in the HEK293T cell line.

CONCLUSION: This study serves as a proof of concept for the utilization of CRISPR-based gene suppression to overcome TMZ resistance and enhance the lethal effect of TMZ in glioblastoma tumor cells.},
}

Humans
*Temozolomide/pharmacology
*Antineoplastic Agents, Alkylating/pharmacology/therapeutic use
*CRISPR-Cas Systems
*Down-Regulation
*Glioblastoma/genetics/drug therapy
DNA Repair Enzymes/genetics/metabolism
Brain Neoplasms/genetics/drug therapy/pathology
DNA Modification Methylases/genetics/metabolism
Tumor Suppressor Proteins/genetics/metabolism
HEK293 Cells
Drug Resistance, Neoplasm/genetics
DNA Methylation/drug effects
Gene Expression Regulation, Neoplastic/drug effects
Cell Line, Tumor
Promoter Regions, Genetic

@article {pmid38718928,
year = {2024},
author = {Tonetto, E and Cucci, A and Follenzi, A and Bernardi, F and Pinotti, M and Balestra, D},
title = {DNA base editing corrects common hemophilia A mutations and restores factor VIII expression in in vitro and ex vivo models.},
journal = {Journal of thrombosis and haemostasis : JTH},
volume = {22},
number = {8},
pages = {2171-2183},
doi = {10.1016/j.jtha.2024.04.020},
pmid = {38718928},
issn = {1538-7836},
mesh = {*Factor VIII/genetics/metabolism ; Humans ; *Hemophilia A/genetics/therapy/blood ; *Gene Editing/methods ; HEK293 Cells ; *Genetic Therapy ; Endothelial Cells/metabolism ; Point Mutation ; Mutation ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Replacement and nonreplacement therapies effectively control bleeding in hemophilia A (HA) but imply lifelong interventions. Authorized gene addition therapy could provide a cure but still poses questions on durability. FVIIIgene correction would definitively restore factor (F)VIII production, as shown in animal models through nuclease-mediated homologous recombination (HR). However, low efficiency and potential off-target double-strand break still limit HR translatability.

OBJECTIVES: To correct common model single point mutations leading to severe HA through the recently developed double-strand break/HR-independent base editing (BE) and prime editing (PE) approaches.

METHODS: Screening for efficacy of BE/PE systems in HEK293T cells transiently expressing FVIII variants and validation at DNA (sequencing) and protein (enzyme-linked immunosorbent assay; activated partial thromboplastin time) level in stable clones. Evaluation of rescue in engineered blood outgrowth endothelial cells by lentiviral-mediated delivery of BE.

RESULTS: Transient assays identified the best-performing BE/PE systems for each variant, with the highest rescue of FVIII expression (up to 25% of wild-type recombinant FVIII) for the p.R2166∗ and p.R2228Q mutations. In stable clones, we demonstrated that the mutation reversion on DNA (∼24%) was consistent with the rescue of FVIII secretion and activity of 20% to 30%. The lentiviral-mediated delivery of the selected BE systems was attempted in engineered blood outgrowth endothelial cells harboring the p.R2166∗ and p.R2228Q variants, which led to an appreciable and dose-dependent rescue of secreted functional FVIII.

CONCLUSION: Overall data provide the first proof-of-concept for effective BE/PE-mediated correction of HA-causing mutations, which encourage studies in mouse models to develop a personalized cure for large cohorts of patients through a single intervention.},
}

*Factor VIII/genetics/metabolism
Humans
*Hemophilia A/genetics/therapy/blood
*Gene Editing/methods
HEK293 Cells
*Genetic Therapy
Endothelial Cells/metabolism
Point Mutation
Mutation
CRISPR-Cas Systems

@article {pmid38708901,
year = {2024},
author = {Mahmood, M and Taufiq, I and Mazhar, S and Hafeez, F and Malik, K and Afzal, S},
title = {Revolutionizing personalized cancer treatment: the synergy of next-generation sequencing and CRISPR/Cas9.},
journal = {Personalized medicine},
volume = {21},
number = {3},
pages = {175-190},
doi = {10.1080/17410541.2024.2341610},
pmid = {38708901},
issn = {1744-828X},
mesh = {Humans ; *Precision Medicine/methods/trends ; *High-Throughput Nucleotide Sequencing/methods ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Mutation/genetics ; Gene Editing/methods ; },
abstract = {In the context of cancer heterogeneity, the synergistic action of next-generation sequencing (NGS) and CRISPR/Cas9 plays a promising role in the personalized treatment of cancer. NGS enables high-throughput genomic profiling of tumors and pinpoints specific mutations that primarily lead to cancer. Oncologists use this information obtained from NGS in the form of DNA profiling or RNA analysis to tailor precision strategies based on an individual's unique molecular signature. Furthermore, the CRISPR technique enables precise editing of cancer-specific mutations, allowing targeted gene modifications. Harnessing the potential insights of NGS and CRISPR/Cas9 heralds a remarkable frontier in cancer therapeutics with unprecedented precision, effectiveness and minimal off-target effects.},
}

Humans
*Precision Medicine/methods/trends
*High-Throughput Nucleotide Sequencing/methods
*Neoplasms/genetics/therapy
*CRISPR-Cas Systems/genetics
Mutation/genetics
Gene Editing/methods

@article {pmid38651662,
year = {2024},
author = {Yao, Z and He, K and Wang, H and Feng, S and Ding, X and Xu, Y and Wang, Q and Xu, X and Wu, Q and Wang, L},
title = {Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification.},
journal = {ACS sensors},
volume = {9},
number = {7},
pages = {3511-3519},
doi = {10.1021/acssensors.3c02485},
pmid = {38651662},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *SARS-CoV-2/genetics ; *Nucleic Acid Amplification Techniques/methods ; COVID-19/diagnosis/virology ; Lactobacillus/genetics ; Humans ; CRISPR-Associated Proteins/genetics ; Recombinases/metabolism ; Endodeoxyribonucleases/genetics ; Bacterial Proteins ; },
abstract = {Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage and isothermal amplification is the primary obstacle to quantitative detection. Though several works have demonstrated enhanced detection sensitivity by reducing the inhibition of CRISPR on amplification in one pot, few paid attention to the amplification process and even dynamic reaction processes between the two. Herein, we find that DNA quantification can be realized by regulating either recombinase polymerase amplification (RPA) efficiency or CRISPR/Cas12a cleaving efficiency (namely, tuning the dynamic reaction balance) in one pot. The sensitive quantification is realized by utilizing dual PAM-free crRNAs for CRISPR/Cas12a recognition. The varied RPA primer concentration with stabilized CRISPR systems significantly affects the amplification efficiency and quantitative performances. Alternatively, quantitative detection can also be achieved by stabilizing the amplification process while regulating the CRISPR/Cas12a concentration. The quantitative capability is proved by detecting DNA targets from Lactobacillus acetotolerans and SARS-CoV-2. The quantitative performance toward real samples is comparable to quantitative real-time PCR for detecting L. acetotolerans spiked in fermented food samples and SARS-CoV-2 clinical samples. We expect that the presented method will be a powerful tool for quantifying other nucleic acid targets.},
}

*CRISPR-Cas Systems/genetics
*SARS-CoV-2/genetics
*Nucleic Acid Amplification Techniques/methods
COVID-19/diagnosis/virology
Lactobacillus/genetics
Humans
CRISPR-Associated Proteins/genetics
Recombinases/metabolism
Endodeoxyribonucleases/genetics
Bacterial Proteins

@article {pmid38402425,
year = {2024},
author = {Qiu, L and Sun, M and Chen, L and Jiang, J and Fu, Z and Wang, Y and Bi, Y and Guo, Q and Bai, H and Chen, S and Gao, L and Chang, G},
title = {Iron-Confined CRISPR/Cas9-Ribonucleoprotein Delivery System for Redox-Responsive Gene Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {20},
number = {30},
pages = {e2309431},
doi = {10.1002/smll.202309431},
pmid = {38402425},
issn = {1613-6829},
support = {22121003//National Natural Science Foundation of China/ ; 81930050//National Natural Science Foundation of China/ ; 32272921//National Natural Science Foundation of China/ ; 2019YFA0709200//National Key R&D Program of China/ ; 2023ZD04053//Major Project of Agricultural Biological Breeding/ ; BE2022341//Jiangsu Provincial Key Research and Development Program/ ; CARS-41//Earmarked Fund for China Agriculture Research System/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Iron/chemistry ; *Oxidation-Reduction ; Humans ; *Ribonucleoproteins/metabolism/chemistry ; Liposomes/chemistry ; Gene Transfer Techniques ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) is a promising gene editing tool to treat diseases at the genetic level. Nonetheless, the challenge of the safe and efficient delivery of CRISPR/Cas9 to host cells constrains its clinical applicability. In the current study, a facile, redox-responsive CRISPR/Cas9-Ribonucleoprotein (RNP) delivery system by combining iron-coordinated aggregation with liposomes (Fe-RNP@L) is reported. The Fe-RNP is formed by the coordination of Fe[3+] with amino and carboxyl groups of Cas9, which modifies the lipophilicity and surface charge of RNP and alters cellular uptake from primary endocytosis to endocytosis and cholesterol-dependent membrane fusion. RNP can be rapidly and reversibly released from Fe-RNP in response to glutathione without loss of structural integrity and enzymatic activity. In addition, iron coordination also improves the stability of RNP and substantially mitigates cytotoxicity. This construct enabled highly efficient cytoplasmic/nuclear delivery (≈90%) and gene-editing efficiency (≈70%) even at low concentrations. The high payload content, high editing efficiency, good stability, low immunogenicity, and ease of production and storage, highlight its potential for diverse genome editing and clinical applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Iron/chemistry
*Oxidation-Reduction
Humans
*Ribonucleoproteins/metabolism/chemistry
Liposomes/chemistry
Gene Transfer Techniques
CRISPR-Associated Protein 9/metabolism

@article {pmid37977820,
year = {2023},
author = {Davies, B and Zhang, G and Moralli, D and Alghadban, S and Biggs, D and Preece, C and Donnelly, P and Hinch, AG},
title = {Characterization of meiotic recombination intermediates through gene knockouts in founder hybrid mice.},
journal = {Genome research},
volume = {33},
number = {11},
pages = {2018-2027},
pmid = {37977820},
issn = {1549-5469},
support = {/WT_/Wellcome Trust/United Kingdom ; NC/R001014/1/NC3RS_/National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; },
mesh = {Animals ; *Meiosis/genetics ; Mice ; *Rad51 Recombinase/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; *Cell Cycle Proteins/genetics/metabolism ; *Mice, Knockout ; DNA Breaks, Double-Stranded ; Phosphate-Binding Proteins/genetics/metabolism ; Replication Protein A/metabolism/genetics ; Recombination, Genetic ; Male ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Female ; Homologous Recombination ; },
abstract = {Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks, which requires choreographed binding of RPA, DMC1, and RAD51 to single-stranded DNA substrates. High-resolution in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F1-hybrid mice, these maps can distinguish the broken chromosome from the chromosome used as template for repair, revealing more mechanistic detail and enabling the structure of the recombination intermediates to be inferred. By applying CRISPR-Cas9 mutagenesis directly on F1-hybrid embryos, we have extended this approach to explore the molecular detail of recombination when a key component is knocked out. As a proof of concept, we have generated hybrid biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in them. DMC1 is essential for meiotic recombination, and comparison of these maps with those from wild-type mice is informative about the structure and timing of critical recombination intermediates. We observe redistribution of RAD51 binding and complete abrogation of D-loop recombination intermediates at a molecular level in Dmc1 mutants. These data provide insight on the configuration of RPA in D-loop intermediates and suggest that stable strand exchange proceeds via multiple rounds of strand invasion with template switching in mouse. Our methodology provides a high-throughput approach for characterization of gene function in meiotic recombination at low animal cost.},
}

Animals
*Meiosis/genetics
Mice
*Rad51 Recombinase/genetics/metabolism
*DNA-Binding Proteins/genetics/metabolism
*Cell Cycle Proteins/genetics/metabolism
*Mice, Knockout
DNA Breaks, Double-Stranded
Phosphate-Binding Proteins/genetics/metabolism
Replication Protein A/metabolism/genetics
Recombination, Genetic
Male
CRISPR-Cas Systems
Gene Knockout Techniques
Female
Homologous Recombination

@article {pmid35066025,
year = {2022},
author = {Pan, X and Luo, Y and Liao, N and Zhang, Y and Xiao, M and Chen, P and Lu, C and Dong, Z},
title = {CRISPR/Cpf1 multiplex genome editing system increases silkworm tolerance to BmNPV.},
journal = {International journal of biological macromolecules},
volume = {200},
number = {},
pages = {566-573},
doi = {10.1016/j.ijbiomac.2022.01.109},
pmid = {35066025},
issn = {1879-0003},
mesh = {Animals ; *Bombyx/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Animals, Genetically Modified ; Nucleopolyhedroviruses/genetics ; },
abstract = {The CRISPR/Cas9 genome editing technology is now widely used in insect studies, but the use of CRISPR can be further increased to improve insect genome engineering. We established a direct mutation at multiple loci in several genes simultaneously used by CRISPR/Cpf1 multiplex genome editing technology to target the BmNPV genome. We constructed a transgenic line that can target the BmNPV ie-1, gp64, and DNApoly genes simultaneously, and hybridized this line with an FnCpf1 transgenic line to obtain an FnCpf1 × gNPVM binary hybrid expression system and to activate the FnCpf1 gene editing system. We showed that the multiple gene editing system introduced deletions, mutations, and insertions at three target sites, and that it did not affect the economic traits of transgenic silkworm lines. The antiviral response of multiplexed genome editing lines increased significantly, and viral gene transcription and replication were significantly affected in the transgenic silkworm lines. This study provides innovative resistance materials for silkworm breeding and also provides a simplified platform for efficient insect multi genome engineering and genetic operation.},
}

Animals
*Bombyx/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Animals, Genetically Modified
Nucleopolyhedroviruses/genetics

@article {pmid35041685,
year = {2022},
author = {Rawsthorne-Manning, H and Calahorro, F and G Izquierdo, P and Tardy, P and Boulin, T and Holden-Dye, L and O'Connor, V and Dillon, J},
title = {Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes.},
journal = {PloS one},
volume = {17},
number = {1},
pages = {e0253351},
pmid = {35041685},
issn = {1932-6203},
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *CRISPR-Cas Systems ; *Caenorhabditis elegans Proteins/genetics ; Gene Editing/methods ; Mutation ; Genotype ; Genetic Markers ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Phenotype ; },
abstract = {Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR 'marker' genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co-CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.},
}

Animals
*Caenorhabditis elegans/genetics
*CRISPR-Cas Systems
*Caenorhabditis elegans Proteins/genetics
Gene Editing/methods
Mutation
Genotype
Genetic Markers
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Phenotype

@article {pmid34997243,
year = {2022},
author = {de Bakker, V and Liu, X and Bravo, AM and Veening, JW},
title = {CRISPRi-seq for genome-wide fitness quantification in bacteria.},
journal = {Nature protocols},
volume = {17},
number = {2},
pages = {252-281},
pmid = {34997243},
issn = {1750-2799},
mesh = {*Streptococcus pneumoniae/genetics ; *Genetic Fitness ; *Genome, Bacterial/genetics ; *Gene Library ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR interference (CRISPRi) is a powerful tool to link essential and nonessential genes to specific phenotypes and to explore their functions. Here we describe a protocol for CRISPRi screenings to assess genome-wide gene fitness in a single sequencing step (CRISPRi-seq). We demonstrate the use of the protocol in Streptococcus pneumoniae, an important human pathogen; however, the protocol can easily be adapted for use in other organisms. The protocol includes a pipeline for single-guide RNA library design, workflows for pooled CRISPRi library construction, growth assays and sequencing steps, a read analysis tool (2FAST2Q) and instructions for fitness quantification. We describe how to make an IPTG-inducible system with small libraries that are easy to handle and cost-effective and overcome bottleneck issues, which can be a problem when using similar, transposon mutagenesis-based methods. Ultimately, the procedure yields a fitness score per single-guide RNA target for any given growth condition. A genome-wide screening can be finished in 1 week with a constructed library. Data analysis and follow-up confirmation experiments can be completed in another 2-3 weeks.},
}

*Streptococcus pneumoniae/genetics
*Genetic Fitness
*Genome, Bacterial/genetics
*Gene Library
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid34939826,
year = {2022},
author = {Johansen, KH},
title = {How CRISPR/Cas9 Gene Editing Is Revolutionizing T Cell Research.},
journal = {DNA and cell biology},
volume = {41},
number = {1},
pages = {53-57},
pmid = {34939826},
issn = {1557-7430},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *T-Lymphocytes/immunology/metabolism ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows for precise gene targeting in mammalian cells, including T cells, allowing scientists to disrupt or edit specific genes of interest. This has enabled immunologists to investigate T cell functions as well as opened the path for novel therapeutics involving gene editing of T cells ex vivo before transferring these back to patients to increase T cell efficacy. This review outlines how CRISPR/Cas9 has transformed T cell research allowing immunologists to rapidly probe the roles of genes in T cells thus paving the way for novel therapeutics. Furthermore, this review describes how these tools reduce the requirement for genetic mouse models, while increasing the translational potential of T cell research.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Animals
*T-Lymphocytes/immunology/metabolism
Humans

@article {pmid34902367,
year = {2022},
author = {Lucero, D and Dikilitas, O and Mendelson, MM and Aligabi, Z and Islam, P and Neufeld, EB and Bansal, AT and Freeman, LA and Vaisman, B and Tang, J and Combs, CA and Li, Y and Voros, S and Kullo, IJ and Remaley, AT},
title = {Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen.},
journal = {Journal of lipid research},
volume = {63},
number = {1},
pages = {100160},
pmid = {34902367},
issn = {1539-7262},
support = {K99 HL136875/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Endocytosis ; Hep G2 Cells ; *CRISPR-Cas Systems/genetics ; *Microfilament Proteins/genetics/metabolism ; *Muscle Proteins/genetics/metabolism ; Receptors, LDL/genetics/metabolism ; Lipoproteins, LDL/metabolism ; Sterol Regulatory Element Binding Protein 2/metabolism/genetics ; Genome-Wide Association Study ; },
abstract = {A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.},
}

Humans
*Endocytosis
Hep G2 Cells
*CRISPR-Cas Systems/genetics
*Microfilament Proteins/genetics/metabolism
*Muscle Proteins/genetics/metabolism
Receptors, LDL/genetics/metabolism
Lipoproteins, LDL/metabolism
Sterol Regulatory Element Binding Protein 2/metabolism/genetics
Genome-Wide Association Study

@article {pmid34849910,
year = {2021},
author = {Christesen, D and Yang, YT and Chen, W and Batterham, P and Perry, T},
title = {Loss of the Dβ1 nicotinic acetylcholine receptor subunit disrupts bursicon-driven wing expansion and diminishes adult viability in Drosophila melanogaster.},
journal = {Genetics},
volume = {219},
number = {1},
pages = {},
pmid = {34849910},
issn = {1943-2631},
mesh = {Animals ; *Drosophila melanogaster/genetics/metabolism ; *Receptors, Nicotinic/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Wings, Animal/metabolism/growth & development ; *Invertebrate Hormones/metabolism/genetics ; Male ; CRISPR-Cas Systems ; Neurons/metabolism ; Female ; Gene Editing ; Longevity/genetics ; },
abstract = {Cholinergic signaling dominates the insect central nervous system, contributing to numerous fundamental pathways and behavioral circuits. However, we are only just beginning to uncover the diverse roles different cholinergic receptors may play. Historically, insect nicotinic acetylcholine receptors have received attention due to several subunits being key insecticide targets. More recently, there has been a focus on teasing apart the roles of these receptors, and their constituent subunits, in native signaling pathways. In this study, we use CRISPR-Cas9 genome editing to generate germline and somatic deletions of the Dβ1 nicotinic acetylcholine receptor subunit and investigate the consequences of loss of function in Drosophila melanogaster. Severe impacts on movement, male courtship, longevity, and wing expansion were found. Loss of Dβ1 was also associated with a reduction in transcript levels for the wing expansion hormone bursicon. Neuron-specific somatic deletion of Dβ1 in bursicon-producing neurons (CCAP-GAL4) was sufficient to disrupt wing expansion. Furthermore, CCAP-GAL4-specific expression of Dβ1 in a germline deletion background was sufficient to rescue the wing phenotype, pinpointing CCAP neurons as the neuronal subset requiring Dβ1 for the wing expansion pathway. Dβ1 is a known target of multiple commercially important insecticides, and the fitness costs exposed here explain why field-isolated target-site resistance has only been reported for amino acid replacements and not loss of function. This work reveals the importance of Dβ1-containing nicotinic acetylcholine receptors in CCAP neurons for robust bursicon-driven wing expansion.},
}

Animals
*Drosophila melanogaster/genetics/metabolism
*Receptors, Nicotinic/genetics/metabolism
*Drosophila Proteins/genetics/metabolism
*Wings, Animal/metabolism/growth & development
*Invertebrate Hormones/metabolism/genetics
Male
CRISPR-Cas Systems
Neurons/metabolism
Female
Gene Editing
Longevity/genetics

@article {pmid34791245,
year = {2022},
author = {Medley, JC and Hebbar, S and Sydzyik, JT and Zinovyeva, AY},
title = {Single nucleotide substitutions effectively block Cas9 and allow for scarless genome editing in Caenorhabditis elegans.},
journal = {Genetics},
volume = {220},
number = {1},
pages = {},
pmid = {34791245},
issn = {1943-2631},
support = {P20 GM103418/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R35 GM124828/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Genome, Helminth ; },
abstract = {In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize "off-target" sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts postinjection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.},
}

Animals
*Caenorhabditis elegans/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/metabolism/genetics
Genome, Helminth

@article {pmid34791226,
year = {2022},
author = {Huang, MY and Joshi, MB and Boucher, MJ and Lee, S and Loza, LC and Gaylord, EA and Doering, TL and Madhani, HD},
title = {Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging.},
journal = {Genetics},
volume = {220},
number = {1},
pages = {},
pmid = {34791226},
issn = {1943-2631},
support = {F31 AI150194/AI/NIAID NIH HHS/United States ; T32 AI060537/AI/NIAID NIH HHS/United States ; F32 AI152270/AI/NIAID NIH HHS/United States ; T32 HL007185/HL/NHLBI NIH HHS/United States ; R01 AI100272/AI/NIAID NIH HHS/United States ; R01 AI135012/AI/NIAID NIH HHS/United States ; R01 AI027243/AI/NIAID NIH HHS/United States ; R01 AI087794/AI/NIAID NIH HHS/United States ; T32 GM007067/GM/NIGMS NIH HHS/United States ; },
mesh = {*Cryptococcus neoformans/genetics/pathogenicity ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Deletion ; Recombinational DNA Repair ; CRISPR-Associated Protein 9/genetics/metabolism ; Homologous Recombination ; Animals ; Fungal Proteins/genetics/metabolism ; },
abstract = {Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized (Cno) Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (Cno CAS9 or "CnoCAS9") reliably enabled genome editing in the widely used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.},
}

*Cryptococcus neoformans/genetics/pathogenicity
*CRISPR-Cas Systems
*Gene Editing/methods
*Gene Deletion
Recombinational DNA Repair
CRISPR-Associated Protein 9/genetics/metabolism
Homologous Recombination
Animals
Fungal Proteins/genetics/metabolism

@article {pmid34791209,
year = {2022},
author = {Lutz, S and Van Dyke, K and Feraru, MA and Albert, FW},
title = {Multiple epistatic DNA variants in a single gene affect gene expression in trans.},
journal = {Genetics},
volume = {220},
number = {1},
pages = {},
pmid = {34791209},
issn = {1943-2631},
support = {R35 GM124676/GM/NIGMS NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *Epistasis, Genetic ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; *Gene Expression Regulation, Fungal ; Genetic Variation ; CRISPR-Cas Systems ; },
abstract = {DNA variants that alter gene expression in trans are important sources of phenotypic variation. Nevertheless, the identity of trans-acting variants remains poorly understood. Single causal variants in several genes have been reported to affect the expression of numerous distant genes in trans. Whether these simple molecular architectures are representative of trans-acting variation is unknown. Here, we studied the large RAS signaling regulator gene IRA2, which contains variants with extensive trans-acting effects on gene expression in the yeast Saccharomyces cerevisiae. We used systematic CRISPR-based genome engineering and a sensitive phenotyping strategy to dissect causal variants to the nucleotide level. In contrast to the simple molecular architectures known so far, IRA2 contained at least seven causal nonsynonymous variants. The effects of these variants were modulated by nonadditive, epistatic interactions. Two variants at the 5'-end affected gene expression and growth only when combined with a third variant that also had no effect in isolation. Our findings indicate that the molecular basis of trans-acting genetic variation may be considerably more complex than previously appreciated.},
}

*Saccharomyces cerevisiae/genetics
*Epistasis, Genetic
*Saccharomyces cerevisiae Proteins/genetics/metabolism
*Gene Expression Regulation, Fungal
Genetic Variation
CRISPR-Cas Systems

@article {pmid34788305,
year = {2021},
author = {Ahmed, S and Roy, MC and Al Baki, MA and Jung, JK and Lee, D and Kim, Y},
title = {CRISPR/Cas9 mutagenesis against sex pheromone biosynthesis leads to loss of female attractiveness in Spodoptera exigua, an insect pestt.},
journal = {PloS one},
volume = {16},
number = {11},
pages = {e0259322},
pmid = {34788305},
issn = {1932-6203},
mesh = {Animals ; *Sex Attractants/metabolism/biosynthesis ; Female ; *Spodoptera/physiology/genetics ; *CRISPR-Cas Systems ; Male ; Sexual Behavior, Animal ; Mutagenesis ; Fatty Acid Desaturases/genetics/metabolism ; },
abstract = {Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones of Spodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12th carbon position. A desaturase of S. exigua (SexiDES5) was proposed to have dual functions by forming double bonds at the 11th and 12th carbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion of SexiDES5 using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components in S. exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.},
}

Animals
*Sex Attractants/metabolism/biosynthesis
Female
*Spodoptera/physiology/genetics
*CRISPR-Cas Systems
Male
Sexual Behavior, Animal
Mutagenesis
Fatty Acid Desaturases/genetics/metabolism

@article {pmid34562094,
year = {2021},
author = {Hasebe, F and Yuba, H and Hashimoto, T and Saito, K and Funa, N and Shoji, T},
title = {CRISPR/Cas9-mediated disruption of the PYRROLIDINE KETIDE SYNTHASE gene reduces the accumulation of tropane alkaloids in Atropa belladonna hairy roots.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {85},
number = {12},
pages = {2404-2409},
doi = {10.1093/bbb/zbab165},
pmid = {34562094},
issn = {1347-6947},
support = {19H05652//Japan Society for the Promotion of Science/ ; },
mesh = {*Plant Roots/metabolism/genetics ; *CRISPR-Cas Systems ; *Tropanes/metabolism ; *Atropa belladonna/metabolism/genetics/enzymology ; Gene Editing ; Pyrrolidines/metabolism ; },
abstract = {Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A noncanonical type III polyketide synthase, pyrrolidine ketide synthase (PYKS) catalyzes a two-step decarboxylative reaction, which involves imine-ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.},
}

*Plant Roots/metabolism/genetics
*CRISPR-Cas Systems
*Tropanes/metabolism
*Atropa belladonna/metabolism/genetics/enzymology
Gene Editing
Pyrrolidines/metabolism

@article {pmid34531148,
year = {2021},
author = {Dai, Z and Li, R and Hou, Y and Li, Q and Zhao, K and Li, T and Li, MJ and Wu, X},
title = {Inducible CRISPRa screen identifies putative enhancers.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {48},
number = {10},
pages = {917-927},
doi = {10.1016/j.jgg.2021.06.012},
pmid = {34531148},
issn = {1673-8527},
mesh = {Animals ; *Enhancer Elements, Genetic/genetics ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; Mouse Embryonic Stem Cells/metabolism/cytology ; Doxycycline/pharmacology ; Cell Differentiation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Chromatin/genetics/metabolism ; },
abstract = {Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here, we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox)-inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible, and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.},
}

Animals
*Enhancer Elements, Genetic/genetics
Mice
*CRISPR-Cas Systems/genetics
Humans
Mouse Embryonic Stem Cells/metabolism/cytology
Doxycycline/pharmacology
Cell Differentiation/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Chromatin/genetics/metabolism

@article {pmid34508260,
year = {2021},
author = {Aregger, M and Xing, K and Gonatopoulos-Pournatzis, T},
title = {Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening.},
journal = {Nature protocols},
volume = {16},
number = {10},
pages = {4722-4765},
pmid = {34508260},
issn = {1750-2799},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Gene Deletion ; Endonucleases/metabolism/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Endodeoxyribonucleases/genetics/metabolism ; Animals ; Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic perturbations, most of which are mediated by a single nuclease and the expression of structurally identical guide backbones from two promoters. In contrast, we have developed CHyMErA (Cas hybrid for multiplexed editing and screening applications), which is based on the co-expression of Cas9 and Cas12a nucleases in conjunction with a hybrid guide RNA (hgRNA) engineered by the fusion of Cas9 and Cas12a guides and expressed from a single U6 promoter. CHyMErA is suitable for the high-throughput deletion of genetic segments including the excision of individual exons. Furthermore, CHyMErA enables the concomitant targeting of two or more genes and can thus be used for the systematic mapping of genetic interactions in mammalian cells. CHyMErA can also be applied for the perturbation of paralogous gene pairs, thereby allowing the capturing of phenotypic roles that would otherwise be masked because of genetic redundancy. Here, we provide instructions for the cloning of hgRNA screening libraries and individual hgRNA constructs and offer guidelines for designing and performing combinatorial pooled genetic screens using CHyMErA. Starting with the generation of Cas9- and Cas12a-expressing cell lines, CHyMErA screening can be implemented within 15-20 weeks.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Proteins/genetics/metabolism
Gene Deletion
Endonucleases/metabolism/genetics
CRISPR-Associated Protein 9/metabolism/genetics
Endodeoxyribonucleases/genetics/metabolism
Animals
Bacterial Proteins/genetics/metabolism

@article {pmid34508259,
year = {2021},
author = {Ward, HN and Aregger, M and Gonatopoulos-Pournatzis, T and Billmann, M and Ohsumi, TK and Brown, KR and Blencowe, BJ and Moffat, J and Myers, CL},
title = {Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.},
journal = {Nature protocols},
volume = {16},
number = {10},
pages = {4766-4798},
pmid = {34508259},
issn = {1750-2799},
support = {R01 HG005084/HG/NHGRI NIH HHS/United States ; R01 HG005853/HG/NHGRI NIH HHS/United States ; MOP-142375//CIHR/Canada ; FDN-148434//CIHR/Canada ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Software ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology/methods ; Gene Editing/methods ; Animals ; },
abstract = {The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.},
}

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

@article {pmid34411150,
year = {2021},
author = {Waldmann, L and Leyhr, J and Zhang, H and Öhman-Mägi, C and Allalou, A and Haitina, T},
title = {The broad role of Nkx3.2 in the development of the zebrafish axial skeleton.},
journal = {PloS one},
volume = {16},
number = {8},
pages = {e0255953},
pmid = {34411150},
issn = {1932-6203},
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Homeodomain Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Bone Development/genetics ; Gene Expression Regulation, Developmental ; Phenotype ; Bone and Bones/metabolism ; },
abstract = {The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knockdown and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while the function of this gene in broader skeletal development is not fully described. We generated a mutant allele of nkx3.2 in zebrafish with CRISPR/Cas9 and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing loss of the jaw joint, fusions in bones of the occiput, morphological changes in the Weberian apparatus, and the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae. Axial phenotypes are reminiscent of Nkx3.2 knockout in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role of nkx3.2 in zebrafish skeletal development and its context-specific functions in different skeletal elements.},
}

Animals
*Zebrafish/genetics
*Zebrafish Proteins/genetics/metabolism
*Homeodomain Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
CRISPR-Cas Systems
Bone Development/genetics
Gene Expression Regulation, Developmental
Phenotype
Bone and Bones/metabolism

@article {pmid34390914,
year = {2021},
author = {Shin, U and Nakhro, K and Oh, CK and Carrington, B and Song, H and Varshney, GK and Kim, Y and Song, H and Jeon, S and Robbins, G and Kim, S and Yoon, S and Choi, YJ and Kim, YJ and Burgess, S and Kang, S and Sood, R and Lee, Y and Myung, K},
title = {Large-scale generation and phenotypic characterization of zebrafish CRISPR mutants of DNA repair genes.},
journal = {DNA repair},
volume = {107},
number = {},
pages = {103173},
doi = {10.1016/j.dnarep.2021.103173},
pmid = {34390914},
issn = {1568-7856},
mesh = {*Zebrafish/genetics ; Animals ; *DNA Repair ; *CRISPR-Cas Systems ; *Phenotype ; *Zebrafish Proteins/genetics/metabolism ; *Mutation ; DNA Damage ; Gene Editing ; },
abstract = {A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental approaches using animal model systems. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. Large-scale phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.},
}

*Zebrafish/genetics
Animals
*DNA Repair
*CRISPR-Cas Systems
*Phenotype
*Zebrafish Proteins/genetics/metabolism
*Mutation
DNA Damage
Gene Editing

@article {pmid34362681,
year = {2021},
author = {Li, Y and Li, W and Li, J},
title = {The CRISPR/Cas9 revolution continues: From base editing to prime editing in plant science.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {48},
number = {8},
pages = {661-670},
doi = {10.1016/j.jgg.2021.05.001},
pmid = {34362681},
issn = {1673-8527},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plants/genetics ; *Genome, Plant/genetics ; },
abstract = {The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing (e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Plants/genetics
*Genome, Plant/genetics

@article {pmid34215255,
year = {2021},
author = {Bensalel, J and Xu, H and Lu, ML and Capobianco, E and Wei, J},
title = {RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells.},
journal = {BMC medical genomics},
volume = {14},
number = {1},
pages = {176},
pmid = {34215255},
issn = {1755-8794},
support = {R01 EB025819/EB/NIBIB NIH HHS/United States ; R21 NS111202/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *Huntingtin Protein/genetics ; *Neuroblastoma/genetics/pathology/metabolism ; *Transcriptome ; Cell Line, Tumor ; RNA-Seq ; Gene Expression Profiling ; Sequence Analysis, RNA ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {BACKGROUND: Huntingtin (Htt) protein is the product of the gene mutated in Huntington's disease (HD), a fatal, autosomal dominant, neurodegenerative disorder. Normal Htt is essential for early embryogenesis and the development of the central nervous system. However, the role of Htt in adult tissues is less defined. Following the recent promising clinical trial in which both normal and mutant Htt mRNA were knocked down in HD patients, there is an urgent need to fully understand the molecular consequences of knocking out/down Htt in adult tissues. Htt has been identified as an important transcriptional regulator. Unbiased investigations of transcriptome changes with RNA-sequencing (RNA-Seq) have been done in multiple cell types in HD, further confirming that transcriptional dysregulation is a central pathogenic mechanism in HD. However, there is lack of direct understanding of the transcriptional regulation by normal Htt.

METHODS: To investigate the transcriptional role of normal Htt, we first knocked out Htt in the human neuroblastoma SH-SY5Y cell line using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing approach. We then performed RNA-seq analysis on Htt-null and wild type SH-SY5Y cells to probe the global transcriptome changes induced by Htt deletion.

RESULTS: In general, Htt has a widespread effect on gene transcription. Functional analysis of the differentially expressed genes (DEGs) using various bioinformatic tools revealed irregularities in pathways related to cell communication and signaling, and more specifically those related to neuron development, neurotransmission and synaptic signaling. We further examined the transcription factors that may regulate these DEGs. Consistent with the disrupted pathways associated with cellular development, we showed that Htt-null cells exhibited slower cell proliferation than wild type cells. We finally validated some of the top DEGS with quantitative RT-PCR.

CONCLUSIONS: The widespread transcriptome changes in Htt-null cells could be directly caused by the loss of Htt-mediated transcriptional regulation or due to the secondary consequences of disruption in the gene regulatory network. Our study therefore provides valuable information about key genes associated with Htt-mediated transcription and improves our understanding of the molecular mechanisms underlying the cellular functions of normal and mutant Htt.},
}

Humans
*Huntingtin Protein/genetics
*Neuroblastoma/genetics/pathology/metabolism
*Transcriptome
Cell Line, Tumor
RNA-Seq
Gene Expression Profiling
Sequence Analysis, RNA
CRISPR-Cas Systems
Gene Knockout Techniques

@article {pmid34046943,
year = {2021},
author = {Shen, J and Lu, Z and Wang, J and Hao, Q and Ji, W and Wu, Y and Peng, H and Zhao, R and Yang, J and Li, Y and Shi, Z and Zhang, X},
title = {Traceable Nano-Biohybrid Complexes by One-Step Synthesis as CRISPR-Chem Vectors for Neurodegenerative Diseases Synergistic Treatment.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {33},
number = {27},
pages = {e2101993},
doi = {10.1002/adma.202101993},
pmid = {34046943},
issn = {1521-4095},
support = {52073287//National Natural Science Foundation of China/ ; L172046//Beijing Natural Science Foundation/ ; 2192057//Beijing Natural Science Foundation/ ; 31771095//National Natural Science Foundation of China/ ; 21875254//National Natural Science Foundation of China/ ; 21905283//National Natural Science Foundation of China/ ; 22075289//National Natural Science Foundation of China/ ; 32071391//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; Neurodegenerative Diseases/therapy ; Amyloid beta-Peptides/metabolism/chemistry ; Alzheimer Disease/therapy ; Gene Editing/methods ; Humans ; Brain/metabolism/pathology ; Plasmids/genetics/metabolism ; Mice, Transgenic ; Nanoparticles/chemistry ; },
abstract = {Abnormal protein aggregations are essential pathological features of neurodegenerative diseases. Eliminating while inhibiting the regeneration of these protein aggregates is considered an effective treatment strategy. Herein, the CRISPR/Cas9 gene-editing tool is employed to inhibit the regeneration of disease-related proteins, while chemical drugs are applied to eliminate the proteins that are produced. To efficiently deliver CRISPR-chem drugs into brain lesions, traceable nano-biohybrid complexes (F-TBIO) are constructed by one-step synthesis and CRISPR/Cas9 plasmids (CF-TBIO) are loaded in a controllable manner. CF-TBIO can knock out the BACE1 gene and reduce the burden of amyloid-β, and thereby significantly improve the cognitive abilities of 2xTg-AD mice. In particular, by prolonging the dosing interval, the pathological damage and behavioral abilities of 2xTg-AD mice are still significantly improved. During the therapeutic process, CF-TBIO with a high relaxation rate provides accurate imaging signals in the complex brain physiological environment. The finding shows that CF-TBIO has great potential to serve as a CRISPR-chem drug-delivery platform for neurodegenerative diseases therapy.},
}

Animals
*CRISPR-Cas Systems
Mice
Neurodegenerative Diseases/therapy
Amyloid beta-Peptides/metabolism/chemistry
Alzheimer Disease/therapy
Gene Editing/methods
Humans
Brain/metabolism/pathology
Plasmids/genetics/metabolism
Mice, Transgenic
Nanoparticles/chemistry

@article {pmid33967119,
year = {2021},
author = {Nishida, K and Tsuchiya, K and Obinata, H and Onodera, S and Honda, Y and Lai, YC and Haruta, N and Sugimoto, A},
title = {Expression Patterns and Levels of All Tubulin Isotypes Analyzed in GFP Knock-In C. elegans Strains.},
journal = {Cell structure and function},
volume = {46},
number = {1},
pages = {51-64},
pmid = {33967119},
issn = {1347-3700},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/metabolism/genetics ; *Tubulin/metabolism/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Protein Isoforms/metabolism/genetics ; Caenorhabditis elegans Proteins/metabolism/genetics ; Microtubules/metabolism ; Gene Knock-In Techniques ; CRISPR-Cas Systems/genetics ; },
abstract = {Most organisms have multiple α- and β-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine α-tubulin isotypes and six β-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes-α-tubulins TBA-1 and TBA-2 and β-tubulins TBB-1 and TBB-2-were expressed in virtually all tissues, with a distinct tissue-specific spectrum. Other isotypes were expressed in specific tissues or cell types at significantly lower levels than the broadly expressed isotypes. Four isotypes (TBA-5, TBA-6, TBA-9, and TBB-4) were expressed in different subsets of ciliated sensory neurons, and TBB-4 was inefficiently incorporated into mitotic spindle MTs. Taken together, we propose that MTs in C. elegans are mainly composed of four broadly expressed tubulin isotypes and that incorporation of a small amount of tissue-specific isotypes may contribute to tissue-specific MT properties. These newly constructed strains will be useful for further elucidating the distinct roles of tubulin isotypes.Key words: tubulin isotypes, microtubules, C. elegans.},
}

Animals
*Caenorhabditis elegans/metabolism/genetics
*Tubulin/metabolism/genetics
*Green Fluorescent Proteins/metabolism/genetics
Protein Isoforms/metabolism/genetics
Caenorhabditis elegans Proteins/metabolism/genetics
Microtubules/metabolism
Gene Knock-In Techniques
CRISPR-Cas Systems/genetics

@article {pmid33939982,
year = {2021},
author = {Green, CD and Weigel, C and Oyeniran, C and James, BN and Davis, D and Mahawar, U and Newton, J and Wattenberg, BW and Maceyka, M and Spiegel, S},
title = {CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism.},
journal = {Journal of lipid research},
volume = {62},
number = {},
pages = {100082},
pmid = {33939982},
issn = {1539-7262},
support = {R01 AI125433/AI/NIAID NIH HHS/United States ; R01 HL131340/HL/NHLBI NIH HHS/United States ; P30 CA016059/CA/NCI NIH HHS/United States ; R25 GM090084/GM/NIGMS NIH HHS/United States ; K99 HD096117/HD/NICHD NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Sphingolipids/metabolism ; Humans ; *Membrane Proteins/genetics/metabolism/deficiency ; Gene Deletion ; Ceramides/metabolism ; Animals ; Serine C-Palmitoyltransferase/genetics/metabolism ; },
abstract = {The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.},
}

*CRISPR-Cas Systems
*Sphingolipids/metabolism
Humans
*Membrane Proteins/genetics/metabolism/deficiency
Gene Deletion
Ceramides/metabolism
Animals
Serine C-Palmitoyltransferase/genetics/metabolism

@article {pmid33850267,
year = {2021},
author = {Choi, M and Yun, JY and Kim, JH and Kim, JS and Kim, ST},
title = {The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {8087},
pmid = {33850267},
issn = {2045-2322},
mesh = {*Arabidopsis/genetics ; *Gene Editing/methods ; *Promoter Regions, Genetic ; *CRISPR-Cas Systems ; *Cytosine/metabolism ; Plants, Genetically Modified/genetics ; Arabidopsis Proteins/genetics ; Animals ; Ribosomal Proteins/genetics ; Rats ; },
abstract = {CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.},
}

*Arabidopsis/genetics
*Gene Editing/methods
*Promoter Regions, Genetic
*CRISPR-Cas Systems
*Cytosine/metabolism
Plants, Genetically Modified/genetics
Arabidopsis Proteins/genetics
Animals
Ribosomal Proteins/genetics
Rats

@article {pmid33846570,
year = {2021},
author = {Jung, HR and Oh, Y and Na, D and Min, S and Kang, J and Jang, D and Shin, S and Kim, J and Lee, SE and Jeong, EM and An, JY and Sung, CO and Lee, WS and Lee, C and Cho, SY},
title = {CRISPR screens identify a novel combination treatment targeting BCL-XL and WNT signaling for KRAS/BRAF-mutated colorectal cancers.},
journal = {Oncogene},
volume = {40},
number = {18},
pages = {3287-3302},
pmid = {33846570},
issn = {1476-5594},
mesh = {Humans ; *Colorectal Neoplasms/genetics/drug therapy/pathology ; *Wnt Signaling Pathway/drug effects/genetics ; *bcl-X Protein/genetics/antagonists & inhibitors ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Proto-Oncogene Proteins B-raf/genetics/antagonists & inhibitors ; Animals ; *Sulfonamides/pharmacology ; Mice ; *Mutation ; Cell Line, Tumor ; *Aniline Compounds/pharmacology/therapeutic use ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm/genetics ; Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; },
abstract = {Metastatic or recurrent colorectal cancer (CRC) patients require systemic chemotherapy, but the therapeutic options of targeted agents remain limited. CRC patients with KRAS or BRAF gene mutations exhibit a worse prognosis and are resistant to anti-EGFR treatment. Previous studies have shown that the expression of anti-apoptotic protein BCL-XL is increased in CRC patients with KRAS/BRAF mutations, suggesting BCL-XL as a therapeutic target for this subgroup. Here, we performed genome-wide CRISPR/Cas9 screens of cell lines with KRAS mutations to investigate the factors required for sensitivity to BCL-XL inhibitor ABT-263 using single-guide RNAs (sgRNAs) that induce loss-of-function mutations. In the presence of ABT-263, sgRNAs targeting negative regulators of WNT signaling (resulting in WNT activation) were enriched, whereas sgRNAs targeting positive regulators of WNT signaling (resulting in WNT inhibition) were depleted in ABT-263-resistant cells. The activation of WNT signaling was highly associated with an increased expression ratio of anti- to pro-apoptotic BCL-2 family genes in CRC samples. Genetic and pharmacologic inhibition of WNT signaling using β-catenin short hairpin RNA or TNIK inhibitor NCB-0846, respectively, augmented ABT-263-induced cell death in KRAS/BRAF-mutated cells. Inhibition of WNT signaling resulted in transcriptional repression of the anti-apoptotic BCL-2 family member, MCL1, via the functional inhibition of the β-catenin-containing complex at the MCL1 promoter. In addition, the combination of ABT-263 and NCB-0846 exhibited synergistic effects in in vivo patient-derived xenograft (PDX) models with KRAS mutations. Our data provide a novel targeted combination treatment strategy for the CRC patient subgroup with KRAS or BRAF mutations.},
}

Humans
*Colorectal Neoplasms/genetics/drug therapy/pathology
*Wnt Signaling Pathway/drug effects/genetics
*bcl-X Protein/genetics/antagonists & inhibitors
*Proto-Oncogene Proteins p21(ras)/genetics
*Proto-Oncogene Proteins B-raf/genetics/antagonists & inhibitors
Animals
*Sulfonamides/pharmacology
Mice
*Mutation
Cell Line, Tumor
*Aniline Compounds/pharmacology/therapeutic use
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Drug Resistance, Neoplasm/genetics
Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use

@article {pmid33660495,
year = {2021},
author = {Baumgarten, N and Schmidt, F and Wegner, M and Hebel, M and Kaulich, M and Schulz, MH},
title = {Computational prediction of CRISPR-impaired non-coding regulatory regions.},
journal = {Biological chemistry},
volume = {402},
number = {8},
pages = {973-982},
doi = {10.1515/hsz-2020-0392},
pmid = {33660495},
issn = {1437-4315},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computational Biology ; CRISPR-Cas Systems/genetics ; Doxorubicin/pharmacology ; Mutation ; },
abstract = {Genome-wide CRISPR screens are becoming more widespread and allow the simultaneous interrogation of thousands of genomic regions. Although recent progress has been made in the analysis of CRISPR screens, it is still an open problem how to interpret CRISPR mutations in non-coding regions of the genome. Most of the tools concentrate on the interpretation of mutations introduced in gene coding regions. We introduce a computational pipeline that uses epigenomic information about regulatory elements for the interpretation of CRISPR mutations in non-coding regions. We illustrate our analysis protocol on the analysis of a genome-wide CRISPR screen in hTERT-RPE1 cells and reveal novel regulatory elements that mediate chemoresistance against doxorubicin in these cells. We infer links to established and to novel chemoresistance genes. Our analysis protocol is general and can be applied on any cell type and with different CRISPR enzymes.},
}

Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Computational Biology
CRISPR-Cas Systems/genetics
Doxorubicin/pharmacology
Mutation

@article {pmid33538047,
year = {2021},
author = {Tang, H and Xu, X and Chen, Y and Xin, H and Wan, T and Li, B and Pan, H and Li, D and Ping, Y},
title = {Reprogramming the Tumor Microenvironment through Second-Near-Infrared-Window Photothermal Genome Editing of PD-L1 Mediated by Supramolecular Gold Nanorods for Enhanced Cancer Immunotherapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {33},
number = {12},
pages = {e2006003},
doi = {10.1002/adma.202006003},
pmid = {33538047},
issn = {1521-4095},
support = {82073779//National Natural Science Foundation of China/ ; 81872807//National Natural Science Foundation of China/ ; 81573003//National Natural Science Foundation of China/ ; 2018XZZX001-14//Fundamental Research Funds for the Central Universities/ ; LR21H300002//Natural Science Foundation of Zhejiang Province/ ; LSY19H160005//Association Foundation of Zhejiang Natural Science Foundation-Zhejiang Society for Mathematical Medicine/ ; 2018YFA0901800//National Key Research and Development Program of China/ ; //Leading Talent of "Ten Thousand Plan" - National High-Level Talents Special Support Plan/ ; },
mesh = {*Tumor Microenvironment/drug effects ; *B7-H1 Antigen/metabolism ; *Gold/chemistry ; *Nanotubes/chemistry ; *Immunotherapy ; Animals ; Mice ; *Gene Editing/methods ; *Infrared Rays ; Humans ; Cell Line, Tumor ; CRISPR-Cas Systems ; Photothermal Therapy ; Neoplasms/therapy ; Immunogenic Cell Death/drug effects ; Immune Checkpoint Inhibitors/pharmacology/chemistry ; },
abstract = {A photothermal genome-editing strategy is described to improve immune checkpoint blockade (ICB) therapy by CRISPR/Cas9-mediated disruption of PD-L1 and mild-hyperthermia-induced activation of immunogenic cell death (ICD). This strategy relies on a supramolecular cationic gold nanorod that not only serves as a carrier to deliver CRISPR/Cas9 targeting PD-L1, but also harvests the second near-infrared-window (NIR-II) light and converts into mild hyperthermia to induce both ICD and gene expression of Cas9. The genomic disruption of PD-L1 significantly augments ICB therapy by improving the conversion of dendritic cells to T cells, followed by promoting the infiltration of cytotoxic T lymphocytes into tumors, thereby reprogramming immunosuppressive tumor microenvironment into immunoactive one. Such a therapeutic modality greatly inhibits the activity of primary and metastatic tumors and exhibits long-term immune memory effects against both rechallenged and recurrent tumors. The current therapeutic strategy for synergistic PD-L1 disruption and ICD activation represents an appealing way for cancer immunotherapy.},
}

*Tumor Microenvironment/drug effects
*B7-H1 Antigen/metabolism
*Gold/chemistry
*Nanotubes/chemistry
*Immunotherapy
Animals
Mice
*Gene Editing/methods
*Infrared Rays
Humans
Cell Line, Tumor
CRISPR-Cas Systems
Photothermal Therapy
Neoplasms/therapy
Immunogenic Cell Death/drug effects
Immune Checkpoint Inhibitors/pharmacology/chemistry

@article {pmid33504736,
year = {2021},
author = {Ijaz, F and Ikegami, K},
title = {Knock-in of Labeled Proteins into 5'UTR Enables Highly Efficient Generation of Stable Cell Lines.},
journal = {Cell structure and function},
volume = {46},
number = {1},
pages = {21-35},
pmid = {33504736},
issn = {1347-3700},
mesh = {*5' Untranslated Regions/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Tubulin/metabolism/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Cell Line ; Luminescent Proteins/genetics/metabolism ; ADP-Ribosylation Factors/metabolism/genetics ; Animals ; },
abstract = {Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biology technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5'UTR of the endogenous gene loci. With this 5'UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high efficiencies ranging from 50 to 80% in antibiotic selected cells. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5'UTR is an amenable site to generate cell lines for the stable expression of tagged proteins from endogenous loci in mammalian cells.Key words: CRISPR/Cas9, knock-in, primary cilium, UTR, tubulin.},
}

*5' Untranslated Regions/genetics
Humans
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Tubulin/metabolism/genetics
Green Fluorescent Proteins/genetics/metabolism
Cell Line
Luminescent Proteins/genetics/metabolism
ADP-Ribosylation Factors/metabolism/genetics
Animals

@article {pmid33109681,
year = {2020},
author = {De Giorgi, M and Jarrett, KE and Burton, JC and Doerfler, AM and Hurley, A and Li, A and Hsu, RH and Furgurson, M and Patel, KR and Han, J and Borchers, CH and Lagor, WR},
title = {Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase.},
journal = {Journal of lipid research},
volume = {61},
number = {12},
pages = {1675-1686},
pmid = {33109681},
issn = {1539-7262},
support = {T32 HL007676/HL/NHLBI NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; R01 DK124477/DK/NIDDK NIH HHS/United States ; 19POST34430092/AHA/American Heart Association-American Stroke Association/United States ; },
mesh = {*Hydroxymethylglutaryl CoA Reductases/metabolism/genetics ; Animals ; *Endoplasmic Reticulum Stress/genetics ; Mice ; *Liver/metabolism ; *Terpenes/metabolism ; Hepatocytes/metabolism ; Gene Deletion ; Apoptosis ; CRISPR-Cas Systems ; Mevalonic Acid/metabolism ; Mice, Knockout ; Dolichols/metabolism ; },
abstract = {HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.},
}

*Hydroxymethylglutaryl CoA Reductases/metabolism/genetics
Animals
*Endoplasmic Reticulum Stress/genetics
Mice
*Liver/metabolism
*Terpenes/metabolism
Hepatocytes/metabolism
Gene Deletion
Apoptosis
CRISPR-Cas Systems
Mevalonic Acid/metabolism
Mice, Knockout
Dolichols/metabolism

@article {pmid39042694,
year = {2024},
author = {Zhou, B and Purmann, C and Guo, H and Shin, G and Huang, Y and Pattni, R and Meng, Q and Greer, SU and Roychowdhury, T and Wood, RN and Ho, M and Dohna, HZ and Abyzov, A and Hallmayer, JF and Wong, WH and Ji, HP and Urban, AE},
title = {Resolving the 22q11.2 deletion using CTLR-Seq reveals chromosomal rearrangement mechanisms and individual variance in breakpoints.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {121},
number = {31},
pages = {e2322834121},
doi = {10.1073/pnas.2322834121},
pmid = {39042694},
issn = {1091-6490},
support = {K01MH129758//HHS | NIH | National Institute of Mental Health (NIMH)/ ; Instructor K Support Award//SU | SOM | Stanford Maternal and Child Health Research Institute (MCHRI)/ ; Uytengsu-Hamilton 22q11 Neuropsychiatry Research Award//SU | SOM | Stanford Maternal and Child Health Research Institute (MCHRI)/ ; R01HG006137//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; P01HG00205ESH//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; U01HG010963//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; P50HG00773506//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; DP2 MH100010-01//HHS | NIH | National Institute of Mental Health (NIMH)/ ; RSG-13-297-01-TBG//American Cancer Society (ACS)/ ; 1807371//National Science Foundation (NSF)/ ; R01MH100900//HHS | NIH | National Institute of Mental Health (NIMH)/ ; HG010359//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *DiGeorge Syndrome/genetics ; CRISPR-Cas Systems ; Chromosome Breakpoints ; Chromosomes, Human, Pair 22/genetics ; Genome, Human ; Gene Rearrangement ; Sequence Analysis, DNA/methods ; Chromosome Deletion ; },
abstract = {We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.},
}

Humans
*DiGeorge Syndrome/genetics
CRISPR-Cas Systems
Chromosome Breakpoints
Chromosomes, Human, Pair 22/genetics
Genome, Human
Gene Rearrangement
Sequence Analysis, DNA/methods
Chromosome Deletion

@article {pmid39039550,
year = {2024},
author = {Song, P and Zhang, Q and Xu, Z and Shi, Y and Jing, R and Luo, D},
title = {Correction: CRISPR/Cas-based CAR-T cells: production and application.},
journal = {Biomarker research},
volume = {12},
number = {1},
pages = {69},
doi = {10.1186/s40364-024-00616-7},
pmid = {39039550},
issn = {2050-7771},
}

@article {pmid39039415,
year = {2024},
author = {Meertens, L and Couture, L and Amara, A},
title = {Genome-Wide CRISPR-Cas9 Screening for the Identification of Host Dependency Factors of Emerging Viruses.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2824},
number = {},
pages = {203-219},
pmid = {39039415},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Rift Valley fever virus/genetics ; Virus Replication/genetics ; Host-Pathogen Interactions/genetics ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Like all the RNA viruses, Rift Valley fever virus (RVFV) encodes only few viral proteins and relies heavily on the host cellular machinery for productive infection. This dependence creates a potential "Achille's heel" that may be exploited to develop new approaches to treat RVFV infection. The recent development of lentiviral sgRNAs pool has enabled the creation of genome-scale CRISPR-Cas9 knockout libraries that has been used to identify host factors required for virus replication. In this chapter, we describe the preparation and execution of a pooled CRISPR-Cas9 loss-of-function screen using virus-induced cell death phenotypic readout. Using this technique, we outline a strategy for the identification of host factors essential for important human emerging viruses such as RVFV.},
}

*CRISPR-Cas Systems
Humans
*Rift Valley fever virus/genetics
Virus Replication/genetics
Host-Pathogen Interactions/genetics
Gene Knockout Techniques
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39037655,
year = {2024},
author = {Cortina, C and Cañellas-Socias, A},
title = {CRISPR Knock-Ins in Organoids to Track Tumor Cell Subpopulations.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2811},
number = {},
pages = {137-154},
pmid = {39037655},
issn = {1940-6029},
mesh = {*Organoids/metabolism/pathology ; *CRISPR-Cas Systems ; Humans ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; Animals ; Neoplasms/genetics/pathology ; Genes, Reporter ; },
abstract = {The integration of CRISPR/Cas9 genome editing techniques with organoid technology has revolutionized the field of tumor modeling, enabling the creation of diverse tumor models with distinct mutational profiles. This protocol details the application of CRISPR knock-ins to engineer tumor organoids with reporter cassettes, which are regulated by endogenous promoters of specific genes of interest. This approach facilitates the precise fluorescent labeling, isolation, and subsequent manipulation of targeted tumor cell subpopulations. The utilization of these knock-in reporter cassettes not only allows the visualization and purification of specific tumor cell subsets but also enables conditional cell ablation and lineage tracing studies. In this chapter, we provide a comprehensive guide for the design, construction, delivery, and validation of CRISPR/Cas9 tools tailored for knock-in reporter cassette integration into specific marker genes of interest. By following this protocol, researchers can harness the potential of engineered tumor organoids to decipher intricate tumor heterogeneity, track metastatic trajectories, and unveil novel therapeutic vulnerabilities linked to specific tumor cell subpopulations.},
}

*Organoids/metabolism/pathology
*CRISPR-Cas Systems
Humans
*Gene Knock-In Techniques/methods
*Gene Editing/methods
Animals
Neoplasms/genetics/pathology
Genes, Reporter

@article {pmid39037483,
year = {2024},
author = {de Carvalho, A and Giambiagi-deMarval, M and Rossi, CC},
title = {Mammaliicoccus sciuri's Pan-Immune System and the Dynamics of Horizontal Gene Transfer Among Staphylococcaceae: a One-Health CRISPR Tale.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {},
number = {},
pages = {},
pmid = {39037483},
issn = {1976-3794},
support = {408564/2023-7//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 304839/2022-1//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; E-26/010.000172/2016; 010.00128/2016; E-26.210.875/2016//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; 101056/2018; 001463/2019; 211.554/2019; 201.071/2020//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; 200.895/2021//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; E-26/204.925/2022//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; APQ-03498-22//Fundação de Amparo à Pesquisa do Estado de Minas Gerais/ ; 23038.002486/2018-26//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; },
abstract = {Recently emancipated from the Staphylococcus genus due to genomic differences, Mammaliicoccus sciuri, previously classified as an occasional pathogen, emerges as a significant player in the landscape of resistance gene dissemination among Staphylococcaceae. Despite its classification, its role remained enigmatic. In this study, we delved into the genomic repertoire of M. sciuri to unravel its contribution to resistance and virulence gene transfer in the context of One Health. Through comprehensive analysis of publicly available genomes, we unveiled a diverse pan-immune system adept at defending against exogenous genetic elements, yet concurrently fostering horizontal gene transfer (HGT). Specifically, exploration of CRISPR-Cas systems, with spacer sequences as molecular signatures, elucidated a global dissemination pattern spanning environmental, animal, and human hosts. Notably, we identified the integration of CRISPR-Cas systems within SCCmecs (Staphylococcal Cassette Chromosome mec), harboring key genes associated with pathogenicity and resistance, especially the methicillin resistance gene mecA, suggesting a strategic adaptation to outcompete other mobile genetic elements. Our findings underscored M. sciuri's active engagement in HGT dynamics and evolutionary trajectories within Staphylococcaceae, emphasizing its central role in shaping microbial communities and highlighting the significance of understanding its implications in the One Health framework, an interdisciplinary approach that recognizes the interconnectedness of human, animal, and environmental health to address global health challenges.},
}

@article {pmid39035353,
year = {2024},
author = {Zuberi, A and Ahmad, N and Ahmad, H and Saeed, M and Ahmad, I},
title = {Beyond antibiotics: CRISPR/Cas9 triumph over biofilm-associated antibiotic resistance infections.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1408569},
pmid = {39035353},
issn = {2235-2988},
mesh = {*Biofilms/drug effects/growth & development ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/pharmacology ; Humans ; Bacteria/drug effects/genetics ; Drug Resistance, Bacterial/genetics ; Bacterial Infections/microbiology/drug therapy ; Gene Editing ; },
abstract = {A complex structure known as a biofilm is formed when a variety of bacterial colonies or a single type of cell in a group sticks to a surface. The extracellular polymeric compounds that encase these cells, often consisting of proteins, eDNA, and polysaccharides, exhibit strong antibiotic resistance. Concerns about biofilm in the pharmaceutical industry, public health, and medical fields have sparked a lot of interest, as antibiotic resistance is a unique capacity exhibited by these biofilm-producing bacteria, which increases morbidity and death. Biofilm formation is a complicated process that is controlled by several variables. Insights into the processes to target for the therapy have been gained from multiple attempts to dissect the biofilm formation process. Targeting pathogens within a biofilm is profitable because the bacterial pathogens become considerably more resistant to drugs in the biofilm state. Although biofilm-mediated infections can be lessened using the currently available medications, there has been a lot of focus on the development of new approaches, such as bioinformatics tools, for both treating and preventing the production of biofilms. Technologies such as transcriptomics, metabolomics, nanotherapeutics and proteomics are also used to develop novel anti-biofilm agents. These techniques help to identify small compounds that can be used to inhibit important biofilm regulators. The field of appropriate control strategies to avoid biofilm formation is expanding quickly because of this spurred study. As a result, the current article addresses our current knowledge of how biofilms form, the mechanisms by which bacteria in biofilms resist antibiotics, and cutting-edge treatment approaches for infections caused by biofilms. Furthermore, we have showcased current ongoing research utilizing the CRISPR/Cas9 gene editing system to combat bacterial biofilm infections, particularly those brought on by lethal drug-resistant pathogens, concluded the article with a novel hypothesis and aspirations, and acknowledged certain limitations.},
}

*Biofilms/drug effects/growth & development
*CRISPR-Cas Systems
*Anti-Bacterial Agents/pharmacology
Humans
Bacteria/drug effects/genetics
Drug Resistance, Bacterial/genetics
Bacterial Infections/microbiology/drug therapy
Gene Editing

@article {pmid39035184,
year = {2024},
author = {Rooholamini, Z and Dianat-Moghadam, H and Esmaeilifallah, M and Khanahmad, H},
title = {From classical approaches to new developments in genetic engineering of live attenuated vaccine against cutaneous leishmaniasis: potential and immunization.},
journal = {Frontiers in public health},
volume = {12},
number = {},
pages = {1382996},
pmid = {39035184},
issn = {2296-2565},
mesh = {*Vaccines, Attenuated/immunology ; *Leishmaniasis, Cutaneous/prevention & control ; Humans ; *Leishmaniasis Vaccines/immunology ; *Genetic Engineering ; *Leishmania major/immunology/genetics ; Animals ; Immunization ; Gene Editing ; },
abstract = {Despite the development of a vaccine against cutaneous leishmaniasis in preclinical and clinical studies, we still do not have a safe and effective vaccine for human use. Given this situation, the search for a new prophylactic alternative to control leishmaniasis should be a global priority. A first-generation vaccine strategy-leishmanization, in which live Leishmania major parasites are inoculated into the skin to protect against reinfection, is taking advantage of this situation. Live attenuated Leishmania vaccine candidates are promising alternatives due to their robust protective immune responses. Importantly, they do not cause disease and could provide long-term protection following challenges with a virulent strain. In addition to physical and chemical methods, genetic tools, including the Cre-loxP system, have enabled the selection of safer null mutant live attenuated Leishmania parasites obtained by gene disruption. This was followed by the discovery and introduction of CRISPR/Cas-based gene editing tools, which can be easily and precisely used to modify genes. Here, we briefly review the immunopathology of L. major parasites and then present the classical methods and their limitations for the production of live attenuated vaccines. We then discuss the potential of current genetic engineering tools to generate live attenuated vaccine strains by targeting key genes involved in L. major pathogenesis and then discuss their discovery and implications for immune responses to control leishmaniasis.},
}

*Vaccines, Attenuated/immunology
*Leishmaniasis, Cutaneous/prevention & control
Humans
*Leishmaniasis Vaccines/immunology
*Genetic Engineering
*Leishmania major/immunology/genetics
Animals
Immunization
Gene Editing

@article {pmid39034763,
year = {2024},
author = {Ou, X and Li, K and Liu, M and Song, J and Zuo, Z and Guo, Y},
title = {EXPAR for biosensing: recent developments and applications.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4an00609g},
pmid = {39034763},
issn = {1364-5528},
abstract = {Emerging as a promising novel amplification technique, the exponential amplification reaction (EXPAR) offers significant advantages due to its potent exponential amplification capability, straightforward reaction design, rapid reaction kinetics, and isothermal operation. The past few years have witnessed swift advancements and refinements in EXPAR-based technologies, with numerous high-performance biosensing systems documented. A deeper understanding of the EXPAR mechanism has facilitated the proposal of novel strategies to overcome limitations inherent to traditional EXPAR. Furthermore, the synergistic integration of EXPAR with diverse amplification methodologies, including the use of a CRISPR/Cas system, metal nanoparticles, aptamers, alternative isothermal amplification techniques, and enzymes, has significantly bolstered analytical efficacy, aiming to enhance specificity, sensitivity, and amplification efficiency. This comprehensive review presents a detailed exposition of the EXPAR mechanism and analyzes its primary challenges. Additionally, we summarize the latest research advancements in the biomedical field concerning the integration of EXPAR with diverse amplification technologies for sensing strategies. Finally, we discuss the challenges and future prospects of EXPAR technology in the realms of biosensing and clinical applications.},
}

@article {pmid39034336,
year = {2024},
author = {Tarafder, E and Nizamani, MM and Karunarathna, SC and Das, D and Zeng, X and Rind, RA and Wang, Y and Tian, F},
title = {Advancements in genetic studies of mushrooms: a comprehensive review.},
journal = {World journal of microbiology & biotechnology},
volume = {40},
number = {9},
pages = {275},
pmid = {39034336},
issn = {1573-0972},
mesh = {*Agaricales/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA Interference ; Genome, Fungal ; Agriculture/methods ; Gene Knockout Techniques ; },
abstract = {Genetic studies in mushrooms, driven by innovations such as CRISPR-Cas9 genome editing and RNA interference, transform our understanding of these enigmatic fungi and their multifaceted roles in agriculture, medicine, and conservation. This comprehensive review explores the rationale and significance of genetic research in mushrooms, delving into the ethical, regulatory, and ecological dimensions of this field. CRISPR-Cas9 emerges as a game-changing technology, enabling precise genome editing, targeted gene knockouts, and pathway manipulation. RNA interference complements these efforts by downregulating genes for improved crop yield and enhanced pest and disease resistance. Genetic studies also contribute to the conservation of rare species and developing more robust mushroom strains, fostering sustainable cultivation practices. Moreover, they unlock the potential for discovering novel medicinal compounds, offering new horizons in pharmaceuticals and nutraceuticals. As emerging technologies and ethical considerations shape the future of mushroom research, these studies promise to revolutionize our relationship with these fungi, paving the way for a more sustainable and innovative world.},
}

*Agaricales/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
RNA Interference
Genome, Fungal
Agriculture/methods
Gene Knockout Techniques

@article {pmid39034325,
year = {2024},
author = {Maurizio, M and Masid, M and Woods, K and Caldelari, R and Doench, JG and Naguleswaran, A and Joly, D and González-Fernández, M and Zemp, J and Borteele, M and Hatzimanikatis, V and Heussler, V and Rottenberg, S and Olias, P},
title = {Host cell CRISPR genomics and modelling reveal shared metabolic vulnerabilities in the intracellular development of Plasmodium falciparum and related hemoparasites.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {6145},
pmid = {39034325},
issn = {2041-1723},
support = {173972//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; CRSII5_198543//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; CRSII5_198543//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; CRSII5_198543//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 189127//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Plasmodium falciparum/genetics ; Humans ; *Hepatocytes/parasitology/metabolism ; *Malaria, Falciparum/parasitology ; *CRISPR-Cas Systems ; *Theileria/genetics ; Genomics/methods ; Heme/metabolism ; Host-Parasite Interactions/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; Gene Knockout Techniques ; },
abstract = {Parasitic diseases, particularly malaria (caused by Plasmodium falciparum) and theileriosis (caused by Theileria spp.), profoundly impact global health and the socioeconomic well-being of lower-income countries. Despite recent advances, identifying host metabolic proteins essential for these auxotrophic pathogens remains challenging. Here, we generate a novel metabolic model of human hepatocytes infected with P. falciparum and integrate it with a genome-wide CRISPR knockout screen targeting Theileria-infected cells to pinpoint shared vulnerabilities. We identify key host metabolic enzymes critical for the intracellular survival of both of these lethal hemoparasites. Remarkably, among the metabolic proteins identified by our synergistic approach, we find that host purine and heme biosynthetic enzymes are essential for the intracellular survival of P. falciparum and Theileria, while other host enzymes are only essential under certain metabolic conditions, highlighting P. falciparum's adaptability and ability to scavenge nutrients selectively. Unexpectedly, host porphyrins emerge as being essential for both parasites. The shared vulnerabilities open new avenues for developing more effective therapies against these debilitating diseases, with the potential for broader applicability in combating apicomplexan infections.},
}

*Plasmodium falciparum/genetics
Humans
*Hepatocytes/parasitology/metabolism
*Malaria, Falciparum/parasitology
*CRISPR-Cas Systems
*Theileria/genetics
Genomics/methods
Heme/metabolism
Host-Parasite Interactions/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Animals
Gene Knockout Techniques

@article {pmid39034177,
year = {2024},
author = {Ye, X and Wu, H and Liu, J and Xiang, J and Feng, Y and Liu, Q},
title = {One-pot diagnostic methods based on CRISPR/Cas and Argonaute nucleases: strategies and perspectives.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2024.06.009},
pmid = {39034177},
issn = {1879-3096},
abstract = {CRISPR/Cas and Argonaute (Ago) proteins, which target specific nucleic acid sequences, can be applied as diagnostic tools. Despite high specificity and efficiency, achieving sensitive detection often necessitates a preamplification step that involves opening the lid and multistep operation, which may elevate the risk of contamination and prove inadequate for point-of-care testing. Hence, various one-pot detection strategies have been developed that enable preamplification and sensing in a single operation. We outline the challenges of one-pot detection with Cas and Ago proteins, present several main implementation strategies, and discuss future prospects. This review offers comprehensive insights into this vital field and explores potential improvements to detection methods that will be beneficial for human health.},
}

@article {pmid39033258,
year = {2024},
author = {Reyhani-Ardabili, M and Fathi, M and Ghafouri-Fard, S},
title = {CRISPR/Cas9 technology in the modeling of and evaluation of possible treatments for Niemann-Pick C.},
journal = {Molecular biology reports},
volume = {51},
number = {1},
pages = {828},
pmid = {39033258},
issn = {1573-4978},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Niemann-Pick Disease, Type C/therapy/genetics/metabolism ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Cholesterol/metabolism ; Animals ; Niemann-Pick C1 Protein ; Disease Models, Animal ; },
abstract = {Niemann-Pick disease type C (NPC) is a rare neurodegenerative condition resulted from mutations in NPC1 and NPC2 genes. This cellular lipid transferring disorder mainly involves endocytosed cholesterol trafficking. The accumulation of cholesterol and glycolipids in late endosomes and lysosomes results in progressive neurodegeneration and death. Recently, genome editing technologies, particularly CRISPR/Cas9 have offered the opportunity to create disease models to screen novel therapeutic options for this disorder. Moreover, these methods have been used for the purpose of gene therapy. This review summarizes the studies that focused on the application of CRISPR/Cas9 technology for exploring the mechanism of intracellular cholesterol transferring, and screening of novel agents for treatment of NPC.},
}

*CRISPR-Cas Systems/genetics
Humans
*Niemann-Pick Disease, Type C/therapy/genetics/metabolism
*Gene Editing/methods
*Genetic Therapy/methods
*Cholesterol/metabolism
Animals
Niemann-Pick C1 Protein
Disease Models, Animal

@article {pmid39033086,
year = {2024},
author = {Zimmerman, E and Sturrock, A and Reilly, CA and Burrell-Gerbers, KL and Warren, K and Mir-Kasimov, M and Zhang, MA and Pierce, MS and Helms, MN and Paine, R},
title = {Aryl Hydrocarbon Receptor Activation in Pulmonary Alveolar Epithelial Cells Limits Inflammation and Preserves Lung Epithelial Cell Integrity.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {},
number = {},
pages = {},
doi = {10.4049/jimmunol.2300325},
pmid = {39033086},
issn = {1550-6606},
support = {5I01BX001777//VA Merit Grant/ ; },
abstract = {The aryl hydrocarbon receptor (AHR) is a receptor/transcription factor widely expressed in the lung. The physiological roles of AHR expressed in the alveolar epithelium remain unclear. In this study, we tested the hypothesis that alveolar epithelial AHR activity plays an important role in modulating inflammatory responses and maintaining alveolar integrity during lung injury and repair. AHR is expressed in alveolar epithelial cells (AECs) and is active. AHR activation with the endogenous AHR ligand, FICZ (5,11-dihydroindolo[3,2-b] carbazole-6-carboxaldehyde), significantly suppressed inflammatory cytokine expression in response to inflammatory stimuli in primary murine AECs and in the MLE-15 epithelial cell line. In an LPS model of acute lung injury in mice, coadministration of FICZ with LPS suppressed protein leak, reduced neutrophil accumulation in BAL fluid, and suppressed inflammatory cytokine expression in lung tissue and BAL fluid. Relevant to healing following inflammatory injury, AHR activation suppressed TGF-β-induced expression of genes associated with epithelial-mesenchymal transition. Knockdown of AHR in primary AECs with shRNA or in CRISPR-Cas-9-induced MLE-15 cells resulted in upregulation of α-smooth muscle actin (αSma), Col1a1, and Fn1 and reduced expression of epithelial genes Col4a1 and Sdc1. MLE-15 clones lacking AHR demonstrated accelerated wound closure in a scratch model. AHR activation with FICZ enhanced barrier function (transepithelial electrical resistance) in primary murine AECs and limited decline of transepithelial electrical resistance following inflammatory injury. AHR activation in AECs preserves alveolar integrity by modulating inflammatory cytokine expression while enhancing barrier function and limiting stress-induced expression of mesenchymal genes.},
}

@article {pmid39032106,
year = {2024},
author = {Bircheneder, M and Schreiber, T and Tissier, A and Parniske, M},
title = {A quantitative assay for the efficiency of RNA-guided genome editing in plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/tpj.16931},
pmid = {39032106},
issn = {1365-313X},
abstract = {RNA-guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double-strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch-on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20-bp spacer either side to preferentially detect full-deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas-mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4-HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms.},
}

@article {pmid39030569,
year = {2024},
author = {Vinceti, A and Iannuzzi, RM and Boyle, I and Trastulla, L and Campbell, CD and Vazquez, F and Dempster, JM and Iorio, F},
title = {A benchmark of computational methods for correcting biases of established and unknown origin in CRISPR-Cas9 screening data.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {192},
pmid = {39030569},
issn = {1474-760X},
support = {28772//Fondazione AIRC per la ricerca sul cancro ETS/ ; 101125051//HORIZON EUROPE European Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Benchmarking ; Computational Biology/methods ; Bias ; },
abstract = {BACKGROUND: CRISPR-Cas9 dropout screens are formidable tools for investigating biology with unprecedented precision and scale. However, biases in data lead to potential confounding effects on interpretation and compromise overall quality. The activity of Cas9 is influenced by structural features of the target site, including copy number amplifications (CN bias). More worryingly, proximal targeted loci tend to generate similar gene-independent responses to CRISPR-Cas9 targeting (proximity bias), possibly due to Cas9-induced whole chromosome-arm truncations or other genomic structural features and different chromatin accessibility levels.

RESULTS: We benchmarked eight computational methods, rigorously evaluating their ability to reduce both CN and proximity bias in the two largest publicly available cell-line-based CRISPR-Cas9 screens to date. We also evaluated the capability of each method to preserve data quality and heterogeneity by assessing the extent to which the processed data allows accurate detection of true positive essential genes, established oncogenetic addictions, and known/novel biomarkers of cancer dependency. Our analysis sheds light on the ability of each method to correct biases under different scenarios. AC-Chronos outperforms other methods in correcting both CN and proximity biases when jointly processing multiple screens of models with available CN information, whereas CRISPRcleanR is the top performing method for individual screens or when CN information is not available. In addition, Chronos and AC-Chronos yield a final dataset better able to recapitulate known sets of essential and non-essential genes.

CONCLUSIONS: Overall, our investigation provides guidance for the selection of the most appropriate bias-correction method, based on its strengths, weaknesses and experimental settings.},
}

*CRISPR-Cas Systems
Humans
*Benchmarking
Computational Biology/methods
Bias

@article {pmid39030018,
year = {2024},
author = {Sun, H and Zhang, X and Ma, H and Zhang, L and Zhang, Y and Sun, R and Zheng, H and Wang, H and Guo, J and Liu, Y and Wang, Y and Qi, Y},
title = {A programmable sensitive platform for pathogen detection based on CRISPR/Cas12a -hybridization chain reaction-poly T-Cu.},
journal = {Analytica chimica acta},
volume = {1317},
number = {},
pages = {342888},
doi = {10.1016/j.aca.2024.342888},
pmid = {39030018},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *Copper/chemistry ; *Nucleic Acid Hybridization ; Poly T/chemistry ; Staphylococcus aureus/isolation & purification/genetics ; DNA, Bacterial ; Limit of Detection ; Fluorescent Dyes/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and sensitive detection of pathogenic bacteria is crucial for disease prevention and control. The CRISPR/Cas12a system with the DNA cleavage capability holds promise in pathogenic bacteria diagnosis. However, the sensitivity of CRISPR-based assays remains a challenge. Herein, we report a versatile and sensitive pathogen sensing platform (HTCas12a) based on the CRISPR/Cas12a system, hybridization chain reaction (HCR) and Poly T-copper fluorescence nanoprobe. The sensitivity is improved by HCR and the Poly-T-Cu reporter probe reduces the overall experiment cost to less than one dollar per sample. Our results demonstrate the specific recognition of target nucleic acid fragments from other pathogens. Furthermore, a good linear correlation between fluorescence intensity and target quantities were achieved with detection limits of 23.36 fM for Target DNA and 4.17 CFU/mL for S.aureus, respectively. The HTCas12a system offers a universal platform for pathogen detection in various fields, including environmental monitoring, clinical diagnosis, and food safety.},
}

*CRISPR-Cas Systems
*Copper/chemistry
*Nucleic Acid Hybridization
Poly T/chemistry
Staphylococcus aureus/isolation & purification/genetics
DNA, Bacterial
Limit of Detection
Fluorescent Dyes/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39028827,
year = {2024},
author = {Patel, RP and Ghilardi, G and Zhang, Y and Chiang, YH and Xie, W and Guruprasad, P and Kim, KH and Chun, I and Angelos, MG and Pajarillo, R and Hong, SJ and Lee, YG and Shestova, O and Shaw, C and Cohen, I and Gupta, A and Vu, T and Qian, D and Yang, S and Nimmagadda, A and Snook, AE and Siciliano, N and Rotolo, A and Inamdar, A and Harris, J and Ugwuanyi, O and Wang, M and Carturan, A and Paruzzo, L and Chen, L and Ballard, HJ and Blanchard, T and Xu, C and Abdel-Mohsen, M and Gabunia, K and Wysocka, M and Linette, GP and Carreno, B and Barrett, DM and Teachey, DT and Posey, AD and Powell, DJ and Sauter, CT and Pileri, S and Pillai, V and Scholler, J and Rook, AH and Schuster, SJ and Barta, SK and Porazzi, P and Ruella, M},
title = {CD5 deletion enhances the antitumor activity of adoptive T cell therapies.},
journal = {Science immunology},
volume = {9},
number = {97},
pages = {eadn6509},
doi = {10.1126/sciimmunol.adn6509},
pmid = {39028827},
issn = {2470-9468},
mesh = {Animals ; *Immunotherapy, Adoptive/methods ; *CD5 Antigens/immunology ; Mice ; Humans ; *T-Lymphocytes/immunology/transplantation ; Receptors, Chimeric Antigen/immunology/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; Female ; },
abstract = {Most patients treated with US Food and Drug Administration (FDA)-approved chimeric antigen receptor (CAR) T cells eventually experience disease progression. Furthermore, CAR T cells have not been curative against solid cancers and several hematological malignancies such as T cell lymphomas, which have very poor prognoses. One of the main barriers to the clinical success of adoptive T cell immunotherapies is CAR T cell dysfunction and lack of expansion and/or persistence after infusion. In this study, we found that CD5 inhibits CAR T cell activation and that knockout (KO) of CD5 using CRISPR-Cas9 enhances the antitumor effect of CAR T cells in multiple hematological and solid cancer models. Mechanistically, CD5 KO drives increased T cell effector function with enhanced cytotoxicity, in vivo expansion, and persistence, without apparent toxicity in preclinical models. These findings indicate that CD5 is a critical inhibitor of T cell function and a potential clinical target for enhancing T cell therapies.},
}

Animals
*Immunotherapy, Adoptive/methods
*CD5 Antigens/immunology
Mice
Humans
*T-Lymphocytes/immunology/transplantation
Receptors, Chimeric Antigen/immunology/genetics
Cell Line, Tumor
CRISPR-Cas Systems
Female

@article {pmid39026343,
year = {2024},
author = {Zhou, H and Ye, P and Xiong, W and Duan, X and Jing, S and He, Y and Zeng, Z and Wei, Y and Ye, Q},
title = {Genome-scale CRISPR-Cas9 screening in stem cells: theories, applications and challenges.},
journal = {Stem cell research & therapy},
volume = {15},
number = {1},
pages = {218},
pmid = {39026343},
issn = {1757-6512},
support = {2022YFC2504200//Chinese Academy of Sciences Key Project/ ; 2022BCA029//Science and Technology Program of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; *Stem Cells/metabolism/cytology ; Animals ; },
abstract = {Due to the rapid development of stem cell technology, there have been tremendous advances in molecular biological and pathological research, cell therapy as well as organoid technologies over the past decades. Advances in genome editing technology, particularly the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related protein 9 (Cas9), have further facilitated the rapid development of stem cell researches. The CRISPR-Cas9 technology now goes beyond creating single gene editing to enable the inhibition or activation of endogenous gene loci by fusing inhibitory (CRISPRi) or activating (CRISPRa) domains with deactivated Cas9 proteins (dCas9). These tools have been utilized in genome-scale CRISPRi/a screen to recognize hereditary modifiers that are synergistic or opposing to malady mutations in an orderly and fair manner, thereby identifying illness mechanisms and discovering novel restorative targets to accelerate medicinal discovery investigation. However, the application of this technique is still relatively rare in stem cell research. There are numerous specialized challenges in applying large-scale useful genomics approaches to differentiated stem cell populations. Here, we present the first comprehensive review on CRISPR-based functional genomics screening in the field of stem cells, as well as practical considerations implemented in a range of scenarios, and exploration of the insights of CRISPR-based screen into cell fates, disease mechanisms and cell treatments in stem cell models. This review will broadly benefit scientists, engineers and medical practitioners in the areas of stem cell research.},
}

*CRISPR-Cas Systems
Humans
*Gene Editing/methods
*Stem Cells/metabolism/cytology
Animals

@article {pmid39025833,
year = {2024},
author = {Ge, W and Gou, S and Zhao, X and Jin, Q and Zhuang, Z and Zhao, Y and Liang, Y and Ouyang, Z and Liu, X and Chen, F and Shi, H and Yan, H and Wu, H and Lai, L and Wang, K},
title = {In vivo evaluation of guide-free Cas9-induced safety risks in a pig model.},
journal = {Signal transduction and targeted therapy},
volume = {9},
number = {1},
pages = {184},
pmid = {39025833},
issn = {2059-3635},
support = {32300426//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Genetic Therapy ; },
abstract = {The CRISPR/Cas9 system has shown great potential for treating human genetic diseases through gene therapy. However, there are concerns about the safety of this system, specifically related to the use of guide-free Cas9. Previous studies have shown that guide-free Cas9 can induce genomic instability in vitro. However, the in vivo safety risks associated with guide-free Cas9 have not been evaluated, which is necessary for the development of gene therapy in clinical settings. In this study, we used doxycycline-inducible Cas9-expressing pigs to evaluate the safety risks of guide-free Cas9 in vivo. Our findings demonstrated that expression of guide-free Cas9 could induce genomic damages and transcriptome changes in vivo. The severity of the genomic damages and transcriptome changes were correlate with the expression levels of Cas9 protein. Moreover, prolonged expression of Cas9 in pigs led to abnormal phenotypes, including a significant decrease in body weight, which may be attributable to genomic damage-induced nutritional absorption and metabolic dysfunction. Furthermore, we observed an increase in whole-genome and tumor driver gene mutations in pigs with long-term Cas9 expression, raising the risk of tumor occurrence. Our in vivo evaluation of guide-free Cas9 in pigs highlights the necessity of considering and monitoring the detrimental effects of Cas9 alone as genome editing via the CRISPR/Cas9 system is implemented in clinical gene therapy. This research emphasizes the importance of further study and implementation of safety measures to ensure the successful and safe application of the CRISPR/Cas9 system in clinical practice.},
}

Animals
Swine
*CRISPR-Cas Systems/genetics
*Gene Editing
*CRISPR-Associated Protein 9/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Genetic Therapy

@article {pmid39024377,
year = {2024},
author = {Yoon, PH and Zhang, Z and Loi, KJ and Adler, BA and Lahiri, A and Vohra, K and Shi, H and Rabelo, DB and Trinidad, M and Boger, RS and Al-Shimary, MJ and Doudna, JA},
title = {Structure-guided discovery of ancestral CRISPR-Cas13 ribonucleases.},
journal = {Science (New York, N.Y.)},
volume = {},
number = {},
pages = {eadq0553},
doi = {10.1126/science.adq0553},
pmid = {39024377},
issn = {1095-9203},
abstract = {The RNA-guided ribonuclease CRISPR-Cas13 enables adaptive immunity in bacteria and programmable RNA manipulation in heterologous systems. Cas13s share limited sequence similarity, hindering discovery of related or ancestral systems. To address this, we developed an automated structural-search pipeline to identify an ancestral clade of Cas13 (Cas13an), and further trace Cas13 origins to defense-associated ribonucleases. Despite being one third the size of other Cas13s, Cas13an mediates robust programmable RNA depletion and defense against diverse bacteriophages. However, unlike its larger counterparts, Cas13an uses a single active site for both CRISPR RNA processing and RNA-guided cleavage, revealing the ancestral nuclease domain has two modes of activity. Discovery of Cas13an deepens our understanding of CRISPR-Cas evolution and expands opportunities for precision RNA editing, showcasing the promise of structure-guided genome mining.},
}

@article {pmid39023769,
year = {2024},
author = {He, X and Deng, L and Zhou, S and Dong, J and Zhu, S and Li, J and Li, X and Huo, D and Hou, C},
title = {CRISPR/Cas12a-coupled multiplexed strand displacement amplification for miRNA155 one-tube detection: via a dual-cavity PCR tube.},
journal = {Mikrochimica acta},
volume = {191},
number = {8},
pages = {470},
pmid = {39023769},
issn = {1436-5073},
support = {2023-K08//Hubei Key Laboratory of Intelligent Geo-Information Processing/ ; CSTB2022NSCO-BHX0727//Chongqing Natural Science Foundation/ ; 2022CDJYGRH-013//Fundamental Research Founds for the Central Universities/ ; },
mesh = {*MicroRNAs/analysis/blood/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/chemistry/genetics ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {A CRISPR/Cas12a-coupled multiplexed strand displacement amplification (CMSDA) for the detection of miR155 has been developed. Non-specific amplification was avoided by designing a single-stranded DNA template with a hairpin structure. The detection target miR155 was used as a primer to initiate a multiple-strand displacement reaction to produce abundant ssDNA. ssDNA was recognized by the Cas12a/CrRNA binary complex, activating the trans-cleaving activity of Cas12a. The multiple-strand displacement reaction is more efficiently detected compared with a single-strand displacement reaction. The detection range is from 250 pM to 1 nM, and the limit of the detection is 6.5 pM. The proposed method showed a good applicability in complex serum environments, indicating that the method has a broad prospect for disease detection and clinical application. In addition, we designed a dual-cavity PCR tube, which realized one-tube detection of miRNA155 and avoided open-cap contamination.},
}

*MicroRNAs/analysis/blood/genetics
Humans
*CRISPR-Cas Systems/genetics
DNA, Single-Stranded/chemistry/genetics
Limit of Detection
Nucleic Acid Amplification Techniques/methods
Polymerase Chain Reaction/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39023219,
year = {2024},
author = {López-Beltrán, A and Botelho, J and Iranzo, J},
title = {Dynamics of CRISPR-mediated virus-host interactions in the human gut microbiome.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wrae134},
pmid = {39023219},
issn = {1751-7370},
abstract = {Arms races between mobile genetic elements and prokaryotic hosts are major drivers of ecological and evolutionary change in microbial communities. Prokaryotic defense systems such as CRISPR-Cas have the potential to regulate microbiome composition by modifying the interactions among bacteria, plasmids, and phages. Here, we used longitudinal metagenomic data from 130 healthy and diseased individuals to study how the interplay of genetic parasites and CRISPR-Cas immunity reflects on the dynamics and composition of the human gut microbiome. Based on the coordinated study of 80 000 CRISPR-Cas loci and their targets, we show that CRISPR-Cas immunity effectively modulates bacteriophage abundances in the gut. Acquisition of CRISPR-Cas immunity typically leads to a decrease in the abundance of lytic phages but does not necessarily cause their complete disappearance. Much smaller effects are observed for lysogenic phages and plasmids. Conversely, phage-CRISPR interactions shape bacterial microdiversity by producing weak selective sweeps that benefit immune host lineages. We also show that distal (and chronologically older) regions of CRISPR arrays are enriched in spacers that are potentially functional and target crass-like phages and local prophages. This suggests that exposure to reactivated prophages and other endemic viruses is a major selective pressure in the gut microbiome that drives the maintenance of long-lasting immune memory.},
}

@article {pmid39023080,
year = {2024},
author = {Sha, L and Yao, J and Yang, S and Hu, M and Zhou, Q and Zhao, J and Bei, Y and Cao, Y},
title = {Collaborative CRISPR-Cas System-Enabled Detection of Circulating Circular RNA for Reliable Monitoring of Acute Myocardial Infarction.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e2402895},
doi = {10.1002/smll.202402895},
pmid = {39023080},
issn = {1613-6829},
support = {81972799//National Natural Science Foundation of China/ ; 81970335//National Natural Science Foundation of China/ ; 82170285//National Natural Science Foundation of China/ ; 20ZR1443300//Natural Science Foundation of Shanghai/ ; 23ZR1421400//Natural Science Foundation of Shanghai/ ; XZ2020ZR-YZ35//Natural Science Foundation of Xizang/ ; },
abstract = {Acute myocardial infarction (AMI) is one of the major causes of death worldwide, posing significant global health challenges. Circular RNA (circRNA) has recently emerged as a potential diagnostic biomarker for AMI, providing valuable information for timely medical care. In this work, a new electrochemical method for circRNA detection by engineering a collaborative CRISPR-Cas system is developed. This system integrates the unique circRNA-targeting ability with cascade trans-cleavage activities of Cas effectors, using an isothermal primer exchange reaction as the bridge. Using cZNF292, a circulating circRNA biomarker for AMI is identified by this group; as a model, the collaborative CRISPR-Cas system-based method exhibits excellent accuracy and sensitivity with a low detection limit of 2.13 × 10[-15] m. Moreover, the method demonstrates a good diagnostic performance for AMI when analyzing whole blood samples. Therefore, the method may provide new insight into the detection of circRNA biomarkers and is expected to have great potential in AMI diagnosis in the future.},
}

@article {pmid39021763,
year = {2024},
author = {Asadbeigi, A and Bakhtiarizadeh, MR and Saffari, M and Modarressi, MH and Sadri, N and Kafi, ZZ and Fazilaty, H and Ghalyanchilangeroudi, A and Esmaeili, H},
title = {Protection of animals against devastating RNA viruses using CRISPR-Cas13s.},
journal = {Molecular therapy. Nucleic acids},
volume = {35},
number = {3},
pages = {102235},
pmid = {39021763},
issn = {2162-2531},
abstract = {The intrinsic nature of CRISPR-Cas in conferring immunity to bacteria and archaea has been repurposed to combat pathogenic agents in mammalian and plant cells. In this regard, CRISPR-Cas13 systems have proved their remarkable potential for single-strand RNA viruses targeting. Here, different types of Cas13 orthologs were applied to knockdown foot-and-mouth disease virus (FMDV), a highly contagious disease of a wide variety of species with genetically diverse strains and is widely geographically distributed. Using programmable CRISPR RNAs capable of targeting conserved regions of the viral genome, all Cas13s from CRISPR system type VI (subtype A/B/D) could comprehensively target and repress different serotypes of FMDV virus. This approach has the potential to destroy all strains of a virus as targets the ultra-conserved regions of genome. We experimentally compared the silencing efficiency of CRISPR and RNAi by designing the most effective short hairpin RNAs according to our developed scoring system and observed comparable results. This study showed successful usage of various Cas13 enzymes for suppression of FMDV, which provides a flexible strategy to battle with other animal infectious RNA viruses, an underdeveloped field in the biotechnology scope.},
}

@article {pmid39020310,
year = {2024},
author = {Collier, TC and Lee, Y and Mathias, DK and Del Amo, VL},
title = {CRISPR-Cas9 and Cas12a target site richness reflects genomic diversity in natural populations of Anopheles gambiae and Aedes aegypti mosquitoes.},
journal = {BMC genomics},
volume = {25},
number = {1},
pages = {700},
pmid = {39020310},
issn = {1471-2164},
mesh = {Animals ; *Anopheles/genetics ; *CRISPR-Cas Systems ; *Aedes/genetics ; Genetic Variation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Genome, Insect ; Mosquito Vectors/genetics ; Gene Editing ; Bacterial Proteins ; },
abstract = {Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.},
}

Animals
*Anopheles/genetics
*CRISPR-Cas Systems
*Aedes/genetics
Genetic Variation
RNA, Guide, CRISPR-Cas Systems/genetics
Endodeoxyribonucleases/genetics/metabolism
CRISPR-Associated Proteins/genetics/metabolism
Genome, Insect
Mosquito Vectors/genetics
Gene Editing
Bacterial Proteins

@article {pmid39019776,
year = {2024},
author = {Nguyen, GT and Schelling, MA and Raju, A and Buscher, KA and Sritharan, A and Sashital, DG},
title = {CRISPR-Cas12a exhibits metal-dependent specificity switching.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae613},
pmid = {39019776},
issn = {1362-4962},
support = {1652661//National Science Foundation/ ; GM140876/NH/NIH HHS/United States ; //Iowa State University/ ; },
abstract = {Cas12a is the immune effector of type V-A CRISPR-Cas systems and has been co-opted for genome editing and other biotechnology tools. The specificity of Cas12a has been the subject of extensive investigation both in vitro and in genome editing experiments. However, in vitro studies have often been performed at high magnesium ion concentrations that are inconsistent with the free Mg2+ concentrations that would be present in cells. By profiling the specificity of Cas12a orthologs at a range of Mg2+ concentrations, we find that Cas12a switches its specificity depending on metal ion concentration. Lowering Mg2+ concentration decreases cleavage defects caused by seed mismatches, while increasing the defects caused by PAM-distal mismatches. We show that Cas12a can bind seed mutant targets more rapidly at low Mg2+ concentrations, resulting in faster cleavage. In contrast, PAM-distal mismatches cause substantial defects in cleavage following formation of the Cas12a-target complex at low Mg2+ concentrations. We observe differences in Cas12a specificity switching between three orthologs that results in variations in the routes of phage escape from Cas12a-mediated immunity. Overall, our results reveal the importance of physiological metal ion conditions on the specificity of Cas effectors that are used in different cellular environments.},
}

@article {pmid39018342,
year = {2024},
author = {Bubeck, F and Grimm, D},
title = {When size matters: A novel compact Cas12a variant for in vivo genome editing.},
journal = {PLoS biology},
volume = {22},
number = {7},
pages = {e3002637},
pmid = {39018342},
issn = {1545-7885},
mesh = {*Gene Editing/methods ; *Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; *Genetic Vectors/genetics ; Animals ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; },
abstract = {A new study characterizes and improves a novel small Cas12a variant before adapting it for in vivo genome editing by delivery via adeno-associated virus (AAV) vectors, showcasing the potential of small CRISPR systems and their compatibility with viral vectors.},
}

*Gene Editing/methods
*Dependovirus/genetics
*CRISPR-Cas Systems/genetics
*Genetic Vectors/genetics
Animals
CRISPR-Associated Proteins/genetics/metabolism
Humans
Bacterial Proteins/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism

@article {pmid39018338,
year = {2024},
author = {Vatanparast, M and Esmaeily, M and Stanley, D and Kim, Y},
title = {A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.},
journal = {PloS one},
volume = {19},
number = {7},
pages = {e0304958},
pmid = {39018338},
issn = {1932-6203},
mesh = {Animals ; *Eicosanoids/metabolism ; *CRISPR-Cas Systems ; *Spodoptera ; *Phospholipases A2/genetics/metabolism ; Signal Transduction ; Larva/genetics/immunology ; Insect Proteins/genetics/metabolism ; Sequence Deletion ; Genes, Insect ; Gene Editing ; Gene Deletion ; },
abstract = {Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.},
}

Animals
*Eicosanoids/metabolism
*CRISPR-Cas Systems
*Spodoptera
*Phospholipases A2/genetics/metabolism
Signal Transduction
Larva/genetics/immunology
Insect Proteins/genetics/metabolism
Sequence Deletion
Genes, Insect
Gene Editing
Gene Deletion

@article {pmid39018310,
year = {2024},
author = {Lee, HY and Min, YH and Lee, DG and Lee, KH and Kim, J and Lee, MK and Byun, JY and Shin, YB},
title = {CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c01246},
pmid = {39018310},
issn = {1520-6882},
abstract = {The clustered regularly interspaced short palindromic repeat/Cas (CRISPR/Cas) system is a powerful tool for nucleic acid detection owing to specific recognition as well as cis- and trans-cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid preamplification, which has several limitations. Here, we present a method for direct nucleic acid detection without preamplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up RNA aptamer transcription. We first designed two DNA templates to transcribe the light-up RNA aptamer and kleptamer (Kb) RNA: the first DNA template encodes a Broccoli RNA aptamer for fluorescence signal generation, and the Kb DNA template comprises a dsDNA T7 promoter sequence and an ssDNA sequence that encodes an antisense strand for the Broccoli RNA aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas12a complex, leading to the catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli DNA template can then produce several Broccoli RNA aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with 22.4 fM detection limit, good accuracy, and stability for determining the target HBV dsDNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.},
}

@article {pmid39017887,
year = {2024},
author = {Anupam, K and Laidlaw, BJ},
title = {In Vivo CRISPR/Cas9-Mediated Gene Ablation in Murine B Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2826},
number = {},
pages = {79-91},
pmid = {39017887},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *B-Lymphocytes/metabolism/immunology ; *Cell Differentiation/genetics ; *Gene Editing/methods ; Germinal Center/immunology/metabolism/cytology ; Gene Deletion ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-Cas9 genome editing is a powerful tool for assessing the functional role of candidate genes. In vitro CRISPR/Cas9 screens have been used to rapidly assess the role of thousands of genes in the differentiation and function of immune populations. However, the physiological relevance of a gene is often dependent on signals received in the tissue microenvironment, such as exposure to growth factors, chemokines, cytokines, and cell contact-dependent signals, which may not be recapitulated in an in vitro setting. Additionally, in vitro approaches are not sufficient to induce the differentiation of all cell populations limiting the cell types that can be screened. This has posed a major barrier to understanding the genes regulating the differentiation of germinal center B cells. Here, we describe an approach to perform an in vivo Crispr-Cas9 screen to specifically ablate genes in activated B cells. Using this approach, we have been able to reveal novel transcriptional regulators of germinal center B cell differentiation following viral infection.},
}

Animals
*CRISPR-Cas Systems
Mice
*B-Lymphocytes/metabolism/immunology
*Cell Differentiation/genetics
*Gene Editing/methods
Germinal Center/immunology/metabolism/cytology
Gene Deletion
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39017845,
year = {2024},
author = {Zheng, G and Orkin, SH},
title = {Transcriptional Repressor BCL11A in Erythroid Cells.},
journal = {Advances in experimental medicine and biology},
volume = {1459},
number = {},
pages = {199-215},
pmid = {39017845},
issn = {0065-2598},
mesh = {*Repressor Proteins/genetics/metabolism ; Humans ; Animals ; *Erythroid Cells/metabolism ; Anemia, Sickle Cell/genetics/metabolism ; Fetal Hemoglobin/genetics/metabolism ; Mice ; beta-Thalassemia/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; Nuclear Proteins/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; gamma-Globins/genetics/metabolism ; Gene Expression Regulation ; Genome-Wide Association Study ; },
abstract = {BCL11A, a zinc finger repressor, is a stage-specific transcription factor that controls the switch from fetal (HbF, α2γ2) to adult (HbA, α2β2) hemoglobin in erythroid cells. While BCL11A was known as a factor critical for B-lymphoid cell development, its relationship to erythroid cells and HbF arose through genome-wide association studies (GWAS). Subsequent work validated its role as a silencer of γ-globin gene expression in cultured cells and mice. Erythroid-specific loss of BCL11A rescues the phenotype of engineered sickle cell disease (SCD) mice, thereby suggesting that downregulation of BCL11A expression might be beneficial in patients with SCD and β-thalassemia. Common genetic variation in GWAS resides in an erythroid-specific enhancer within the BCL11A gene that is required for its own expression. CRISPR/Cas9 gene editing of the enhancer revealed a GATA-binding site that confers a large portion of its regulatory function. Disruption of the GATA site leads to robust HbF reactivation. Advancement of a guide RNA targeting the GATA-binding site in clinical trials has recently led to approval of first-in-man use of ex vivo CRISPR editing of hematopoietic stem/progenitor cells (HSPCs) as therapy of SCD and β-thalassemia. Future challenges include expanding access and infrastructure for delivery of genetic therapy to eligible patients, reducing potential toxicity and costs, exploring prospects for in vivo targeting of hematopoietic stem cells (HSCs), and developing small molecule drugs that impair function of BCL11A protein as an alternative option.},
}

*Repressor Proteins/genetics/metabolism
Humans
Animals
*Erythroid Cells/metabolism
Anemia, Sickle Cell/genetics/metabolism
Fetal Hemoglobin/genetics/metabolism
Mice
beta-Thalassemia/genetics/metabolism
CRISPR-Cas Systems
Gene Editing/methods
Nuclear Proteins/genetics/metabolism
Carrier Proteins/genetics/metabolism
gamma-Globins/genetics/metabolism
Gene Expression Regulation
Genome-Wide Association Study

@article {pmid39017814,
year = {2024},
author = {He, Q and Chen, Q and Lian, L and Qu, J and Yuan, X and Wang, C and Xu, L and Wei, J and Zeng, S and Yu, D and Dong, Y and Zhang, Y and Deng, L and Du, K and Zhang, C and Pandey, V and Gul, I and Qin, P},
title = {Unraveling the influence of CRISPR/Cas13a reaction components on enhancing trans-cleavage activity for ultrasensitive on-chip RNA detection.},
journal = {Mikrochimica acta},
volume = {191},
number = {8},
pages = {466},
pmid = {39017814},
issn = {1436-5073},
support = {31970752//National Natural Science Foundation of China/ ; 32350410397//National Natural Science Foundation of China/ ; JCYJ20190809180003689//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JSGG20191129110812708//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JSGG20200225150707332; JCYJ20220530143014032//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; JCYJ20230807113017035//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; WDZC20200820173710001//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; SZBL2020090501004//Shenzhen Bay Laboratory Open Funding/ ; 2020M680023//China Postdoctoral Science Foundation/ ; D2301002//Shenzhen Medical Research Funds/ ; DCE-iBHE-2022-3//Department of Chemical Engineering-iBHE special cooperation joint fund project/ ; JC2022009//Tsinghua Shenzhen International Graduate School/ ; (2022) 207//Ministry of Land and Resources of the People's Republic of China/ ; },
mesh = {*SARS-CoV-2/genetics ; *CRISPR-Cas Systems ; *RNA, Viral/analysis/genetics ; Humans ; COVID-19/diagnosis/virology ; Biosensing Techniques/methods ; CRISPR-Associated Proteins ; Microscopy, Fluorescence ; Lab-On-A-Chip Devices ; Limit of Detection ; Magnesium/chemistry ; COVID-19 Nucleic Acid Testing/methods ; },
abstract = {The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg[2+] concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.},
}

*SARS-CoV-2/genetics
*CRISPR-Cas Systems
*RNA, Viral/analysis/genetics
Humans
COVID-19/diagnosis/virology
Biosensing Techniques/methods
CRISPR-Associated Proteins
Microscopy, Fluorescence
Lab-On-A-Chip Devices
Limit of Detection
Magnesium/chemistry
COVID-19 Nucleic Acid Testing/methods

@article {pmid39017499,
year = {2024},
author = {Makin, S},
title = {Rapid Answers: Gene-editing technology gives diagnostic tests a boost.},
journal = {Scientific American},
volume = {331},
number = {1},
pages = {12},
doi = {10.1038/scientificamerican072024-2MXPoUcSWyso9xhGc3BU5U},
pmid = {39017499},
issn = {0036-8733},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; },
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems

@article {pmid39014928,
year = {2024},
author = {Huang, L and Li, N and Song, Y and Gao, J and Nian, L and Zhou, J and Zhang, B and Liu, Z and Zheng, Y},
title = {Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi.},
journal = {Biotechnology journal},
volume = {19},
number = {7},
pages = {e2400164},
doi = {10.1002/biot.202400164},
pmid = {39014928},
issn = {1860-7314},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Fusarium/genetics ; Plasmids/genetics ; Metabolic Engineering/methods ; Genetic Markers/genetics ; },
abstract = {Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. Thus, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system has emerged as a promising tool for precise genome editing in this organism. Some integrated CRISPR/Cas9 strategies have been used to engineer F. fujikuroi to improve GA3 production capabilities, but low editing efficiency and possible genomic instability became the major obstacle. Herein, we developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi. This system, based on an autonomously replicating sequence, demonstrated the capability of a single plasmid harboring all editing components to achieve 100%, 75%, and 37.5% editing efficiency for single, double, and triple gene targets, respectively. Remarkably, even with a reduction in homologous arms to 50 bp, we achieved a 12.5% gene editing efficiency. By employing this system, we successfully achieved multicopy integration of the truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene (tHMGR), leading to enhanced GA3 production. A key advantage of our plasmid-based gene editing approach was the ability to recycle selective markers through a simplified protoplast preparation and recovery process, which eliminated the need for additional genetic markers. These findings demonstrated that the single-plasmid CRISPR/Cas9 system enables rapid and precise multiple gene deletions/integrations, laying a solid foundation for future metabolic engineering efforts aimed at industrial GA3 production.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Fusarium/genetics
Plasmids/genetics
Metabolic Engineering/methods
Genetic Markers/genetics

@article {pmid39012602,
year = {2024},
author = {Sarno, F and Koncz, M and Eilers, RE and Verschure, PJ and Rots, MG},
title = {Generation of Cell Lines Stably Expressing a dCas9-Fusion or sgRNA to Address Dynamics of Long-Term Effects of Epigenetic Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {289-307},
pmid = {39012602},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; },
abstract = {Epigenetic modifications play a crucial role in regulating gene expression patterns. Through epigenetic editing approaches, the chromatin structure is modified and the activity of the targeted gene can be reprogrammed without altering the DNA sequence. By using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic repeats) platform with nuclease-deactivated dCas9 proteins to direct epigenetic effector domains (EDs) to genomic regulatory regions, the expression of the targeted gene can be modulated. However, the long-term stability of these effects, although demonstrated, remains unpredictable. The versatility and flexibility of (co-)targeting different genes with multiple epigenetic effectors has made the CRISPR/dCas9 platform the most widely used gene modulating technology currently available. Efficient delivery of large dCas9-ED fusion constructs into target cells, however, is challenging. An approach to overcome this limitation is to generate cells that stably express sgRNA(s) or dCas9-ED constructs. The sgRNA(s) or dCas9-ED stable cell lines can be used to study the mechanisms underlying sustained gene expression reprogramming by transiently expressing the other of the two constructs. Here, we describe a detailed protocol for the engineering of cells that stably express CRISPR/dCas9 or sgRNA. Creating a system where one component of the CRISPR/dCas9 is stably expressed while the other is transiently expressed offers a versatile platform for investigating the dynamics of epigenetic reprogramming.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Epigenesis, Genetic
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
Cell Line
CRISPR-Associated Protein 9/metabolism/genetics
HEK293 Cells

@article {pmid39012601,
year = {2024},
author = {Woodward, EA and Wang, E and Wallis, C and Sharma, R and Tie, AWJ and Murthy, N and Blancafort, P},
title = {Protocol for Delivery of CRISPR/dCas9 Systems for Epigenetic Editing into Solid Tumors Using Lipid Nanoparticles Encapsulating RNA.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {267-287},
pmid = {39012601},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles/chemistry ; Animals ; *Neoplasms/genetics/therapy ; Epigenesis, Genetic ; Mice ; RNA, Guide, CRISPR-Cas Systems/genetics ; Liposomes/chemistry ; Cell Line, Tumor ; Lipids/chemistry ; CRISPR-Associated Protein 9/genetics/metabolism ; Genetic Therapy/methods ; Gene Transfer Techniques ; },
abstract = {Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
*Nanoparticles/chemistry
Animals
*Neoplasms/genetics/therapy
Epigenesis, Genetic
Mice
RNA, Guide, CRISPR-Cas Systems/genetics
Liposomes/chemistry
Cell Line, Tumor
Lipids/chemistry
CRISPR-Associated Protein 9/genetics/metabolism
Genetic Therapy/methods
Gene Transfer Techniques

@article {pmid39012600,
year = {2024},
author = {van den Berg van Saparoea, ACH and van Loosen, QC and Sarno, F and Ntini, E and Rots, MG and Gjaltema, RAF and Verschure, PJ},
title = {Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {255-265},
pmid = {39012600},
issn = {1940-6029},
mesh = {Humans ; *Plasmids/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; HEK293 Cells ; *Transfection/methods ; *Epigenesis, Genetic ; *Single-Cell Analysis/methods ; Epigenomics/methods ; Flow Cytometry ; },
abstract = {To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.},
}

Humans
*Plasmids/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
HEK293 Cells
*Transfection/methods
*Epigenesis, Genetic
*Single-Cell Analysis/methods
Epigenomics/methods
Flow Cytometry

@article {pmid39012598,
year = {2024},
author = {Azcona, MSR and Mussolino, C},
title = {Protocol for Efficient Generation of Chimeric Antigen Receptor T Cells with Multiplexed Gene Silencing by Epigenome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {209-223},
pmid = {39012598},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Humans ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Gene Silencing ; T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; Programmed Cell Death 1 Receptor/genetics ; Epigenome ; },
abstract = {Multiplex gene regulation enables the controlled and simultaneous alteration of the expression levels of multiple genes and is generally pursued to precisely alter complex cellular pathways with a single intervention. Thus far, this has been typically exploited in combination with genome editing tools (i.e., base-/prime-editing, designer nucleases) to enable simultaneous genetic alterations and modulate complex physiologic cellular pathways. In the field of cancer immunotherapy, multiplex genome editing has been used to simultaneously inactivate three genes (i.e., TRAC, B2M, and PDCD1) and generate universal chimeric antigen receptor (CAR) T cells resistant to the inhibitory activity of the PD-1 ligand. However, the intrinsic risk of genomic aberrations driven by such tools poses concerns because of the generation of multiple single-strand or double-strand DNA breaks followed by DNA repair. Modulating gene expression without DNA damage using epigenome editing promises a safer and efficient approach to alter gene expression. This method enables for simultaneous activation and/or repression of target genes, offering superior fine-tuning capabilities with reduced off-targeting effects and potential reversibility as compared to genome editing. Here we describe a detailed protocol for achieving multiplexed and sustainable gene silencing in CAR T cells. In an exemplary approach, we use designer epigenome modifiers (DEMs) for the simultaneous inactivation of two T cell inhibitory genes, PDCD1 and LAG3 to generate CAR T cells with increased resistance to tumor-induced exhaustion.},
}

*Gene Editing/methods
Humans
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Gene Silencing
T-Lymphocytes/immunology/metabolism
CRISPR-Cas Systems
Immunotherapy, Adoptive/methods
Programmed Cell Death 1 Receptor/genetics
Epigenome

@article {pmid39012597,
year = {2024},
author = {Oberkofler, V and Bäurle, I},
title = {Plant Epigenetic Editing to Analyze the Function of Histone Modifications in Gene-Specific Regulation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {193-207},
pmid = {39012597},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Histones/metabolism/genetics ; *Epigenesis, Genetic ; *Gene Expression Regulation, Plant ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Histone Code ; Chromatin/genetics/metabolism ; Promoter Regions, Genetic ; Plants, Genetically Modified/genetics ; Plants/genetics/metabolism ; Arabidopsis/genetics/metabolism ; },
abstract = {Epigenetic editing enables the locus-specific manipulation of chromatin modifications. It allows the functional analysis of interactions between chromatin modifications and epigenetically stable gene expression states, thus establishing causal relationships, where previously correlations were suspected. Here, we describe the procedures for gene-specific epigenetic editing in plants that are based on targeting a histone modifier using an inactive dCas9 fusion protein that is recruited by a set of three distinct single guide RNAs (sgRNAs) that all target a region within the promoter of the target gene. We outline design principles and emphasize the need for suitable control constructs. In summary, the protocol will be widely useful for plant scientists looking to manipulate chromatin modifications in a locus-specific manner.},
}

*Gene Editing/methods
*Histones/metabolism/genetics
*Epigenesis, Genetic
*Gene Expression Regulation, Plant
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Histone Code
Chromatin/genetics/metabolism
Promoter Regions, Genetic
Plants, Genetically Modified/genetics
Plants/genetics/metabolism
Arabidopsis/genetics/metabolism

@article {pmid39012596,
year = {2024},
author = {Rajaram, N and Bashtrykov, P and Jeltsch, A},
title = {Protocol for Allele-Specific Epigenome Editing Using CRISPR/dCas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {179-192},
pmid = {39012596},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Alleles ; *DNA Methylation ; HEK293 Cells ; *Epigenome ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epigenomics/methods ; Epigenesis, Genetic ; },
abstract = {The discovery and adaptation of CRISPR/Cas systema for epigenome editing has allowed for a straightforward design of targeting modules that can direct epigenome editors to virtually any genomic site. This advancement in DNA-targeting technology brings allele-specific epigenome editing into reach, a "super-specific" variation of epigenome editing whose goal is an alteration of chromatin marks at only one selected allele of the genomic target locus. This technology could be useful for the treatment of diseases caused by a mutant allele with a dominant effect, because allele-specific epigenome editing allows the specific silencing of the mutated allele leaving the healthy counterpart expressed. Moreover, it may allow the direct correction of aberrant imprints in imprinting disorders where editing of DNA methylation is required exclusively in a single allele. Here, we describe a basic protocol for the design and application of allele-specific epigenome editing systems using allele-specific DNA methylation at the NARF gene in HEK293 cells as an example. An sgRNA/dCas9 unit is used for allele-specific binding to the target locus containing a SNP in the seed region of the sgRNA or the PAM region. The dCas9 protein is connected to a SunTag allowing to recruit up to 10 DNMT3A/3L units fused to a single-chain Fv fragment, which specifically binds to the SunTag peptide sequence. The plasmids expressing dCas9-10x SunTag, scFv-DNMT3A/3L, and sgRNA, each of them co-expressing a fluorophore, are introduced into cells by co-transfection. Cells containing all three plasmids are enriched by FACS, cultivated, and later the genomic DNA and RNA can be retrieved for DNA methylation and gene expression analysis.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Alleles
*DNA Methylation
HEK293 Cells
*Epigenome
RNA, Guide, CRISPR-Cas Systems/genetics
Epigenomics/methods
Epigenesis, Genetic

@article {pmid39012595,
year = {2024},
author = {Sekita, Y and Kimura, T},
title = {Protocol for DNA Methylation Editing of Imprinted Loci and Assessment of the Effects.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {167-178},
pmid = {39012595},
issn = {1940-6029},
mesh = {*Genomic Imprinting ; *DNA Methylation ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; Mice ; Genetic Loci ; Calcium-Binding Proteins/genetics/metabolism ; Iodide Peroxidase/genetics ; Alleles ; Humans ; },
abstract = {In this chapter, we present an experimental protocol to conduct DNA methylation editing experiments, that is, to induce loss or gain of DNA methylation, targeting Dlk1-Dio3 imprinted domain, a well-studied imprinted locus, in ES cells. In this protocol, plasmid vectors expressing the DNA methylation editing tools, combining the CRISPR/dCas9 system and the SunTag system coupled to a DNA methyltransferase or a TET enzyme, are introduced into cells for transient expression. By employing this strategy, researchers can effectively investigate a distinct DNA methylation signature that has an impact on the imprinting status, including gene expression and histone modifications, across the entire domain. We also describe strategies for allele-specific quantitative analyses of DNA methylation, gene expression, and histone modifications and binding protein levels for assessing the imprinting state of the locus.},
}

*Genomic Imprinting
*DNA Methylation
*Gene Editing/methods
Animals
*CRISPR-Cas Systems
Mice
Genetic Loci
Calcium-Binding Proteins/genetics/metabolism
Iodide Peroxidase/genetics
Alleles
Humans

@article {pmid39012594,
year = {2024},
author = {Morita, S and Horii, T and Hatada, I},
title = {Optimized Protocol for the Regulation of DNA Methylation and Gene Expression Using Modified dCas9-SunTag Platforms.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {155-165},
pmid = {39012594},
issn = {1940-6029},
mesh = {*DNA Methylation ; Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Gene Expression Regulation ; Epigenesis, Genetic ; Mixed Function Oxygenases/genetics/metabolism ; Proto-Oncogene Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.},
}

*DNA Methylation
Humans
*CRISPR-Cas Systems
Gene Editing/methods
Gene Expression Regulation
Epigenesis, Genetic
Mixed Function Oxygenases/genetics/metabolism
Proto-Oncogene Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39012593,
year = {2024},
author = {Hamilton, PJ and Lim, CJ and Nestler, EJ and Heller, EA},
title = {Neuroepigenetic Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {129-152},
pmid = {39012593},
issn = {1940-6029},
mesh = {*Epigenesis, Genetic ; Animals ; Humans ; *Gene Editing/methods ; Neurons/metabolism ; Brain/metabolism ; Histones/metabolism ; Chromatin/genetics/metabolism ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Epigenetic regulation is intrinsic to basic neurobiological function as well as neurological disease. Regulation of chromatin-modifying enzymes in the brain is critical during both development and adulthood and in response to external stimuli. Biochemical studies are complemented by numerous next-generation sequencing (NGS) studies that quantify global changes in gene expression, chromatin accessibility, histone and DNA modifications in neurons and glial cells. Neuroepigenetic editing tools are essential to distinguish between the mere presence and functional relevance of histone and DNA modifications to gene transcription in the brain and animal behavior. This review discusses current advances in neuroepigenetic editing, highlighting methodological considerations pertinent to neuroscience, such as delivery methods and the spatiotemporal specificity of editing and it demonstrates the enormous potential of epigenetic editing for basic neurobiological research and therapeutic application.},
}

*Epigenesis, Genetic
Animals
Humans
*Gene Editing/methods
Neurons/metabolism
Brain/metabolism
Histones/metabolism
Chromatin/genetics/metabolism
CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing/methods

@article {pmid39012592,
year = {2024},
author = {Kim, SH and Haynes, KA},
title = {Reader-Effectors as Actuators of Epigenome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2842},
number = {},
pages = {103-127},
pmid = {39012592},
issn = {1940-6029},
mesh = {Animals ; Humans ; Chromatin/genetics/metabolism ; Chromatin Assembly and Disassembly ; CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Epigenome ; Epigenomics/methods ; *Gene Editing/methods ; Gene Expression Regulation ; },
abstract = {Epigenome editing applications are gaining broader use for targeted transcriptional control as more enzymes with diverse chromatin-modifying functions are being incorporated into fusion proteins. Development of these fusion proteins, called epigenome editors, has outpaced the study of proteins that interact with edited chromatin. One type of protein that acts downstream of chromatin editing is the reader-effector, which bridges epigenetic marks with biological effects like gene regulation. As the name suggests, a reader-effector protein is generally composed of a reader domain and an effector domain. Reader domains directly bind epigenetic marks, while effector domains often recruit protein complexes that mediate transcription, chromatin remodeling, and DNA repair. In this chapter, we discuss the role of reader-effectors in driving the outputs of epigenome editing and highlight instances where abnormal and context-specific reader-effectors might impair the effects of epigenome editing. Lastly, we discuss how engineered reader-effectors may complement the epigenome editing toolbox to achieve robust and reliable gene regulation.},
}

Animals
Humans
Chromatin/genetics/metabolism
Chromatin Assembly and Disassembly
CRISPR-Cas Systems
*Epigenesis, Genetic
*Epigenome
Epigenomics/methods
*Gene Editing/methods
Gene Expression Regulation