@article {pmid33479216,
year = {2021},
author = {Li, S and Akrap, N and Cerboni, S and Porritt, MJ and Wimberger, S and Lundin, A and Möller, C and Firth, M and Gordon, E and Lazovic, B and Sieńska, A and Pane, LS and Coelho, MA and Ciotta, G and Pellegrini, G and Sini, M and Xu, X and Mitra, S and Bohlooly-Y, M and Taylor, BJM and Sienski, G and Maresca, M},
title = {Universal toxin-based selection for precise genome engineering in human cells.},
journal = {Nature communications},
volume = {12},
number = {1},
pages = {497},
pmid = {33479216},
issn = {2041-1723},
abstract = {Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.},
}

@article {pmid33478937,
year = {2020},
author = {Nakato, M and Shiranaga, N and Tomioka, M and Watanabe, H and Kurisu, J and Kengaku, M and Komura, N and Ando, H and Kimura, Y and Kioka, N and Ueda, K},
title = {ABCA13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking.},
journal = {The Journal of biological chemistry},
volume = {296},
number = {},
pages = {100166},
doi = {10.1074/jbc.RA120.015997},
pmid = {33478937},
issn = {1083-351X},
abstract = {ATP-binding cassette subfamily A member 13 (ABCA13) is predicted to be the largest ABC protein, consisting of 5058 amino acids and a long N-terminal region. Mutations in the ABCA13 gene were reported to increase the susceptibility to schizophrenia, bipolar disorder, and major depression. However, little is known about the molecular functions of ABCA13 or how they associate with psychiatric disorders. Here, we examined the biochemical activity of ABCA13 using HEK293 cells transfected with mouse ABCA13. The expression of ABCA13 induced the internalization of cholesterol and gangliosides from the plasma membrane to intracellular vesicles. Cholesterol internalization by ABCA13 required the long N-terminal region and ATP hydrolysis. To examine the physiological roles of ABCA13, we generated Abca13 KO mice using CRISPR/Cas and found that these mice exhibited deficits of prepulse inhibition. Vesicular cholesterol accumulation and synaptic vesicle endocytosis were impaired in primary cultures of Abca13 KO cortical neurons. Furthermore, mutations in ABCA13 gene associated with psychiatric disorders disrupted the protein's subcellular localization and impaired cholesterol trafficking. These findings suggest that ABCA13 accelerates cholesterol internalization by endocytic retrograde transport in neurons and that loss of this function is associated with the pathophysiology of psychiatric disorders.},
}

@article {pmid33478513,
year = {2021},
author = {Asemoloye, MD and Marchisio, MA and Gupta, VK and Pecoraro, L},
title = {Genome-based engineering of ligninolytic enzymes in fungi.},
journal = {Microbial cell factories},
volume = {20},
number = {1},
pages = {20},
pmid = {33478513},
issn = {1475-2859},
abstract = {BACKGROUND: Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes.

SHORT CONCLUSION: Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.},
}

@article {pmid33478128,
year = {2021},
author = {Varanda, CM and Félix, MDR and Campos, MD and Patanita, M and Materatski, P},
title = {Plant Viruses: From Targets to Tools for CRISPR.},
journal = {Viruses},
volume = {13},
number = {1},
pages = {},
doi = {10.3390/v13010141},
pmid = {33478128},
issn = {1999-4915},
support = {PTDC/ASP-PLA/28266/2017//Fundação para a Ciência e a Tecnologia/ ; PTDC/ASP-PLA/28263/2017//Fundação para a Ciência e a Tecnologia/ ; UIDB/05183/2020//Fundação para a Ciência e a Tecnologia/ ; ALT20-03-0145-FEDER-028266//European Regional Development Fund/ ; ALT20-03-0145-FEDER-028263//European Regional Development Fund/ ; SFRH/BD/145321/2019//Fundação para a Ciência e a Tecnologia/ ; },
abstract = {Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present, there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virus-resistant varieties, but which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for 'clustered regularly interspaced short palindromic repeats' and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology can been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection; or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different, beneficial perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry, and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology; namely, to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing some of the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects.},
}

@article {pmid33471982,
year = {2021},
author = {Malech, HL},
title = {Treatment by CRISPR-Cas9 Gene Editing - A Proof of Principle.},
journal = {The New England journal of medicine},
volume = {384},
number = {3},
pages = {286-287},
doi = {10.1056/NEJMe2034624},
pmid = {33471982},
issn = {1533-4406},
mesh = {*Anemia, Sickle Cell ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; *beta-Thalassemia ; },
}

*Anemia, Sickle Cell
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Humans
*beta-Thalassemia

@article {pmid33471981,
year = {2021},
author = {Walters, MC},
title = {Induction of Fetal Hemoglobin by Gene Therapy.},
journal = {The New England journal of medicine},
volume = {384},
number = {3},
pages = {284-285},
doi = {10.1056/NEJMe2034338},
pmid = {33471981},
issn = {1533-4406},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Fetal Hemoglobin/genetics ; Gene Editing ; Genetic Therapy ; Humans ; *beta-Thalassemia/genetics ; },
}

*Anemia, Sickle Cell/genetics/therapy
CRISPR-Cas Systems
Fetal Hemoglobin/genetics
Gene Editing
Genetic Therapy
Humans
*beta-Thalassemia/genetics

@article {pmid33346188,
year = {2020},
author = {Li, H and Qin, H and Zhang, N and Zhao, J and Xin, J and Perez-Campo, FM and Liu, H},
title = {Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {166},
pages = {},
doi = {10.3791/59639},
pmid = {33346188},
issn = {1940-087X},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA/metabolism ; DNA Repair ; *Gene Knockout Techniques ; *Genes, Reporter ; Genetic Vectors/metabolism ; Luciferases/genetics/*metabolism ; Mammals/*metabolism ; Oligonucleotides/metabolism ; Plasmids/*genetics ; RNA, Guide/genetics ; Reproducibility of Results ; Sheep ; Transformation, Genetic ; },
abstract = {Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).},
}

Animals
Base Sequence
CRISPR-Cas Systems/*genetics
Cell Line
DNA/metabolism
DNA Repair
*Gene Knockout Techniques
*Genes, Reporter
Genetic Vectors/metabolism
Luciferases/genetics/*metabolism
Mammals/*metabolism
Oligonucleotides/metabolism
Plasmids/*genetics
RNA, Guide/genetics
Reproducibility of Results
Sheep
Transformation, Genetic

@article {pmid33305314,
year = {2021},
author = {Levi, O and Arava, YS},
title = {Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase.},
journal = {Nucleic acids research},
volume = {49},
number = {1},
pages = {432-443},
pmid = {33305314},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; *Gene Expression Regulation, Fungal ; Methionine/metabolism ; Methionine-tRNA Ligase/*metabolism ; Peptide Elongation Factors/*biosynthesis/genetics ; Polyribosomes/metabolism ; Protein Binding ; *Protein Biosynthesis ; Pseudouridine/*physiology ; RNA Processing, Post-Transcriptional ; RNA, Fungal/*genetics ; RNA, Messenger/*genetics ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/biosynthesis/genetics/*metabolism ; },
abstract = {Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNAMet synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNAMet and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 'writes' modifications on tRNA and mRNA, and both types of RNAs are 'read' by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.},
}

CRISPR-Cas Systems
*Gene Expression Regulation, Fungal
Methionine/metabolism
Methionine-tRNA Ligase/*metabolism
Peptide Elongation Factors/*biosynthesis/genetics
Polyribosomes/metabolism
Protein Binding
*Protein Biosynthesis
Pseudouridine/*physiology
RNA Processing, Post-Transcriptional
RNA, Fungal/*genetics
RNA, Messenger/*genetics
Saccharomyces cerevisiae/genetics/*metabolism
Saccharomyces cerevisiae Proteins/biosynthesis/genetics/*metabolism

@article {pmid33283228,
year = {2021},
author = {Cipullo, M and Pearce, SF and Lopez Sanchez, IG and Gopalakrishna, S and Krüger, A and Schober, F and Busch, JD and Li, X and Wredenberg, A and Atanassov, I and Rorbach, J},
title = {Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly.},
journal = {Nucleic acids research},
volume = {49},
number = {1},
pages = {354-370},
pmid = {33283228},
issn = {1362-4962},
mesh = {Bone Neoplasms/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Regulation ; Gene Knockout Techniques ; Guanosine Triphosphate/metabolism ; HEK293 Cells ; Humans ; Mitochondrial Proteins/*metabolism ; Mitochondrial Ribosomes/*metabolism ; Monomeric GTP-Binding Proteins/*physiology ; Osteosarcoma/pathology ; Oxidative Phosphorylation ; Protein Interaction Mapping ; Ribosomal Proteins/*metabolism ; Ribosome Subunits, Large, Eukaryotic/*metabolism ; },
abstract = {Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.},
}

Bone Neoplasms/pathology
CRISPR-Cas Systems
Cell Line, Tumor
Gene Expression Regulation
Gene Knockout Techniques
Guanosine Triphosphate/metabolism
HEK293 Cells
Humans
Mitochondrial Proteins/*metabolism
Mitochondrial Ribosomes/*metabolism
Monomeric GTP-Binding Proteins/*physiology
Osteosarcoma/pathology
Oxidative Phosphorylation
Protein Interaction Mapping
Ribosomal Proteins/*metabolism
Ribosome Subunits, Large, Eukaryotic/*metabolism

@article {pmid32563448,
year = {2020},
author = {Navarro-Serna, S and Vilarino, M and Park, I and Gadea, J and Ross, PJ},
title = {Livestock Gene Editing by One-step Embryo Manipulation.},
journal = {Journal of equine veterinary science},
volume = {89},
number = {},
pages = {103025},
doi = {10.1016/j.jevs.2020.103025},
pmid = {32563448},
issn = {0737-0806},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/veterinary ; *Livestock ; },
abstract = {The breakthrough and rapid advance of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has enabled the efficient generation of gene-edited animals by one-step embryo manipulation. Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 delivery to the livestock embryos has been typically achieved by intracytoplasmic microinjection; however, recent studies show that electroporation may be a reliable, efficient, and practical method for CRISPR/Cas9 delivery. The source of embryos used to generate gene-edited animals varies from in vivo to in vitro produced, depending mostly on the species of interest. In addition, different Cas9 and gRNA reagents can be used for embryo editing, ranging from Cas9-coding plasmid or messenger RNA to Cas9 recombinant protein, which can be combined with in vitro transcribed or synthetic guide RNAs. Mosaicism is reported as one of the main problems with generation of animals by embryo editing. On the other hand, off-target mutations are rarely found in livestock derived from one-step editing. In this review, we discussed these and other aspects of generating gene-edited animals by single-step embryo manipulation.},
}

Animals
CRISPR-Associated Protein 9
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing/veterinary
*Livestock

@article {pmid32458346,
year = {2020},
author = {Chen, B and Deng, S and Ge, T and Ye, M and Yu, J and Lin, S and Ma, W and Songyang, Z},
title = {Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in.},
journal = {Protein & cell},
volume = {11},
number = {9},
pages = {641-660},
pmid = {32458346},
issn = {1674-8018},
mesh = {*CRISPR-Cas Systems ; *Cell Tracking ; *Gene Expression Profiling ; *Gene Knock-In Techniques ; HEK293 Cells ; Humans ; *Proteomics ; RNA, Long Noncoding/genetics/*metabolism ; },
abstract = {In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.},
}

*CRISPR-Cas Systems
*Cell Tracking
*Gene Expression Profiling
*Gene Knock-In Techniques
HEK293 Cells
Humans
*Proteomics
RNA, Long Noncoding/genetics/*metabolism

@article {pmid32246439,
year = {2020},
author = {Jia, F and Li, X and Zhang, C and Tang, X},
title = {The expanded development and application of CRISPR system for sensitive nucleotide detection.},
journal = {Protein & cell},
volume = {11},
number = {9},
pages = {624-629},
pmid = {32246439},
issn = {1674-8018},
mesh = {Animals ; *CRISPR-Cas Systems ; Humans ; RNA, Viral/*genetics ; Viruses/*genetics ; },
}

Animals
*CRISPR-Cas Systems
Humans
RNA, Viral/*genetics
Viruses/*genetics

@article {pmid32004507,
year = {2020},
author = {Papanicolaou, KN and Ashok, D and Liu, T and Bauer, TM and Sun, J and Li, Z and da Costa, E and D'Orleans, CC and Nathan, S and Lefer, DJ and Murphy, E and Paolocci, N and Foster, DB and O'Rourke, B},
title = {Global knockout of ROMK potassium channel worsens cardiac ischemia-reperfusion injury but cardiomyocyte-specific knockout does not: Implications for the identity of mitoKATP.},
journal = {Journal of molecular and cellular cardiology},
volume = {139},
number = {},
pages = {176-189},
doi = {10.1016/j.yjmcc.2020.01.010},
pmid = {32004507},
issn = {1095-8584},
support = {ZIA HL002066/ImNIH/Intramural NIH HHS/United States ; R01 HL136918/HL/NHLBI NIH HHS/United States ; K12 HL141952/HL/NHLBI NIH HHS/United States ; F31 HL134198/HL/NHLBI NIH HHS/United States ; R01 HL137259/HL/NHLBI NIH HHS/United States ; R01 HL092141/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Electrophysiological Phenomena ; Gene Editing ; Gene Knockout Techniques ; Hemodynamics ; Ischemic Preconditioning, Myocardial ; Mice, Knockout ; Mitochondria, Heart/metabolism ; Myocardial Reperfusion Injury/*metabolism/pathology/physiopathology ; Myocardium/metabolism/pathology ; Myocytes, Cardiac/*metabolism/pathology ; Organ Specificity ; Perfusion ; Phenotype ; Potassium Channels/*metabolism ; Potassium Channels, Inwardly Rectifying/*deficiency/metabolism ; },
abstract = {The renal-outer-medullary‑potassium (ROMK) channel, mutated in Bartter's syndrome, regulates ion exchange in kidney, but its extra-renal functions remain unknown. Additionally, ROMK was postulated to be the pore-forming subunit of the mitochondrial ATP-sensitive K+ channel (mitoKATP), a mediator of cardioprotection. Using global and cardiomyocyte-specific knockout mice (ROMK-GKO and ROMK-CKO respectively), we characterize the effects of ROMK knockout on mitochondrial ion handling, the response to pharmacological KATP channel modulators, and ischemia/reperfusion (I/R) injury. Mitochondria from ROMK-GKO hearts exhibited a lower threshold for Ca2+-triggered permeability transition pore (mPTP) opening but normal matrix volume changes during oxidative phosphorylation. Isolated perfused ROMK-GKO hearts exhibited impaired functional recovery and increased infarct size when I/R was preceded by an ischemic preconditioning (IPC) protocol. Because ROMK-GKO mice exhibited severe renal defects and cardiac remodeling, we further characterized ROMK-CKO hearts to avoid confounding systemic effects. Mitochondria from ROMK-CKO hearts had unchanged matrix volume responses during oxidative phosphorylation and still swelled upon addition of a mitoKATP opener, but exhibited a lower threshold for mPTP opening, similar to GKO mitochondria. Nevertheless, I/R induced damage was not exacerbated in ROMK-CKO hearts, either ex vivo or in vivo. Lastly, we examined the response of ROMK-CKO hearts to ex vivo I/R injury with or without IPC and found that IPC still protected these hearts, suggesting that cardiomyocyte ROMK does not participate significantly in the cardioprotective pathway elicited by IPC. Collectively, our findings from these novel strains of mice suggest that cardiomyocyte ROMK is not a central mediator of mitoKATP function, although it can affect mPTP activation threshold.},
}

Animals
Animals, Newborn
CRISPR-Cas Systems/genetics
Calcium/metabolism
Electrophysiological Phenomena
Gene Editing
Gene Knockout Techniques
Hemodynamics
Ischemic Preconditioning, Myocardial
Mice, Knockout
Mitochondria, Heart/metabolism
Myocardial Reperfusion Injury/*metabolism/pathology/physiopathology
Myocardium/metabolism/pathology
Myocytes, Cardiac/*metabolism/pathology
Organ Specificity
Perfusion
Phenotype
Potassium Channels/*metabolism
Potassium Channels, Inwardly Rectifying/*deficiency/metabolism

@article {pmid31895004,
year = {2020},
author = {Bennett, HW and Gustavsson, AK and Bayas, CA and Petrov, PN and Mooney, N and Moerner, WE and Jackson, PK},
title = {Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy.},
journal = {Molecular biology of the cell},
volume = {31},
number = {7},
pages = {619-639},
pmid = {31895004},
issn = {1939-4586},
support = {R35 GM118067/GM/NIGMS NIH HHS/United States ; R01 GM121565/GM/NIGMS NIH HHS/United States ; R01 GM114276/GM/NIGMS NIH HHS/United States ; K99 GM134187/GM/NIGMS NIH HHS/United States ; T32 AG047126/AG/NIA NIH HHS/United States ; },
mesh = {Biomarkers/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line ; Cilia/*metabolism ; Green Fluorescent Proteins/metabolism ; Humans ; *Imaging, Three-Dimensional ; Kinesin/metabolism ; *Microscopy ; Models, Biological ; Mutation/genetics ; NIMA-Related Kinases/metabolism ; Nuclear Proteins/metabolism ; Protein Transport ; *Single Molecule Imaging ; Transcription Factors/*metabolism ; },
abstract = {Primary cilia in many cell types contain a periaxonemal subcompartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development, including left-right asymmetry and renal tissue homeostasis. Here we combine superresolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein-protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by three-dimensional single-molecule superresolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein-protein interactions.},
}

Biomarkers/metabolism
CRISPR-Cas Systems/genetics
Cell Line
Cilia/*metabolism
Green Fluorescent Proteins/metabolism
Humans
*Imaging, Three-Dimensional
Kinesin/metabolism
*Microscopy
Models, Biological
Mutation/genetics
NIMA-Related Kinases/metabolism
Nuclear Proteins/metabolism
Protein Transport
*Single Molecule Imaging
Transcription Factors/*metabolism

@article {pmid33475257,
year = {2021},
author = {Vavassori, V and Mercuri, E and Marcovecchio, GE and Castiello, MC and Schiroli, G and Albano, L and Margulies, C and Buquicchio, F and Fontana, E and Beretta, S and Merelli, I and Cappelleri, A and Rancoita, PM and Lougaris, V and Plebani, A and Kanariou, M and Lankester, A and Ferrua, F and Scanziani, E and Cotta-Ramusino, C and Villa, A and Naldini, L and Genovese, P},
title = {Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome.},
journal = {EMBO molecular medicine},
volume = {},
number = {},
pages = {e13545},
doi = {10.15252/emmm.202013545},
pmid = {33475257},
issn = {1757-4684},
support = {TIGET-E3//Fondazione Telethon (Telethon Foundation)/ ; GR-2013-02358956//Ministero della Salute (Ministry of Health, Italy)/ ; GR-2016-02364847//Ministero della Salute (Ministry of Health, Italy)/ ; PE-2016-02363691//Ministero della Salute (Ministry of Health, Italy)/ ; E-Rare-3 JTC 2017//Ministero della Salute (Ministry of Health, Italy)/ ; //Banca d'Italia (Bank of Italy)/ ; PRIN 2017 Prot. 20175XHBPN//Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR)/ ; //Editas Medicine/ ; //EU Horizon 2020 Program/ ; //Liberal contribution/ ; //Louis-Jeantet Foundation/ ; //Jeantet-Collen Prize for Translational Medicine/ ; },
abstract = {Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X-linked hyper-IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one-size-fits-all editing strategy for effective T-cell correction, selection, and depletion and investigated the therapeutic potential of T-cell and HSPC therapies in the HIGM1 mouse model. Edited patients' derived CD4 T cells restored physiologically regulated CD40L expression and contact-dependent B-cell helper function. Adoptive transfer of wild-type T cells into conditioned HIGM1 mice rescued antigen-specific IgG responses and protected mice from a disease-relevant pathogen. We then obtained ~ 25% CD40LG editing in long-term repopulating human HSPC. Transplanting such proportion of wild-type HSPC in HIGM1 mice rescued immune functions similarly to T-cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.},
}

@article {pmid33474893,
year = {2021},
author = {He, XY and Zhang, AQ and Gong, T and Li, YQ},
title = {[Transcriptomic Analysis of csn2 Gene Mutant Strains of Streptococcus mutans CRISPR-Cas9 System].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {52},
number = {1},
pages = {76-81},
doi = {10.12182/20210160505},
pmid = {33474893},
issn = {1672-173X},
abstract = {Objective: To explore the differences in transcriptional levels between mutant strains of csn2 gene of CRISPR-Cas9 system of Streptococcus mutans(S. mutans) and wild-type strains.

Methods: The S. mutans UA159, csn2-gene-deleted strains (Δ csn2) and csn2-gene-covering strains (Δ csn2/pDL278- csn2) of S. mutans were cultivated. Total RNA was extracted, and high-throughput sequencing technology was used for transcriptome sequencing. Based on the GO analysis and the KEGG analysis of the differentially expressed genes, the biological processes involved were thoroughly examined. The qRT-PCR method was used to verify the transcriptome sequencing results.

Results: The transcriptome results showed that, compared with UA159, there were 176 genes in Δ csn2 whose gene expression changed more than one fold (P<0.05), of which 72 were up-regulated and 104 were down-regulated. The GO enrichment analysis and the KEGG enrichment analysis revealed that both the up-regulated and down-regulated differentially expressed genes (DEG) were involved in amino acid transport and metabolism. In addition, the biological processes that up-regulated DEGs participated in were mainly related to carbohydrate metabolism, energy production and conversion, and transcription; down-regulated DEGs were mainly related to lipid metabolism, DNA replication, recombination and repair, signal transduction mechanisms, nucleotide transport and metabolism. The functions of some DEGs were still unclear. Results of qRT-PCR verified that the expressions of leuA, leuC and leuD(genes related to the formation of branched-chain amino acids) were significantly down-regulated in Δ csn2 when compared with UA159 and Δ csn2/pDL278- csn2.

Conclusion: Through transcriptome sequencing and qRT-PCR verification, it was found that the expression of genes related to branched-chain amino acid synthesis and cell membrane permeability in Δ csn2 changed significantly.},
}

@article {pmid33472516,
year = {2021},
author = {Hanson, B and Wood, MJA and Roberts, TC},
title = {Molecular correction of Duchenne muscular dystrophy by splice modulation and gene editing.},
journal = {RNA biology},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/15476286.2021.1874161},
pmid = {33472516},
issn = {1555-8584},
abstract = {Duchenne muscular dystrophy (DMD) is a currently incurable X-linked neuromuscular disorder, characterized by progressive muscle wasting and premature death, typically as a consequence of cardiac failure. DMD-causing mutations in the dystrophin gene are highly diverse, meaning that the development of a universally-applicable therapy to treat all patients is very challenging. The leading therapeutic strategy for DMD is antisense oligonucleotide-mediated splice modulation, whereby one or more specific exons are excluded from the mature dystrophin mRNA in order to correct the translation reading frame. Indeed, three exon skipping oligonucleotides have received FDA approval for use in DMD patients. Second-generation exon skipping drugs (i.e. peptide-antisense oligonucleotide conjugates) exhibit enhanced potency, and also induce dystrophin restoration in the heart. Similarly, multiple additional antisense oligonucleotide drugs targeting various exons are in clinical development in order to treat a greater proportion of DMD patient mutations. Relatively recent advances in the field of genome engineering (specifically, the development of the CRISPR/Cas system) have provided multiple promising therapeutic approaches for the RNA-directed genetic correction of DMD, including exon excision, exon reframing via the introduction of insertion/deletion mutations, disruption of splice signals to promote exon skipping, and the templated correction of point mutations by seamless homology directed repair or base editing technology. Potential limitations to the clinical translation of the splice modulation and gene editing approaches are discussed, including drug delivery, the importance of uniform dystrophin expression in corrected myofibres, safety issues (e.g. renal toxicity, viral vector immunogenicity, and off-target gene editing), and the high cost of therapy.},
}

@article {pmid33367730,
year = {2020},
author = {Li, HL and Wang, XY and Zheng, XL and Lu, W},
title = {Research Progress on Oviposition-Related Genes in Insects.},
journal = {Journal of insect science (Online)},
volume = {20},
number = {6},
pages = {},
pmid = {33367730},
issn = {1536-2442},
mesh = {Animals ; CRISPR-Cas Systems ; Egg Proteins/genetics ; Female ; Gene Expression ; Genes, Insect ; Insect Control/methods ; Insecta/*genetics ; Oogenesis/genetics ; Oviposition/*genetics ; RNA Interference ; Receptors, Cell Surface/genetics ; Vitellogenins/genetics ; },
abstract = {Oviposition-related genes have remained a consistent focus of insect molecular biology. Previous research has gradually clarified our mechanistic understanding of oviposition-related genes, including those related to oviposition-gland-related genes, oogenesis-related genes, oviposition-site-selection-related genes, and genes related to ovulation and hatching. Moreover, some of this research has revealed how the expression of single oviposition-related genes affects the expression of related genes, and more importantly, how individual node genes function to link the expression of upstream and downstream genes. However, the research to date is not sufficient to completely explain the overall interactions among the genes of the insect oviposition system. Through a literature review of a large number of studies, this review provides references for future research on oviposition-related genes in insects and the use of RNAi or CRISPR/Cas9 technology to verify the functions of oviposition-related genes and to prevent and control harmful insects.},
}

Animals
CRISPR-Cas Systems
Egg Proteins/genetics
Female
Gene Expression
Genes, Insect
Insect Control/methods
Insecta/*genetics
Oogenesis/genetics
Oviposition/*genetics
RNA Interference
Receptors, Cell Surface/genetics
Vitellogenins/genetics

@article {pmid33315895,
year = {2020},
author = {Ghoshal, B and Vong, B and Picard, CL and Feng, S and Tam, JM and Jacobsen, SE},
title = {A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana.},
journal = {PLoS genetics},
volume = {16},
number = {12},
pages = {e1008983},
pmid = {33315895},
issn = {1553-7404},
support = {R35 GM130272/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Arabidopsis ; Arabidopsis Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *DNA Methylation ; Epigenome ; Gene Editing/*methods ; Gene Targeting/*methods ; Plant Viruses/*genetics ; RNA, Guide/*genetics ; RNA, Transfer/genetics ; Transcriptional Activation ; },
abstract = {Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.},
}

Arabidopsis
Arabidopsis Proteins/*genetics/metabolism
*CRISPR-Cas Systems
*DNA Methylation
Epigenome
Gene Editing/*methods
Gene Targeting/*methods
Plant Viruses/*genetics
RNA, Guide/*genetics
RNA, Transfer/genetics
Transcriptional Activation

@article {pmid33214258,
year = {2020},
author = {Pennisi, E},
title = {Like CRISPR, mystery gene editor began as a virus fighter.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6519},
pages = {898-899},
doi = {10.1126/science.370.6519.898},
pmid = {33214258},
issn = {1095-9203},
mesh = {Bacteria/*virology ; Bacteriophages/*physiology ; *CRISPR-Cas Systems ; DNA, Bacterial/genetics ; *Gene Editing ; RNA, Bacterial/genetics ; RNA-Directed DNA Polymerase/physiology ; },
}

Bacteria/*virology
Bacteriophages/*physiology
*CRISPR-Cas Systems
DNA, Bacterial/genetics
*Gene Editing
RNA, Bacterial/genetics
RNA-Directed DNA Polymerase/physiology

@article {pmid33208534,
year = {2020},
author = {Alphey, LS and Crisanti, A and Randazzo, FF and Akbari, OS},
title = {Opinion: Standardizing the definition of gene drive.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {49},
pages = {30864-30867},
pmid = {33208534},
issn = {1091-6490},
support = {DP2 AI152071/AI/NIAID NIH HHS/United States ; BBS/E/I/00007033/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/I/00007034/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Drive Technology/*standards ; Gene Frequency/genetics ; Humans ; Reference Standards ; },
}

CRISPR-Cas Systems/genetics
Gene Drive Technology/*standards
Gene Frequency/genetics
Humans
Reference Standards

@article {pmid33122427,
year = {2020},
author = {Esk, C and Lindenhofer, D and Haendeler, S and Wester, RA and Pflug, F and Schroeder, B and Bagley, JA and Elling, U and Zuber, J and von Haeseler, A and Knoblich, JA},
title = {A human tissue screen identifies a regulator of ER secretion as a brain-size determinant.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6519},
pages = {935-941},
doi = {10.1126/science.abb5390},
pmid = {33122427},
issn = {1095-9203},
mesh = {Brain/*growth & development/metabolism ; CRISPR-Cas Systems ; Carrier Proteins/genetics/*physiology ; Cell Line ; Cell Lineage ; Endoplasmic Reticulum/*metabolism ; Extracellular Matrix Proteins/*metabolism ; Gene Knockout Techniques ; Genetic Testing/*methods ; Humans ; Membrane Proteins/genetics/*physiology ; Microcephaly/*genetics ; Organ Size ; Organoids/growth & development/metabolism ; },
abstract = {Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional cell cultures, which hinders the testing of gene functions requiring tissue context. Here, we present CRISPR-lineage tracing at cellular resolution in heterogeneous tissue (CRISPR-LICHT), which enables parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes, revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized IER3IP1, which regulates the endoplasmic reticulum (ER) function and extracellular matrix protein secretion crucial for tissue integrity, the dysregulation of which results in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain-size control.},
}

Brain/*growth & development/metabolism
CRISPR-Cas Systems
Carrier Proteins/genetics/*physiology
Cell Line
Cell Lineage
Endoplasmic Reticulum/*metabolism
Extracellular Matrix Proteins/*metabolism
Gene Knockout Techniques
Genetic Testing/*methods
Humans
Membrane Proteins/genetics/*physiology
Microcephaly/*genetics
Organ Size
Organoids/growth & development/metabolism

@article {pmid32778842,
year = {2020},
author = {Mathiasen, S and Palmisano, T and Perry, NA and Stoveken, HM and Vizurraga, A and McEwen, DP and Okashah, N and Langenhan, T and Inoue, A and Lambert, NA and Tall, GG and Javitch, JA},
title = {G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.},
journal = {Nature chemical biology},
volume = {16},
number = {12},
pages = {1343-1350},
pmid = {32778842},
issn = {1552-4469},
support = {R01 GM120110/GM/NIGMS NIH HHS/United States ; R01 NS103946/NS/NINDS NIH HHS/United States ; F30 GM131672/GM/NIGMS NIH HHS/United States ; R01 GM130142/GM/NIGMS NIH HHS/United States ; T32 GM007315/GM/NIGMS NIH HHS/United States ; },
mesh = {Activating Transcription Factor 6/agonists/chemistry/genetics/*metabolism ; Animals ; Arrestin/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; Cell Engineering ; GTP-Binding Protein alpha Subunits, G12-G13/chemistry/genetics/*metabolism ; GTP-Binding Protein alpha Subunits, Gq-G11/chemistry/genetics/metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Kinetics ; Mice ; Mitogen-Activated Protein Kinase 1/chemistry/genetics/metabolism ; Mitogen-Activated Protein Kinase 3/chemistry/genetics/metabolism ; Peptides/chemistry/*metabolism/pharmacology ; Protein Binding ; Receptors, G-Protein-Coupled/chemistry/genetics/*metabolism ; Receptors, Peptide/chemistry/genetics/*metabolism ; Recombinant Proteins/chemistry/genetics/metabolism ; Signal Transduction ; },
abstract = {The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.},
}

Activating Transcription Factor 6/agonists/chemistry/genetics/*metabolism
Animals
Arrestin/chemistry/genetics/metabolism
CRISPR-Cas Systems
Cell Engineering
GTP-Binding Protein alpha Subunits, G12-G13/chemistry/genetics/*metabolism
GTP-Binding Protein alpha Subunits, Gq-G11/chemistry/genetics/metabolism
Gene Expression
HEK293 Cells
Humans
Kinetics
Mice
Mitogen-Activated Protein Kinase 1/chemistry/genetics/metabolism
Mitogen-Activated Protein Kinase 3/chemistry/genetics/metabolism
Peptides/chemistry/*metabolism/pharmacology
Protein Binding
Receptors, G-Protein-Coupled/chemistry/genetics/*metabolism
Receptors, Peptide/chemistry/genetics/*metabolism
Recombinant Proteins/chemistry/genetics/metabolism
Signal Transduction

@article {pmid32544385,
year = {2020},
author = {Lowey, B and Whiteley, AT and Keszei, AFA and Morehouse, BR and Mathews, IT and Antine, SP and Cabrera, VJ and Kashin, D and Niemann, P and Jain, M and Schwede, F and Mekalanos, JJ and Shao, S and Lee, ASY and Kranzusch, PJ},
title = {CBASS Immunity Uses CARF-Related Effectors to Sense 3'-5'- and 2'-5'-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection.},
journal = {Cell},
volume = {182},
number = {1},
pages = {38-49.e17},
doi = {10.1016/j.cell.2020.05.019},
pmid = {32544385},
issn = {1097-4172},
support = {R01 AI026289/AI/NIAID NIH HHS/United States ; R01 ES027595/ES/NIEHS NIH HHS/United States ; P42 ES010337/ES/NIEHS NIH HHS/United States ; F31 CA236405/CA/NCI NIH HHS/United States ; T32 CA207021/CA/NCI NIH HHS/United States ; R01 AI018045/AI/NIAID NIH HHS/United States ; F32 GM133063/GM/NIGMS NIH HHS/United States ; S10 OD020025/OD/NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Bacteria/*virology ; Bacterial Proteins/chemistry/metabolism ; Bacteriophages/*metabolism ; *CRISPR-Cas Systems ; Deoxyribonuclease I/metabolism ; *Immunity ; Ligands ; Mutagenesis/genetics ; Nucleotidyltransferases/metabolism ; Oligonucleotides/*metabolism ; Protein Binding ; Second Messenger Systems ; *Signal Transduction ; },
abstract = {cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.},
}

Amino Acid Sequence
Bacteria/*virology
Bacterial Proteins/chemistry/metabolism
Bacteriophages/*metabolism
*CRISPR-Cas Systems
Deoxyribonuclease I/metabolism
*Immunity
Ligands
Mutagenesis/genetics
Nucleotidyltransferases/metabolism
Oligonucleotides/*metabolism
Protein Binding
Second Messenger Systems
*Signal Transduction

@article {pmid32539998,
year = {2020},
author = {Miyamoto, T and Takada, R and Tobimatsu, Y and Suzuki, S and Yamamura, M and Osakabe, K and Osakabe, Y and Sakamoto, M and Umezawa, T},
title = {Double knockout of OsWRKY36 and OsWRKY102 boosts lignification with altering culm morphology of rice.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {296},
number = {},
pages = {110466},
doi = {10.1016/j.plantsci.2020.110466},
pmid = {32539998},
issn = {1873-2259},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Gene Editing ; Gene Knockout Techniques ; Lignin/*metabolism ; Magnetic Resonance Spectroscopy ; Oryza/*anatomy & histology/genetics/metabolism ; Phylogeny ; Plant Proteins/metabolism/*physiology ; Plant Stems/*anatomy & histology/genetics/metabolism ; Plants, Genetically Modified ; Transcription Factors/metabolism/*physiology ; },
abstract = {Breeding to enrich lignin, a major component of lignocelluloses, in plants contributes to enhanced applications of lignocellulosic biomass into solid biofuels and valuable aromatic chemicals. To collect information on enhancing lignin deposition in grass species, important lignocellulose feedstocks, we generated rice (Oryza sativa) transgenic lines deficient in OsWRKY36 and OsWRKY102, which encode putative transcriptional repressors for secondary cell wall formation. We used CRISPR/Cas9-mediated targeted mutagenesis and closely characterized their altered cell walls using chemical and nuclear magnetic resonance (NMR) methods. Both OsWRKY36 and OsWRKY102 mutations significantly increased lignin content by up to 28 % and 32 %, respectively. Additionally, OsWRKY36/OsWRKY102-double-mutant lines displayed lignin enrichment of cell walls (by up to 41 %) with substantially altered culm morphology over the single-mutant lines as well as the wild-type controls. Our chemical and NMR analyses showed that relative abundances of guaiacyl and p-coumarate units were slightly higher and lower, respectively, in the WRKY mutant lignins compared with those in the wild-type lignins. Our results provide evidence that both OsWRKY36 and OsWRKY102 are associated with repression of rice lignification.},
}

CRISPR-Associated Protein 9
CRISPR-Cas Systems
Cell Wall/metabolism
Gene Editing
Gene Knockout Techniques
Lignin/*metabolism
Magnetic Resonance Spectroscopy
Oryza/*anatomy & histology/genetics/metabolism
Phylogeny
Plant Proteins/metabolism/*physiology
Plant Stems/*anatomy & histology/genetics/metabolism
Plants, Genetically Modified
Transcription Factors/metabolism/*physiology

@article {pmid32488144,
year = {2020},
author = {Eskandari-Shahraki, M and Prud'homme, B and Bergeron, F and Manjunath, P},
title = {Epididymal proteins Binder of SPerm Homologs 1 and 2 (BSPH1/2) are dispensable for male fertility and sperm motility in mice.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {8982},
pmid = {32488144},
issn = {2045-2322},
support = {MOP-130274//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/International ; },
mesh = {Animals ; Animals, Newborn ; Body Weight/genetics ; *CRISPR-Cas Systems ; Female ; Fertility/*genetics ; Male ; Mice, Knockout/*genetics ; Seminal Vesicle Secretory Proteins/*genetics/*physiology ; Sperm Motility/*genetics ; },
abstract = {The binder of sperm family of proteins has been reported to be indispensable for sperm maturation and capacitation. However, their physiological functions in fertility have only been studied in vitro. CRISPR/Cas9 genome editing was utilized to generate double knockout (DKO) mice by simultaneously targeting the two murine binder of sperm genes, Bsph1 and Bsph2. To confirm that the homologous genes and proteins were completely eliminated in the DKO mice, different methods such as reverse transcription polymerase chain reaction, digital droplet-polymerase chain reaction and liquid chromatography tandem mass spectrometry were applied. Bsph1/2 DKO male mice were bred by intercrossing. Compared to wild type counterparts, male Bsph1/2 null mice, lacking BSPH1/2 proteins, were fertile with no differences in sperm motility and sperm count. However, the weights of male pups were significantly increased in Bsph1/2 double knockout mice in a time dependent manner spanning days 6 and 21, as well as 6 weeks of age. No change was detected in the weights of female pups during the same period. Taken together, these data indicate that BSPH1/2 proteins are dispensable for male fertility in mice but may influence growth.},
}

Animals
Animals, Newborn
Body Weight/genetics
*CRISPR-Cas Systems
Female
Fertility/*genetics
Male
Mice, Knockout/*genetics
Seminal Vesicle Secretory Proteins/*genetics/*physiology
Sperm Motility/*genetics

@article {pmid32408049,
year = {2020},
author = {Ramongolalaina, C},
title = {Dual-luciferase assay and siRNA silencing for nodD1 to study the competitiveness of Bradyrhizobium diazoefficiens USDA110 in soybean nodulation.},
journal = {Microbiological research},
volume = {237},
number = {},
pages = {126488},
doi = {10.1016/j.micres.2020.126488},
pmid = {32408049},
issn = {1618-0623},
mesh = {Bacterial Proteins/*genetics ; Bradyrhizobium/*genetics ; CRISPR-Cas Systems ; Fluorescent Dyes/analysis ; Genes, Bacterial ; Luciferases, Renilla ; Nitrogen Fixation/genetics ; Plant Development ; *Plant Root Nodulation ; Plant Roots/microbiology ; RNA, Small Interfering ; Soil Microbiology ; Soybeans/*microbiology ; Symbiosis ; Transformation, Bacterial ; },
abstract = {The symbiosis of soybean with Bradyrhizobium diazoefficiens USDA110, which always competes with other rhizobia in the field, is of great agronomic and environmental importance. Herein, a dual-luciferase reporter assay was utilized to monitor the dynamics of two dominant bradyrhizobia infecting roots of soybean. More explicitly, luciferase-tagged B. diazoefficiens USDA110 (USDA110-FLuc) and Bradyrhizobium elkanii USDA 94 (USDA94-RLuc) were designed, co-inoculated into soybean seeds, and observed for their colonization in root nodules by bioluminescence imaging. The results showed that USDA110-FLuc initiated infection earlier than USDA94-RLuc, but its occupancy in the nodules decreased as the plant grew. A nodulation test showed that nodD1 mutant USDA110 strains, including CRISPR engineered mutants, were less competitive than wild type. I constructed siRNAs to knockdown nodD1 at different target sites and transformed them into the bacteria. Surprisingly, although siRNAs - with 3' end target sites - were able to repress up to 65% of nodD1 expression, the profiling of total RNAs with a bioanalyzer revealed that 23S/16S-rRNA ratios of siRNA-transformed and wild type USDA110 strains were similar, but lower than that of nodD1 mutant. In short, the current work - while reporting the competitiveness of B. diazoefficiens USDA110 in early occupancy of soybean nodules and the gene nodD1 as a key determinant of this infection - gives an insight on siRNA silencing in microbes, and demonstrates a highly efficient imaging approach that could entail many new avenues for many biological research fields.},
}

Bacterial Proteins/*genetics
Bradyrhizobium/*genetics
CRISPR-Cas Systems
Fluorescent Dyes/analysis
Genes, Bacterial
Luciferases, Renilla
Nitrogen Fixation/genetics
Plant Development
*Plant Root Nodulation
Plant Roots/microbiology
RNA, Small Interfering
Soil Microbiology
Soybeans/*microbiology
Symbiosis
Transformation, Bacterial

@article {pmid32326099,
year = {2020},
author = {Becskei, A},
title = {Tuning up Transcription Factors for Therapy.},
journal = {Molecules (Basel, Switzerland)},
volume = {25},
number = {8},
pages = {},
pmid = {32326099},
issn = {1420-3049},
support = {310030_185001//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; },
mesh = {Animals ; Biotechnology/methods ; CRISPR-Cas Systems ; Clinical Trials as Topic ; DNA-Binding Proteins/genetics/metabolism ; Endonucleases/genetics/metabolism ; *Gene Expression Regulation ; Gene Transfer Techniques ; *Genetic Therapy/methods ; Humans ; Protein Binding ; Repressor Proteins/genetics/metabolism ; Transcription Factors/chemistry/*genetics/*metabolism ; *Transcription, Genetic ; },
abstract = {The recent developments in the delivery and design of transcription factors put their therapeutic applications within reach, exemplified by cell replacement, cancer differentiation and T-cell based cancer therapies. The success of such applications depends on the efficacy and precision in the action of transcription factors. The biophysical and genetic characterization of the paradigmatic prokaryotic repressors, LacI and TetR and the designer transcription factors, transcription activator-like effector (TALE) and CRISPR-dCas9 revealed common principles behind their efficacy, which can aid the optimization of transcriptional activators and repressors. Further studies will be required to analyze the linkage between dissociation constants and enzymatic activity, the role of phase separation and squelching in activation and repression and the long-range interaction of transcription factors with epigenetic regulators in the context of the chromosomes. Understanding these mechanisms will help to tailor natural and synthetic transcription factors to the needs of specific applications.},
}

Animals
Biotechnology/methods
CRISPR-Cas Systems
Clinical Trials as Topic
DNA-Binding Proteins/genetics/metabolism
Endonucleases/genetics/metabolism
*Gene Expression Regulation
Gene Transfer Techniques
*Genetic Therapy/methods
Humans
Protein Binding
Repressor Proteins/genetics/metabolism
Transcription Factors/chemistry/*genetics/*metabolism
*Transcription, Genetic

@article {pmid31978714,
year = {2020},
author = {Xu, C and Zhou, Z and Liu, C and Kang, X and Zhong, X and Zhang, Q and Xu, Y},
title = {Generation of a DAPK1 knockout first (conditional ready) human embryonic stem cell line (ZSSYe001-A) by CRISPR-Cas9 technology.},
journal = {Stem cell research},
volume = {43},
number = {},
pages = {101693},
doi = {10.1016/j.scr.2019.101693},
pmid = {31978714},
issn = {1876-7753},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Death-Associated Protein Kinases/*genetics ; Human Embryonic Stem Cells/*metabolism ; Humans ; Male ; },
abstract = {Death-associated protein kinase 1 (DAPK1) is a Ca2+/calmodulin regulated Ser/Thr kinase involved in various cellular processes including cell death, autophagy and inflammation. Its dysregulation has been linked to tumour metastasis, anti-viral responses, Alzheimer's disease and other neurological disorders. To further investigate the role of DAPK1 in these processes, we generated a DAPK1 knockout first (conditional ready) human embryonic stem (hES) cell line in which the endogenous DAPK1 can be easily restored with expression of FLPe. This cell line provides an ideal model to study the role of DAPK1 in human development and various pathologies related to DAPK1 dysregulation in vitro.},
}

CRISPR-Cas Systems/*genetics
Cell Line
Death-Associated Protein Kinases/*genetics
Human Embryonic Stem Cells/*metabolism
Humans
Male

@article {pmid33472057,
year = {2021},
author = {Wang, B and Zhang, T and Yin, J and Yu, Y and Xu, W and Ding, J and Patel, DJ and Yang, H},
title = {Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2020.12.033},
pmid = {33472057},
issn = {1097-4164},
abstract = {Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.},
}

@article {pmid33471332,
year = {2021},
author = {Tang, X and Qi, Y and Zhang, Y},
title = {Single Transcript Unit CRISPR 2.0 Systems for Genome Editing in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {193-204},
pmid = {33471332},
issn = {1940-6029},
abstract = {CRISPR-Cas9 and Cas12a (formerly Cpf1), RNA-guided DNA endonucleases found from adaptive immune system in prokaryotes, have been engineered and widely adopted as two of the most powerful genome editing systems in plants. Recently, we developed a single transcript unit (STU) CRISPR 2.0 toolbox for applications in plants, which contains two STU-Cas9 systems and one STU-Cas12a system. Here, we describe a detailed protocol about using the STU CRISPR 2.0 systems to achieve single and multiplex genome editing in rice.},
}

@article {pmid33471330,
year = {2021},
author = {Liu, G and Qi, Y and Zhang, T},
title = {Analysis of Off-Target Mutations in CRISPR-Edited Rice Plants Using Whole-Genome Sequencing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {145-172},
pmid = {33471330},
issn = {1940-6029},
abstract = {The CRISPR/Cas systems have become the most widely used tool for genome editing in plants and beyond. However, CRISPR/Cas systems may cause unexpected off-target mutations due to sgRNA recognizing highly homologous DNA sequence elsewhere in the genome. Whole-genome sequencing (WGS) can be used to identify on- and off-target mutation. Here, we describe a pipeline of analyzing WGS data using a series of open source software for analysis of off-target mutations in CRISPR-edited rice plants. In this pipeline, the adapter is trimmed using SKEWER. Then, the cleaned reads are mapped to reference genome by applying BWA. To avoid mapping bias, the GATK is used to realign reads near indels (insertions and deletions) and recalibrate base quality controls. Whole-genome single nucleotide variations (SNVs) and indels are detected by LoFreq*, Mutect2, VarScan2, and Pindel. Last, SNVs and indels are compared with in silico off-target sites using Cas-OFFinder.},
}

@article {pmid33471328,
year = {2021},
author = {Das, A and Ghana, P and Rudrappa, B and Gandhi, R and Tavva, VS and Mohanty, A},
title = {Genome Editing of Rice by CRISPR-Cas: End-to-End Pipeline for Crop Improvement.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2238},
number = {},
pages = {115-134},
pmid = {33471328},
issn = {1940-6029},
abstract = {CRISPR-Cas resonates a revolutionary genome editing technology applicable through a horizon spreading across microbial organism to higher plant and animal. This technology can be harnessed with ease to understand the basic genetics of a living system by altering sequence of individual genes and characterizing their functions. The precision of this technology is unparallel. It allows very precise and targeted base pair level edits in the genome. Here, in the current chapter, we have provided end-to-end process outline on how to generate genome edited plants in crops like rice to evaluate for agronomic traits associated with yield, disease resistance and abiotic stress tolerance, etc. Genome editing process includes designing of gene editing strategy, vector construction, plant transformation, molecular screening, and phenotyping under control environment conditions. Furthermore, its application for development of commercial crop product may require additional processes, including field trials in the target geography for evaluation of product efficacy. Evaluation of genome edited lines in controlled greenhouse/net house or open field condition requires few generations for outcrossing with wild-type parent to eliminate and/or reduce any potential pleiotropic effect in the edited genome which may arise during the process. The genome edited plant selected for advancement shall harbor the genome with only the intended changes, which can be analyzed by various molecular techniques, advanced sequencing methods, and genomic data analysis tools. CRISPR-Cas-based genome editing has opened a plethora of opportunities in agriculture as well as human health.},
}

@article {pmid33469138,
year = {2021},
author = {Fernandes, LGV and Hornsby, RL and Nascimento, ALTO and Nally, JE},
title = {Genetic manipulation of pathogenic Leptospira: CRISPR interference (CRISPRi)-mediated gene silencing and rapid mutant recovery at 37 °C.},
journal = {Scientific reports},
volume = {11},
number = {1},
pages = {1768},
pmid = {33469138},
issn = {2045-2322},
support = {2019/20302-8//Fapesp/ ; 2014/50981-0//Fapesp/ ; },
abstract = {Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes CRISPR/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major outer membrane proteins of pathogenic L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO2 within 8 days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.},
}

@article {pmid33468705,
year = {2021},
author = {Singh, A and Gaur, M and Sharma, V and Khanna, P and Bothra, A and Bhaduri, A and Mondal, AK and Dash, D and Singh, Y and Misra, R},
title = {Comparative Genomic Analysis of Mycobacteriaceae Reveals Horizontal Gene Transfer-Mediated Evolution of the CRISPR-Cas System in the Mycobacterium tuberculosis Complex.},
journal = {mSystems},
volume = {6},
number = {1},
pages = {},
pmid = {33468705},
issn = {2379-5077},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are conserved genetic elements in many prokaryotes, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Although knowledge of CRISPR locus variability has been utilized in M. tuberculosis strain genotyping, its evolutionary path in Mycobacteriaceae is not well understood. In this study, we have performed a comparative analysis of 141 mycobacterial genomes and identified the exclusive presence of the CRISPR-Cas type III-A system in M. tuberculosis complex (MTBC). Our global phylogenetic analysis of CRISPR repeats and Cas10 proteins offers evidence of horizontal gene transfer (HGT) of the CRISPR-Cas module in the last common ancestor of MTBC and Mycobacterium canettii from a Streptococcus-like environmental bacterium. Additionally, our results show that the variation of CRISPR-Cas organization in M. tuberculosis lineages, especially in the Beijing sublineage of lineage 2, is due to the transposition of insertion sequence IS6110 The direct repeat (DR) region of the CRISPR-Cas locus acts as a hot spot for IS6110 insertion. We show in M. tuberculosis H37Rv that the repeat at the 5' end of CRISPR1 of the forward strand is an atypical repeat made up partly of IS-terminal inverted repeat and partly CRISPR DR. By tracing an undetectable spacer sequence in the DR region, the two CRISPR loci could theoretically be joined to reconstruct the ancestral single CRISPR-Cas locus organization, as seen in M. canettii This study retracing the evolutionary events of HGT and IS6110-driven genomic deletions helps us to better understand the strain-specific variations in M. tuberculosis lineages.IMPORTANCE Comparative genomic analysis of prokaryotes has led to a better understanding of the biology of several pathogenic microorganisms. One such clinically important pathogen is M. tuberculosis, the leading cause of bacterial infection worldwide. Recent evidence on the functionality of the CRISPR-Cas system in M. tuberculosis has brought back focus on these conserved genetic elements, present in many prokaryotes. Our study advances understanding of mycobacterial CRISPR-Cas origin and its diversity among the different species. We provide phylogenetic evidence of acquisition of CRISPR-Cas type III-A in the last common ancestor shared between MTBC and M. canettii, by HGT-mediated events. The most likely source of HGT was an environmental Firmicutes bacterium. Genomic mapping of the CRISPR loci showed the IS6110 transposition-driven variations in M. tuberculosis strains. Thus, this study offers insights into events related to the evolution of CRISPR-Cas in M. tuberculosis lineages.},
}

@article {pmid33468685,
year = {2021},
author = {Xiao, H and Wyler, E and Milek, M and Grewe, B and Kirchner, P and Ekici, A and Silva, ABOV and Jungnickl, D and Full, F and Thomas, M and Landthaler, M and Ensser, A and Überla, K},
title = {CRNKL1 Is a Highly Selective Regulator of Intron-Retaining HIV-1 and Cellular mRNAs.},
journal = {mBio},
volume = {12},
number = {1},
pages = {},
pmid = {33468685},
issn = {2150-7511},
abstract = {The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern.IMPORTANCE To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.},
}

@article {pmid33349126,
year = {2021},
author = {Cornu, TI and Mussolino, C and Müller, MC and Wehr, C and Kern, WV and Cathomen, T},
title = {HIV Gene Therapy: An Update.},
journal = {Human gene therapy},
volume = {32},
number = {1-2},
pages = {52-65},
doi = {10.1089/hum.2020.159},
pmid = {33349126},
issn = {1557-7422},
abstract = {Progress in antiretroviral therapy has considerably reduced mortality and notably improved the quality of life of individuals infected with HIV since the pandemic began some 40 years ago. However, drug resistance, treatment-associated toxicity, adherence to medication, and the need for lifelong therapy have remained major challenges. While the development of an HIV vaccine has remained elusive, considerable progress in developing innovative cell and gene therapies to treat HIV infection has been made. This includes immune cell therapies, such as chimeric antigen receptor T cells to target HIV infected cells, as well as gene therapies and genome editing strategies to render the patient's immune system resistant to HIV. Nonetheless, all of these attempts to achieve a functional cure in HIV patients have failed thus far. This review introduces the clinical as well as the technical challenges of treating HIV infection, and summarizes the most promising cell and gene therapy concepts that have aspired to bring about functional cure for people living with HIV. It further discusses socioeconomic aspects as well as future directions for developing cell and gene therapies with a potential to be an effective one-time treatment with minimal toxicity.},
}

@article {pmid33454599,
year = {2021},
author = {Ashraf, MU and Salman, HM and Khalid, MF and Khan, MHF and Anwar, S and Afzal, S and Idrees, M and Chaudhary, SU},
title = {CRISPR-Cas13a mediated targeting of hepatitis C virus internal-ribosomal entry site (IRES) as an effective antiviral strategy.},
journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie},
volume = {136},
number = {},
pages = {111239},
doi = {10.1016/j.biopha.2021.111239},
pmid = {33454599},
issn = {1950-6007},
abstract = {Hepatitis C is an inflammatory liver disease caused by the single-stranded RNA (ssRNA) hepatitis C virus (HCV). The genetic diversity of the virus and quasispecies produced during replication have resulted in viral resistance to direct-acting antivirals (DAAs) as well as impediments in vaccine development. The recent adaptation of CRISPR-Cas as an alternative antiviral approach has demonstrated degradation of viral nucleic acids in eukaryotes. In particular, the CRISPR-effector Cas13 enzyme has been shown to target ssRNA viruses effectively. In this work, we have employed Cas13a to knockdown HCV in mammalian cells. Using a computational screen, we identified several potential Cas13a target sites within highly conserved regions of the HCV internal ribosomal entry site (IRES). Our results demonstrate significant inhibition of HCV replication as well as translation in huh-7.5 cells with minimal effects on cell viability. These findings were validated using a multi-modality approach involving qRT-PCR, luciferase assay, and MTT cell viability assay. In conclusion, the CRISPR-Cas13a system efficiently targets HCV in vitro, suggesting its potential as a programmable therapeutic antiviral strategy.},
}

@article {pmid33453817,
year = {2020},
author = {Pinci, F and Gaidt, MM and Jung, C and Kuut, G and Jackson, MA and Bauernfried, S and Hornung, V},
title = {C-tag TNF: a reporter system to study TNF shedding.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {52},
pages = {18065-18075},
doi = {10.1074/jbc.RA120.015248},
pmid = {33453817},
issn = {1083-351X},
abstract = {TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.},
}

@article {pmid33461211,
year = {2021},
author = {Rostøl, JT and Xie, W and Kuryavyi, V and Maguin, P and Kao, K and Froom, R and Patel, DJ and Marraffini, LA},
title = {The Card1 nuclease provides defence during type-III CRISPR immunity.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-021-03206-x},
pmid = {33461211},
issn = {1476-4687},
abstract = {During the prokaryotic type III CRISPR-Cas immune response, infection triggers the production of cyclic oligoadenylates, which bind and activate CARF domain-containing proteins1,2. Many type III loci are associated with proteins in which the CARF domain is fused to an endonuclease-like domain3,4; however, with the exception of the well-characterized Csm6/Csx1 RNases5,6, whether and how these inducible effectors provide defense is not known. Here we investigated one of such type III CRISPR accessory proteins, Card1. Card1 forms a symmetrical dimer with a large central cavity between its CARF and restriction endonuclease (REase) domains that binds cA4. Ligand binding results in a conformational change where individual monomers rotate relative to each other to form a more compact dimeric scaffold wherein a Mn cation coordinates to the catalytic residues and activates the cleavage of single, but not double, stranded nucleic acids (DNA and RNA). In vivo, Card1 activation induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used by CRISPR systems to provide immunity.},
}

@article {pmid33243861,
year = {2020},
author = {Jin, X and Simmons, SK and Guo, A and Shetty, AS and Ko, M and Nguyen, L and Jokhi, V and Robinson, E and Oyler, P and Curry, N and Deangeli, G and Lodato, S and Levin, JZ and Regev, A and Zhang, F and Arlotta, P},
title = {In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes.},
journal = {Science (New York, N.Y.)},
volume = {370},
number = {6520},
pages = {},
doi = {10.1126/science.aaz6063},
pmid = {33243861},
issn = {1095-9203},
support = {U01 MH115727/MH/NIMH NIH HHS/United States ; R01 MH096066/MH/NIMH NIH HHS/United States ; P50 MH094271/MH/NIMH NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; //Howard Hughes Medical Insititue/International ; },
mesh = {Animals ; Ankyrins/genetics/metabolism ; Autistic Disorder/*genetics/*pathology ; Brain/*abnormalities ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Frameshift Mutation ; Gene Expression Profiling ; Genetic Loci ; Humans ; Mice ; Neuroglia/metabolism/*pathology ; Neurons/metabolism/*pathology ; Repressor Proteins/genetics ; Risk ; Transcription Factors/genetics ; },
abstract = {The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA-sequencing of perturbed cells in the postnatal brain. We identified cell type-specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.},
}

Animals
Ankyrins/genetics/metabolism
Autistic Disorder/*genetics/*pathology
Brain/*abnormalities
CRISPR-Cas Systems
DNA-Binding Proteins/genetics
Frameshift Mutation
Gene Expression Profiling
Genetic Loci
Humans
Mice
Neuroglia/metabolism/*pathology
Neurons/metabolism/*pathology
Repressor Proteins/genetics
Risk
Transcription Factors/genetics

@article {pmid33220342,
year = {2021},
author = {Katsuma, S and Shoji, K and Suzuki, Y and Kiuchi, T},
title = {CRISPR/Cas9-mediated mutagenesis of Ago2 and Siwi in silkworm cultured cells.},
journal = {Gene},
volume = {768},
number = {},
pages = {145314},
doi = {10.1016/j.gene.2020.145314},
pmid = {33220342},
issn = {1879-0038},
mesh = {Animals ; Argonaute Proteins/*genetics ; Bombyx/*cytology/genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Cells, Cultured ; Gene Editing ; Insect Proteins/genetics ; Loss of Function Mutation ; Mutagenesis, Site-Directed/*methods ; RNA, Small Interfering/genetics ; RNA, Viral/genetics ; Signal Transduction ; Tymoviridae/*genetics ; },
abstract = {The BmN-4 cell line, originated from the silkworm Bombyx mori ovary, possesses endogenous small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways. We performed CRISPR/Cas9-mediated genome editing of Ago2 and Siwi, which are the core factors for siRNA and piRNA pathways, respectively, to understand the importance of the two distinct small RNA pathways in this cell line. We found that approximately half of the alleles contained loss-of-function mutations in both Ago2- and Siwi-mutated cells. The mutated cells grew at a slower rate compared to the control cells, strongly suggesting that the siRNA and piRNA pathways are both crucial for the normal growth of BmN-4 cells. The amounts of piRNAs decreased markedly in the Siwi-mutated cells, but global de-repression of transposable elements was not observed. Although the RNA amount of latently infected RNA virus, Bombyx mori macula-like virus (BmLV), increased in both Ago2- and Siwi-mutated cells, the siRNA and piRNA pathways showed a bias toward targeting BmLV genomic and subgenomic RNA, respectively. These results indicate the common, specific, and crucial roles of the two small RNA pathways in B. mori cultured cells.},
}

Animals
Argonaute Proteins/*genetics
Bombyx/*cytology/genetics
CRISPR-Cas Systems
Cell Proliferation
Cells, Cultured
Gene Editing
Insect Proteins/genetics
Loss of Function Mutation
Mutagenesis, Site-Directed/*methods
RNA, Small Interfering/genetics
RNA, Viral/genetics
Signal Transduction
Tymoviridae/*genetics

@article {pmid33172989,
year = {2020},
author = {Zhang, H and Zoued, A and Liu, X and Sit, B and Waldor, MK},
title = {Type I interferon remodels lysosome function and modifies intestinal epithelial defense.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {47},
pages = {29862-29871},
pmid = {33172989},
issn = {1091-6490},
support = {/HHMI/Howard Hughes Medical Institute/United States ; R01 AI042347/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Epithelial Cells/chemistry/cytology/*immunology/metabolism ; Gene Expression Regulation, Bacterial/immunology ; HT29 Cells ; Host-Pathogen Interactions/genetics/immunology ; Humans ; Hydrogen-Ion Concentration ; Immunity, Innate ; Interferon Type I/*metabolism ; Intestinal Mucosa/cytology/*immunology/microbiology ; Lysosomes/chemistry/immunology/*metabolism ; Mice ; Mice, Knockout ; Necroptosis/immunology ; Peptide Hydrolases/metabolism ; Proteomics ; Receptor, Interferon alpha-beta/genetics/metabolism ; Salmonella Infections/*immunology/microbiology ; Salmonella typhimurium/immunology/pathogenicity ; Signal Transduction/immunology ; Virulence/immunology ; Virulence Factors/genetics/metabolism ; },
abstract = {Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I IFN (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity, and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including IFITM3, SLC15A3, and CNP contribute to lysosome acidification. IFN-I-dependent lysosome acidification was associated with elevated intracellular Salmonella virulence gene expression, rupture of the Salmonella-containing vacuole, and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium where Salmonella initiates infection, indicating that IFN-I signaling can modify innate defense in the epithelial compartment. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.},
}

Animals
Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Disease Models, Animal
Epithelial Cells/chemistry/cytology/*immunology/metabolism
Gene Expression Regulation, Bacterial/immunology
HT29 Cells
Host-Pathogen Interactions/genetics/immunology
Humans
Hydrogen-Ion Concentration
Immunity, Innate
Interferon Type I/*metabolism
Intestinal Mucosa/cytology/*immunology/microbiology
Lysosomes/chemistry/immunology/*metabolism
Mice
Mice, Knockout
Necroptosis/immunology
Peptide Hydrolases/metabolism
Proteomics
Receptor, Interferon alpha-beta/genetics/metabolism
Salmonella Infections/*immunology/microbiology
Salmonella typhimurium/immunology/pathogenicity
Signal Transduction/immunology
Virulence/immunology
Virulence Factors/genetics/metabolism

@article {pmid33168709,
year = {2020},
author = {Yu, X and Zhao, Q and Li, X and Chen, Y and Tian, Y and Liu, S and Xiong, W and Huang, P},
title = {Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {47},
pages = {29894-29903},
pmid = {33168709},
issn = {1091-6490},
mesh = {Animals ; COS Cells ; CRISPR-Cas Systems/genetics ; Chlorocebus aethiops ; Deafness/*genetics/pathology ; Disease Models, Animal ; Gene Knock-In Techniques ; HEK293 Cells ; Hair Cells, Auditory, Inner/metabolism/*pathology ; Humans ; Mechanotransduction, Cellular/*genetics ; Membrane Proteins/*genetics/isolation & purification/*metabolism ; Mice ; Mice, Transgenic ; Point Mutation ; Protein Binding/genetics ; Two-Hybrid System Techniques ; },
abstract = {Transmembrane channel-like protein 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) are recognized as two critical components of the mechanotransduction complex in inner-ear hair cells. However, the physical and functional interactions of TMC1 and LHFPL5 remain largely unexplored. We examined the interaction between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay. We demonstrate that LHFPL5 physically interacts with and stabilizes TMC1 in both heterologous expression systems and in the soma and hair bundle of hair cells. Moreover, the semidominant deafness mutation D572N in human TMC1 (D569N in mouse TMC1) severely disrupted LHFPL5 binding and destabilized TMC1 expression. Thus, our findings reveal previously unrecognized physical and functional interactions of TMC1 and LHFPL5 and provide insights into the molecular mechanism by which the D572N mutation causes deafness. Notably, these findings identify a missing link in the currently known physical organization of the mechanotransduction macromolecular complex. Furthermore, this study has demonstrated the power of the microbead-based SiMPull assay for biochemical investigation of rare cells such as hair cells.},
}

Animals
COS Cells
CRISPR-Cas Systems/genetics
Chlorocebus aethiops
Deafness/*genetics/pathology
Disease Models, Animal
Gene Knock-In Techniques
HEK293 Cells
Hair Cells, Auditory, Inner/metabolism/*pathology
Humans
Mechanotransduction, Cellular/*genetics
Membrane Proteins/*genetics/isolation & purification/*metabolism
Mice
Mice, Transgenic
Point Mutation
Protein Binding/genetics
Two-Hybrid System Techniques

@article {pmid32887745,
year = {2020},
author = {Llamosas, N and Arora, V and Vij, R and Kilinc, M and Bijoch, L and Rojas, C and Reich, A and Sridharan, B and Willems, E and Piper, DR and Scampavia, L and Spicer, TP and Miller, CA and Holder, JL and Rumbaugh, G},
title = {SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons.},
journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
volume = {40},
number = {41},
pages = {7980-7994},
pmid = {32887745},
issn = {1529-2401},
support = {R01 MH096847/MH/NIMH NIH HHS/United States ; R01 MH113648/MH/NIMH NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Size ; Cells, Cultured ; Dendrites/*physiology ; Excitatory Postsynaptic Potentials/genetics ; Female ; Gene Deletion ; Humans ; Nerve Net/*physiology ; Nervous System/*growth & development ; Neurodevelopmental Disorders/genetics ; Pluripotent Stem Cells ; Synapses/*physiology ; ras GTPase-Activating Proteins/*genetics/*physiology ; },
abstract = {SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.},
}

CRISPR-Cas Systems
Cell Differentiation/genetics
Cell Size
Cells, Cultured
Dendrites/*physiology
Excitatory Postsynaptic Potentials/genetics
Female
Gene Deletion
Humans
Nerve Net/*physiology
Nervous System/*growth & development
Neurodevelopmental Disorders/genetics
Pluripotent Stem Cells
Synapses/*physiology
ras GTPase-Activating Proteins/*genetics/*physiology

@article {pmid32745561,
year = {2020},
author = {Yan, Y and Ziemek, J and Schetelig, MF},
title = {CRISPR/Cas9 mediated disruption of the white gene leads to pigmentation deficiency and copulation failure in Drosophila suzukii.},
journal = {Journal of insect physiology},
volume = {126},
number = {},
pages = {104091},
doi = {10.1016/j.jinsphys.2020.104091},
pmid = {32745561},
issn = {1879-1611},
mesh = {ATP-Binding Cassette Transporters/*genetics ; Animals ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Copulation ; Drosophila/*genetics/physiology ; Drosophila Proteins/*genetics ; Eye Proteins/*genetics ; Female ; Genes, Insect ; Insect Control/methods ; Male ; Mutation ; Pigmentation/*genetics ; *Sexual Behavior, Animal ; },
abstract = {The Spotted-wing Drosophila (Drosophila suzukii) is a devastating invasive pest of fruit crops. In D. melanogaster, the white (w) gene was associated with pigmentation and mating behavior, which are also important aspects to understand the invasion biology as well as to develop control strategies for D. suzukii. Here, we show that the generation of D. suzukii white-eyed mutants by CRISPR/Cas9 mutagenesis of the w gene resulted in the complete failure of copulation when w- males were individually paired with w- females in small circular arenas (diameter 0.7 cm) for 24 h. Further analysis showed that the mating defect was associated with w- males and could not be rectified by two years of inbreeding by crossing sibling w- females with w+ males, dim red illumination, male-female sexual training, changing to large arenas (diameter 3.5 cm), or different sex ratios. Profound pigmentation deficiency was detected in the compound eyes, ocelli, Malpighian tubules and testis sheaths in the w- flies. Specifically, testis imaging showed that w- males failed to deposit any pigments into pigment cells of the testis sheath, and produced smaller sperms and less seminal fluid compared to those from wildtype males. Together these observations suggest that the w gene plays an essential role in the regulation of sexual behavior and reproduction in D. suzukii. The similarities and differences in w gene function between D. suzukii and D. melanogaster in the context of pigmentation and mating behavior are discussed.},
}

ATP-Binding Cassette Transporters/*genetics
Animals
CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Copulation
Drosophila/*genetics/physiology
Drosophila Proteins/*genetics
Eye Proteins/*genetics
Female
Genes, Insect
Insect Control/methods
Male
Mutation
Pigmentation/*genetics
*Sexual Behavior, Animal

@article {pmid32518161,
year = {2020},
author = {Hu, L and Li, H and Chi, Z and He, J},
title = {Loss of the RNA-binding protein Rbm15 disrupts liver maturation in zebrafish.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {33},
pages = {11466-11472},
pmid = {32518161},
issn = {1083-351X},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; *Gene Deletion ; *Gene Expression Regulation, Developmental ; Hepatocytes/cytology/metabolism ; Liver/cytology/*embryology ; Zebrafish/*embryology/genetics ; },
abstract = {Liver organogenesis begins with hepatic precursors in the foregut endoderm, followed by hepatoblast specification, differentiation, outgrowth, and maturation for the formation of functional hepatocytes. Although several signaling pathways and critical factors that regulate liver specification, differentiation, and proliferation have been identified, little is known about how liver maturation is regulated. Here, we used a screen for mutations affecting liver development in zebrafish and identified a cq96 mutant that exhibits a specific defect in liver maturation. Results from positional cloning revealed that cq96 encodes an RNA-binding protein, Rbm15, which is an evolutionarily conserved Spen family protein and known to play a crucial role in RNA m6A modification, nuclear export, and alternative splicing. However, a function of Rbm15 in embryonic liver development has not been reported. We found that Rbm15 is specifically expressed in the liver after its differentiation. CRISPR/Cas9-mediated loss of rbm15 repressed hepatic maturation, but did not affect hepatoblast specification, differentiation, and hepatocyte proliferation and apoptosis. Additional experiments disclosed that the mTOR complex 1 (mTORC1) pathway is highly activated in rbm15-deficient hepatocytes. Moreover, rapamycin treatment partially restored normal hepatic gene expression as well as the nuclear location of the transcription factor Hnf4a. Taken together, these results reveal an unexpected role of Rbm15 in liver maturation.},
}

Animals
Apoptosis
CRISPR-Cas Systems
Cell Differentiation
Cell Proliferation
*Gene Deletion
*Gene Expression Regulation, Developmental
Hepatocytes/cytology/metabolism
Liver/cytology/*embryology
Zebrafish/*embryology/genetics

@article {pmid32349563,
year = {2020},
author = {Tromp, TR and Stroes, ESG and Hovingh, GK},
title = {Gene-based therapy in lipid management: the winding road from promise to practice.},
journal = {Expert opinion on investigational drugs},
volume = {29},
number = {5},
pages = {483-493},
doi = {10.1080/13543784.2020.1757070},
pmid = {32349563},
issn = {1744-7658},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cardiovascular Diseases/etiology/*prevention & control ; Genetic Therapy/*methods ; Heart Disease Risk Factors ; Humans ; Hypercholesterolemia/complications/genetics/*therapy ; Lipid Metabolism/genetics ; Oligonucleotides, Antisense/administration & dosage ; },
abstract = {INTRODUCTION: Cardiovascular disease (CVD) is a leading cause of morbidity and mortality. High plasma low-density lipoprotein cholesterol (LDL-C) levels are a key CVD-risk factor. Triglyceride-rich remnant particles and lipoprotein(a) (Lp[a]) are also causally related to CVD. Consequently, therapeutic strategies for lowering LDL-C and triglyceride levels are widely used in routine clinical practice; however, specific Lp(a) lowering agents are not available. Many patients do not achieve guideline-recommended lipid levels with currently available therapies; hence, novel targets and treatment modalities are eagerly sought.

AREAS COVERED: We discuss the milestones on the trajectory toward the full application of gene-based therapies in daily clinical practice. We describe the different methods, ranging from antisense oligonucleotides to liver-directed gene therapy and Crispr-cas9 modification to target the pivotal players in lipid metabolism: PCSK9, APOB, ANGPTL3, Lp(a), LDLR, and apoC-III.

EXPERT OPINION: While acknowledging their different stages of development, gene-based therapies are likely to invoke a paradigm shift in lipid management because they allow us to target previously undruggable targets. Moreover, their low dosing frequency, high target selectivity, and relatively predictable adverse event profile are considered major advantages over current lipid-lowering therapies.},
}

Animals
CRISPR-Cas Systems/genetics
Cardiovascular Diseases/etiology/*prevention & control
Genetic Therapy/*methods
Heart Disease Risk Factors
Humans
Hypercholesterolemia/complications/genetics/*therapy
Lipid Metabolism/genetics
Oligonucleotides, Antisense/administration & dosage

@article {pmid32307772,
year = {2020},
author = {Chiu, YW and Hori, Y and Ebinuma, I and Sato, H and Hara, N and Ikeuchi, T and Tomita, T},
title = {Identification of calcium and integrin-binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9-based screening system.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {6},
pages = {7661-7674},
doi = {10.1096/fj.201902966RR},
pmid = {32307772},
issn = {1530-6860},
mesh = {Alzheimer Disease/metabolism ; Amyloid Precursor Protein Secretases/metabolism ; Amyloid beta-Peptides/*metabolism ; Amyloid beta-Protein Precursor/metabolism ; Animals ; Brain/metabolism ; CRISPR-Cas Systems/*physiology ; Calcium-Binding Proteins/*metabolism ; Carrier Proteins/metabolism ; Cell Line ; Cell Line, Tumor ; Cell Membrane/metabolism ; HEK293 Cells ; Humans ; Mice ; Neurons/metabolism ; Protein Binding/physiology ; Protein Transport/physiology ; Synapsins/metabolism ; Up-Regulation/physiology ; },
abstract = {The aberrant metabolism of amyloid β peptide (Aβ) has been implicated in the etiology of Alzheimer disease (AD). Aβ is produced via the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. However, the precise regulatory mechanism of Aβ generation still remains unclear. To gain a better understanding of the molecular mechanism of Aβ production, we established a genetic screening method based on the CRISPR/Cas9 system to identify novel regulators of Aβ production. We successfully identified calcium and integrin-binding protein 1 (CIB1) as a potential negative regulator of Aβ production. The disruption of Cib1 significantly upregulated Aβ levels. In addition, immunoprecipitation experiments demonstrated that CIB1 interacts with the γ-secretase complex. Moreover, the disruption of Cib1 specifically reduced the cell-surface localization of mature Nicastrin (Nct), which is a component of the γ-secretase complex, without changing the intrinsic activity of γ-secretase. Finally, we confirmed using the single-cell RNA-seq data in human that CIB1 mRNA level in neuron was decreased in the early stage of AD. Taken together, our results indicate that CIB1 regulates Aβ production via controlling the subcellular localization of γ-secretase, suggesting CIB1 is involved in the development of AD.},
}

Alzheimer Disease/metabolism
Amyloid Precursor Protein Secretases/metabolism
Amyloid beta-Peptides/*metabolism
Amyloid beta-Protein Precursor/metabolism
Animals
Brain/metabolism
CRISPR-Cas Systems/*physiology
Calcium-Binding Proteins/*metabolism
Carrier Proteins/metabolism
Cell Line
Cell Line, Tumor
Cell Membrane/metabolism
HEK293 Cells
Humans
Mice
Neurons/metabolism
Protein Binding/physiology
Protein Transport/physiology
Synapsins/metabolism
Up-Regulation/physiology

@article {pmid32302524,
year = {2020},
author = {Tian, S and Liu, Y and Wu, H and Liu, H and Zeng, J and Choi, MY and Chen, H and Gerhard, R and Dong, M},
title = {Genome-Wide CRISPR Screen Identifies Semaphorin 6A and 6B as Receptors for Paeniclostridium sordellii Toxin TcsL.},
journal = {Cell host & microbe},
volume = {27},
number = {5},
pages = {782-792.e7},
pmid = {32302524},
issn = {1934-6069},
support = {R21 NS106159/NS/NINDS NIH HHS/United States ; R01 NS080833/NS/NINDS NIH HHS/United States ; R01 HL146134/HL/NHLBI NIH HHS/United States ; R01 HL093242/HL/NHLBI NIH HHS/United States ; R01 AI132387/AI/NIAID NIH HHS/United States ; R01 AI139087/AI/NIAID NIH HHS/United States ; R01 HL130845/HL/NHLBI NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; },
mesh = {A549 Cells ; Animals ; Bacterial Proteins ; Bacterial Toxins/*metabolism ; Binding Sites ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clostridium sordellii/*genetics/*metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endothelial Cells/metabolism ; Female ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Lung Neoplasms ; Male ; Mice ; Protein Binding ; Semaphorins/*genetics/*isolation & purification/metabolism ; Virulence Factors/metabolism ; },
abstract = {The exotoxin TcsL is a major virulence factor in Paeniclostridium (Clostridium) sordellii and responsible for the high lethality rate associated with P. sordellii infection. Here, we present a genome-wide CRISPR-Cas9-mediated screen using a human lung carcinoma cell line and identify semaphorin (SEMA) 6A and 6B as receptors for TcsL. Disrupting SEMA6A/6B expression in several distinct human cell lines and primary human endothelial cells results in reduced TcsL sensitivity, while SEMA6A/6B over-expression increases their sensitivity. TcsL recognizes the extracellular domain (ECD) of SEMA6A/6B via a region homologous to the receptor-binding site in Clostridioides difficile toxin B (TcdB), which binds the human receptor Frizzled. Exchanging the receptor-binding interfaces between TcsL and TcdB switches their receptor-binding specificity. Finally, administration of SEMA6A-ECD proteins protects human cells from TcsL toxicity and reduces TcsL-induced damage to lung tissues and the lethality rate in mice. These findings establish SEMA6A and 6B as pathophysiologically relevant receptors for TcsL.},
}

A549 Cells
Animals
Bacterial Proteins
Bacterial Toxins/*metabolism
Binding Sites
CRISPR-Cas Systems
Cell Line, Tumor
Clostridium sordellii/*genetics/*metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Endothelial Cells/metabolism
Female
Gene Knockout Techniques
HeLa Cells
Humans
Lung Neoplasms
Male
Mice
Protein Binding
Semaphorins/*genetics/*isolation & purification/metabolism
Virulence Factors/metabolism

@article {pmid32277852,
year = {2020},
author = {Przanowska, RK and Sobierajska, E and Su, Z and Jensen, K and Przanowski, P and Nagdas, S and Kashatus, JA and Kashatus, DF and Bhatnagar, S and Lukens, JR and Dutta, A},
title = {miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {6},
pages = {7687-7702},
pmid = {32277852},
issn = {1530-6860},
support = {18PRE33990261/AHA/American Heart Association-American Stroke Association/United States ; R01 AR067712/AR/NIAMS NIH HHS/United States ; T32 CA009109/CA/NCI NIH HHS/United States ; T32 GM007267/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; Cell Line ; Cell Proliferation/genetics ; HEK293 Cells ; Humans ; Mice ; Mice, Knockout ; MicroRNAs/*genetics ; Mitochondria/*genetics ; Muscle Development/*genetics ; Muscle, Skeletal/*physiology ; Muscular Diseases/genetics ; Myoblasts, Skeletal/*physiology ; },
abstract = {miR-206, miR-1a-1, and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this miRNA family is hypothesized to play an essential role in differentiation, a triple knock-out (tKO) of the three genes has not been done to test this hypothesis. We report that tKO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected derepression of the miRNA targets. Surprisingly, their mitochondrial function is diminished. tKO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two tKO mice survive and grow normally to adulthood with smaller myofiber diameter, diminished physical performance, and an increase in PAX7 positive satellite cells. Thus, unlike other miRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Differentiation/genetics
Cell Line
Cell Proliferation/genetics
HEK293 Cells
Humans
Mice
Mice, Knockout
MicroRNAs/*genetics
Mitochondria/*genetics
Muscle Development/*genetics
Muscle, Skeletal/*physiology
Muscular Diseases/genetics
Myoblasts, Skeletal/*physiology

@article {pmid32213923,
year = {2020},
author = {Lim, KRQ and Nguyen, Q and Dzierlega, K and Huang, Y and Yokota, T},
title = {CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy.},
journal = {Genes},
volume = {11},
number = {3},
pages = {},
pmid = {32213923},
issn = {2073-4425},
support = {FDN 143251//CIHR/Canada ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Gene Editing/methods ; Haplorhini ; Murinae ; Muscular Dystrophy, Duchenne/*genetics/pathology ; Rabbits ; Swine ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.},
}

Animals
*CRISPR-Cas Systems
*Disease Models, Animal
Gene Editing/methods
Haplorhini
Murinae
Muscular Dystrophy, Duchenne/*genetics/pathology
Rabbits
Swine

@article {pmid32164255,
year = {2020},
author = {Lanigan, TM and Kopera, HC and Saunders, TL},
title = {Principles of Genetic Engineering.},
journal = {Genes},
volume = {11},
number = {3},
pages = {},
pmid = {32164255},
issn = {2073-4425},
mesh = {Animals ; CRISPR-Cas Systems ; Gene Targeting/methods ; Gene Transfer Techniques ; Genetic Engineering/*methods/standards/trends ; Humans ; },
abstract = {Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.},
}

Animals
CRISPR-Cas Systems
Gene Targeting/methods
Gene Transfer Techniques
Genetic Engineering/*methods/standards/trends
Humans

@article {pmid32100378,
year = {2020},
author = {Kim, GD and Lee, JH and Song, S and Kim, SW and Han, JS and Shin, SP and Park, BC and Park, TS},
title = {Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {4},
pages = {5688-5696},
doi = {10.1096/fj.201903035R},
pmid = {32100378},
issn = {1530-6860},
mesh = {Animals ; Animals, Genetically Modified/genetics/growth & development/*metabolism ; *CRISPR-Cas Systems ; Chickens ; *Gene Editing ; Germ Cells/cytology/*metabolism ; Muscle, Skeletal/cytology/*metabolism ; Myostatin/antagonists & inhibitors/*physiology ; Phenotype ; },
abstract = {Many studies have been conducted to improve economically important livestock traits such as feed efficiency and muscle growth. Genome editing technologies represent a major advancement for both basic research and agronomic biotechnology development. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technical platform is a powerful tool used to engineer specific targeted loci. However, the potential occurrence of off-target effects, including the cleavage of unintended targets, limits the practical applications of Cas9-mediated genome editing. In this study, to minimize the off-target effects of this technology, we utilized D10A-Cas9 nickase to generate myostatin-knockout (MSTN KO) chickens via primordial germ cells. D10A-Cas9 nickase (Cas9n)-mediated MSTN KO chickens exhibited significantly larger skeletal muscles in the breast and leg. Degrees of skeletal muscle hypertrophy and hyperplasia induced by myostatin deletion differed by sex and muscle type. The abdominal fat deposition was dramatically lower in MSTN KO chickens than in wild-type chickens. Our results demonstrate that the D10A-Cas9 technical platform can facilitate precise and efficient targeted genome engineering and may broaden the range of applications for genome-edited chickens in practical industrialization and as animal models of human diseases.},
}

Animals
Animals, Genetically Modified/genetics/growth & development/*metabolism
*CRISPR-Cas Systems
Chickens
*Gene Editing
Germ Cells/cytology/*metabolism
Muscle, Skeletal/cytology/*metabolism
Myostatin/antagonists & inhibitors/*physiology
Phenotype

@article {pmid31999448,
year = {2020},
author = {Siemon, T and Wang, Z and Bian, G and Seitz, T and Ye, Z and Lu, Y and Cheng, S and Ding, Y and Huang, Y and Deng, Z and Liu, T and Christmann, M},
title = {Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {6},
pages = {2760-2765},
doi = {10.1021/jacs.9b12940},
pmid = {31999448},
issn = {1520-5126},
mesh = {CRISPR-Cas Systems ; Escherichia coli/genetics ; *Metabolic Engineering ; Plants/*chemistry ; Saccharomyces cerevisiae/genetics ; Sesquiterpenes, Guaiane/chemical synthesis/*chemistry ; },
abstract = {Herein, we report a short semisynthesis of the potent transient receptor potential canonical (TRPC) channel agonist englerin A (EA) and the related guaianes oxyphyllol and orientalol E. The guaia-6,10(14)-diene starting material was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and was produced with high titers. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis providing an efficient and economical method for producing EA and analogues.},
}

CRISPR-Cas Systems
Escherichia coli/genetics
*Metabolic Engineering
Plants/*chemistry
Saccharomyces cerevisiae/genetics
Sesquiterpenes, Guaiane/chemical synthesis/*chemistry

@article {pmid31931564,
year = {2020},
author = {Alyami, MZ and Alsaiari, SK and Li, Y and Qutub, SS and Aleisa, FA and Sougrat, R and Merzaban, JS and Khashab, NM},
title = {Cell-Type-Specific CRISPR/Cas9 Delivery by Biomimetic Metal Organic Frameworks.},
journal = {Journal of the American Chemical Society},
volume = {142},
number = {4},
pages = {1715-1720},
doi = {10.1021/jacs.9b11638},
pmid = {31931564},
issn = {1520-5126},
mesh = {Animals ; *Biomimetics ; *CRISPR-Cas Systems ; HeLa Cells ; Heterografts ; Humans ; MCF-7 Cells ; Metal-Organic Frameworks/*chemistry ; Mice ; },
abstract = {Effective and cell-type-specific delivery of CRISPR/Cas9 gene editing elements remains a challenging open problem. Here we report the development of biomimetic cancer cell coated zeolitic imidazolate frameworks (ZIFs) for targeted and cell-specific delivery of this genome editing machinery. Coating ZIF-8 that is encapsulating CRISPR/Cas9 (CC-ZIF) with a cancer cell membrane resulted in the uniformly covered C3-ZIF(cell membrane type). Incubation of C3-ZIFMCF with MCF-7, HeLa, HDFn, and aTC cell lines showed the highest uptake by MCF-7 cells and negligible uptake by the healthy cells (i.e., HDFn and aTC). As to genome editing, a 3-fold repression in the EGFP expression was observed when MCF-7 were transfected with C3-ZIFMCF compared to 1-fold repression in the EGFP expression when MCF-7 were transfected with C3-ZIFHELA. In vivo testing confirmed the selectivity of C3-ZIFMCF to accumulate in MCF-7 tumor cells. This supports the ability of this biomimetic approach to match the needs of cell-specific targeting, which is unquestionably the most critical step in the future translation of genome editing technologies.},
}

Animals
*Biomimetics
*CRISPR-Cas Systems
HeLa Cells
Heterografts
Humans
MCF-7 Cells
Metal-Organic Frameworks/*chemistry
Mice

@article {pmid31893458,
year = {2020},
author = {Bao, A and Tran, LP and Cao, D},
title = {CRISPR/Cas9-Based Gene Editing in Soybean.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2107},
number = {},
pages = {349-364},
doi = {10.1007/978-1-0716-0235-5_19},
pmid = {31893458},
issn = {1940-6029},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Cotyledon/genetics/*growth & development ; Gene Editing/*methods ; Mutation ; Plant Breeding ; Soybeans/genetics/*growth & development ; Tissue Culture Techniques ; Transformation, Genetic ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean, a number of laboratories have obtained mutants by CRISPR/Cas9 system; however, there has been not yet a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single guide RNA) transgenes.},
}

Agrobacterium/genetics
CRISPR-Cas Systems
Cotyledon/genetics/*growth & development
Gene Editing/*methods
Mutation
Plant Breeding
Soybeans/genetics/*growth & development
Tissue Culture Techniques
Transformation, Genetic

@article {pmid31882399,
year = {2020},
author = {Ianiri, G and Fang, YF and Dahlmann, TA and Clancey, SA and Janbon, G and Kück, U and Heitman, J},
title = {Mating-Type-Specific Ribosomal Proteins Control Aspects of Sexual Reproduction in Cryptococcus neoformans.},
journal = {Genetics},
volume = {214},
number = {3},
pages = {635-649},
pmid = {31882399},
issn = {1943-2631},
support = {R01 AI039115/AI/NIAID NIH HHS/United States ; R01 AI050113/AI/NIAID NIH HHS/United States ; R37 AI039115/AI/NIAID NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Cryptococcus neoformans/*genetics/growth & development ; Fungal Proteins/genetics ; Genes, Mating Type, Fungal/*genetics ; Haploidy ; Phenotype ; Reproduction/*genetics ; Ribosomal Proteins/*genetics ; },
abstract = {The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100 kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, and RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the α and a alleles of the ribosomal genes RPL22 and RPL39 Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22αΔ or RPL22α/rpl22aΔ mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology, the RPL22 alleles were exchanged in haploid MATα and MATa strains of C. neoformans These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidating functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.},
}

Alleles
CRISPR-Cas Systems/genetics
Cryptococcus neoformans/*genetics/growth & development
Fungal Proteins/genetics
Genes, Mating Type, Fungal/*genetics
Haploidy
Phenotype
Reproduction/*genetics
Ribosomal Proteins/*genetics

@article {pmid33453283,
year = {2021},
author = {Koay, TW and Osterhof, C and Orlando, IMC and Keppner, A and Andre, D and Yousefian, S and Alonso, MS and Correia, M and Markworth, R and Schödel, J and Hankeln, T and Hoogewijs, D},
title = {Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {100291},
doi = {10.1016/j.jbc.2021.100291},
pmid = {33453283},
issn = {1083-351X},
abstract = {Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell type-specific expression, regulation and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that -in addition to testes- ADGB is prominently expressed in the female reproductive tract, lungs and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested co-regulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.},
}

@article {pmid33452819,
year = {2021},
author = {Barbour, A and Glogauer, J and Grinfeld, L and Ostadsharif Memar, R and Fine, N and Tenenbaum, H and Glogauer, M},
title = {The role of CRISPR-Cas in advancing precision periodontics.},
journal = {Journal of periodontal research},
volume = {},
number = {},
pages = {},
doi = {10.1111/jre.12846},
pmid = {33452819},
issn = {1600-0765},
abstract = {The significant advancement of molecular biology has revolutionized medicine and provided important technologies to further clinical research development. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are DNA sequences derived from bacteriophages which have previously infected the bacterial species. The CRISPR-Cas system plays a key role in bacterial defense by detecting and destroying DNA fragments during subsequent bacteriophage invasions. The Cas9 enzyme recognizes and cleaves new invading CRISPR-complementary DNA sequences. Researchers have taken advantage of this biological device to manipulate microbes' genes and develop novel therapeutics to tackle systemic disease. In this review, we discuss the potential of utilizing CRISPR-Cas systems in the periodontal field to develop personalized periodontal care. We summarize promising attempts to bring this technology to the clinical setting. Finally, we provide insights regarding future developments to best utilize the CRISPR-Cas systems to advance precision periodontics. Although further research is imperative to evaluate the safety and potential of using CRISPR-Cas to develop precision periodontics approaches, few studies showed promising data to support the investment into this important technology in the dental sector. CRISPR-Cas9 can be a useful tool to create knockouts in vitro and in vivo as a screening tool to identify cellular pathways involved in the pathogenesis of periodontitis. Alternative CRISPR systems such as CRISPRa, CRISPRi, and Cas13 can be used to modify the transcriptome and gene expression of genes involved in periodontitis progression. CRISPR systems such as Cas3 can be used to target the periodontal biofilm and to develop new strategies to reduce or eliminate periodontal pathogens. Currently, the utility of CRISPR-Cas applications in clinical settings is limited. Through this review, we hope to foster further discussion in the periodontal research and clinical communities with respect to the potential clinical application of novel, CRISPR-Cas based, therapeutics for periodontitis.},
}

@article {pmid33449167,
year = {2021},
author = {Brandes, RP and Dueck, A and Engelhardt, S and Kaulich, M and Kupatt, C and De Angelis, MT and Leisegang, MS and le Noble, F and Moretti, A and Müller, OJ and Skryabin, BV and Thum, T and Wurst, W},
title = {DGK and DZHK position paper on genome editing: basic science applications and future perspective.},
journal = {Basic research in cardiology},
volume = {116},
number = {1},
pages = {2},
pmid = {33449167},
issn = {1435-1803},
abstract = {For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.},
}

@article {pmid33338421,
year = {2021},
author = {Hoffmann, HH and Schneider, WM and Rozen-Gagnon, K and Miles, LA and Schuster, F and Razooky, B and Jacobson, E and Wu, X and Yi, S and Rudin, CM and MacDonald, MR and McMullan, LK and Poirier, JT and Rice, CM},
title = {TMEM41B Is a Pan-flavivirus Host Factor.},
journal = {Cell},
volume = {184},
number = {1},
pages = {133-148.e20},
doi = {10.1016/j.cell.2020.12.005},
pmid = {33338421},
issn = {1097-4172},
support = {R01 AI124690/AI/NIAID NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Asian Continental Ancestry Group/genetics ; Autophagy ; COVID-19/genetics/metabolism/virology ; CRISPR-Cas Systems ; Cell Line ; Flavivirus/*physiology ; Flavivirus Infections/*genetics/immunology/metabolism/virology ; Gene Knockout Techniques ; Genome-Wide Association Study ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Membrane Proteins/genetics/*metabolism ; Polymorphism, Single Nucleotide ; SARS-CoV-2/physiology ; Virus Replication ; Yellow fever virus/physiology ; Zika Virus/physiology ; },
abstract = {Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.},
}

Animals
Asian Continental Ancestry Group/genetics
Autophagy
COVID-19/genetics/metabolism/virology
CRISPR-Cas Systems
Cell Line
Flavivirus/*physiology
Flavivirus Infections/*genetics/immunology/metabolism/virology
Gene Knockout Techniques
Genome-Wide Association Study
Host-Pathogen Interactions
Humans
Immunity, Innate
Membrane Proteins/genetics/*metabolism
Polymorphism, Single Nucleotide
SARS-CoV-2/physiology
Virus Replication
Yellow fever virus/physiology
Zika Virus/physiology

@article {pmid33139551,
year = {2020},
author = {Takao, T and Sato, M and Maruyama, T},
title = {Optogenetic regulation of embryo implantation in mice using photoactivatable CRISPR-Cas9.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {46},
pages = {28579-28581},
pmid = {33139551},
issn = {1091-6490},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; *Embryo Implantation ; Fertility ; Leukemia Inhibitory Factor/genetics/*metabolism ; Mice, Inbred ICR ; *Optogenetics ; },
abstract = {Embryo implantation is achieved upon successful interaction between a fertilized egg and receptive endometrium and is mediated by spatiotemporal expression of implantation-associated molecules including leukemia inhibitory factor (LIF). Here we demonstrate, in mice, that LIF knockdown via a photoactivatable CRISPR-Cas9 gene editing system and illumination with a light-emitting diode can spatiotemporally disrupt fertility. This system enables dissection of spatiotemporal molecular mechanisms associated with embryo implantation and provides a therapeutic strategy for temporal control of reproductive functions in vivo.},
}

Animals
CRISPR-Associated Protein 9
CRISPR-Cas Systems
*Embryo Implantation
Fertility
Leukemia Inhibitory Factor/genetics/*metabolism
Mice, Inbred ICR
*Optogenetics

@article {pmid33075436,
year = {2020},
author = {Murphy, ZC and Getman, MR and Myers, JA and Burgos Villar, KN and Leshen, E and Kurita, R and Nakamura, Y and Steiner, LA},
title = {Codanin-1 mutations engineered in human erythroid cells demonstrate role of CDAN1 in terminal erythroid maturation.},
journal = {Experimental hematology},
volume = {91},
number = {},
pages = {32-38.e6},
pmid = {33075436},
issn = {1873-2399},
support = {R01 DK104920/DK/NIDDK NIH HHS/United States ; },
mesh = {Acetylation ; Anemia, Dyserythropoietic, Congenital/blood/*genetics ; CRISPR-Cas Systems ; Cell Line ; Cell Nucleus/ultrastructure ; Cell Survival ; Chromatin/ultrastructure ; Erythroid Cells/*cytology/metabolism ; Erythropoiesis/*genetics/physiology ; Exons/genetics ; Gene Editing ; Glycoproteins/deficiency/*genetics/physiology ; Histone Code ; Humans ; Nuclear Proteins/deficiency/*genetics/physiology ; Phenotype ; Protein Processing, Post-Translational ; },
abstract = {The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by the lack of in vitro models that recapitulate key features of the disease, and most studies on CDAN1 function have been done in nonerythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability and increased intercellular bridges and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insights into CDA-I pathology.},
}

Acetylation
Anemia, Dyserythropoietic, Congenital/blood/*genetics
CRISPR-Cas Systems
Cell Line
Cell Nucleus/ultrastructure
Cell Survival
Chromatin/ultrastructure
Erythroid Cells/*cytology/metabolism
Erythropoiesis/*genetics/physiology
Exons/genetics
Gene Editing
Glycoproteins/deficiency/*genetics/physiology
Histone Code
Humans
Nuclear Proteins/deficiency/*genetics/physiology
Phenotype
Protein Processing, Post-Translational

@article {pmid33020616,
year = {2020},
author = {Matharu, N and Ahituv, N},
title = {Modulating gene regulation to treat genetic disorders.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {11},
pages = {757-775},
doi = {10.1038/s41573-020-0083-7},
pmid = {33020616},
issn = {1474-1784},
mesh = {Animals ; CRISPR-Cas Systems/drug effects/genetics ; Gene Expression Regulation/*drug effects/genetics ; Genetic Diseases, Inborn/*drug therapy/genetics ; Humans ; Mutation/drug effects/genetics ; Pharmaceutical Preparations/*administration & dosage ; },
abstract = {Over a thousand diseases are caused by mutations that alter gene expression levels. The potential of nuclease-deficient zinc fingers, TALEs or CRISPR fusion systems to treat these diseases by modulating gene expression has recently emerged. These systems can be applied to modify the activity of gene-regulatory elements - promoters, enhancers, silencers and insulators, subsequently changing their target gene expression levels to achieve therapeutic benefits - an approach termed cis-regulation therapy (CRT). Here, we review emerging CRT technologies and assess their therapeutic potential for treating a wide range of diseases caused by abnormal gene dosage. The challenges facing the translation of CRT into the clinic are discussed.},
}

Animals
CRISPR-Cas Systems/drug effects/genetics
Gene Expression Regulation/*drug effects/genetics
Genetic Diseases, Inborn/*drug therapy/genetics
Humans
Mutation/drug effects/genetics
Pharmaceutical Preparations/*administration & dosage

@article {pmid33020605,
year = {2020},
author = {Leech, R and Sampath, K},
title = {A CRISPR cut for messenger RNAs.},
journal = {Lab animal},
volume = {49},
number = {11},
pages = {317-319},
doi = {10.1038/s41684-020-00661-3},
pmid = {33020605},
issn = {1548-4475},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; RNA, Messenger/genetics ; },
}

Animals
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
RNA, Messenger/genetics

@article {pmid32601291,
year = {2020},
author = {Kamble, PG and Hetty, S and Vranic, M and Almby, K and Castillejo-López, C and Abalo, XM and Pereira, MJ and Eriksson, JW},
title = {Proof-of-concept for CRISPR/Cas9 gene editing in human preadipocytes: Deletion of FKBP5 and PPARG and effects on adipocyte differentiation and metabolism.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10565},
pmid = {32601291},
issn = {2045-2322},
mesh = {Adipocytes/*metabolism ; Adipogenesis/genetics ; Adipose Tissue/metabolism ; Adult ; Aged ; CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Female ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Humans ; Middle Aged ; PPAR gamma/genetics ; Proof of Concept Study ; Tacrolimus Binding Proteins/genetics ; },
abstract = {CRISPR/Cas9 has revolutionized the genome-editing field. So far, successful application in human adipose tissue has not been convincingly shown. We present a method for gene knockout using electroporation in preadipocytes from human adipose tissue that achieved at least 90% efficiency without any need for selection of edited cells or clonal isolation. We knocked out the FKBP5 and PPARG genes in preadipocytes and studied the resulting phenotypes. PPARG knockout prevented differentiation into adipocytes. Conversely, deletion of FKBP51, the protein coded by the FKBP5 gene, did not affect adipogenesis. Instead, it markedly modulated glucocorticoid effects on adipocyte glucose metabolism and, furthermore, we show some evidence of altered transcriptional activity of glucocorticoid receptors. This has potential implications for the development of insulin resistance and type 2 diabetes. The reported method is simple, easy to adapt, and enables the use of human primary preadipocytes instead of animal adipose cell models to assess the role of key genes and their products in adipose tissue development, metabolism and pathobiology.},
}

Adipocytes/*metabolism
Adipogenesis/genetics
Adipose Tissue/metabolism
Adult
Aged
CRISPR-Cas Systems/genetics
Cell Differentiation/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Female
Gene Editing/*methods
Gene Knockout Techniques/methods
Humans
Middle Aged
PPAR gamma/genetics
Proof of Concept Study
Tacrolimus Binding Proteins/genetics

@article {pmid32576837,
year = {2020},
author = {Lamsfus-Calle, A and Daniel-Moreno, A and Antony, JS and Epting, T and Heumos, L and Baskaran, P and Admard, J and Casadei, N and Latifi, N and Siegmund, DM and Kormann, MSD and Handgretinger, R and Mezger, M},
title = {Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34+ HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {10133},
pmid = {32576837},
issn = {2045-2322},
mesh = {Anemia, Sickle Cell/*genetics/therapy ; Antigens, CD34 ; *CRISPR-Cas Systems ; Cells, Cultured ; Fetal Hemoglobin/*genetics ; Gene Editing/*methods ; Gene Expression/genetics ; Humans ; Kruppel-Like Transcription Factors/*genetics ; Molecular Targeted Therapy ; Mutation ; Repressor Proteins/*genetics ; gamma-Globins/*genetics ; },
abstract = {β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.},
}

Anemia, Sickle Cell/*genetics/therapy
Antigens, CD34
*CRISPR-Cas Systems
Cells, Cultured
Fetal Hemoglobin/*genetics
Gene Editing/*methods
Gene Expression/genetics
Humans
Kruppel-Like Transcription Factors/*genetics
Molecular Targeted Therapy
Mutation
Repressor Proteins/*genetics
gamma-Globins/*genetics

@article {pmid32527526,
year = {2020},
author = {Yang, Y and Liu, G and Chen, X and Liu, M and Zhan, C and Liu, X and Bai, Z},
title = {High efficiency CRISPR/Cas9 genome editing system with an eliminable episomal sgRNA plasmid in Pichia pastoris.},
journal = {Enzyme and microbial technology},
volume = {138},
number = {},
pages = {109556},
doi = {10.1016/j.enzmictec.2020.109556},
pmid = {32527526},
issn = {1879-0909},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Fungal ; Genome, Fungal/genetics ; Nucleotide Motifs ; Plasmids/*genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/chemistry/*genetics ; Saccharomycetales/*genetics/growth & development ; },
abstract = {Pichia pastoris is a methylotrophic yeast in which host heterologous expression of proteins has been developed owing to the strong inducible alcohol oxidase promoter (PAOX1). However, it is difficult to manipulate the genome in P. pastoris. Based on previous attempts to apply the CRISPR/Cas9 system in P. pastoris, a CRISPR/Cas9 system with episomal sgRNA plasmid was developed and 100 % genome editing efficiency, high multicopy gene editing and stable multigene editing were obtained without a sharp decline caused by multi-sgRNA. And 28/34 (∼82 %) sgRNAs tested were effective. The CGG may have a slightly higher and more stable cleavage efficiency than the other three NGG motifs, and a low GC content may be preferable for higher cleavage efficiency. This provides researchers with a stable genome editing tool that shows a high editing efficiency, shortening the experimentation period. Furthermore, we introduced dCas9 into P. pastoris and achieved target gene interference, expanding the CRISPR/Cas9 toolbox in P. pastoris.},
}

CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems
Fungal Proteins/genetics
Gene Editing/*methods
Gene Expression Regulation, Fungal
Genome, Fungal/genetics
Nucleotide Motifs
Plasmids/*genetics
Promoter Regions, Genetic/genetics
RNA, Guide/chemistry/*genetics
Saccharomycetales/*genetics/growth & development

@article {pmid32321775,
year = {2020},
author = {Kagoya, Y and Guo, T and Yeung, B and Saso, K and Anczurowski, M and Wang, CH and Murata, K and Sugata, K and Saijo, H and Matsunaga, Y and Ohashi, Y and Butler, MO and Hirano, N},
title = {Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.},
journal = {Cancer immunology research},
volume = {8},
number = {7},
pages = {926-936},
doi = {10.1158/2326-6066.CIR-18-0508},
pmid = {32321775},
issn = {2326-6074},
mesh = {Allografts ; Animals ; Antigens, CD19/immunology ; CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Histocompatibility Antigens Class I/*chemistry/genetics ; Histocompatibility Antigens Class II/*chemistry/genetics ; Humans ; Immunotherapy, Adoptive/*methods ; Leukocytes, Mononuclear ; Lymphocyte Activation ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasms/immunology/metabolism/*therapy ; Receptors, Antigen, T-Cell/antagonists & inhibitors/genetics/*immunology ; Receptors, Chimeric Antigen/*immunology ; },
abstract = {Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.},
}

Allografts
Animals
Antigens, CD19/immunology
CRISPR-Cas Systems
Cells, Cultured
Disease Models, Animal
Histocompatibility Antigens Class I/*chemistry/genetics
Histocompatibility Antigens Class II/*chemistry/genetics
Humans
Immunotherapy, Adoptive/*methods
Leukocytes, Mononuclear
Lymphocyte Activation
Mice
Mice, Inbred NOD
Mice, SCID
Neoplasms/immunology/metabolism/*therapy
Receptors, Antigen, T-Cell/antagonists & inhibitors/genetics/*immunology
Receptors, Chimeric Antigen/*immunology

@article {pmid33445135,
year = {2021},
author = {Zuo, F and Marcotte, H},
title = {Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing.},
journal = {Current opinion in biotechnology},
volume = {70},
number = {},
pages = {75-82},
doi = {10.1016/j.copbio.2020.12.015},
pmid = {33445135},
issn = {1879-0429},
abstract = {Typical traditional probiotics lactobacilli and bifidobacteria are gaining great interest to be developed as living diagnostics and therapeutics for improving human health. However, the mechanistic basis underlying their inherent health beneficial property remain incompletely understood which can slow down the translational pipeline in the functional food and pharmaceutical field. Efficient genome editing will advance the understanding of the molecular mechanism of the probiotics' physiological properties and their interaction with the host and the host microbiota, thereby further promote the development of next-generation designer probiotics with improved robustness and tailored functionalities. With the expansion of genome editing strategies such as CRISPR-Cas-based tools and IPSD assisted genome engineering as well as other synthetic biology technologies, the research and application of these health-promoting bacteria for the food and pharmaceutical industry will be further enhanced.},
}

@article {pmid33444542,
year = {2020},
author = {Li, Y and Bondy-Denomy, J},
title = {Anti-CRISPRs go viral: the infection biology of CRISPR-Cas inhibitors.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2020.12.007},
pmid = {33444542},
issn = {1934-6069},
abstract = {Bacteriophages encode diverse anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas immunity during infection of their bacterial hosts. Although detailed mechanisms have been characterized for multiple Acr proteins, an understanding of their role in phage infection biology is just emerging. Here, we review recent work in this area and propose a framework of "phage autonomy" to evaluate CRISPR-immune evasion strategies. During phage infection, Acr proteins are deployed by a tightly regulated "fast on-fast off" transcriptional burst, which is necessary, but insufficient, for CRISPR-Cas inactivation. Instead of a single phage shutting down CRISPR-Cas immunity, a community of acr-carrying phages cooperate to suppress bacterial immunity, displaying low phage autonomy. Enzymatic Acr proteins with novel mechanisms have been recently revealed and are predicted to enhance phage autonomy, while phage DNA protective measures offer the highest phage autonomy observed. These varied Acr mechanisms and strengths also have unexpected impacts on the bacterial populations and competing phages.},
}

@article {pmid33443157,
year = {2021},
author = {Kurtz, S and Lucas-Hahn, A and Schlegelberger, B and Göhring, G and Niemann, H and Mettenleiter, TC and Petersen, B},
title = {Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {118},
number = {2},
pages = {},
doi = {10.1073/pnas.2008743118},
pmid = {33443157},
issn = {1091-6490},
abstract = {The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.},
}

@article {pmid33441159,
year = {2021},
author = {Scott, TA and Morris, KV},
title = {Designer nucleases to treat malignant cancers driven by viral oncogenes.},
journal = {Virology journal},
volume = {18},
number = {1},
pages = {18},
pmid = {33441159},
issn = {1743-422X},
support = {R01 113407-01/MH/NIMH NIH HHS/United States ; },
abstract = {Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specific small-molecule drug screens or the broad-acting toxic effects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efficacy in this role. Although many challenges remain for the clinical application of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specific and permanent inactivation by nucleases, argues for their development as next-generation treatments for this aggressive group of cancers.},
}

@article {pmid32868402,
year = {2020},
author = {Guan, J and Bondy-Denomy, J},
title = {Intracellular Organization by Jumbo Bacteriophages.},
journal = {Journal of bacteriology},
volume = {203},
number = {2},
pages = {},
pmid = {32868402},
issn = {1098-5530},
abstract = {Since their discovery more than 100 years ago, the viruses that infect bacteria (bacteriophages) have been widely studied as model systems. Largely overlooked, however, have been "jumbo phages," with genome sizes ranging from 200 to 500 kbp. Jumbo phages generally have large virions with complex structures and a broad host spectrum. While the majority of jumbo phage genes are poorly functionally characterized, recent work has discovered many unique biological features, including a conserved tubulin homolog that coordinates a proteinaceous nucleus-like compartment that houses and segregates phage DNA. The tubulin spindle displays dynamic instability and centers the phage nucleus within the bacterial host during phage infection for optimal reproduction. The shell provides robust physical protection for the enclosed phage genomes against attack from DNA-targeting bacterial immune systems, thereby endowing jumbo phages with broad resistance. In this review, we focus on the current knowledge of the cytoskeletal elements and the specialized nuclear compartment derived from jumbo phages, and we highlight their importance in facilitating spatial and temporal organization over the viral life cycle. Additionally, we discuss the evolutionary relationships between jumbo phages and eukaryotic viruses, as well as the therapeutic potential and drawbacks of jumbo phages as antimicrobial agents in phage therapy.},
}

@article {pmid32471865,
year = {2020},
author = {Das, S and Banerjee, A and Kamran, M and Ejazi, SA and Asad, M and Ali, N and Chakrabarti, S},
title = {A chemical inhibitor of heat shock protein 78 (HSP78) from Leishmania donovani represents a potential antileishmanial drug candidate.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {29},
pages = {9934-9947},
pmid = {32471865},
issn = {1083-351X},
mesh = {Animals ; Antiprotozoal Agents/*pharmacology ; CRISPR-Cas Systems ; Cricetinae ; Dinucleoside Phosphates/*pharmacology ; Gene Knockout Techniques ; Heat-Shock Proteins/*antagonists & inhibitors/genetics/metabolism ; Humans ; Leishmania donovani/genetics/*metabolism ; Leishmaniasis, Visceral/*drug therapy/genetics/metabolism ; Macrophages/metabolism/*parasitology ; Mice ; Protozoan Proteins/*antagonists & inhibitors/genetics/metabolism ; },
abstract = {The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 (LdHSP78+/-) and Leishmania mexicana HSP78 (LmxHSP78+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78+/- parasites, compared with WT parasites. Virulence of the LdHSP78+/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P1,P5-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.},
}

Animals
Antiprotozoal Agents/*pharmacology
CRISPR-Cas Systems
Cricetinae
Dinucleoside Phosphates/*pharmacology
Gene Knockout Techniques
Heat-Shock Proteins/*antagonists & inhibitors/genetics/metabolism
Humans
Leishmania donovani/genetics/*metabolism
Leishmaniasis, Visceral/*drug therapy/genetics/metabolism
Macrophages/metabolism/*parasitology
Mice
Protozoan Proteins/*antagonists & inhibitors/genetics/metabolism

@article {pmid31959747,
year = {2020},
author = {Huang, T and Liu, Z and Zheng, Y and Feng, T and Gao, Q and Zeng, W},
title = {YTHDF2 promotes spermagonial adhesion through modulating MMPs decay via m6A/mRNA pathway.},
journal = {Cell death & disease},
volume = {11},
number = {1},
pages = {37},
pmid = {31959747},
issn = {2041-4889},
mesh = {Adenosine/*analogs & derivatives/metabolism ; Animals ; Apoptosis ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems/genetics ; Cell Adhesion/genetics ; Cell Cycle ; Cell Line ; Cell Movement ; Cell Proliferation ; Extracellular Matrix/metabolism ; Gene Deletion ; Gene Expression Regulation ; Male ; Matrix Metalloproteinases/*metabolism ; Mice, Knockout ; Phenotype ; RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/genetics/*metabolism ; Spermatogonia/*cytology/metabolism ; },
abstract = {As the foundation of male fertility, spermatogenesis is a complicated and highly controlled process. YTHDF2 plays regulatory roles in biological processes through accelerating the degradation of target mRNAs. However, the function of YTHDF2 in spermatogenesis remains elusive. Here, we knocked out Ythdf2 in mouse spermatogonia via CRISPR/Cas9, and found that depletion of Ythdf2 mainly downregulated the expression of matrix metallopeptidase (MMPs), thus affecting cell adhesion and proliferation. m6A-IP-PCR and RIP-PCR analysis showed that Mmp3, Mmp13, Adamts1 and Adamts9 were modified with m6A and simultaneously interacted with YTHDF2. Moreover, inhibition of Mmp13 partially rescued the phenotypes in Ythdf2-KO cells. Taken together, YTHDF2 regulates cell-matrix adhesion and proliferation through modulating the expression of Mmps by the m6A/mRNA degradation pathway.},
}

Adenosine/*analogs & derivatives/metabolism
Animals
Apoptosis
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems/genetics
Cell Adhesion/genetics
Cell Cycle
Cell Line
Cell Movement
Cell Proliferation
Extracellular Matrix/metabolism
Gene Deletion
Gene Expression Regulation
Male
Matrix Metalloproteinases/*metabolism
Mice, Knockout
Phenotype
RNA, Messenger/genetics/metabolism
RNA-Binding Proteins/genetics/*metabolism
Spermatogonia/*cytology/metabolism

@article {pmid33391497,
year = {2021},
author = {Wang, L and Zhou, J and Wang, Q and Wang, Y and Kang, C},
title = {Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein.},
journal = {Theranostics},
volume = {11},
number = {2},
pages = {649-664},
pmid = {33391497},
issn = {1838-7640},
mesh = {Antiviral Agents/*administration & dosage ; COVID-19/*drug therapy/virology ; CRISPR-Cas Systems/genetics ; Computational Biology ; Drug Evaluation, Preclinical ; Genetic Vectors/administration & dosage/genetics ; Hep G2 Cells ; Humans ; Molecular Docking Simulation ; RNA, Guide/*genetics ; SARS-CoV-2/genetics ; Sequence Homology, Amino Acid ; Spike Glycoprotein, Coronavirus/*genetics ; },
abstract = {The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.},
}

Antiviral Agents/*administration & dosage
COVID-19/*drug therapy/virology
CRISPR-Cas Systems/genetics
Computational Biology
Drug Evaluation, Preclinical
Genetic Vectors/administration & dosage/genetics
Hep G2 Cells
Humans
Molecular Docking Simulation
RNA, Guide/*genetics
SARS-CoV-2/genetics
Sequence Homology, Amino Acid
Spike Glycoprotein, Coronavirus/*genetics

@article {pmid33303323,
year = {2021},
author = {Dronina, J and Bubniene, US and Ramanavicius, A},
title = {The application of DNA polymerases and Cas9 as representative of DNA-modifying enzymes group in DNA sensor design (review).},
journal = {Biosensors & bioelectronics},
volume = {175},
number = {},
pages = {112867},
doi = {10.1016/j.bios.2020.112867},
pmid = {33303323},
issn = {1873-4235},
mesh = {*Biosensing Techniques ; COVID-19/*diagnosis/genetics/virology ; CRISPR-Cas Systems/genetics ; Diagnostic Tests, Routine ; Humans ; RNA, Viral/genetics/*isolation & purification ; SARS-CoV-2/*isolation & purification/pathogenicity ; },
abstract = {Rapid detection of nucleic acids (DNA or RNA) by inexpensive, selective, accurate, and highly sensitive methods is very important for biosensors. DNA-sensors based on DNA-modifying enzymes for fast determination and monitoring of pathogenic (Zika, Dengue, SARS-Cov-2 (inducer of COVID-19), human papillomavirus, HIV, etc.) viruses and diagnosis of virus-induced diseases is a key factor of this overview. Recently, DNA-modifying enzymes (Taq DNA polymerase, Phi29 DNA polymerase) have been widely used for the diagnosis of virus or pathogenic disease by gold standard (PCR, qPCR, RT-qPCR) methods, therefore, alternative methods have been reviewed. The main mechanisms of DNA metabolism (replication cycle, amplification) and the genomeediting tool CRISPR-Cas9 are purposefully discussed in order to address strategic possibility to design DNA-sensors based on immobilized DNA-enzymes. However, the immobilization of biologically active proteins on a gold carrier technique with the ability to detect viral or bacterial nucleic acids is individual for each DNA-modifying enzyme group, due to a different number of active sites, C and N terminal locations and arrangement, therefore, individual protocols based on the 'masking' of active sites should be elaborated for each enzyme.},
}

*Biosensing Techniques
COVID-19/*diagnosis/genetics/virology
CRISPR-Cas Systems/genetics
Diagnostic Tests, Routine
Humans
RNA, Viral/genetics/*isolation & purification
SARS-CoV-2/*isolation & purification/pathogenicity

@article {pmid33196851,
year = {2020},
author = {Sgro, A and Blancafort, P},
title = {Epigenome engineering: new technologies for precision medicine.},
journal = {Nucleic acids research},
volume = {48},
number = {22},
pages = {12453-12482},
pmid = {33196851},
issn = {1362-4962},
support = {R01 CA170370/CA/NCI NIH HHS/United States ; R01 DA036906/DA/NIDA NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromatin/*genetics ; DNA Methylation/*genetics ; Epigenome/*genetics ; *Gene Editing ; Genetic Engineering ; Humans ; Precision Medicine/trends ; },
abstract = {Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more 'normal-like state', having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.},
}

CRISPR-Cas Systems/genetics
Chromatin/*genetics
DNA Methylation/*genetics
Epigenome/*genetics
*Gene Editing
Genetic Engineering
Humans
Precision Medicine/trends

@article {pmid33176167,
year = {2020},
author = {Mehta, HM and Corey, SJ},
title = {Getting Back to Normal: Correcting SCN by Universal or Precision Strikes.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {28},
number = {12},
pages = {2525-2526},
pmid = {33176167},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Congenital Bone Marrow Failure Syndromes ; *Hematopoietic Stem Cell Transplantation ; Mutation ; Neutropenia/congenital ; },
}

*CRISPR-Cas Systems
Congenital Bone Marrow Failure Syndromes
*Hematopoietic Stem Cell Transplantation
Mutation
Neutropenia/congenital

@article {pmid33152068,
year = {2020},
author = {Cooper, SE and Schwartzentruber, J and Bello, E and Coomber, EL and Bassett, AR},
title = {Screening for functional transcriptional and splicing regulatory variants with GenIE.},
journal = {Nucleic acids research},
volume = {48},
number = {22},
pages = {e131},
pmid = {33152068},
issn = {1362-4962},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Alleles ; Alternative Splicing/genetics ; Alzheimer Disease/*genetics/pathology/therapy ; CRISPR-Cas Systems/genetics ; Clusterin/*genetics ; Enhancer Elements, Genetic/*genetics ; Gene Editing ; Genetic Variation/genetics ; Genome-Wide Association Study ; Humans ; Induced Pluripotent Stem Cells/metabolism/transplantation ; Mutation ; Polymorphism, Single Nucleotide/genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; },
abstract = {Genome-wide association studies (GWAS) have identified numerous genetic loci underlying human diseases, but a fundamental challenge remains to accurately identify the underlying causal genes and variants. Here, we describe an arrayed CRISPR screening method, Genome engineering-based Interrogation of Enhancers (GenIE), which assesses the effects of defined alleles on transcription or splicing when introduced in their endogenous genomic locations. We use this sensitive assay to validate the activity of transcriptional enhancers and splice regulatory elements in human induced pluripotent stem cells (hiPSCs), and develop a software package (rgenie) to analyse the data. We screen the 99% credible set of Alzheimer's disease (AD) GWAS variants identified at the clusterin (CLU) locus to identify a subset of likely causal variants, and employ GenIE to understand the impact of specific mutations on splicing efficiency. We thus establish GenIE as an efficient tool to rapidly screen for the role of transcribed variants on gene expression.},
}

Alleles
Alternative Splicing/genetics
Alzheimer Disease/*genetics/pathology/therapy
CRISPR-Cas Systems/genetics
Clusterin/*genetics
Enhancer Elements, Genetic/*genetics
Gene Editing
Genetic Variation/genetics
Genome-Wide Association Study
Humans
Induced Pluripotent Stem Cells/metabolism/transplantation
Mutation
Polymorphism, Single Nucleotide/genetics
Regulatory Sequences, Nucleic Acid/*genetics

@article {pmid33104788,
year = {2020},
author = {Xu, H and Wang, J and Liang, Y and Fu, Y and Li, S and Huang, J and Xu, H and Zou, W and Chen, B},
title = {TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells.},
journal = {Nucleic acids research},
volume = {48},
number = {22},
pages = {e127},
pmid = {33104788},
issn = {1362-4962},
mesh = {Alleles ; Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Chromatin/*genetics ; Fluorescent Antibody Technique/methods ; Gene Expression Regulation/genetics ; Gene Regulatory Networks/*genetics ; Humans ; *Molecular Imaging ; *Single-Cell Analysis ; Transcription, Genetic ; },
abstract = {A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.},
}

Alleles
Aptamers, Nucleotide/genetics
CRISPR-Cas Systems/genetics
Cell Cycle/genetics
Chromatin/*genetics
Fluorescent Antibody Technique/methods
Gene Expression Regulation/genetics
Gene Regulatory Networks/*genetics
Humans
*Molecular Imaging
*Single-Cell Analysis
Transcription, Genetic

@article {pmid32534122,
year = {2020},
author = {Li, G and Zhang, X and Wang, H and Liu, D and Li, Z and Wu, Z and Yang, H},
title = {Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors.},
journal = {The international journal of biochemistry & cell biology},
volume = {125},
number = {},
pages = {105790},
doi = {10.1016/j.biocel.2020.105790},
pmid = {32534122},
issn = {1878-5875},
mesh = {Acetylation ; Animals ; Benzofurans/*pharmacology ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; Cells, Cultured ; Chromatin Immunoprecipitation ; DNA End-Joining Repair/*drug effects ; DNA Repair/drug effects ; Gene Editing/*methods ; HEK293 Cells ; Histone Deacetylase Inhibitors/*pharmacology ; Humans ; Hydroxamic Acids/*pharmacology ; Ku Autoantigen/metabolism ; Poly (ADP-Ribose) Polymerase-1/metabolism ; Rad51 Recombinase/genetics/metabolism ; Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; Recombinational DNA Repair/*drug effects ; Swine ; },
abstract = {Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries.},
}

Acetylation
Animals
Benzofurans/*pharmacology
CRISPR-Cas Systems
Cell Cycle/drug effects
Cells, Cultured
Chromatin Immunoprecipitation
DNA End-Joining Repair/*drug effects
DNA Repair/drug effects
Gene Editing/*methods
HEK293 Cells
Histone Deacetylase Inhibitors/*pharmacology
Humans
Hydroxamic Acids/*pharmacology
Ku Autoantigen/metabolism
Poly (ADP-Ribose) Polymerase-1/metabolism
Rad51 Recombinase/genetics/metabolism
Rad52 DNA Repair and Recombination Protein/genetics/metabolism
Recombinational DNA Repair/*drug effects
Swine

@article {pmid32470463,
year = {2020},
author = {Strezoska, Ž and Dickerson, SM and Maksimova, E and Chou, E and Gross, MM and Hemphill, K and Hardcastle, T and Perkett, M and Stombaugh, J and Miller, GW and Anderson, EM and Vermeulen, A and Smith, AVB},
title = {CRISPR-mediated transcriptional activation with synthetic guide RNA.},
journal = {Journal of biotechnology},
volume = {319},
number = {},
pages = {25-35},
doi = {10.1016/j.jbiotec.2020.05.005},
pmid = {32470463},
issn = {1873-4863},
mesh = {Animals ; Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Mice ; NIH 3T3 Cells ; *RNA, Guide ; Transcriptional Activation/*genetics ; },
abstract = {The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format.},
}

Animals
Aptamers, Nucleotide/genetics
*CRISPR-Cas Systems
Gene Editing/*methods
HEK293 Cells
Humans
Mice
NIH 3T3 Cells
*RNA, Guide
Transcriptional Activation/*genetics

@article {pmid32460957,
year = {2020},
author = {Zhang, Q and Fu, Y and Thakur, C and Bi, Z and Wadgaonkar, P and Qiu, Y and Xu, L and Rice, M and Zhang, W and Almutairy, B and Chen, F},
title = {CRISPR-Cas9 gene editing causes alternative splicing of the targeting mRNA.},
journal = {Biochemical and biophysical research communications},
volume = {528},
number = {1},
pages = {54-61},
pmid = {32460957},
issn = {1090-2104},
support = {P30 ES020957/ES/NIEHS NIH HHS/United States ; R01 ES028263/ES/NIEHS NIH HHS/United States ; R01 ES028335/ES/NIEHS NIH HHS/United States ; },
mesh = {Alternative Splicing/*genetics ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; Dioxygenases/genetics ; Exons/genetics ; *Gene Editing ; Histone Demethylases/genetics ; Humans ; Nuclear Proteins/genetics ; Polymorphism, Single Nucleotide/genetics ; RNA, Guide/genetics ; RNA, Messenger/genetics/metabolism ; Sequence Deletion ; },
abstract = {The technique of CRISPR-Cas9 gene editing has been widely used to specifically delete the selected target genes through generating double strand breaks (DSBs) and inducing insertion and/or deletion (indel) of the genomic DNAs in the cells. We recently applied this technique to disrupt mineral dust-induced gene (mdig), a potential oncogene as previously reported, by single guide RNA (sgRNA) targeting the third exon of mdig gene in several cell types, including human bronchial epithelial cell line BEAS-2B, lung cancer cell line A549, and human triple negative breast cancer cell line MDA-MB-231 cells. In addition to the successful knockout of mdig gene in these cells, we unexpectedly noted generation of several alternatively spliced mdig mRNAs. Amplification of the mdig mRNAs during the screening of knockout clones by reverse transcription-polymerase chain reaction (RT-PCR) and the subsequent sanger sequencing of DNA revealed deletion and alternative splicing of mdig mRNAs induced by CRISPR-Cas9 gene editing. The most common deletions include nine and twenty-four nucleotides deletion around the DSBs. In addition, interestingly, some mdig mRNAs showed skipping of the entire exon 3, or alternative splicing between exon 2 and exon 8 using the new donor and accept splicing sites, leading to deletion of exons 3, 4, 5, 6, and 7. Accordingly, cautions should be taken when using CRISPR-Cas9 strategy to edit human genes due to the unintended alterative splicing of the target mRNAs. It is very likely that new proteins, some of which may be highly oncogenic, may be generated from CRISPR-Cas9 gene editing.},
}

Alternative Splicing/*genetics
Base Sequence
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Cell Line
Dioxygenases/genetics
Exons/genetics
*Gene Editing
Histone Demethylases/genetics
Humans
Nuclear Proteins/genetics
Polymorphism, Single Nucleotide/genetics
RNA, Guide/genetics
RNA, Messenger/genetics/metabolism
Sequence Deletion

@article {pmid32446977,
year = {2020},
author = {Tyagi, S and Kumar, R and Das, A and Won, SY and Shukla, P},
title = {CRISPR-Cas9 system: A genome-editing tool with endless possibilities.},
journal = {Journal of biotechnology},
volume = {319},
number = {},
pages = {36-53},
doi = {10.1016/j.jbiotec.2020.05.008},
pmid = {32446977},
issn = {1873-4863},
mesh = {Animals ; Biomedical Research ; *CRISPR-Cas Systems ; *Gene Editing/ethics/methods/standards ; Humans ; Plants, Genetically Modified ; },
abstract = {The discovery of CRISPR: Cas9 and its application as a powerful gene-editing tool has transformed the world of basic and applied science, especially the molecular biology dome. Also, the smooth, quick, flexible, and very efficient nature of this technology has enabled the biologists to alter the genome of prokaryotes to complex eukaryotic systems, including plants and animals. Using CRISPR and associated tools, investigation, control, and modification of significant biological events have been more accessible than before. These biological scissors are now being used to accelerate breeding programs of crop and livestock, engineer new antimicrobials, and control disease-carrying pathogens. However, like other techniques, these cutters emerged as a double-edged sword and put several challenges to the scientific society. Here in this review article, we summarized the beneficial application of the CRISPR: Cas9 system and unsafe perception to the society if handled carelessly. We also discussed the limitations and ethical issues related to CRISPR: Cas9 technology.},
}

Animals
Biomedical Research
*CRISPR-Cas Systems
*Gene Editing/ethics/methods/standards
Humans
Plants, Genetically Modified

@article {pmid32408895,
year = {2020},
author = {Thom, CS and Jobaliya, CD and Lorenz, K and Maguire, JA and Gagne, A and Gadue, P and French, DL and Voight, BF},
title = {Tropomyosin 1 genetically constrains in vitro hematopoiesis.},
journal = {BMC biology},
volume = {18},
number = {1},
pages = {52},
pmid = {32408895},
issn = {1741-7007},
support = {R56 DK101478/DK/NIDDK NIH HHS/United States ; R01 HL130698/HL/NHLBI NIH HHS/United States ; T32HD043021//National Institute of Child Health and Human Development/International ; R01 DK101478/DK/NIDDK NIH HHS/United States ; R01DK101478/DK/NIDDK NIH HHS/United States ; T32 HD043021/HD/NICHD NIH HHS/United States ; },
mesh = {Blood Platelets/*metabolism ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Hematopoiesis/*genetics ; Hematopoietic Stem Cells/*metabolism ; Humans ; In Vitro Techniques ; Tropomyosin/deficiency/*metabolism ; },
abstract = {BACKGROUND: Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits.

RESULTS: Our model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields.

CONCLUSIONS: Our findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.},
}

Blood Platelets/*metabolism
CRISPR-Cas Systems
Genome-Wide Association Study
Hematopoiesis/*genetics
Hematopoietic Stem Cells/*metabolism
Humans
In Vitro Techniques
Tropomyosin/deficiency/*metabolism

@article {pmid32354746,
year = {2020},
author = {Chen, X and Gao, YQ and Zheng, YY and Wang, W and Wang, P and Liang, J and Zhao, W and Tao, T and Sun, J and Wei, L and Li, Y and Zhou, Y and Gan, Z and Zhang, X and Chen, HQ and Zhu, MS},
title = {The intragenic microRNA miR199A1 in the dynamin 2 gene contributes to the pathology of X-linked centronuclear myopathy.},
journal = {The Journal of biological chemistry},
volume = {295},
number = {26},
pages = {8656-8667},
pmid = {32354746},
issn = {1083-351X},
mesh = {Animals ; CRISPR-Cas Systems ; Dynamin II/analysis/*genetics ; Female ; Longevity ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/analysis/*genetics ; Muscle Strength ; Muscle, Skeletal/metabolism/pathology ; Myopathies, Structural, Congenital/*genetics/pathology ; },
abstract = {Mutations in the myotubularin 1 (MTM1) gene can cause the fatal disease X-linked centronuclear myopathy (XLCNM), but the underlying mechanism is incompletely understood. In this report, using an Mtm1-/y disease model, we found that expression of the intragenic microRNA miR-199a-1 is up-regulated along with that of its host gene, dynamin 2 (Dnm2), in XLCNM skeletal muscle. To assess the role of miR-199a-1 in XLCNM, we crossed miR-199a-1-/- with Mtm1-/y mice and found that the resultant miR-199a-1-Mtm1 double-knockout mice display markers of improved health, as evidenced by lifespans prolonged by 30% and improved muscle strength and histology. Mechanistic analyses showed that miR-199a-1 directly targets nonmuscle myosin IIA (NM IIA) expression and, hence, inhibits muscle postnatal development as well as muscle maturation. Further analysis revealed that increased expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) up-regulates Dnm2/miR-199a-1 expression in XLCNM muscle. Our results suggest that miR-199a-1 has a critical role in XLCNM pathology and imply that this microRNA could be targeted in therapies to manage XLCNM.},
}

Animals
CRISPR-Cas Systems
Dynamin II/analysis/*genetics
Female
Longevity
Male
Mice
Mice, Inbred C57BL
Mice, Knockout
MicroRNAs/analysis/*genetics
Muscle Strength
Muscle, Skeletal/metabolism/pathology
Myopathies, Structural, Congenital/*genetics/pathology

@article {pmid32245271,
year = {2020},
author = {Vidyanti, AN and Hsieh, JY and Lin, KJ and Fang, YC and Setyopranoto, I and Hu, CJ},
title = {Role of HMGB1 in an Animal Model of Vascular Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion.},
journal = {International journal of molecular sciences},
volume = {21},
number = {6},
pages = {},
pmid = {32245271},
issn = {1422-0067},
support = {MOST 106-2314-B-038-038-MY2//Ministry of Science and Technology, Taiwan/ ; },
mesh = {Amyloid beta-Peptides/metabolism ; Animals ; Behavior Rating Scale ; Brain Ischemia/diagnostic imaging/genetics/*metabolism/physiopathology ; CRISPR-Cas Systems ; Carotid Stenosis ; *Cerebrovascular Circulation ; Chronic Disease ; Dementia, Vascular/physiopathology ; Disease Models, Animal ; Gene Knockout Techniques ; HMGB1 Protein/genetics/*metabolism ; Hippocampus/diagnostic imaging/pathology/physiopathology ; Interleukin-1beta/metabolism ; Interleukin-6/metabolism ; Magnetic Resonance Imaging ; Male ; Mice ; Mice, Inbred C57BL ; Psychomotor Performance ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {The pathophysiology of vascular cognitive impairment (VCI) is associated with chronic cerebral hypoperfusion (CCH). Increased high-mobility group box protein 1 (HMGB1), a nonhistone protein involved in injury and inflammation, has been established in the acute phase of CCH. However, the role of HMGB1 in the chronic phase of CCH remains unclear. We developed a novel animal model of CCH with a modified bilateral common carotid artery occlusion (BCCAO) in C57BL/6 mice. Cerebral blood flow (CBF) reduction, the expression of HMGB1 and its proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, and IL-6), and brain pathology were assessed. Furthermore, we evaluated the effect of HMGB1 suppression through bilateral intrahippocampus injection with the CRISPR/Cas9 knockout plasmid. Three months after CCH induction, CBF decreased to 30-50% with significant cognitive decline in BCCAO mice. The 7T-aMRI showed hippocampal atrophy, but amyloid positron imaging tomography showed nonsignificant amyloid-beta accumulation. Increased levels of HMGB1, TNF-α, IL-1β, and IL-6 were observed 3 months after BCCAO. HMGB1 suppression with CRISPR/Cas9 knockout plasmid restored TNF-α, IL-1β, and IL-6 and attenuated hippocampal atrophy and cognitive decline. We believe that HMGB1 plays a pivotal role in CCH-induced VCI pathophysiology and can be a potential therapeutic target of VCI.},
}

Amyloid beta-Peptides/metabolism
Animals
Behavior Rating Scale
Brain Ischemia/diagnostic imaging/genetics/*metabolism/physiopathology
CRISPR-Cas Systems
Carotid Stenosis
*Cerebrovascular Circulation
Chronic Disease
Dementia, Vascular/physiopathology
Disease Models, Animal
Gene Knockout Techniques
HMGB1 Protein/genetics/*metabolism
Hippocampus/diagnostic imaging/pathology/physiopathology
Interleukin-1beta/metabolism
Interleukin-6/metabolism
Magnetic Resonance Imaging
Male
Mice
Mice, Inbred C57BL
Psychomotor Performance
Tumor Necrosis Factor-alpha/metabolism

@article {pmid31859532,
year = {2020},
author = {Martin, J and Free, T},
title = {A look back at 2019 in BioTechniques.},
journal = {BioTechniques},
volume = {68},
number = {1},
pages = {2-3},
doi = {10.2144/btn-2019-0164},
pmid = {31859532},
issn = {1940-9818},
mesh = {CRISPR-Cas Systems ; Humans ; *Polymerase Chain Reaction ; *Serial Publications ; Social Media ; Urine Specimen Collection/*methods ; },
}

CRISPR-Cas Systems
Humans
*Polymerase Chain Reaction
*Serial Publications
Social Media
Urine Specimen Collection/*methods

@article {pmid33428981,
year = {2021},
author = {Safari, F and Afarid, M and Rastegari, B and Haghighi, AB and Barekati-Mowahed, M and Behbahani, AB},
title = {CRISPR systems: Novel approaches for detection and combating COVID-19.},
journal = {Virus research},
volume = {},
number = {},
pages = {198282},
doi = {10.1016/j.virusres.2020.198282},
pmid = {33428981},
issn = {1872-7492},
abstract = {Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than fifty million people worldwide with near 1.3 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2. In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.},
}

@article {pmid33428900,
year = {2021},
author = {Palaz, F and Kerem Kalkan, A and Tozluyurt, A and Ozsoz, M},
title = {CRISPR-based tools: alternative methods for the diagnosis of COVID-19.},
journal = {Clinical biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.clinbiochem.2020.12.011},
pmid = {33428900},
issn = {1873-2933},
abstract = {The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spread all over the world rapidly and caused a global pandemic. To prevent the virus from spreading to more individuals, it is of great importance to identify and isolate infected individuals through testing. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard method for the diagnosis of coronavirus disease (COVID-19) worldwide. However, performing RT-qPCR is limited to centralized laboratories because of the need for sophisticated laboratory equipment and skilled personnel. Further, it can sometimes give false negative or uncertain results. Recently, new methods have been developed for nucleic acid detection and pathogen diagnosis using CRISPR-Cas systems. These methods present rapid and cost-effective diagnostic platforms that provide high sensitivity and specificity without the need for complex instrumentation. Using the CRISPR-based SARS-CoV-2 detection methods, it is possible to increase the number of daily tests in existing laboratories, reduce false negative or uncertain result rates obtained with RT-qPCR, and perform testing in resource-limited settings or at points of need where performing RT-qPCR is not feasible. Here, we briefly describe the RT-qPCR method, and discuss its limitations in meeting the current diagnostic needs. We explain how the unique properties of various CRISPR-associated enzymes are utilized for nucleic acid detection and pathogen diagnosis. Then, we highlight the important features of CRISPR-based diagnostic methods developed for SARS-CoV-2 detection. Finally, we examine the advantages and limitations of these methods, and discuss how they can contribute to improving the efficiency of the current testing systems for combating SARS-CoV-2.},
}

@article {pmid33425987,
year = {2020},
author = {Srivastava, S and Upadhyay, DJ and Srivastava, A},
title = {Next-Generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks.},
journal = {Frontiers in molecular biosciences},
volume = {7},
number = {},
pages = {582499},
doi = {10.3389/fmolb.2020.582499},
pmid = {33425987},
issn = {2296-889X},
abstract = {Virus disease spreads effortlessly mechanically or through minute insect vectors that are extremely challenging to avoid. Emergence and reemergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), H1N1 influenza virus, avian influenza virus, dengue virus, Citrus tristeza virus, and Tomato yellow leaf curl virus have paralyzed the economy of many countries. The cure for major viral diseases is not feasible; however, early detection and surveillance of the disease can obstruct their spread. Therefore, advances in the field of virus diagnosis and the development of new point-of-care testing kits become necessary globally. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is an emerging technology for gene editing and diagnostics development. Several rapid nucleic acid diagnostic kits have been developed and validated using Cas9, Cas12, and Cas13 proteins. This review summarizes the CRISPR/Cas-based next-generation molecular diagnostic techniques and portability of devices for field-based utilization.},
}

@article {pmid33332350,
year = {2020},
author = {House, NCM and Parasuram, R and Layer, JV and Price, BD},
title = {Site-specific targeting of a light activated dCas9-KillerRed fusion protein generates transient, localized regions of oxidative DNA damage.},
journal = {PloS one},
volume = {15},
number = {12},
pages = {e0237759},
pmid = {33332350},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Chromatin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/*genetics ; DNA Breaks, Double-Stranded ; DNA Damage/*genetics ; DNA Repair/*genetics ; Endonucleases/genetics ; Genome/genetics ; HEK293 Cells ; Humans ; Light ; Oxidative Stress/*genetics ; RNA, Guide/genetics ; },
abstract = {DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in "clustered" oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in "real time" by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.},
}

CRISPR-Cas Systems/*genetics
Cell Line
Chromatin/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
DNA/*genetics
DNA Breaks, Double-Stranded
DNA Damage/*genetics
DNA Repair/*genetics
Endonucleases/genetics
Genome/genetics
HEK293 Cells
Humans
Light
Oxidative Stress/*genetics
RNA, Guide/genetics

@article {pmid33046744,
year = {2020},
author = {Kang, D and Shin, W and Yoo, H and Kim, S and Lee, S and Rhee, K},
title = {Cep215 is essential for morphological differentiation of astrocytes.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {17000},
pmid = {33046744},
issn = {2045-2322},
mesh = {Animals ; Astrocytes/*physiology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/*metabolism ; Cell Differentiation ; Cell Line ; Mice ; Microtubules/*metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; Neuroglia/*physiology ; RNA, Small Interfering/genetics ; },
abstract = {Cep215 (also known as Cdk5rap2) is a centrosome protein which is involved in microtubule organization. Cep215 is also placed at specific subcellular locations and organizes microtubules outside the centrosome. Here, we report that Cep215 is involved in morphological differentiation of astrocytes. Cep215 was specifically localized at the glial processes as well as centrosomes in developing astrocytes. Morphological differentiation of astrocytes was suppressed in the Cep215-deleted P19 cells and in the Cep215-depleted embryonic hippocampal culture. We confirm that the microtubule organizing function of Cep215 is critical for the glial process formation. However, Cep215 is not involved in the regulation of cell proliferation nor cell specification. Based on the results, we propose that Cep215 organizes microtubules for glial process formation during astrocyte differentiation.},
}

Animals
Astrocytes/*physiology
CRISPR-Cas Systems
Cell Cycle Proteins/genetics/*metabolism
Cell Differentiation
Cell Line
Mice
Microtubules/*metabolism
Nerve Tissue Proteins/genetics/*metabolism
Neuroglia/*physiology
RNA, Small Interfering/genetics

@article {pmid32876764,
year = {2020},
author = {Zhao, N and Li, L and Luo, G and Xie, S and Lin, Y and Han, S and Huang, Y and Zheng, S},
title = {Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.},
journal = {Journal of industrial microbiology & biotechnology},
volume = {47},
number = {8},
pages = {599-608},
doi = {10.1007/s10295-020-02304-5},
pmid = {32876764},
issn = {1476-5535},
support = {31671840//National Natural Science Foundation of China/ ; 2018YFA0901700//National Key R&D Program of China/ ; 2019M661676//Postdoctoral Research Foundation of China/ ; },
mesh = {CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Corynebacterium glutamicum/enzymology/*genetics/metabolism ; DNA, Bacterial/*genetics ; *Gene Deletion ; Gene Editing/*methods ; Metabolic Engineering/methods ; },
abstract = {Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the - 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.},
}

CRISPR-Associated Proteins/*genetics
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Corynebacterium glutamicum/enzymology/*genetics/metabolism
DNA, Bacterial/*genetics
*Gene Deletion
Gene Editing/*methods
Metabolic Engineering/methods

@article {pmid32788672,
year = {2020},
author = {Akkaya, M and Bansal, A and Sheehan, PW and Pena, M and Cimperman, CK and Qi, CF and Yazew, T and Otto, TD and Billker, O and Miller, LH and Pierce, SK},
title = {Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {13630},
pmid = {32788672},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Extracellular Matrix/*parasitology ; Female ; Malaria, Cerebral/*parasitology ; Mice ; Mice, Inbred C57BL ; Plasmodium berghei/*genetics/growth & development/physiology ; *Polymorphism, Single Nucleotide ; Protozoan Proteins/antagonists & inhibitors/*genetics ; Virulence Factors/antagonists & inhibitors/*genetics ; },
abstract = {Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.},
}

Animals
CRISPR-Cas Systems/*genetics
Extracellular Matrix/*parasitology
Female
Malaria, Cerebral/*parasitology
Mice
Mice, Inbred C57BL
Plasmodium berghei/*genetics/growth & development/physiology
*Polymorphism, Single Nucleotide
Protozoan Proteins/antagonists & inhibitors/*genetics
Virulence Factors/antagonists & inhibitors/*genetics

@article {pmid32651405,
year = {2020},
author = {Gans, I and Hartig, EI and Zhu, S and Tilden, AR and Hutchins, LN and Maki, NJ and Graber, JH and Coffman, JA},
title = {Klf9 is a key feedforward regulator of the transcriptomic response to glucocorticoid receptor activity.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {11415},
pmid = {32651405},
issn = {2045-2322},
support = {R03 HD099468/HD/NICHD NIH HHS/United States ; P20 GM104318/GM/NIGMS NIH HHS/United States ; P20 GM103423/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Exons ; *Frameshift Mutation ; Gene Deletion ; Gene Expression Regulation ; Homozygote ; Humans ; Hydrocortisone/metabolism ; Inflammation ; Kruppel-Like Transcription Factors/*metabolism ; Larva ; Mutation ; RNA-Seq ; Receptors, Glucocorticoid/*metabolism ; Receptors, Mineralocorticoid/metabolism ; Signal Transduction ; *Transcriptome ; Up-Regulation ; Zebrafish/genetics ; Zebrafish Proteins/*metabolism ; },
abstract = {The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR369- larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously.},
}

Animals
*CRISPR-Cas Systems
Exons
*Frameshift Mutation
Gene Deletion
Gene Expression Regulation
Homozygote
Humans
Hydrocortisone/metabolism
Inflammation
Kruppel-Like Transcription Factors/*metabolism
Larva
Mutation
RNA-Seq
Receptors, Glucocorticoid/*metabolism
Receptors, Mineralocorticoid/metabolism
Signal Transduction
*Transcriptome
Up-Regulation
Zebrafish/genetics
Zebrafish Proteins/*metabolism

@article {pmid32403926,
year = {2020},
author = {Modell, AE and Siriwardena, SU and Choudhary, A},
title = {A Jumbo Phage Forms a Nucleus-like Compartment to Evade Bacterial Defense Systems.},
journal = {Biochemistry},
volume = {59},
number = {20},
pages = {1869-1870},
doi = {10.1021/acs.biochem.0c00273},
pmid = {32403926},
issn = {1520-4995},
mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Viral ; Immune System ; },
}

*Bacteriophages/genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Genome, Viral
Immune System