@article {pmid32102684,
year = {2020},
author = {Cui, YR and Wang, SJ and Chen, J and Li, J and Chen, W and Wang, S and Meng, B and Zhu, W and Zhang, Z and Yang, B and Jiang, B and Yang, G and Ma, P and Liu, J},
title = {Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides.},
journal = {Genome biology},
volume = {21},
number = {1},
pages = {51},
doi = {10.1186/s13059-020-01956-x},
pmid = {32102684},
issn = {1474-760X},
support = {31600686//National Natural Science Foundation of China/ ; 31500632//National Natural Science Foundation of China/ ; },
abstract = {BACKGROUND: CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies.

RESULTS: We report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity.

CONCLUSION: G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.},
}

@article {pmid31970591,
year = {2019},
author = {Knott, MML and Hölting, TLB and Ohmura, S and Kirchner, T and Cidre-Aranaz, F and Grünewald, TGP},
title = {Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies.},
journal = {Cancer metastasis reviews},
volume = {38},
number = {4},
pages = {625-642},
pmid = {31970591},
issn = {1573-7233},
mesh = {Animals ; Bone Neoplasms/drug therapy/genetics/metabolism/*therapy ; CRISPR-Cas Systems ; Child ; Genetic Therapy/*methods ; Humans ; Oncogene Proteins, Fusion/*genetics/*metabolism ; Pediatrics/methods ; Rhabdomyosarcoma, Alveolar/drug therapy/metabolism/therapy ; Sarcoma/drug therapy/genetics/metabolism/*therapy ; Sarcoma, Ewing/genetics/metabolism/*therapy ; },
abstract = {While sarcomas account for approximately 1% of malignant tumors of adults, they are particularly more common in children and adolescents affected by cancer. In contrast to malignancies that occur in later stages of life, childhood tumors, including sarcoma, are characterized by a striking paucity of somatic mutations. However, entity-defining fusion oncogenes acting as the main oncogenic driver mutations are frequently found in pediatric bone and soft-tissue sarcomas such as Ewing sarcoma (EWSR1-FLI1), alveolar rhabdomyosarcoma (PAX3/7-FOXO1), and synovial sarcoma (SS18-SSX1/2/4). Since strong oncogene-dependency has been demonstrated in these entities, direct pharmacological targeting of these fusion oncogenes has been excessively attempted, thus far, with limited success. Despite apparent challenges, our increasing understanding of the neomorphic features of these fusion oncogenes in conjunction with rapid technological advances will likely enable the development of new strategies to therapeutically exploit these neomorphic features and to ultimately turn the "undruggable" into first-line target structures. In this review, we provide a broad overview of the current literature on targeting neomorphic features of fusion oncogenes found in Ewing sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma, and give a perspective for future developments. Graphical abstract Scheme depicting the different targeting strategies of fusion oncogenes in pediatric fusion-driven sarcomas. Fusion oncogenes can be targeted on their DNA level (1), RNA level (2), protein level (3), and by targeting downstream functions and interaction partners (4).},
}

Animals
Bone Neoplasms/drug therapy/genetics/metabolism/*therapy
CRISPR-Cas Systems
Child
Genetic Therapy/*methods
Humans
Oncogene Proteins, Fusion/*genetics/*metabolism
Pediatrics/methods
Rhabdomyosarcoma, Alveolar/drug therapy/metabolism/therapy
Sarcoma/drug therapy/genetics/metabolism/*therapy
Sarcoma, Ewing/genetics/metabolism/*therapy

@article {pmid31609975,
year = {2019},
author = {Zhang, W and Chen, Z and Zhang, D and Zhao, B and Liu, L and Xie, Z and Yao, Y and Zheng, P},
title = {KHDC3L mutation causes recurrent pregnancy loss by inducing genomic instability of human early embryonic cells.},
journal = {PLoS biology},
volume = {17},
number = {10},
pages = {e3000468},
pmid = {31609975},
issn = {1545-7885},
mesh = {Abortion, Habitual/*genetics/metabolism/pathology ; Animals ; Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; DNA Damage ; Embryo, Mammalian ; Female ; Gene Editing ; Gene Expression Regulation ; *Genomic Instability ; Human Embryonic Stem Cells/*metabolism/pathology ; Humans ; Karyotype ; Mice ; Mice, SCID ; Micronuclei, Chromosome-Defective ; Poly (ADP-Ribose) Polymerase-1/*genetics/metabolism ; Pregnancy ; Proteins/*genetics/metabolism ; *Recombinational DNA Repair ; Signal Transduction ; },
abstract = {Recurrent pregnancy loss (RPL) is an important complication in reproductive health. About 50% of RPL cases are unexplained, and understanding the genetic basis is essential for its diagnosis and prognosis. Herein, we report causal KH domain containing 3 like (KHDC3L) mutations in RPL. KHDC3L is expressed in human epiblast cells and ensures their genome stability and viability. Mechanistically, KHDC3L binds to poly(ADP-ribose) polymerase 1 (PARP1) to stimulate its activity. In response to DNA damage, KHDC3L also localizes to DNA damage sites and facilitates homologous recombination (HR)-mediated DNA repair. KHDC3L dysfunction causes PARP1 inhibition and HR repair deficiency, which is synthetically lethal. Notably, we identified two critical residues, Thr145 and Thr156, whose phosphorylation by Ataxia-telangiectasia mutated (ATM) is essential for KHDC3L's functions. Importantly, two deletions of KHDC3L (p.E150_V160del and p.E150_V172del) were detected in female RPL patients, both of which harbor a common loss of Thr156 and are impaired in PARP1 activation and HR repair. In summary, our study reveals both KHDC3L as a new RPL risk gene and its critical function in DNA damage repair pathways.},
}

Abortion, Habitual/*genetics/metabolism/pathology
Animals
Ataxia Telangiectasia Mutated Proteins/genetics/metabolism
CRISPR-Cas Systems
Cells, Cultured
DNA Damage
Embryo, Mammalian
Female
Gene Editing
Gene Expression Regulation
*Genomic Instability
Human Embryonic Stem Cells/*metabolism/pathology
Humans
Karyotype
Mice
Mice, SCID
Micronuclei, Chromosome-Defective
Poly (ADP-Ribose) Polymerase-1/*genetics/metabolism
Pregnancy
Proteins/*genetics/metabolism
*Recombinational DNA Repair
Signal Transduction

@article {pmid31600188,
year = {2019},
author = {Graziano, BR and Town, JP and Sitarska, E and Nagy, TL and Fošnarič, M and Penič, S and Iglič, A and Kralj-Iglič, V and Gov, NS and Diz-Muñoz, A and Weiner, OD},
title = {Cell confinement reveals a branched-actin independent circuit for neutrophil polarity.},
journal = {PLoS biology},
volume = {17},
number = {10},
pages = {e3000457},
pmid = {31600188},
issn = {1545-7885},
support = {R35 GM118167/GM/NIGMS NIH HHS/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; },
mesh = {Actin-Related Protein 2-3 Complex/*genetics/metabolism ; Actins/*genetics/metabolism ; Biomechanical Phenomena ; CRISPR-Cas Systems ; Cell Adhesion/drug effects ; Cell Membrane/drug effects/metabolism/ultrastructure ; Cell Polarity/drug effects/*genetics ; Chemotactic Factors/pharmacology ; Chemotaxis/drug effects/*genetics ; Gene Editing ; Gene Expression Regulation ; HEK293 Cells ; HL-60 Cells ; Humans ; Microscopy, Atomic Force ; N-Formylmethionine Leucyl-Phenylalanine/pharmacology ; Pseudopodia/drug effects/metabolism/ultrastructure ; Signal Transduction ; Surface Properties ; Wiskott-Aldrich Syndrome Protein Family/deficiency/*genetics ; },
abstract = {Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.},
}

Actin-Related Protein 2-3 Complex/*genetics/metabolism
Actins/*genetics/metabolism
Biomechanical Phenomena
CRISPR-Cas Systems
Cell Adhesion/drug effects
Cell Membrane/drug effects/metabolism/ultrastructure
Cell Polarity/drug effects/*genetics
Chemotactic Factors/pharmacology
Chemotaxis/drug effects/*genetics
Gene Editing
Gene Expression Regulation
HEK293 Cells
HL-60 Cells
Humans
Microscopy, Atomic Force
N-Formylmethionine Leucyl-Phenylalanine/pharmacology
Pseudopodia/drug effects/metabolism/ultrastructure
Signal Transduction
Surface Properties
Wiskott-Aldrich Syndrome Protein Family/deficiency/*genetics

@article {pmid31206994,
year = {2019},
author = {Schachtsiek, J and Stehle, F},
title = {Nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9.},
journal = {Plant biotechnology journal},
volume = {17},
number = {12},
pages = {2228-2230},
pmid = {31206994},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Nicotine ; Tobacco/*chemistry/*genetics ; },
}

*CRISPR-Cas Systems
*Gene Editing
Nicotine
Tobacco/*chemistry/*genetics

@article {pmid31148128,
year = {2019},
author = {Kumari, P and Yusuf, F and Gaur, NA},
title = {Novel Microbial Modification Tools to Convert Lipids into Other Value-Added Products.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1995},
number = {},
pages = {161-171},
doi = {10.1007/978-1-4939-9484-7_10},
pmid = {31148128},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Cloning, Molecular/methods ; Gene Editing/*methods ; Genetic Vectors/genetics ; Lipids/*genetics ; RNA, Guide/genetics ; Transformation, Genetic ; Yeasts/*genetics ; },
abstract = {CRISPR-Cas9 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) is a microbial adaptive immune system that has revolutionized the field of molecular biology and genome engineering. The Type II CRISPR system consists of Cas9 nuclease of Streptococcus pyogenes and the RNA complex that guides Cas9 nuclease to a specific sequence of DNA in the genome. The CRISPR-Cas9 technology has reformed our ability to edit DNA and to regulate expression levels of genes of interest to high precision and accuracy. It is a powerful technology, which is used for genome engineering of a wide range of organisms for various applications. Here, we describe a method involving CRISPR-Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms for biotechnological applications in yeast. The complete procedure of genome editing including target sequence selection, cloning gRNA with a target sequence, transformation, and verification of the desired mutation/deletion or insertion can be achieved within 2-3 weeks in yeast.},
}

*CRISPR-Cas Systems
Cloning, Molecular/methods
Gene Editing/*methods
Genetic Vectors/genetics
Lipids/*genetics
RNA, Guide/genetics
Transformation, Genetic
Yeasts/*genetics

@article {pmid31131534,
year = {2019},
author = {Nomura, T and Inoue, K and Uehara-Yamaguchi, Y and Yamada, K and Iwata, O and Suzuki, K and Mochida, K},
title = {Highly efficient transgene-free targeted mutagenesis and single-stranded oligodeoxynucleotide-mediated precise knock-in in the industrial microalga Euglena gracilis using Cas9 ribonucleoproteins.},
journal = {Plant biotechnology journal},
volume = {17},
number = {11},
pages = {2032-2034},
pmid = {31131534},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Euglena gracilis/*growth & development ; *Gene Editing ; Gene Knock-In Techniques ; Microalgae/*genetics ; *Mutagenesis ; Oligodeoxyribonucleotides ; Ribonucleoproteins ; },
}

*CRISPR-Cas Systems
Euglena gracilis/*growth & development
*Gene Editing
Gene Knock-In Techniques
Microalgae/*genetics
*Mutagenesis
Oligodeoxyribonucleotides
Ribonucleoproteins

@article {pmid31002444,
year = {2019},
author = {Zhu, Y and Lin, Y and Chen, S and Liu, H and Chen, Z and Fan, M and Hu, T and Mei, F and Chen, J and Chen, L and Wang, F},
title = {CRISPR/Cas9-mediated functional recovery of the recessive rc allele to develop red rice.},
journal = {Plant biotechnology journal},
volume = {17},
number = {11},
pages = {2096-2105},
pmid = {31002444},
issn = {1467-7652},
mesh = {*Alleles ; *CRISPR-Cas Systems ; Frameshift Mutation ; Genes, Plant ; Oryza/*genetics ; *Pigmentation ; Sequence Deletion ; },
abstract = {Red rice contains high levels of proanthocyanidins and anthocyanins, which have been recognized as health-promoting nutrients. The red coloration of rice grains is controlled by two complementary genes, Rc and Rd. The RcRd genotype produces red pericarp in wild species Oryza rufipogon, whereas most cultivated rice varieties produce white grains resulted from a 14-bp frame-shift deletion in the seventh exon of the Rc gene. In the present study, we developed a CRISPR/Cas9-mediated method to functionally restore the recessive rc allele through reverting the 14-bp frame-shift deletion to in-frame mutations in which the deletions were in multiples of three bases, and successfully converted three elite white pericarp rice varieties into red ones. Rice seeds from T1 in-frame Rc lines were measured for proanthocyanidins and anthocyanidins, and high accumulation levels of proanthocyanidins and anthocyanidins were observed in red grains from the mutants. Moreover, there was no significant difference between wild-type and in-frame Rc mutants in major agronomic traits, indicating that restoration of Rc function had no negative effect on important agronomic traits in rice. Given that most white pericarp rice varieties are resulted from the 14-bp deletion in Rc, it is conceivable that our method could be applied to most white pericarp rice varieties and would greatly accelerate the breeding of new red rice varieties with elite agronomic traits. In addition, our study demonstrates an effective approach to restore recessive frame-shift alleles for crop improvement.},
}

*Alleles
*CRISPR-Cas Systems
Frameshift Mutation
Genes, Plant
Oryza/*genetics
*Pigmentation
Sequence Deletion

@article {pmid30972865,
year = {2019},
author = {Liu, Y and Wang, Y and Xu, S and Tang, X and Zhao, J and Yu, C and He, G and Xu, H and Wang, S and Tang, Y and Fu, C and Ma, Y and Zhou, G},
title = {Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis.},
journal = {Plant biotechnology journal},
volume = {17},
number = {11},
pages = {2143-2152},
pmid = {30972865},
issn = {1467-7652},
mesh = {Agrobacterium ; Araceae/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Transformation, Genetic ; },
abstract = {The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium-mediated transformation to 5-6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9-mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9-mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.},
}

Agrobacterium
Araceae/*genetics
*CRISPR-Cas Systems
*Gene Editing
Genome, Plant
Transformation, Genetic

@article {pmid30910031,
year = {2019},
author = {Leto, DE and Kopito, RR},
title = {Methods for genetic analysis of mammalian ER-associated degradation.},
journal = {Methods in enzymology},
volume = {619},
number = {},
pages = {97-120},
doi = {10.1016/bs.mie.2019.01.006},
pmid = {30910031},
issn = {1557-7988},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; *Endoplasmic Reticulum-Associated Degradation ; Gene Editing/methods ; Humans ; RNA, Guide/genetics ; Transduction, Genetic ; },
abstract = {Identification and degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is crucial for maintaining proteostasis, but only a handful of UPS components have been linked to the recognition of specific substrates. Studies in Saccharomyces cerevisiae using systematic perturbation of nonessential genes have uncovered UPS components that recognize and ubiquitylate model substrates of the UPS; however, similar analyses in metazoans have been limited. In this chapter, we describe methods for using CRISPR/Cas9 technology combined with genome-wide high complexity single guide (sgRNA) libraries and a transcriptional shutoff strategy for phenotypic selection based on kinetic measurements of protein turnover to identify the genes required to degrade model clients of the mammalian ER-associated degradation system. We also discuss considerations for screen design, execution, and interpretation.},
}

Animals
*CRISPR-Cas Systems
Cell Line
*Endoplasmic Reticulum-Associated Degradation
Gene Editing/methods
Humans
RNA, Guide/genetics
Transduction, Genetic

@article {pmid30847946,
year = {2019},
author = {Ishii, T and Schubert, V and Khosravi, S and Dreissig, S and Metje-Sprink, J and Sprink, T and Fuchs, J and Meister, A and Houben, A},
title = {RNA-guided endonuclease - in situ labelling (RGEN-ISL): a fast CRISPR/Cas9-based method to label genomic sequences in various species.},
journal = {The New phytologist},
volume = {222},
number = {3},
pages = {1652-1661},
doi = {10.1111/nph.15720},
pmid = {30847946},
issn = {1469-8137},
support = {//CSIRO/International ; Ho1779/28-1//Deutsche Forschungsgemeinschaft/International ; },
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Centromere/metabolism ; Endonucleases/*metabolism ; *Genomics ; RNA, Guide/*metabolism ; Species Specificity ; *Staining and Labeling ; },
abstract = {Visualising the spatio-temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two-part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN-ISL (RNA-guided endonuclease - in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans-activating crRNAs allows the multiplexing of RGEN-ISL. Moreover, this technique is combinable with immunohistochemistry. Real-time visualisation of the CRISPR/Cas9-mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN-ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology.},
}

Base Sequence
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Centromere/metabolism
Endonucleases/*metabolism
*Genomics
RNA, Guide/*metabolism
Species Specificity
*Staining and Labeling

@article {pmid30636294,
year = {2019},
author = {Mara, K and Charlot, F and Guyon-Debast, A and Schaefer, DG and Collonnier, C and Grelon, M and Nogué, F},
title = {POLQ plays a key role in the repair of CRISPR/Cas9-induced double-stranded breaks in the moss Physcomitrella patens.},
journal = {The New phytologist},
volume = {222},
number = {3},
pages = {1380-1391},
doi = {10.1111/nph.15680},
pmid = {30636294},
issn = {1469-8137},
support = {ANR-10-LABX-0040-SPS//Agence Nationale de la Recherche/International ; ANR11-BTBR-0001-GENIUS//Agence Nationale de la Recherche/International ; },
mesh = {Base Sequence ; Bleomycin/pharmacology ; Bryopsida/drug effects/*genetics/radiation effects ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cisplatin/pharmacology ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *DNA Repair ; DNA-Directed DNA Polymerase/genetics/*metabolism ; Genomic Instability ; Homologous Recombination/drug effects/radiation effects ; Methyl Methanesulfonate/pharmacology ; Mutation/genetics ; Mutation Rate ; Phenotype ; Ultraviolet Rays ; },
abstract = {Double-stranded breaks can be repaired by different mechanisms such as homologous recombination (HR), classical nonhomologous end joining (C-NHEJ) and alternative end joining (Alt-EJ). Polymerase Q (POLQ) has been proposed to be the main factor involved in Alt-EJ-mediated DNA repair. Here we describe the role of POLQ in DNA repair and gene targeting in Physcomitrella patens. The disruption of the POLQ gene does not influence the genetic stability of P. patens nor its development. The polq mutant shows the same sensitivity as wild-type towards most of the genotoxic agents tested (ultraviolet (UV), methyl methanesulfonate (MMS) and cisplatin) with the notable exception of bleomycin for which it shows less sensitivity than the wild-type. Furthermore, we show that POLQ is involved in the repair of CRISPR-Cas9-induced double-stranded breaks in P. patens. We also demonstrate that POLQ is a potential competitor and/or inhibitor of the HR repair pathway. This finding has a consequence in terms of genetic engineering, as in the absence of POLQ the frequency of gene targeting is significantly increased and the number of clean two-sided HR-mediated insertions is enhanced. Therefore, the control of POLQ activity in plants could be a useful strategy to optimize the tools of genome engineering for plant breeding.},
}

Base Sequence
Bleomycin/pharmacology
Bryopsida/drug effects/*genetics/radiation effects
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Cisplatin/pharmacology
*DNA Breaks, Double-Stranded
DNA End-Joining Repair
*DNA Repair
DNA-Directed DNA Polymerase/genetics/*metabolism
Genomic Instability
Homologous Recombination/drug effects/radiation effects
Methyl Methanesulfonate/pharmacology
Mutation/genetics
Mutation Rate
Phenotype
Ultraviolet Rays

@article {pmid32101273,
year = {2020},
author = {Zhou, Q and Zhang, Y and Zou, Y and Yin, T and Yang, J},
title = {Human embryo gene editing: God's scalpel or Pandora's box?.},
journal = {Briefings in functional genomics},
volume = {},
number = {},
pages = {},
doi = {10.1093/bfgp/elz025},
pmid = {32101273},
issn = {2041-2657},
abstract = {Gene editing refers to the site-specific modification of the genome, which mainly focuses on basic research, model organism construction and treatment and prevention of disease. Since the first application of CRISPR/Cas9 on the human embryo genome in 2015, the controversy over embryo gene editing (abbreviated as EGE in the following text) has never stopped. At present, the main contradictions focus on (1) ideal application prospects and immature technologies; (2) scientific progress and ethical supervision; and (3) definition of reasonable application scope. In fact, whether the EGE is 'God's scalpel' or 'Pandora's box' depends on the maturity of the technology and ethical supervision. This non-systematic review included English articles in NCBI, technical documents from the Human Fertilization and Embryology Authority as well as reports in the media, which performed from 1980 to 2018 with the following search terms: 'gene editing, human embryo, sequence-specific nuclease (SSN) (CRISPR/Cas, TALENT, ZFN), ethical consideration, gene therapy.' Based on the research status of EGE, this paper summarizes the technical defects and ethical controversies, enumerates the optimization measures and looks forward to the application prospect, aimed at providing some suggestions for the development trend. We should regard the research and development of EGE optimistically, improve and innovate the technology boldly and apply its clinical practice carefully.},
}

@article {pmid32098761,
year = {2020},
author = {Sandoval, A and Elahi, H and Ploski, JE},
title = {Genetically Engineering the Nervous System with CRISPR-Cas.},
journal = {eNeuro},
volume = {},
number = {},
pages = {},
doi = {10.1523/ENEURO.0419-19.2020},
pmid = {32098761},
issn = {2373-2822},
abstract = {The multitude of neuronal subtypes and extensive interconnectivity of the mammalian brain presents a substantial challenge to those seeking to decipher its functions. While the molecular mechanisms of several neuronal functions remain poorly characterized, advances in Next-Generation Sequencing (NGS) and gene-editing technology have begun to close this gap. The Clustered Regularly Interspaced Palindromic Repeats - CRISPR Associated Protein (CRISPR-Cas) system has emerged as a powerful genetic tool capable of manipulating the genome of essentially any organism and cell type, an attribute which has advanced our understanding of complex neurological diseases by enabling the rapid generation of novel, disease-relevant in vitro and transgenic animal models. In this review, we discuss recent developments in the rapidly accelerating field of CRISPR-mediated genome engineering. We begin with an overview of the canonical function of the CRISPR platform, followed by a functional review of its many adaptations, with an emphasis on its applications for genetic interrogation of the normal and diseased nervous system. Additionally, we discuss limitations of the CRISPR editing system and suggest how future modifications to existing platforms may advance our understanding of the brain.},
}

@article {pmid32098042,
year = {2020},
author = {Plavec, TV and Berlec, A},
title = {Safety Aspects of Genetically Modified Lactic Acid Bacteria.},
journal = {Microorganisms},
volume = {8},
number = {2},
pages = {},
doi = {10.3390/microorganisms8020297},
pmid = {32098042},
issn = {2076-2607},
support = {J4-9327, P4-0127//Slovenian Research Agency/ ; },
abstract = {Lactic acid bacteria (LAB) have a long history of use in the food industry. Some species are part of the normal human microbiota and have beneficial properties for human health. Their long-standing use and considerable biotechnological potential have led to the development of various systems for their engineering. Together with novel approaches such as CRISPR-Cas, the established systems for engineering now allow significant improvements to LAB strains. Nevertheless, genetically modified LAB (GM-LAB) still encounter disapproval and are under extensive regulatory requirements. This review presents data on the prospects for LAB to obtain 'generally recognized as safe' (GRAS) status. Genetic modification of LAB is discussed, together with problems that can arise from their engineering, including their dissemination into the environment and the spread of antibiotic resistance markers. Possible solutions that would allow the use of GM-LAB are described, such as biocontainment, alternative selection markers, and use of homologous DNA. The use of GM-LAB as cell factories in closed systems that prevent their environmental release is the least problematic aspect, and this is also discussed.},
}

@article {pmid32058903,
year = {2020},
author = {Chang, KS and Kim, J and Park, H and Hong, SJ and Lee, CG and Jin, E},
title = {Enhanced lipid productivity in AGP knockout marine microalga Tetraselmis sp. using a DNA-free CRISPR-Cas9 RNP method.},
journal = {Bioresource technology},
volume = {303},
number = {},
pages = {122932},
doi = {10.1016/j.biortech.2020.122932},
pmid = {32058903},
issn = {1873-2976},
mesh = {CRISPR-Cas Systems ; Glucose-1-Phosphate Adenylyltransferase ; Lipids ; *Microalgae ; Ribonucleoproteins ; },
abstract = {A marine green microalga, Tetraselmis sp., has been studied for the production of biomass and lipids in seawater culture. Since carbohydrate and lipid biosynthesis are competitive metabolic pathways, we attempted to increase lipid synthesis in Tetraselmis by inhibiting carbohydrate synthesis. The main regulatory enzyme in the starch synthesis pathway is ADP-glucose pyrophosphorylase (AGP). AGP loss-of-function mutants were developed using the CRISPR-Cas9 ribonucleoprotein (RNP) delivery system. AGP mutants showed a slight decrease in growth. However, the lipid content in two AGP mutants was significantly enhanced by 2.7 and 3.1 fold (21.1% and 24.1% of DCW), respectively, compared to that in the wild type (7.68% of DCW) under nitrogen starvation. This study is an example of metabolic engineering by genetic editing using the CRISPR-Cas9 RNP method in marine green microalgae. Consequently, starchless Tetraselmis mutants might be considered potential producers of lipids in seawater cultures.},
}

CRISPR-Cas Systems
Glucose-1-Phosphate Adenylyltransferase
Lipids
*Microalgae
Ribonucleoproteins

@article {pmid31642890,
year = {2019},
author = {Apura, P and Domingues, S and Viegas, SC and Arraiano, CM},
title = {Reprogramming bacteria with RNA regulators.},
journal = {Biochemical Society transactions},
volume = {47},
number = {5},
pages = {1279-1289},
doi = {10.1042/BST20190173},
pmid = {31642890},
issn = {1470-8752},
mesh = {5' Untranslated Regions ; *Bacterial Physiological Phenomena ; Base Pairing ; CRISPR-Cas Systems ; RNA, Bacterial/genetics ; Riboswitch ; *Synthetic Biology ; },
abstract = {The revolution of genomics and growth of systems biology urged the creation of synthetic biology, an engineering discipline aiming at recreating and reprogramming cellular functions for industrial needs. There has been a huge effort in synthetic biology to develop versatile and programmable genetic regulators that would enable the precise control of gene expression. Synthetic RNA components have emerged as a solution, offering a diverse range of programmable functions, including signal sensing, gene regulation and the modulation of molecular interactions. Owing to their compactness, structure and way of action, several types of RNA devices that act on DNA, RNA and protein have been characterized and applied in synthetic biology. RNA-based approaches are more 'economical' for the cell, since they are generally not translated. These RNA-based strategies act on a much shorter time scale than transcription-based ones and can be more efficient than protein-based mechanisms. In this review, we explore these RNA components as building blocks in the RNA synthetic biology field, first by explaining their natural mode of action and secondly discussing how these RNA components have been exploited to rewire bacterial regulatory circuitry.},
}

5' Untranslated Regions
*Bacterial Physiological Phenomena
Base Pairing
CRISPR-Cas Systems
RNA, Bacterial/genetics
Riboswitch
*Synthetic Biology

@article {pmid31637258,
year = {2019},
author = {Zhang, J and Niu, H and Zhao, ZJ and Fu, X and Wang, Y and Zhang, X and Zhang, F and Zeng, L},
title = {CRISPR/Cas9 Knockout of Bak Mediates Bax Translocation to Mitochondria in response to TNFα/CHX-induced Apoptosis.},
journal = {BioMed research international},
volume = {2019},
number = {},
pages = {9071297},
pmid = {31637258},
issn = {2314-6141},
mesh = {Apoptosis/drug effects/*genetics ; CRISPR-Cas Systems/genetics ; Caspases/*genetics ; Cell Line, Tumor ; Cycloheximide/pharmacology ; Cytosol/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; Humans ; Mitochondria/drug effects/genetics ; Proto-Oncogene Proteins c-bcl-2/genetics ; RNA, Small Interfering/genetics ; Tumor Necrosis Factor-alpha/genetics/pharmacology ; bcl-2 Homologous Antagonist-Killer Protein/*genetics ; bcl-2-Associated X Protein/*genetics ; },
abstract = {TNFα/CHX-induced apoptosis is dependent on caspase-8 activation and regulated by Bcl-2. However, the specific participants and precise mechanisms underlying this apoptotic pathway are poorly understood. The proapoptotic proteins Bak and Bax-members of the Bcl-2 family-are essential for the functioning of the mitochondrial apoptotic pathway. In this study, we used the CRISPR/Cas9 system to knockout Bak in the human SH-SY5Y cell line and determined the effects of this knockout on TNFα/CHX-induced apoptosis. Our data showed that overexpression of Bcl-2 dramatically prevented TNFα/CHX-induced apoptosis, and then pro-apoptotic protein Bak was downregulated and became more resistant to TNFα/CHX-induced apoptosis, because both TNFα/CHX-induced PARP cleavage and caspase activation were blocked in BAK-/- cells or using specific siRNA, whereas Bax was dispensable in TNFα/CHX-induced apoptosis, as evidenced using specific siRNA. Bax translocated from the cytosol into the mitochondria in response to TNFα/CHX, and CRISPR/Cas9 knockout of Bak significantly decreased this translocation. These results indicate that TNFα/CHX-induced apoptosis does not occur in Bak-/- cells, suggesting that TNFα/CHX-induced apoptosis is Bak-dependent but Bax-independent.},
}

Apoptosis/drug effects/*genetics
CRISPR-Cas Systems/genetics
Caspases/*genetics
Cell Line, Tumor
Cycloheximide/pharmacology
Cytosol/drug effects
Gene Expression Regulation, Neoplastic/drug effects
Gene Knockout Techniques
Humans
Mitochondria/drug effects/genetics
Proto-Oncogene Proteins c-bcl-2/genetics
RNA, Small Interfering/genetics
Tumor Necrosis Factor-alpha/genetics/pharmacology
bcl-2 Homologous Antagonist-Killer Protein/*genetics
bcl-2-Associated X Protein/*genetics

@article {pmid31484740,
year = {2019},
author = {Shrivastava, R and Tupperwar, N and Drory-Retwitzer, M and Shapira, M},
title = {Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of Leishmania.},
journal = {mSphere},
volume = {4},
number = {5},
pages = {},
doi = {10.1128/mSphere.00450-19},
pmid = {31484740},
issn = {2379-5042},
mesh = {*Alleles ; Animals ; *CRISPR-Cas Systems ; *Gene Deletion ; Leishmania mexicana/genetics/*pathogenicity ; Macrophages/parasitology ; Mice ; Mutation ; Protozoan Proteins/*genetics ; RAW 264.7 Cells ; },
abstract = {The genomes of Leishmania and trypanosomes encode six paralogs of the eIF4E cap-binding protein, known in other eukaryotes to anchor the translation initiation complex. In line with the heteroxenous nature of these parasites, the different LeishIF4E paralogs vary in their biophysical features and their biological behavior. We therefore hypothesize that each has a specialized function, not limited to protein synthesis. Of the six paralogs, LeishIF4E-3 has a weak cap-binding activity. It participates in the assembly of granules that store inactive transcripts and ribosomal proteins during nutritional stress that is experienced in the sand fly. We investigated the role of LeishIF4E-3 in Leishmania mexicana promastigotes using the CRISPR-Cas9 system. We deleted one of the two LeishIF4E-3 alleles, generating a heterologous deletion mutant with reduced LeishIF4E-3 expression. The mutant showed a decline in de novo protein synthesis and growth kinetics, altered morphology, and impaired infectivity. The mutant cells were rounded and failed to transform into the nectomonad-like form, in response to purine starvation. Furthermore, the infectivity of macrophage cells by the LeishIF4E-3(+/-) mutant was severely reduced. These phenotypic features were not observed in the addback cells, in which expression of LeishIF4E-3 was restored. The observed phenotypic changes correlated with the profile of transcripts associated with LeishIF4E-3. These were enriched for cytoskeleton- and flagellum-encoding genes, along with genes for RNA binding proteins. Our data illustrate the importance of LeishIF4E-3 in translation and in the parasite virulence.IMPORTANCELeishmania species are the causative agents of a spectrum of diseases. Available drug treatment is toxic and expensive, with drug resistance a growing concern. Leishmania parasites migrate between transmitting sand flies and mammalian hosts, experiencing unfavorable extreme conditions. The parasites therefore developed unique mechanisms for promoting a stage-specific program for gene expression, with translation playing a central role. There are six paralogs of the cap-binding protein eIF4E, which vary in their function, expression profiles, and assemblages. Using the CRISPR-Cas9 system for Leishmania, we deleted one of the two LeishIF4E-3 alleles. Expression of LeishIF4E-3 in the deletion mutant was low, leading to reduction in global translation and growth of the mutant cells. Cell morphology also changed, affecting flagellum growth, cell shape, and infectivity. The importance of this study is in highlighting that LeishIF4E-3 is essential for completion of the parasite life cycle. Our study gives new insight into how parasite virulence is determined.},
}

*Alleles
Animals
*CRISPR-Cas Systems
*Gene Deletion
Leishmania mexicana/genetics/*pathogenicity
Macrophages/parasitology
Mice
Mutation
Protozoan Proteins/*genetics
RAW 264.7 Cells

@article {pmid31406246,
year = {2019},
author = {Gobbi, G and Donati, B and Do Valle, IF and Reggiani, F and Torricelli, F and Remondini, D and Castellani, G and Ambrosetti, DC and Ciarrocchi, A and Sancisi, V},
title = {The Hippo pathway modulates resistance to BET proteins inhibitors in lung cancer cells.},
journal = {Oncogene},
volume = {38},
number = {42},
pages = {6801-6817},
pmid = {31406246},
issn = {1476-5594},
mesh = {A549 Cells ; Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*metabolism/pathology ; Cell Nucleus/metabolism ; *Drug Resistance, Neoplasm ; Humans ; Lung Neoplasms/*metabolism/pathology ; Neoplasm Proteins/*antagonists & inhibitors/metabolism ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; },
abstract = {Inhibitors of BET proteins (BETi) are anti-cancer drugs that have shown efficacy in pre-clinical settings and are currently in clinical trials for different types of cancer, including non-small cell lung cancer (NSCLC). Currently, no predictive biomarker is available to identify patients that may benefit from this treatment. To uncover the mechanisms of resistance to BETi, we performed a genome-scale CRISPR/Cas9 screening in lung cancer cells. We identified three Hippo pathway genes, LATS2, TAOK1, and NF2, as key determinants for sensitivity to BETi. The knockout of these genes induces resistance to BETi, by promoting TAZ nuclear localization and transcriptional activity. Conversely, TAZ expression promotes resistance to these drugs. We also showed that TAZ, YAP, and their partner TEAD are direct targets of BRD4 and that treatment with BETi downregulates their expression. Noticeably, molecular alterations in one or more of these genes are present in a large fraction of NSCLC patients and TAZ amplification or overexpression correlates with a worse outcome in lung adenocarcinoma. Our data define the central role of Hippo pathway in mediating resistance to BETi and provide a rationale for using BETi to counter-act YAP/TAZ-mediated pro-oncogenic activity.},
}

A549 Cells
Antineoplastic Agents/*pharmacology
CRISPR-Cas Systems
Carcinoma, Non-Small-Cell Lung/*metabolism/pathology
Cell Nucleus/metabolism
*Drug Resistance, Neoplasm
Humans
Lung Neoplasms/*metabolism/pathology
Neoplasm Proteins/*antagonists & inhibitors/metabolism
Protein-Serine-Threonine Kinases/genetics/*metabolism

@article {pmid31391551,
year = {2019},
author = {Zhong, Z and Sepramaniam, S and Chew, XH and Wood, K and Lee, MA and Madan, B and Virshup, DM},
title = {PORCN inhibition synergizes with PI3K/mTOR inhibition in Wnt-addicted cancers.},
journal = {Oncogene},
volume = {38},
number = {40},
pages = {6662-6677},
pmid = {31391551},
issn = {1476-5594},
mesh = {Acyltransferases/*genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Carcinoma, Pancreatic Ductal/*metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation ; Glucose/metabolism ; Heterografts ; Humans ; Loss of Function Mutation ; Membrane Proteins/*genetics/metabolism ; Mice ; Pancreatic Neoplasms/*metabolism/pathology ; Phosphatidylinositol 3-Kinases/*genetics/metabolism ; TOR Serine-Threonine Kinases/*genetics/metabolism ; Wnt Proteins/*metabolism ; },
abstract = {Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is aggressive and lethal. Although there is an urgent need for effective therapeutics in treating pancreatic cancer, none of the targeted therapies tested in clinical trials to date significantly improve its outcome. PORCN inhibitors show efficacy in preclinical models of Wnt-addicted cancers, including RNF43-mutant pancreatic cancers and have advanced to clinical trials. In this study, we aimed to develop drug combination strategies to further enhance the therapeutic efficacy of the PORCN inhibitor ETC-159. To identify additional druggable vulnerabilities in Wnt-driven pancreatic cancers, we performed an in vivo CRISPR loss-of-function screen. CTNNB1, KRAS, and MYC were reidentified as key oncogenic drivers. Notably, glucose metabolism pathway genes were important in vivo but less so in vitro. Knockout of multiple genes regulating PI3K/mTOR signaling impacted the growth of Wnt-driven pancreatic cancer cells in vivo. Importantly, multiple PI3K/mTOR pathway inhibitors in combination with ETC-159 synergistically suppressed the growth of multiple Wnt-addicted cancer cell lines in soft agar. Furthermore, the combination of the PORCN inhibitor ETC-159 and the pan-PI3K inhibitor GDC-0941 potently suppressed the in vivo growth of RNF43-mutant pancreatic cancer xenografts. This was largely due to enhanced suppressive effects on both cell proliferation and glucose metabolism. These findings demonstrate that dual PORCN and PI3K/mTOR inhibition is a potential strategy for treating Wnt-driven pancreatic cancers.},
}

Acyltransferases/*genetics/metabolism
Animals
CRISPR-Cas Systems
Carcinoma, Pancreatic Ductal/*metabolism/pathology
Cell Line, Tumor
Cell Proliferation
Glucose/metabolism
Heterografts
Humans
Loss of Function Mutation
Membrane Proteins/*genetics/metabolism
Mice
Pancreatic Neoplasms/*metabolism/pathology
Phosphatidylinositol 3-Kinases/*genetics/metabolism
TOR Serine-Threonine Kinases/*genetics/metabolism
Wnt Proteins/*metabolism

@article {pmid30924368,
year = {2019},
author = {Jiang, W and Lian, W and Chen, J and Li, W and Huang, J and Lai, B and Li, L and Huang, Z and Xu, J},
title = {Rapid identification of genome-edited mesenchymal stem cell colonies via Cas9.},
journal = {BioTechniques},
volume = {66},
number = {5},
pages = {231-234},
doi = {10.2144/btn-2018-0183},
pmid = {30924368},
issn = {1940-9818},
mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Humans ; Interleukin-10/genetics ; Mesenchymal Stem Cells/*microbiology ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {Mesenchymal stem cells (MSCs) have been intensively investigated and widely applied in regenerative medicine and immune modulation. However, their efficacy declines during the aging or disease process. Thus, genome-edited MSCs with over-expression or inhibition of specific genes hold a great deal of promise in terms of their therapeutic application. Here we optimized the direct PCR approach for rapid identification of genome-edited MSCs with only ten cells required, which reduces the time and labor to expand the MSC colonies. Combined with our previously optimized guide RNA structure and plasmid construction strategy for Cas9, we successfully identified MSC colonies over-expressing IL-10 in the AAVS1 locus.},
}

CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/genetics
*Gene Editing
Humans
Interleukin-10/genetics
Mesenchymal Stem Cells/*microbiology
Plasmids/genetics
RNA, Guide/genetics

@article {pmid30582405,
year = {2019},
author = {Ng, LY and Ma, HT and Liu, JCY and Huang, X and Lee, N and Poon, RYC},
title = {Conditional gene inactivation by combining tetracycline-mediated transcriptional repression and auxin-inducible degron-mediated degradation.},
journal = {Cell cycle (Georgetown, Tex.)},
volume = {18},
number = {2},
pages = {238-248},
doi = {10.1080/15384101.2018.1563395},
pmid = {30582405},
issn = {1551-4005},
mesh = {ATPases Associated with Diverse Cellular Activities/genetics ; CRISPR-Cas Systems/*genetics ; Cell Cycle Proteins/genetics ; Clone Cells ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cyclin A/genetics ; Cyclin-Dependent Kinase 2/genetics ; Gene Expression ; Gene Knockout Techniques ; HeLa Cells ; Humans ; Indoleacetic Acids/*metabolism ; *Proteolysis ; Response Elements/genetics ; Retroviridae/genetics ; Tetracyclines/*metabolism ; Transcriptional Activation/*genetics ; Transfection ; },
abstract = {Characterizing the functions of essential cell cycle control genes requires tight and rapid inducible gene inactivation. Drawbacks of current conditional depletion approaches include slow responses and incomplete depletion. We demonstrated that by integrating the tetracycline-controlled promoter system and the auxin-inducible degron (AID) system together, AID-tagged proteins can be downregulated more efficiently than the individual technology alone. When used in conjunction with CRISPR-Cas9-mediated disruption of the endogenous locus, this system facilitates the analysis of essential genes by allowing rapid and tight conditional depletion, as we have demonstrated using several cell cycle-regulatory genes including cyclin A, CDK2, and TRIP13. The vectors constructed in this study allow expression of AID-fusion proteins under the control of tetracycline-controlled promoters and should be useful in studies requiring rapid and tight suppression of gene expression in mammalian cells.},
}

ATPases Associated with Diverse Cellular Activities/genetics
CRISPR-Cas Systems/*genetics
Cell Cycle Proteins/genetics
Clone Cells
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cyclin A/genetics
Cyclin-Dependent Kinase 2/genetics
Gene Expression
Gene Knockout Techniques
HeLa Cells
Humans
Indoleacetic Acids/*metabolism
*Proteolysis
Response Elements/genetics
Retroviridae/genetics
Tetracyclines/*metabolism
Transcriptional Activation/*genetics
Transfection

@article {pmid30576956,
year = {2019},
author = {Carlin, AF and Shresta, S},
title = {Genome-wide approaches to unravelling host-virus interactions in Dengue and Zika infections.},
journal = {Current opinion in virology},
volume = {34},
number = {},
pages = {29-38},
doi = {10.1016/j.coviro.2018.11.010},
pmid = {30576956},
issn = {1879-6265},
support = {KL2 TR001444/TR/NCATS NIH HHS/United States ; R01 AI116813/AI/NIAID NIH HHS/United States ; R21 AI127988/AI/NIAID NIH HHS/United States ; R21 NS100477/NS/NINDS NIH HHS/United States ; R21 AI140063/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Dengue/*genetics/virology ; Dengue Virus/*genetics/pathogenicity ; Genomics ; *Host-Pathogen Interactions ; Humans ; Zika Virus/*genetics/pathogenicity ; Zika Virus Infection/*genetics/virology ; },
abstract = {Genomics approaches are increasingly utilized to probe host-viral interactions and identify mechanisms of viral pathogenesis and host-subversion. Here we review recent studies that utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 screens, transcriptomics and epigenomics to gain insight into Dengue and Zika virus infections in humans. We discuss the benefits and limitations of recently utilized techniques that separate virally infected cells from neighboring uninfected cells to identify the mechanisms by which these viruses regulate host responses. We conclude by discussing how these approaches can best advance our understanding of Dengue and Zika virus pathogenesis in humans.},
}

CRISPR-Cas Systems
Dengue/*genetics/virology
Dengue Virus/*genetics/pathogenicity
Genomics
*Host-Pathogen Interactions
Humans
Zika Virus/*genetics/pathogenicity
Zika Virus Infection/*genetics/virology

@article {pmid32097710,
year = {2020},
author = {Chen, S and Yao, Y and Zhang, Y and Fan, G},
title = {CRISPR system: Discovery, development and off-target detection.},
journal = {Cellular signalling},
volume = {},
number = {},
pages = {109577},
doi = {10.1016/j.cellsig.2020.109577},
pmid = {32097710},
issn = {1873-3913},
abstract = {As a revolutionary gene editing tool based on the adaptive immune defense mechanism of bacteria and archaea against exogenous DNA invasion, CRISPR/Cas system shows many remarkable characteristics over ZFNs and TALENs. However, off-target effect remains as one of the major imperfection of CRISPR/Cas system, hindering its further application in translational research. In this review, we highlight major breakthrough cross the development/application of this powerful toolkit, and summarize feasible methods for detecting potential off-target effects during genetic manipulation. We hope this review will assist scientists for accurate genomic editing in their future research.},
}

@article {pmid32095891,
year = {2020},
author = {Ding, Y and Han, D and Cui, HL},
title = {Halorussus halophilus sp. nov., A Novel Halophilic Archaeon Isolated from a Marine Solar Saltern.},
journal = {Current microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00284-020-01921-8},
pmid = {32095891},
issn = {1432-0991},
support = {31770005//National Natural Science Foundation of China/ ; 2017FY100302//National Science & Technology Infrastructure Program of China/ ; },
abstract = {The halophilic archaeal strain ZS-3T (= CGMCC 1.12866T = JCM 30239T) was isolated from a sediment sample of Zhoushan marine solar saltern, P. R. China. Phylogenetic analyses based on 16S rRNA, rpoB' genes and the concatenation of 738 protein sequences reveal that strain ZS-3T was related to members of the genus Halorussus. The OrthoANI and in silico DDH values between strain ZS-3T and the current Halorussus members are much lower than the threshold values proposed as the species boundary (ANI 95-96% and in silico DDH 70%), suggesting that strain ZS-3T represents a novel species of Halorussus (Halorussus halophilus sp. nov.). Diverse phenotypic characteristics differentiate strain ZS-3T from current Halorussus members. Since the strain expressed diverse hydrolyzing enzyme activity, its complete genome was sequenced. The genome of strain ZS-3T was found to be 4,450,731 bp with total GC content of 61.51%, and comprises one chromosome and three plasmids. A total of 4694 protein coding genes, 43 tRNA genes and 6 rRNA genes were predicted. A CRISPR-Cas system was also detected. The genome encodes sixteen putative glycoside hydrolases, nine extracellular proteases, seventeen aminopeptidases, seven carboxypeptidases, one esterase and one nitrite reductase. The exploration of the hydrolase genes may expand our understanding of adapted mechanism of halophilic archaea surviving optimally in hypersaline environments where containing organic matter. Meanwhile, various hydrolyzing enzymes may extend this microorganism for further applications in salt-based fermentation.},
}

@article {pmid31616098,
year = {2019},
author = {Callaway, E},
title = {Geneticists retract study suggesting first CRISPR babies might die early.},
journal = {Nature},
volume = {574},
number = {7778},
pages = {307},
doi = {10.1038/d41586-019-03032-2},
pmid = {31616098},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/*genetics ; Databases, Factual ; Female ; *Gene Editing/ethics ; Germ-Line Mutation/genetics ; HIV Infections/genetics/prevention & control ; Heterozygote ; Humans ; Iceland ; *Patient Safety ; Receptors, CCR5/genetics ; Reproducibility of Results ; *Research Design ; *Research Personnel ; *Retraction of Publication as Topic ; Social Media ; Twins/*genetics ; United Kingdom ; },
}

CRISPR-Cas Systems/*genetics
Databases, Factual
Female
*Gene Editing/ethics
Germ-Line Mutation/genetics
HIV Infections/genetics/prevention & control
Heterozygote
Humans
Iceland
*Patient Safety
Receptors, CCR5/genetics
Reproducibility of Results
*Research Design
*Research Personnel
*Retraction of Publication as Topic
Social Media
Twins/*genetics
United Kingdom

@article {pmid31613873,
year = {2019},
author = {You, ST and Jhou, YT and Kao, CF and Leu, JY},
title = {Experimental evolution reveals a general role for the methyltransferase Hmt1 in noise buffering.},
journal = {PLoS biology},
volume = {17},
number = {10},
pages = {e3000433},
pmid = {31613873},
issn = {1545-7885},
mesh = {CRISPR-Cas Systems ; Directed Molecular Evolution ; Ethyl Methanesulfonate/pharmacology ; Gene Editing ; *Gene Expression Regulation, Fungal ; Genes, Reporter ; *Genetic Heterogeneity ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; Methylation ; Mutation ; *Protein Processing, Post-Translational ; Protein-Arginine N-Methyltransferases/*genetics/metabolism ; Repressor Proteins/*genetics/metabolism ; Saccharomyces cerevisiae/drug effects/*genetics/growth & development/metabolism ; Saccharomyces cerevisiae Proteins/*genetics/metabolism ; },
abstract = {Cell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Although general features and the origins of cellular noise have been revealed, details of the molecular pathways underlying noise regulation remain elusive. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise. By combining bulk segregant analysis and CRISPR/Cas9-based reconstitution, we identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 methylation activity is critical for the evolved phenotype, and we also show that two of the Hmt1 methylation targets can suppress noise. Hmt1 functions as an environmental sensor to adjust noise levels in response to environmental cues. Moreover, Hmt1-mediated noise buffering is conserved in an evolutionarily distant yeast species, suggesting broad significance of noise regulation.},
}

CRISPR-Cas Systems
Directed Molecular Evolution
Ethyl Methanesulfonate/pharmacology
Gene Editing
*Gene Expression Regulation, Fungal
Genes, Reporter
*Genetic Heterogeneity
Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics/metabolism
Green Fluorescent Proteins/genetics/metabolism
Methylation
Mutation
*Protein Processing, Post-Translational
Protein-Arginine N-Methyltransferases/*genetics/metabolism
Repressor Proteins/*genetics/metabolism
Saccharomyces cerevisiae/drug effects/*genetics/growth & development/metabolism
Saccharomyces cerevisiae Proteins/*genetics/metabolism

@article {pmid31562218,
year = {2019},
author = {Lockhart, J},
title = {Introducing CRISPR-TSKO: A Breakthrough in Precision Gene Editing.},
journal = {The Plant cell},
volume = {31},
number = {12},
pages = {2831-2832},
pmid = {31562218},
issn = {1532-298X},
mesh = {*Arabidopsis ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Mutagenesis ; },
}

*Arabidopsis
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Mutagenesis

@article {pmid31541694,
year = {2020},
author = {Xu, X and Wang, Y and Bi, H and Xu, J and Liu, Z and Niu, C and He, L and James, AA and Li, K and Huang, Y},
title = {Mutation of the seminal protease gene, serine protease 2, results in male sterility in diverse lepidopterans.},
journal = {Insect biochemistry and molecular biology},
volume = {116},
number = {},
pages = {103243},
doi = {10.1016/j.ibmb.2019.103243},
pmid = {31541694},
issn = {1879-0240},
mesh = {Animals ; Bombyx/genetics/physiology ; CRISPR-Cas Systems ; Infertility, Male/genetics ; Insect Proteins/*genetics/metabolism ; Male ; Moths/genetics/*physiology ; Mutation ; Reproduction/genetics ; Serine Proteases/*genetics/metabolism ; },
abstract = {Sterile insect technology (SIT) is an environmentally friendly method for pest control. As part of our efforts to develop a strategy that results in engineered male-sterile strains with minimum effects on viability and mating competition, we used CRISPR/Cas9 technology to disrupt Ser2, which encodes a seminal fluid protein, in the model lepidopteran insect, Bombyx mori, and an important agricultural pest, Plutella xylostella. Disruption of Ser2 resulted in dominant heritable male sterility. Wild-type females mated with Ser2-deficient males laid eggs normally, but the eggs did not hatch. We detected no differences in other reproductive behaviors in the mutant males. These results support the conclusion that Ser2 gene is necessary for male reproductive success in diverse lepidopterans. Targeting Ser2 gene has the potential to form the basis for a new strategy for pest control.},
}

Animals
Bombyx/genetics/physiology
CRISPR-Cas Systems
Infertility, Male/genetics
Insect Proteins/*genetics/metabolism
Male
Moths/genetics/*physiology
Mutation
Reproduction/genetics
Serine Proteases/*genetics/metabolism

@article {pmid31299065,
year = {2019},
author = {Sowińska, W and Wawro, M and Solecka, A and Kasza, A},
title = {Potential limitations of the Sleeping Beauty transposon use in gene expression studies.},
journal = {Acta biochimica Polonica},
volume = {66},
number = {3},
pages = {263-268},
doi = {10.18388/abp.2019_2839},
pmid = {31299065},
issn = {1734-154X},
mesh = {Aged ; Brain Neoplasms/genetics/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; DNA Transposable Elements/genetics ; Gene Editing ; Gene Knockout Techniques ; Glioblastoma/genetics/pathology ; Humans ; Interleukin-6/*genetics ; Male ; Mutagenesis, Insertional/*methods ; RNA, Messenger/genetics ; Ribonucleases/*genetics ; Transcription Factors/*genetics ; Transcriptome/*genetics ; Transfection ; Vascular Endothelial Growth Factor A/*genetics ; },
abstract = {MCPIP2 is the least known member of the MCPIP family of proteins. Recently we have found that it is a new RNase involved in transcript turnover. However, the full spectrum of its cellular targets is still unidentified. To discover transcripts which are regulated by this protein we have employed Sleeping Beauty transposons. This tool allows for rapid generation of a stable transgenic cell line with inducible expression of the desired gene. In this study, we analysed how the Sleeping Beauty system itself influences expression of chosen genes, namely IL-6, Regnase-1 and VEGF. We found that the system alone may influence expression of IL-6. Our results indicate that Sleeping Beauty transposons should be used with caution in studies that are focused on changes in the transcript level.},
}

Aged
Brain Neoplasms/genetics/pathology
CRISPR-Cas Systems/genetics
Cell Line, Tumor
DNA Transposable Elements/genetics
Gene Editing
Gene Knockout Techniques
Glioblastoma/genetics/pathology
Humans
Interleukin-6/*genetics
Male
Mutagenesis, Insertional/*methods
RNA, Messenger/genetics
Ribonucleases/*genetics
Transcription Factors/*genetics
Transcriptome/*genetics
Transfection
Vascular Endothelial Growth Factor A/*genetics

@article {pmid31204998,
year = {2019},
author = {Roberts, AW and Popov, LM and Mitchell, G and Ching, KL and Licht, DJ and Golovkine, G and Barton, GM and Cox, JS},
title = {Cas9+ conditionally-immortalized macrophages as a tool for bacterial pathogenesis and beyond.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
doi = {10.7554/eLife.45957},
pmid = {31204998},
issn = {2050-084X},
support = {Open Philanthropy Fellow//Life Sciences Research Foundation/International ; U19 AI135990/AI/NIAID NIH HHS/United States ; R01AI072429/NH/NIH HHS/United States ; P01AI063302/NH/NIH HHS/United States ; U19AI135990/NH/NIH HHS/United States ; DP1AI24619/NH/NIH HHS/United States ; DP1 AI124619/AI/NIAID NIH HHS/United States ; P01 AI063302/AI/NIAID NIH HHS/United States ; R01 AI072429/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Bone Marrow Cells/immunology/metabolism/microbiology ; *CRISPR-Cas Systems ; Cell Line ; Cells, Cultured ; Gene Editing/*methods ; Homeodomain Proteins/genetics/immunology/metabolism ; Host-Pathogen Interactions/immunology ; Humans ; Listeria monocytogenes/*immunology/physiology ; Macrophages/*immunology/*metabolism/microbiology ; Mice, Transgenic ; Mycobacterium tuberculosis/*immunology/physiology ; Stem Cells/immunology/metabolism ; },
abstract = {Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis.},
}

Animals
Bone Marrow Cells/immunology/metabolism/microbiology
*CRISPR-Cas Systems
Cell Line
Cells, Cultured
Gene Editing/*methods
Homeodomain Proteins/genetics/immunology/metabolism
Host-Pathogen Interactions/immunology
Humans
Listeria monocytogenes/*immunology/physiology
Macrophages/*immunology/*metabolism/microbiology
Mice, Transgenic
Mycobacterium tuberculosis/*immunology/physiology
Stem Cells/immunology/metabolism

@article {pmid31149896,
year = {2019},
author = {Torres, SE and Gallagher, CM and Plate, L and Gupta, M and Liem, CR and Guo, X and Tian, R and Stroud, RM and Kampmann, M and Weissman, JS and Walter, P},
title = {Ceapins block the unfolded protein response sensor ATF6α by inducing a neomorphic inter-organelle tether.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
doi = {10.7554/eLife.46595},
pmid = {31149896},
issn = {2050-084X},
support = {P01 GM111126/GM/NIGMS NIH HHS/United States ; R01 GM024485/GM/NIGMS NIH HHS/United States ; GM111126/NH/NIH HHS/United States ; DP2 OD021007/NH/NIH HHS/United States ; },
mesh = {ATP-Binding Cassette Transporters/metabolism ; Activating Transcription Factor 6/*antagonists & inhibitors/metabolism ; CRISPR-Cas Systems/genetics ; Endoplasmic Reticulum/drug effects/metabolism ; HEK293 Cells ; Hep G2 Cells ; Humans ; Organelles/drug effects/*metabolism ; Peroxisomes/drug effects/metabolism ; Phenotype ; Protein Binding/drug effects ; Small Molecule Libraries/*pharmacology ; *Unfolded Protein Response/drug effects ; },
abstract = {The unfolded protein response (UPR) detects and restores deficits in the endoplasmic reticulum (ER) protein folding capacity. Ceapins specifically inhibit the UPR sensor ATF6α, an ER-tethered transcription factor, by retaining it at the ER through an unknown mechanism. Our genome-wide CRISPR interference (CRISPRi) screen reveals that Ceapins function is completely dependent on the ABCD3 peroxisomal transporter. Proteomics studies establish that ABCD3 physically associates with ER-resident ATF6α in cells and in vitro in a Ceapin-dependent manner. Ceapins induce the neomorphic association of ER and peroxisomes by directly tethering the cytosolic domain of ATF6α to ABCD3's transmembrane regions without inhibiting or depending on ABCD3 transporter activity. Thus, our studies reveal that Ceapins function by chemical-induced misdirection which explains their remarkable specificity and opens up new mechanistic routes for drug development and synthetic biology.},
}

ATP-Binding Cassette Transporters/metabolism
Activating Transcription Factor 6/*antagonists & inhibitors/metabolism
CRISPR-Cas Systems/genetics
Endoplasmic Reticulum/drug effects/metabolism
HEK293 Cells
Hep G2 Cells
Humans
Organelles/drug effects/*metabolism
Peroxisomes/drug effects/metabolism
Phenotype
Protein Binding/drug effects
Small Molecule Libraries/*pharmacology
*Unfolded Protein Response/drug effects

@article {pmid30932914,
year = {2019},
author = {Neill, US},
title = {A conversation with George Church.},
journal = {The Journal of clinical investigation},
volume = {129},
number = {4},
pages = {1403-1404},
doi = {10.1172/JCI128550},
pmid = {30932914},
issn = {1558-8238},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*history ; History, 20th Century ; History, 21st Century ; Humans ; Sequence Analysis, DNA/*history ; },
}

*CRISPR-Cas Systems
Gene Editing/*history
History, 20th Century
History, 21st Century
Humans
Sequence Analysis, DNA/*history

@article {pmid30918971,
year = {2019},
author = {Wand, NO and Smith, DA and Wilkinson, AA and Rushton, AE and Busby, SJW and Styles, IB and Neely, RK},
title = {DNA barcodes for rapid, whole genome, single-molecule analyses.},
journal = {Nucleic acids research},
volume = {47},
number = {12},
pages = {e68},
doi = {10.1093/nar/gkz212},
pmid = {30918971},
issn = {1362-4962},
mesh = {Adenoviruses, Human/genetics/isolation & purification ; Bacteriophage lambda/genetics ; Base Sequence ; CRISPR-Cas Systems ; Computer Simulation ; DNA/*chemistry ; DNA, Bacterial/chemistry ; DNA, Viral/chemistry ; Escherichia coli/genetics/isolation & purification ; Fluorescent Dyes ; Genomics/*methods ; Humans ; Klebsiella pneumoniae/genetics ; },
abstract = {We report an approach for visualizing DNA sequence and using these 'DNA barcodes' to search complex mixtures of genomic material for DNA molecules of interest. We demonstrate three applications of this methodology; identifying specific molecules of interest from a dataset containing gigabasepairs of genome; identification of a bacterium from such a dataset and, finally, by locating infecting virus molecules in a background of human genomic material. As a result of the dense fluorescent labelling of the DNA, individual barcodes of the order 40 kb pairs in length can be reliably identified. This means DNA can be prepared for imaging using standard handling and purification techniques. The recorded dataset provides stable physical and electronic records of the total genomic content of a sample that can be readily searched for a molecule or region of interest.},
}

Adenoviruses, Human/genetics/isolation & purification
Bacteriophage lambda/genetics
Base Sequence
CRISPR-Cas Systems
Computer Simulation
DNA/*chemistry
DNA, Bacterial/chemistry
DNA, Viral/chemistry
Escherichia coli/genetics/isolation & purification
Fluorescent Dyes
Genomics/*methods
Humans
Klebsiella pneumoniae/genetics

@article {pmid30633425,
year = {2019},
author = {Kis, A and Hamar, É and Tholt, G and Bán, R and Havelda, Z},
title = {Creating highly efficient resistance against wheat dwarf virus in barley by employing CRISPR/Cas9 system.},
journal = {Plant biotechnology journal},
volume = {17},
number = {6},
pages = {1004-1006},
doi = {10.1111/pbi.13077},
pmid = {30633425},
issn = {1467-7652},
mesh = {*CRISPR-Cas Systems ; Disease Resistance/*genetics ; Geminiviridae/*physiology ; Hordeum/*genetics/*virology ; },
}

*CRISPR-Cas Systems
Disease Resistance/*genetics
Geminiviridae/*physiology
Hordeum/*genetics/*virology

@article {pmid32090878,
year = {2020},
author = {Xu, W and Jin, T and Dai, Y and Liu, CC},
title = {Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPR Cas systems.},
journal = {Biosensors & bioelectronics},
volume = {155},
number = {},
pages = {112100},
doi = {10.1016/j.bios.2020.112100},
pmid = {32090878},
issn = {1873-4235},
abstract = {Robust developments of personalized medicine for next-generation healthcare highlight the need for sensitive and accurate point-of-care platforms for quantification of disease biomarkers. Broad presentations of clustered regularly interspaced short palindromic repeats (CRISPR) as an accurate gene editing tool also indicate that the high-specificity and programmability of CRISPR system can be utilized for the development of biosensing systems. Herein, we present a CRISPR Cas system enhanced electrochemical DNA (E-DNA) sensor with unprecedented sensitivity and accuracy. The principle of the E-DNA sensor is the target induced conformational change of the surface signaling probe (containing an electrochemical tag), leading to the variation of the electron transfer rate of the electrochemical tag. With the introduction of CRISPR cleavage activity into the E-DNA sensor, a more apparent signal change between with and without the presence of the target can be achieved. We compared the performance of Cas9 and Cas12a enhanced E-DNA sensor and optimized the chemical environment of CRISPR, achieving a femto-molar detection limit without enzymatic amplification. Moreover, we correlated the CRISPR cleavage signal with the original E-DNA signal as a strategy to indicate potential mismatches in the target sequence. Comparing with classic DNA electrochemistry based mutation detection strategy, CRISPR enhanced E-DNA sensor can determine the presence of a single mutation at an unknown concentration condition. Overall, we believe that the CRISPR enhanced E-DNA sensing strategy will be of especially high utility for point-of-care systems owing to the programmability, modularity, high-sensitivity and high-accuracy.},
}

@article {pmid31613223,
year = {2019},
author = {Schlichting, M and Díaz, MM and Xin, J and Rosbash, M},
title = {Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31613223},
issn = {2050-084X},
mesh = {Animals ; Basic-Leucine Zipper Transcription Factors/deficiency/genetics ; Brain/cytology/*metabolism/radiation effects ; CRISPR-Cas Systems ; Cell Communication ; Cell Lineage/genetics ; Circadian Clocks/drug effects/*genetics ; Circadian Rhythm/drug effects/*genetics ; Darkness ; Drosophila Proteins/deficiency/genetics ; Drosophila melanogaster/*genetics/metabolism/radiation effects ; Feedback, Physiological ; Gene Editing ; *Gene Expression Regulation ; Light Signal Transduction/*genetics ; Nerve Net/metabolism/radiation effects ; Neurons/cytology/*metabolism/radiation effects ; Neuropeptides/deficiency/genetics ; Period Circadian Proteins/deficiency/genetics ; Transcription Factors/deficiency/genetics ; },
abstract = {Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, we assayed behavior as well as molecular rhythms within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. We also generated CRISPR-mediated neuron-specific circadian clock knockouts. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light.},
}

Animals
Basic-Leucine Zipper Transcription Factors/deficiency/genetics
Brain/cytology/*metabolism/radiation effects
CRISPR-Cas Systems
Cell Communication
Cell Lineage/genetics
Circadian Clocks/drug effects/*genetics
Circadian Rhythm/drug effects/*genetics
Darkness
Drosophila Proteins/deficiency/genetics
Drosophila melanogaster/*genetics/metabolism/radiation effects
Feedback, Physiological
Gene Editing
*Gene Expression Regulation
Light Signal Transduction/*genetics
Nerve Net/metabolism/radiation effects
Neurons/cytology/*metabolism/radiation effects
Neuropeptides/deficiency/genetics
Period Circadian Proteins/deficiency/genetics
Transcription Factors/deficiency/genetics

@article {pmid31613218,
year = {2019},
author = {Delventhal, R and O'Connor, RM and Pantalia, MM and Ulgherait, M and Kim, HX and Basturk, MK and Canman, JC and Shirasu-Hiza, M},
title = {Dissection of central clock function in Drosophila through cell-specific CRISPR-mediated clock gene disruption.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31613218},
issn = {2050-084X},
support = {T32 HL120826/HL/NHLBI NIH HHS/United States ; R01GM117407/NH/NIH HHS/United States ; T32 GM007367/GM/NIGMS NIH HHS/United States ; 5T32DK007328/NH/NIH HHS/United States ; R35GM127049/NH/NIH HHS/United States ; R01GM105775/NH/NIH HHS/United States ; R01 GM105775/GM/NIGMS NIH HHS/United States ; 2T32GM007367-42/NH/NIH HHS/United States ; R01 AG045842/AG/NIA NIH HHS/United States ; R01 GM117407/GM/NIGMS NIH HHS/United States ; 5T32HL120826/NH/NIH HHS/United States ; T32 GM007088/GM/NIGMS NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; T32 DK007328/DK/NIDDK NIH HHS/United States ; R35 GM127049/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Brain/cytology/metabolism/radiation effects ; CRISPR-Cas Systems ; Cell Communication ; Cell Lineage/genetics ; Circadian Clocks/drug effects/*genetics ; Circadian Rhythm/drug effects/*genetics ; Darkness ; Drosophila Proteins/deficiency/*genetics ; Drosophila melanogaster/*genetics/metabolism/radiation effects ; Feedback, Physiological ; Gene Editing ; Gene Expression Regulation ; Light Signal Transduction/genetics ; Locomotion/genetics/radiation effects ; Nerve Net/metabolism/radiation effects ; Neurons/cytology/*metabolism/radiation effects ; Neuropeptides/deficiency/*genetics ; Period Circadian Proteins/deficiency/*genetics ; Transcription Factors/deficiency/genetics ; },
abstract = {In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called 'master pacemakers' because they are essential for circadian rhythms. A subset of Pdf+ neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf+ neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf+ neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.},
}

Animals
Brain/cytology/metabolism/radiation effects
CRISPR-Cas Systems
Cell Communication
Cell Lineage/genetics
Circadian Clocks/drug effects/*genetics
Circadian Rhythm/drug effects/*genetics
Darkness
Drosophila Proteins/deficiency/*genetics
Drosophila melanogaster/*genetics/metabolism/radiation effects
Feedback, Physiological
Gene Editing
Gene Expression Regulation
Light Signal Transduction/genetics
Locomotion/genetics/radiation effects
Nerve Net/metabolism/radiation effects
Neurons/cytology/*metabolism/radiation effects
Neuropeptides/deficiency/*genetics
Period Circadian Proteins/deficiency/*genetics
Transcription Factors/deficiency/genetics

@article {pmid31612854,
year = {2019},
author = {Laflamme, C and McKeever, PM and Kumar, R and Schwartz, J and Kolahdouzan, M and Chen, CX and You, Z and Benaliouad, F and Gileadi, O and McBride, HM and Durcan, TM and Edwards, AM and Healy, LM and Robertson, J and McPherson, PS},
title = {Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31612854},
issn = {2050-084X},
support = {Hudson Translational Grant//ALS Society of Canada/International ; ALS RAP//ALS Society of Canada/International ; ALS RAP//ALS Therapy Alliance/International ; ALS RAP/MNDA_/Motor Neurone Disease Association/United Kingdom ; },
mesh = {Amyotrophic Lateral Sclerosis/*diagnosis/genetics/immunology/metabolism ; Animals ; Antibodies, Monoclonal/*chemistry/classification/immunology ; Biomarkers/metabolism ; C9orf72 Protein/*genetics/immunology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Frontotemporal Dementia/*diagnosis/genetics/immunology/metabolism ; Gene Editing ; Gene Expression ; HEK293 Cells ; Humans ; Immunohistochemistry/*standards ; Lysosomes/genetics/metabolism/ultrastructure ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Osteoblasts/metabolism/ultrastructure ; Phagosomes/genetics/metabolism/ultrastructure ; RAW 264.7 Cells ; },
abstract = {Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.},
}

Amyotrophic Lateral Sclerosis/*diagnosis/genetics/immunology/metabolism
Animals
Antibodies, Monoclonal/*chemistry/classification/immunology
Biomarkers/metabolism
C9orf72 Protein/*genetics/immunology
CRISPR-Cas Systems
Cell Line, Tumor
Frontotemporal Dementia/*diagnosis/genetics/immunology/metabolism
Gene Editing
Gene Expression
HEK293 Cells
Humans
Immunohistochemistry/*standards
Lysosomes/genetics/metabolism/ultrastructure
Mice
Mice, Inbred C57BL
Mice, Transgenic
Osteoblasts/metabolism/ultrastructure
Phagosomes/genetics/metabolism/ultrastructure
RAW 264.7 Cells

@article {pmid31467027,
year = {2019},
author = {Chung, JY and Ain, QU and Song, Y and Yong, SB and Kim, YH},
title = {Targeted delivery of CRISPR interference system against Fabp4 to white adipocytes ameliorates obesity, inflammation, hepatic steatosis, and insulin resistance.},
journal = {Genome research},
volume = {29},
number = {9},
pages = {1442-1452},
doi = {10.1101/gr.246900.118},
pmid = {31467027},
issn = {1549-5469},
mesh = {3T3 Cells ; Adipocytes, White/chemistry/cytology ; Animals ; CRISPR-Cas Systems ; Cytokines/metabolism ; Diabetes Mellitus, Type 2/*drug therapy/genetics ; Disease Models, Animal ; Fatty Acid-Binding Proteins/antagonists & inhibitors/*genetics ; Fatty Liver/*drug therapy/genetics ; Gene Expression Regulation ; Gene Knockdown Techniques ; Insulin Resistance ; Mice ; Molecular Targeted Therapy ; Obesity/*drug therapy/genetics ; RNA, Guide/*administration & dosage/pharmacology ; },
abstract = {Obesity is an increasing pathophysiological problem in developed societies. Despite all major progress in understanding molecular mechanisms of obesity, currently available anti-obesity drugs have shown limited efficacy with severe side effects. CRISPR interference (CRISPRi) mechanism based on catalytically dead Cas9 (dCas9) and single guide RNA (sgRNA) was combined with a targeted nonviral gene delivery system to treat obesity and obesity-induced type 2 diabetes. A fusion peptide targeting a vascular and cellular marker of adipose tissue, prohibitin, was developed by conjugation of adipocyte targeting sequence (CKGGRAKDC) to 9-mer arginine (ATS-9R). (dCas9/sgFabp4) + ATS-9R oligoplexes showed effective condensation and selective delivery into mature adipocytes. Targeted delivery of the CRISPRi system against Fabp4 to white adipocytes by ATS-9R induced effective silencing of Fabp4, resulting in reduction of body weight and inflammation and restoration of hepatic steatosis in obese mice. This RNA-guided DNA recognition platform provides a simple and safe approach to regress and treat obesity and obesity-induced metabolic syndromes.},
}

3T3 Cells
Adipocytes, White/chemistry/cytology
Animals
CRISPR-Cas Systems
Cytokines/metabolism
Diabetes Mellitus, Type 2/*drug therapy/genetics
Disease Models, Animal
Fatty Acid-Binding Proteins/antagonists & inhibitors/*genetics
Fatty Liver/*drug therapy/genetics
Gene Expression Regulation
Gene Knockdown Techniques
Insulin Resistance
Mice
Molecular Targeted Therapy
Obesity/*drug therapy/genetics
RNA, Guide/*administration & dosage/pharmacology

@article {pmid31362937,
year = {2019},
author = {Blanchard, Z and Vahrenkamp, JM and Berrett, KC and Arnesen, S and Gertz, J},
title = {Estrogen-independent molecular actions of mutant estrogen receptor 1 in endometrial cancer.},
journal = {Genome research},
volume = {29},
number = {9},
pages = {1429-1441},
doi = {10.1101/gr.244780.118},
pmid = {31362937},
issn = {1549-5469},
support = {P30 CA042014/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Endometrial Neoplasms/*genetics/metabolism ; Estrogen Receptor alpha/*genetics/metabolism ; Estrogens/metabolism ; Female ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks ; Humans ; *Mutation ; },
abstract = {Estrogen receptor 1 (ESR1) mutations have been identified in hormone therapy-resistant breast cancer and primary endometrial cancer. Analyses in breast cancer suggest that mutant ESR1 exhibits estrogen-independent activity. In endometrial cancer, ESR1 mutations are associated with worse outcomes and less obesity, however, experimental investigation of these mutations has not been performed. Using a unique CRISPR/Cas9 strategy, we introduced the D538G mutation, a common endometrial cancer mutation that alters the ligand binding domain of ESR1, while epitope tagging the endogenous locus. We discovered estrogen-independent mutant ESR1 genomic binding that is significantly altered from wild-type ESR1. The D538G mutation impacted expression, including a large set of nonestrogen-regulated genes, and chromatin accessibility, with most affected loci bound by mutant ESR1. Mutant ESR1 is distinct from constitutive ESR1 activity because mutant-specific changes are not recapitulated with prolonged estrogen exposure. Overall, the D538G mutant ESR1 confers estrogen-independent activity while causing additional regulatory changes in endometrial cancer cells that are distinct from breast cancer cells.},
}

CRISPR-Cas Systems
Cell Line, Tumor
Cell Movement
Cell Proliferation
Endometrial Neoplasms/*genetics/metabolism
Estrogen Receptor alpha/*genetics/metabolism
Estrogens/metabolism
Female
Gene Expression Profiling/*methods
Gene Expression Regulation, Neoplastic
Gene Regulatory Networks
Humans
*Mutation

@article {pmid31315906,
year = {2019},
author = {Zhang, S and Wang, Y and Jia, L and Wen, X and Du, Z and Wang, C and Hao, Y and Yu, D and Zhou, L and Chen, N and Chen, J and Chen, H and Zhang, H and Celik, I and Gülsoy, G and Luo, J and Qin, B and Cui, X and Liu, Z and Zhang, S and Esteban, MA and Ay, F and Xu, W and Chen, R and Li, W and Hoffman, AR and Hu, JF and Cui, J},
title = {Profiling the long noncoding RNA interaction network in the regulatory elements of target genes by chromatin in situ reverse transcription sequencing.},
journal = {Genome research},
volume = {29},
number = {9},
pages = {1521-1532},
doi = {10.1101/gr.244996.118},
pmid = {31315906},
issn = {1549-5469},
support = {I01 BX002905/BX/BLRD VA/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cellular Reprogramming ; Chromatin/*genetics ; Fibroblasts/cytology/metabolism ; Mice ; Octamer Transcription Factor-3/*genetics ; Pluripotent Stem Cells/cytology/metabolism ; Promoter Regions, Genetic ; RNA, Long Noncoding/*genetics ; Regulatory Sequences, Nucleic Acid ; SOXB1 Transcription Factors/*genetics ; Sequence Analysis, RNA/*methods ; },
abstract = {Long noncoding RNAs (lncRNAs) can regulate the activity of target genes by participating in the organization of chromatin architecture. We have devised a "chromatin-RNA in situ reverse transcription sequencing" (CRIST-seq) approach to profile the lncRNA interaction network in gene regulatory elements by combining the simplicity of RNA biotin labeling with the specificity of the CRISPR/Cas9 system. Using gene-specific gRNAs, we describe a pluripotency-specific lncRNA interacting network in the promoters of Sox2 and Pou5f1, two critical stem cell factors that are required for the maintenance of pluripotency. The promoter-interacting lncRNAs were specifically activated during reprogramming into pluripotency. Knockdown of these lncRNAs caused the stem cells to exit from pluripotency. In contrast, overexpression of the pluripotency-associated lncRNA activated the promoters of core stem cell factor genes and enhanced fibroblast reprogramming into pluripotency. These CRIST-seq data suggest that the Sox2 and Pou5f1 promoters are organized within a unique lncRNA interaction network that determines the fate of pluripotency during reprogramming. This CRIST approach may be broadly used to map lncRNA interaction networks at target loci across the genome.},
}

Animals
CRISPR-Cas Systems
Cell Line
Cellular Reprogramming
Chromatin/*genetics
Fibroblasts/cytology/metabolism
Mice
Octamer Transcription Factor-3/*genetics
Pluripotent Stem Cells/cytology/metabolism
Promoter Regions, Genetic
RNA, Long Noncoding/*genetics
Regulatory Sequences, Nucleic Acid
SOXB1 Transcription Factors/*genetics
Sequence Analysis, RNA/*methods

@article {pmid31300729,
year = {2019},
author = {Schneider-Futschik, EK},
title = {Beyond cystic fibrosis transmembrane conductance regulator therapy: a perspective on gene therapy and small molecule treatment for cystic fibrosis.},
journal = {Gene therapy},
volume = {26},
number = {9},
pages = {354-362},
doi = {10.1038/s41434-019-0092-5},
pmid = {31300729},
issn = {1476-5462},
mesh = {Aminophenols/therapeutic use ; Animals ; CRISPR-Cas Systems ; Cystic Fibrosis/genetics/metabolism/*therapy ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics/*metabolism ; Gene Transfer Techniques ; *Genetic Therapy ; Genetic Vectors ; Humans ; Quinolones/therapeutic use ; },
abstract = {Cystic fibrosis (CF) is a life-limiting disease caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) activity. The recent advent of the FDA-approved CFTR modulator drug ivacaftor, alone or in combination with lumacaftor or tezacaftor, has enabled treatment of the majority of patients suffering from CF. Even before the identification of the CFTR gene, gene therapy was put forward as a viable treatment option for this genetic condition. However, initial enthusiasm has been hampered as CFTR gene delivery to the lungs has proven to be more challenging than expected. This review covers the contemporary clinical and scientific knowledge base for small molecule CFTR modulator drug therapy, gene delivery vectors and CRISPR/Cas9 gene editing and highlights the prospect of these technologies for future treatment options.},
}

Aminophenols/therapeutic use
Animals
CRISPR-Cas Systems
Cystic Fibrosis/genetics/metabolism/*therapy
Cystic Fibrosis Transmembrane Conductance Regulator/genetics/*metabolism
Gene Transfer Techniques
*Genetic Therapy
Genetic Vectors
Humans
Quinolones/therapeutic use

@article {pmid31287821,
year = {2019},
author = {Nord, H and Dennhag, N and Tydinger, H and von Hofsten, J},
title = {The zebrafish HGF receptor met controls migration of myogenic progenitor cells in appendicular development.},
journal = {PloS one},
volume = {14},
number = {7},
pages = {e0219259},
doi = {10.1371/journal.pone.0219259},
pmid = {31287821},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Movement/physiology ; Extremities/growth & development ; Gene Expression Regulation, Developmental/genetics ; Muscle Development/physiology ; Muscle Fibers, Skeletal/metabolism ; Muscle, Skeletal/metabolism ; Muscles/metabolism ; Proto-Oncogene Proteins c-met/*genetics/*metabolism ; Stem Cells/metabolism ; Zebrafish/embryology/genetics ; Zebrafish Proteins/metabolism ; },
abstract = {The hepatocyte growth factor receptor C-met plays an important role in cellular migration, which is crucial for many developmental processes as well as for cancer cell metastasis. C-met has been linked to the development of mammalian appendicular muscle, which are derived from migrating muscle progenitor cells (MMPs) from within the somite. Mammalian limbs are homologous to the teleost pectoral and pelvic fins. In this study we used Crispr/Cas9 to mutate the zebrafish met gene and found that the MMP derived musculature of the paired appendages was severely affected. The mutation resulted in a reduced muscle fibre number, in particular in the pectoral abductor, and in a disturbed pectoral fin function. Other MMP derived muscles, such as the sternohyoid muscle and posterior hypaxial muscle were also affected in met mutants. This indicates that the role of met in MMP function and appendicular myogenesis is conserved within vertebrates.},
}

Animals
CRISPR-Cas Systems
Cell Movement/physiology
Extremities/growth & development
Gene Expression Regulation, Developmental/genetics
Muscle Development/physiology
Muscle Fibers, Skeletal/metabolism
Muscle, Skeletal/metabolism
Muscles/metabolism
Proto-Oncogene Proteins c-met/*genetics/*metabolism
Stem Cells/metabolism
Zebrafish/embryology/genetics
Zebrafish Proteins/metabolism

@article {pmid31237422,
year = {2019},
author = {Li, Z and Wang, F and Li, JF},
title = {Targeted Transcriptional Activation in Plants Using a Potent Dead Cas9-Derived Synthetic Gene Activator.},
journal = {Current protocols in molecular biology},
volume = {127},
number = {1},
pages = {e89},
doi = {10.1002/cpmb.89},
pmid = {31237422},
issn = {1934-3647},
mesh = {Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Gene Targeting/*methods ; Genes, Plant/genetics ; Genes, Synthetic/*genetics ; Oryza/*genetics ; Promoter Regions, Genetic ; Protoplasts/metabolism ; RNA, Guide/genetics ; *Transcriptional Activation ; },
abstract = {Genetic tools for specific perturbation of endogenous gene expression are highly desirable for interrogation of plant gene functions and improvement of crop traits. Synthetic transcriptional activators derived from the CRISPR/Cas9 system are emerging as powerful new tools for activating the endogenous expression of genes of interest in plants. These synthetic constructs, generated by tethering transcriptional activation domains to a nuclease-dead Cas9 (dCas9), can be directed to the promoters of endogenous target genes by single guide RNAs (sgRNAs) to activate transcription. Here, we provide a detailed protocol for targeted transcriptional activation in plants using a recently developed, highly potent dCas9 gene activator construct referred to as dCas9-TV. This protocol covers selection of sgRNA targets, construction of sgRNA expression cassettes, and screening for an optimal sgRNA using a protoplast-based promoter-luciferase assay. Finally, the dCas9-TV gene activator coupled with the optimal sgRNA is delivered into plants via Agrobacterium-mediated transformation, thereby enabling robust upregulation of target gene expression in transgenic Arabidopsis and rice plants. © 2019 by John Wiley & Sons, Inc.},
}

Arabidopsis/*genetics
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/genetics
Gene Targeting/*methods
Genes, Plant/genetics
Genes, Synthetic/*genetics
Oryza/*genetics
Promoter Regions, Genetic
Protoplasts/metabolism
RNA, Guide/genetics
*Transcriptional Activation

@article {pmid31128790,
year = {2019},
author = {Zhao, C and Wei, S and Wang, Y},
title = {A guide for drug inducible transcriptional activation with HIT systems.},
journal = {Methods in enzymology},
volume = {621},
number = {},
pages = {69-86},
doi = {10.1016/bs.mie.2019.02.032},
pmid = {31128790},
issn = {1557-7988},
mesh = {CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; Humans ; Lentivirus/genetics ; RNA, Guide/genetics ; Transcriptional Activation/*drug effects ; },
abstract = {Precise investigation and manipulation of gene function often require modulation in a controlled and dynamic manner. In this chapter, we describe the methods to apply HIT systems for drug inducible transcriptional activation or simultaneous activation and genome editing in human cells. Together with those for editing, which are described in another chapter, HIT systems herein provide a valuable toolbox toward many biological applications, especially when precision and dynamics are required for a functional perturbation.},
}

CRISPR-Cas Systems
Cell Line
Gene Editing/*methods
Humans
Lentivirus/genetics
RNA, Guide/genetics
Transcriptional Activation/*drug effects

@article {pmid31128789,
year = {2019},
author = {Zhao, C and Wei, S and Wang, Y},
title = {A guide for drug inducible genome editing with HIT systems.},
journal = {Methods in enzymology},
volume = {621},
number = {},
pages = {53-68},
doi = {10.1016/bs.mie.2019.02.031},
pmid = {31128789},
issn = {1557-7988},
mesh = {*CRISPR-Cas Systems ; Cell Line ; Gene Editing/*methods ; Humans ; Lentivirus/genetics ; Pharmacology/methods ; Transcription Activator-Like Effector Nucleases/genetics ; Transcription Activator-Like Effectors/genetics ; Transfection/methods ; },
abstract = {Technologies toward precise control of biological events are desired for biomedical research and potential clinical applications. Our recently reported HIT systems based on CRISPR/Cas9 and TAL effectors can achieve temporal and dose dependent drug control for genome editing. Methods are presented for the application of these optimized HIT systems in human cells in this chapter.},
}

*CRISPR-Cas Systems
Cell Line
Gene Editing/*methods
Humans
Lentivirus/genetics
Pharmacology/methods
Transcription Activator-Like Effector Nucleases/genetics
Transcription Activator-Like Effectors/genetics
Transfection/methods

@article {pmid30973754,
year = {2019},
author = {Chen, G and Ribeiro, CMP and Sun, L and Okuda, K and Kato, T and Gilmore, RC and Martino, MB and Dang, H and Abzhanova, A and Lin, JM and Hull-Ryde, EA and Volmer, AS and Randell, SH and Livraghi-Butrico, A and Deng, Y and Scherer, PE and Stripp, BR and O'Neal, WK and Boucher, RC},
title = {XBP1S Regulates MUC5B in a Promoter Variant-Dependent Pathway in Idiopathic Pulmonary Fibrosis Airway Epithelia.},
journal = {American journal of respiratory and critical care medicine},
volume = {200},
number = {2},
pages = {220-234},
doi = {10.1164/rccm.201810-1972OC},
pmid = {30973754},
issn = {1535-4970},
support = {P01 HL110873/HL/NHLBI NIH HHS/United States ; P50 HL060280/HL/NHLBI NIH HHS/United States ; P50 HL107168/HL/NHLBI NIH HHS/United States ; R01 HL080396/HL/NHLBI NIH HHS/United States ; P50 HL084934/HL/NHLBI NIH HHS/United States ; UH3 HL123645/HL/NHLBI NIH HHS/United States ; UH2 HL123645/HL/NHLBI NIH HHS/United States ; P01 HL108808/HL/NHLBI NIH HHS/United States ; P30 DK065988/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Endoplasmic Reticulum Stress/genetics ; Endoribonucleases/*genetics/metabolism ; Gene Expression Regulation ; Humans ; Idiopathic Pulmonary Fibrosis/*genetics/metabolism ; Membrane Proteins/*genetics/metabolism ; Mice ; Mice, Transgenic ; Polymorphism, Genetic ; Primary Cell Culture ; Promoter Regions, Genetic ; Protein-Serine-Threonine Kinases/*genetics/metabolism ; Respiratory Mucosa/*metabolism ; X-Box Binding Protein 1/*genetics/metabolism ; },
abstract = {Rationale: The goal was to connect elements of idiopathic pulmonary fibrosis (IPF) pathogenesis, including chronic endoplasmic reticulum stress in respiratory epithelia associated with injury/inflammation and remodeling, distal airway mucus obstruction and honeycomb cyst formation with accumulation of MUC5B (mucin 5B), and associations between IPF risk and polymorphisms in the MUC5B promoter. Objectives: To test whether the endoplasmic reticulum (ER) stress sensor protein ERN2 (ER-to-nucleus signaling 2) and its downstream effector, the spliced form of XBP1S (X-box-binding protein 1), regulate MUC5B expression and differentially activate the MUC5B promoter variant in respiratory epithelia. Methods: Primary human airway epithelial (HAE) cells, transgenic mouse models, human IPF lung tissues, and cell lines expressing XBP1S and MUC5B promoters were used to explore relationships between the ERN2/XBP1S pathway and MUC5B. An inhibitor of the pathway, KIRA6, and XBP1 CRISPR-Cas9 were used in HAE cells to explore therapeutic potential. Measurements and Main Results: ERN2 regulated MUC5B and MUC5AC mRNAs. Downstream XBP1S selectively promoted MUC5B expression in vitro and in distal murine airway epithelia in vivo. XBP1S bound to the proximal region of the MUC5B promoter and differentially upregulated MUC5B expression in the context of the MUC5B promoter rs35705950 variant. High levels of ERN2 and XBP1S were associated with excessive MUC5B mRNAs in distal airways of human IPF lungs. Cytokine-induced MUC5B expression in HAE cells was inhibited by KIRA6 and XBP1 CRISPR-Cas9. Conclusions: A positive feedback bistable ERN2-XBP1S pathway regulates MUC5B-dominated mucus obstruction in IPF, providing an unfolded protein response-dependent mechanism linking the MUC5B promoter rs35705950 polymorphism with IPF pathogenesis. Inhibiting ERN2-dependent pathways/elements may provide a therapeutic option for IPF.},
}

Animals
CRISPR-Cas Systems
Cell Line
Endoplasmic Reticulum Stress/genetics
Endoribonucleases/*genetics/metabolism
Gene Expression Regulation
Humans
Idiopathic Pulmonary Fibrosis/*genetics/metabolism
Membrane Proteins/*genetics/metabolism
Mice
Mice, Transgenic
Polymorphism, Genetic
Primary Cell Culture
Promoter Regions, Genetic
Protein-Serine-Threonine Kinases/*genetics/metabolism
Respiratory Mucosa/*metabolism
X-Box Binding Protein 1/*genetics/metabolism

@article {pmid30745225,
year = {2019},
author = {Soni, S and Kohn, DB},
title = {Chemistry, manufacturing and controls for gene modified hematopoietic stem cells.},
journal = {Cytotherapy},
volume = {21},
number = {3},
pages = {358-366},
doi = {10.1016/j.jcyt.2018.12.001},
pmid = {30745225},
issn = {1477-2566},
mesh = {Blood Component Removal/methods ; CRISPR-Cas Systems/genetics ; Cell Survival ; Endotoxins/analysis ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors ; HEK293 Cells ; *Hematopoietic Stem Cells ; Humans ; Lentivirus/genetics ; Limulus Test ; Mycoplasma ; *Quality Control ; Transduction, Genetic/*methods ; },
abstract = {Gene modification of hematopoietic stem cells is increasingly becoming popular as a therapeutic approach, given the recent approvals and the number of new applications for clinical trials targeting monogenetic and immunodeficiency disorders. Technological advances in stem cell selection, culture, transduction and gene editing now allow for efficient ex vivo genetic manipulation of stem cells. Gene-addition techniques using viral vectors (mainly retrovirus- and lentivirus-based) and gene editing using various targeted nuclease platforms (e.g., Zinc finger, TALEN and Crispr/Cas9) are being applied to the treatment of multiple genetic and immunodeficiency disorders. Herein, the current state of the art in manufacturing and critical assays that are required for ex vivo manipulation of stem cells are addressed. Important quality control and safety assays that need to be planned early in the process development phase of these products for regulatory approval are also highlighted.},
}

Blood Component Removal/methods
CRISPR-Cas Systems/genetics
Cell Survival
Endotoxins/analysis
Gene Editing/*methods
Genetic Therapy/*methods
Genetic Vectors
HEK293 Cells
*Hematopoietic Stem Cells
Humans
Lentivirus/genetics
Limulus Test
Mycoplasma
*Quality Control
Transduction, Genetic/*methods

@article {pmid29618844,
year = {2018},
author = {Rhee, JW and Wu, JC},
title = {Dyslipidaemia: In vivo genome editing of ANGPTL3: a therapy for atherosclerosis?.},
journal = {Nature reviews. Cardiology},
volume = {15},
number = {5},
pages = {259-260},
pmid = {29618844},
issn = {1759-5010},
support = {F32 HL134221/HL/NHLBI NIH HHS/United States ; R01 HL113006/HL/NHLBI NIH HHS/United States ; R01 HL126527/HL/NHLBI NIH HHS/United States ; R01 HL130020/HL/NHLBI NIH HHS/United States ; },
mesh = {Angiopoietin-like Proteins ; *Atherosclerosis ; CRISPR-Cas Systems ; *Dyslipidemias ; Gene Editing ; Humans ; Lipids ; },
}

Angiopoietin-like Proteins
*Atherosclerosis
CRISPR-Cas Systems
*Dyslipidemias
Gene Editing
Humans
Lipids

@article {pmid32085579,
year = {2020},
author = {Hao, M and Wang, Z and Qiao, H and Yin, P and Qiao, J and Qi, H},
title = {Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli.},
journal = {Cells},
volume = {9},
number = {2},
pages = {},
doi = {10.3390/cells9020467},
pmid = {32085579},
issn = {2073-4409},
support = {21476167; .21778039; 21621004//National Science Foundation of China/ ; },
abstract = {As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.},
}

@article {pmid32084428,
year = {2020},
author = {Deem, TL and Collins, JB and DeVost, MH and Parker, CO and Saroka, SC and Zoldork, RJ and Gutierrez, F and Russell, JM and Lantz, CS},
title = {Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice.},
journal = {Immunology letters},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.imlet.2020.02.006},
pmid = {32084428},
issn = {1879-0542},
abstract = {Interleukin-3 (IL-3) is an important hematopoietic growth factor and immunregulatory cytokine. Although activated T helper cells represent a main source of IL-3, other cell types have been reported to express this cytokine. However, precise identification and quantification of the cells that produce IL-3 in vivo have not been performed. Therefore, we used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. To characterize these novel reporter mice, we first examined ZsGreen1 expression by CD4 T cells subsets primed and activated in vitro. We found that activated Th1 cells expressed ∼4-fold higher levels of ZsGreen1 as compared to Th0 and Th2 cells. Endogenous IL-3 expression remained intact although reporter Th1 cells secreted ∼33% less IL-3 than similarly activated wild-type cells. To characterize the ability of reporter mice to accurately mark IL-3-producing cells in vivo, we infected mice with Nippostrongylus brasiliensis. Low but significant numbers of ZsGreen1+ CD4 T cells were detected in the mesenteric lymph nodes and lung following both primary and secondary infection. No difference in basophil and intestinal mast cell numbers were observed between infected reporter and wild-type mice indicating that reporter mice secreted IL-3 levels in vivo that results in IL-3-driven biological activities which are indistinguishable from those observed in corresponding wild-type mice. These IL-3 reporter mice will be a valuable resource to investigate IL-3-dependent immune responses in vivo.},
}

@article {pmid32080068,
year = {2020},
author = {Frith, KH},
title = {CRISPR-Cas: What Is It and Why Should Nurses Care?.},
journal = {Nursing education perspectives},
volume = {41},
number = {2},
pages = {136-137},
doi = {10.1097/01.NEP.0000000000000644},
pmid = {32080068},
issn = {1536-5026},
}

@article {pmid30169558,
year = {2019},
author = {Zhang, S and Li, X and Lin, Q and Wong, KC},
title = {Synergizing CRISPR/Cas9 off-target predictions for ensemble insights and practical applications.},
journal = {Bioinformatics (Oxford, England)},
volume = {35},
number = {7},
pages = {1108-1115},
doi = {10.1093/bioinformatics/bty748},
pmid = {30169558},
issn = {1367-4811},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genomics ; RNA, Guide ; },
abstract = {MOTIVATION: The RNA-guided CRISPR/Cas9 system has been widely applied to genome editing. CRISPR/Cas9 system can effectively edit the on-target genes. Nonetheless, it has recently been demonstrated that many homologous off-target genomic sequences could be mutated, leading to unexpected gene-editing outcomes. Therefore, a plethora of tools were proposed for the prediction of off-target activities of CRISPR/Cas9. Nonetheless, each computational tool has its own advantages and drawbacks under diverse conditions. It is hardly believed that a single tool is optimal for all conditions. Hence, we would like to explore the ensemble learning potential on synergizing multiple tools with genomic annotations together to enhance its predictive abilities.

RESULTS: We proposed an ensemble learning framework which synergizes multiple tools together to predict the off-target activities of CRISPR/Cas9 in different combinations. Interestingly, the ensemble learning using AdaBoost outperformed other individual off-target predictive tools. We also investigated the effect of evolutionary conservation (PhyloP and PhastCons) and chromatin annotations (ChromHMM and Segway) and found that only PhyloP can enhance the predictive capabilities further. Case studies are conducted to reveal ensemble insights into the off-target predictions, demonstrating how the current study can be applied in different genomic contexts. The best prediction predicted by AdaBoost is up to 0.9383 (AUC) and 0.2998 (PRC) that outperforms other classifiers. This is ascribable to the fact that AdaBoost introduces a new weak classifier (i.e. decision stump) in each iteration to learn the DNA sequences that were misclassified as off-targets until a small error rate is reached iteratively.

The source codes are freely available on GitHub at https://github.com/Alexzsx/CRISPR.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Genomics
RNA, Guide

@article {pmid29869114,
year = {2019},
author = {Mu, W and Zhang, Y and Xue, X and Liu, L and Wei, X and Wang, H},
title = {5' capped and 3' polyA-tailed sgRNAs enhance the efficiency of CRISPR-Cas9 system.},
journal = {Protein & cell},
volume = {10},
number = {3},
pages = {223-228},
doi = {10.1007/s13238-018-0552-5},
pmid = {29869114},
issn = {1674-8018},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Humans ; K562 Cells ; RNA, Guide/*genetics ; RNA, Messenger/*genetics ; },
}

CRISPR-Cas Systems/*genetics
Gene Editing
Humans
K562 Cells
RNA, Guide/*genetics
RNA, Messenger/*genetics

@article {pmid32076642,
year = {2020},
author = {Aschenbrenner, S and Kallenberger, SM and Hoffmann, MD and Huck, A and Eils, R and Niopek, D},
title = {Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.},
journal = {Science advances},
volume = {6},
number = {6},
pages = {eaay0187},
doi = {10.1126/sciadv.aay0187},
pmid = {32076642},
issn = {2375-2548},
abstract = {The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.},
}

@article {pmid32076262,
year = {2020},
author = {Kim, S and Loeff, L and Colombo, S and Jergic, S and Brouns, SJJ and Joo, C},
title = {Selective loading and processing of prespacers for precise CRISPR adaptation.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-020-2018-1},
pmid = {32076262},
issn = {1476-4687},
abstract = {CRISPR-Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.},
}

@article {pmid32075512,
year = {2020},
author = {Bor, B and Collins, AJ and Murugkar, PP and Balasubramanian, S and To, TT and Hendrickson, EL and Bedree, JK and Bidlack, FB and Johnston, CD and Shi, W and McLean, JS and He, X and Dewhirst, FE},
title = {Insights Obtained by Culturing Saccharibacteria With Their Bacterial Hosts.},
journal = {Journal of dental research},
volume = {},
number = {},
pages = {22034520905792},
doi = {10.1177/0022034520905792},
pmid = {32075512},
issn = {1544-0591},
abstract = {Oral microbiome research has moved from asking "Who's there?" to "What are they doing?" Understanding what microbes "do" involves multiple approaches, including obtaining genomic information and examining the interspecies interactions. Recently we isolated a human oral Saccharibacteria (TM7) bacterium, HMT-952, strain TM7x, which is an ultrasmall parasite of the oral bacterium Actinomyces odontolyticus. The host-parasite interactions, such as phage-bacterium or Saccharibacteria-host bacterium, are understudied areas with large potential for insight. The Saccharibacteria phylum is a member of Candidate Phyla Radiation, a large lineage previously devoid of cultivated members. However, expanding our understanding of Saccharibacteria-host interactions requires examining multiple phylogenetically distinct Saccharibacteria-host pairs. Here we report the isolation of 3 additional Saccharibacteria species from the human oral cavity in binary coculture with their bacterial hosts. They were obtained by filtering ultrasmall Saccharibacteria cells free of other larger bacteria and inoculating them into cultures of potential host bacteria. The binary cocultures obtained could be stably passaged and studied. Complete closed genomes were obtained and allowed full genome analyses. All have small genomes (<1 Mb) characteristic of parasitic species and dramatically limited de novo synthetic pathway capabilities but include either restriction modification or CRISPR-Cas systems as part of an innate defense against foreign DNA. High levels of gene synteny exist among Saccharibacteria species. Having isolates growing in coculture with their hosts allowed time course studies of growth and parasite-host interactions by phase contrast, fluorescence in situ hybridization, and scanning electron microscopy. The cells of the 4 oral Saccharibacteria species are ultrasmall and could be seen attached to their larger Actinobacteria hosts. Parasite attachment appears to lead to host cell death and lysis. The successful cultivation of Saccharibacteria species has significantly expanded our understanding of these ultrasmall Candidate Phyla Radiation bacteria.},
}

@article {pmid31959180,
year = {2020},
author = {Jo, A and Ringel-Scaia, VM and McDaniel, DK and Thomas, CA and Zhang, R and Riffle, JS and Allen, IC and Davis, RM},
title = {Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR-Cas9 plasmid.},
journal = {Journal of nanobiotechnology},
volume = {18},
number = {1},
pages = {16},
pmid = {31959180},
issn = {1477-3155},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; DNA/chemistry ; Fluorescent Dyes/chemistry ; Gene Transfer Techniques ; Macrophages/metabolism ; Mice ; Nanoparticles/*chemistry ; Organosilicon Compounds/chemistry ; Plasmids ; Polylactic Acid-Polyglycolic Acid Copolymer/*chemistry ; Transfection ; },
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR-Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells.

RESULTS: Using poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR-Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160 nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6 wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24 h and corresponded to ~ 2-3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells.

CONCLUSIONS: In this work, plasmids for the CRISPR-Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR-Cas9 system.},
}

Animals
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
DNA/chemistry
Fluorescent Dyes/chemistry
Gene Transfer Techniques
Macrophages/metabolism
Mice
Nanoparticles/*chemistry
Organosilicon Compounds/chemistry
Plasmids
Polylactic Acid-Polyglycolic Acid Copolymer/*chemistry
Transfection

@article {pmid31434745,
year = {2019},
author = {Zhang, WW and Matlashewski, G},
title = {Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania.},
journal = {mSphere},
volume = {4},
number = {4},
pages = {},
doi = {10.1128/mSphere.00408-19},
pmid = {31434745},
issn = {2379-5042},
support = {//CIHR/Canada ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA Repair ; DNA, Protozoan/metabolism ; DNA, Single-Stranded/chemistry ; Gene Editing/methods ; *Genome, Protozoan ; Leishmania/*genetics ; Recombination, Genetic ; Staphylococcus aureus ; },
abstract = {CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite Leishmania lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that Leishmania predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the Leishmania donovani LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 Leishmania genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in Leishmania, which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the Leishmania genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Leishmania Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in Leishmania In addition, we developed a novel Staphylococcus aureus Cas9 constitutive expression vector (pLdSaCN) for gene targeting in LeishmaniaIMPORTANCE Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania.},
}

CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
*DNA Breaks, Double-Stranded
*DNA Repair
DNA, Protozoan/metabolism
DNA, Single-Stranded/chemistry
Gene Editing/methods
*Genome, Protozoan
Leishmania/*genetics
Recombination, Genetic
Staphylococcus aureus

@article {pmid31278903,
year = {2019},
author = {Chen, N and Hu, Z and Yang, Y and Han, H and Lei, H},
title = {Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells.},
journal = {Experimental eye research},
volume = {186},
number = {},
pages = {107716},
doi = {10.1016/j.exer.2019.107716},
pmid = {31278903},
issn = {1096-0007},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Cell Proliferation/physiology ; Cell Survival/physiology ; Cells, Cultured ; Epithelial Cells/*physiology ; Humans ; Mice ; Molecular Targeted Therapy/methods ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins c-mdm2/*metabolism ; Retinal Pigment Epithelium/*physiology ; Tumor Suppressor Protein p53/metabolism ; Vitreoretinopathy, Proliferative/physiopathology ; Vitreous Body/*metabolism ; },
abstract = {Mouse double minute (MDM)2 single nucleotide polymorphism (SNP) 309G allele in the second promoter of MDM2 enhances vitreous-induced expression of Mdm2 and degradation of the tumor suppressor protein p53. This MDM2SNP309G contributes to certain cancer development and experimental proliferative vitreoretinopathy. The goal of this study is to discover a novel strategy to only block vitreous-induced expression of Mdm2 for preventing vitreous-induced cell proliferation and survival and thus find a potential novel strategy to treat proliferation-related diseases. We created two mutations (D10A and H840A) in Streptococcus pyogenes (Sp)Cas9 within the nuclease domains (RuvC1 and HNH, respectively) to render this SpCas9 nuclease dead named as dCas9 in a lentiCRISPR v2 vector. Then an MDM2-sgRNA targeting the second promoter of human MDM2 gene was cloned into this vector for producing lentivirus to infect human retinal pigment epithelial (RPE) cells with, which carry a heterozygous genotype of MDM2SNP309 T/G. lacZ-sgRNA was used as a control. As a result, we discovered that vitreous from experimental rabbits induced a 1.9 ± 0.2 fold increase in Mdm2 and a 2.0 ± 0.2 fold decrease in p53 in the RPE cells with dCas9/lacZ-sgRNA compared to those with dCas9/MDM2-sgRNA, suggesting that dCas9 under the guidance of the MDM2-sgRNA prevented RV-stimulated increase in Mdm2. In addition, we found that the rabbit vitreous significantly enhanced cell proliferation (1.5 ± 0.2 fold), survival against apoptosis (2.2 ± 0.2 fold), migration (10 ± 1.5%) and contraction (112.7 ± 14.1 mm2) of the cells with dCas9/lacZ-sgRNA compared with those with dCas9/MDM2-sgRNA. These results indicated that application of the dCas9 targeted to the P2 of MDM2 is a potential therapeutic approach to diseases due to the P2-driven aberrant expression of Mdm2 - such as proliferative vitreoretinopathy.},
}

Animals
CRISPR-Cas Systems/*physiology
Cell Proliferation/physiology
Cell Survival/physiology
Cells, Cultured
Epithelial Cells/*physiology
Humans
Mice
Molecular Targeted Therapy/methods
Polymorphism, Single Nucleotide
Proto-Oncogene Proteins c-mdm2/*metabolism
Retinal Pigment Epithelium/*physiology
Tumor Suppressor Protein p53/metabolism
Vitreoretinopathy, Proliferative/physiopathology
Vitreous Body/*metabolism

@article {pmid31175335,
year = {2019},
author = {},
title = {Clever chip designs for diagnostics.},
journal = {Nature biomedical engineering},
volume = {3},
number = {6},
pages = {417-418},
doi = {10.1038/s41551-019-0418-z},
pmid = {31175335},
issn = {2157-846X},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Graphite ; Macrophages ; Oligonucleotide Array Sequence Analysis ; },
}

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Graphite
Macrophages
Oligonucleotide Array Sequence Analysis

@article {pmid31175332,
year = {2019},
author = {Bruch, R and Urban, GA and Dincer, C},
title = {Unamplified gene sensing via Cas9 on graphene.},
journal = {Nature biomedical engineering},
volume = {3},
number = {6},
pages = {419-420},
doi = {10.1038/s41551-019-0413-4},
pmid = {31175332},
issn = {2157-846X},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Graphite ; },
}

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Graphite

@article {pmid31015728,
year = {2018},
author = {Bae, S and Kim, JS},
title = {Machine learning finds Cas9-edited genotypes.},
journal = {Nature biomedical engineering},
volume = {2},
number = {12},
pages = {892-893},
doi = {10.1038/s41551-018-0327-6},
pmid = {31015728},
issn = {2157-846X},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genotype ; Machine Learning ; },
}

*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Genotype
Machine Learning

@article {pmid31015617,
year = {2018},
author = {Duan, D},
title = {CRISPR alleviates muscular dystrophy in dogs.},
journal = {Nature biomedical engineering},
volume = {2},
number = {11},
pages = {795-796},
doi = {10.1038/s41551-018-0320-0},
pmid = {31015617},
issn = {2157-846X},
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dogs ; *Dystrophin ; Gene Editing ; *Muscular Dystrophy, Duchenne ; },
}

Animals
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Dogs
*Dystrophin
Gene Editing
*Muscular Dystrophy, Duchenne

@article {pmid30944435,
year = {2019},
author = {Hsu, MN and Hu, YC},
title = {Local magnetic activation of CRISPR.},
journal = {Nature biomedical engineering},
volume = {3},
number = {2},
pages = {83-84},
doi = {10.1038/s41551-019-0354-y},
pmid = {30944435},
issn = {2157-846X},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Magnetic Phenomena ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Magnetic Phenomena

@article {pmid30406958,
year = {2019},
author = {Ortega-Escalante, JA and Jasper, R and Miller, SM},
title = {CRISPR/Cas9 mutagenesis in Volvox carteri.},
journal = {The Plant journal : for cell and molecular biology},
volume = {97},
number = {4},
pages = {661-672},
doi = {10.1111/tpj.14149},
pmid = {30406958},
issn = {1365-313X},
support = {/NH/NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Gene Editing/methods ; Mutagenesis/genetics/physiology ; Volvox/*genetics ; },
abstract = {Volvox carteri and other volvocine green algae comprise an excellent model for investigating developmental complexity and its origins. Here we describe a method for targeted mutagenesis in V. carteri using CRISPR/Cas9 components expressed from transgenes. We used V. carteri nitrate reductase gene (nitA) regulatory sequences to conditionally express Streptococcus pyogenes Cas9, and V. carteri U6 RNA gene regulatory sequences to constitutively express single-guide RNA (sgRNA) transcripts. Volvox carteri was bombarded with both Cas9 vector and one of several sgRNA vectors programmed to target different test genes (glsA, regA and invA), and transformants were selected for expression of a hygromycin-resistance marker present on the sgRNA vector. Hygromycin-resistant transformants grown with nitrate as sole nitrogen source (inducing for nitA) were tested for Cas9 and sgRNA expression, and for the ability to generate progeny with expected mutant phenotypes. Some transformants of a somatic regenerator (Reg) mutant strain receiving sgRNA plasmid with glsA protospacer sequence yielded progeny (at a rate of ~0.01%) with a gonidialess (Gls) phenotype similar to that observed for previously described glsA mutants, and sequencing of the glsA gene in independent mutants revealed short deletions within the targeted region of glsA, indicative of Cas9-directed non-homologous end joining. Similarly, bombardment of a morphologically wild-type strain with the Cas9 plasmid and sgRNA plasmids targeting regA or invA yielded regA and invA mutant transformants/progeny, respectively (at rates of 0.1-100%). The capacity to make precisely directed frameshift mutations should greatly accelerate the molecular genetic analysis of development in V. carteri, and of developmental novelty in the volvocine algae.},
}

CRISPR-Cas Systems/genetics/*physiology
Gene Editing/methods
Mutagenesis/genetics/physiology
Volvox/*genetics

@article {pmid32073884,
year = {2020},
author = {Deng, M and Liu, Z and Chen, B and Cai, Y and Wan, Y and Wang, F},
title = {Locus-Specific Regulation of Xist Expression Using the CRISPR-Cas9-Based System.},
journal = {DNA and cell biology},
volume = {},
number = {},
pages = {},
doi = {10.1089/dna.2019.4945},
pmid = {32073884},
issn = {1557-7430},
abstract = {DNA methylation inhibitor or loss and gain of function of DNA methylation key players were widely used to investigate the regulation of X inactive-specific transcript (Xist) expression by DNA methylation, which results in global change of DNA methylation. Here, we reported a novel method for regulation of Xist using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. First, Xist expression was increased in 5-aza-2'-deoxycytidine-treated female goat fibroblast cells. Second, three single-guide RNAs (sgRNAs) that target the Xist differential methylation region (DMR) were inserted to deactivated Cas9 (dCas9) nuclease and the catalytic domain of the DNA methyltransferase Dnmt3a coexpression plasmid. Bisulfite PCR analysis and quantitative real-time PCR revealed that the methylation level of the DMR was significantly increased, while the expression of Xist was downregulated in all three sgRNAs, compared with the mock-transfected cells. Third, the methylation activity at the sites of 37 bp from the protospacer-adjacent motif sequence showed the strong change relative to the mock-transfected cells. Furthermore, genome-wide DNA methylation and expression of the DNA methylation key players were not statistically changed in all three sgRNAs. Therefore, we confirmed that Xist expression was regulated by DNA methylation, and directed DNA methylation of Xist DMR at locus-specific solution decreased Xist expression.},
}

@article {pmid32072789,
year = {2020},
author = {Li, J and Liu, Y and Wang, Y and Yu, P and Zheng, P and Wang, M},
title = {[Optimization of base editing in Corynebacterium glutamicum].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {1},
pages = {143-151},
doi = {10.13345/j.cjb.190192},
pmid = {32072789},
issn = {1000-3061},
abstract = {In recent years, CRISPR/Cas9-mediated base editing has been developed to a powerful genome editing tool, providing advantages such as without introducing double-stranded DNA break, a donor template and relying on host homologous recombination repair pathway, and has been widely applied in animals, plants, yeast and bacteria. In previous study, our group developed a multiplex automated base editing method (MACBETH) in the important industrial model strain Corynebacterium glutamicum. In this study, to further optimize the method and improve the base editing efficiency in C. glutamicum, we first constructed a green fluorescent protein (GFP) reporter-based detection system. The point mutation in the inactivated GFP protein can be edited to restore the GFP fluorescence. By combining with flow cytometry analysis, the base-editing efficiency can be quickly calculated. Then, the base editor with the target gRNA was constructed, and the editing efficiency with the initial editing condition was (13.11±0.21)%. Based on this result, the editing conditions were optimized and the result indicated that the best medium is CGXII, the best initial OD₆₀₀ of induction is 0.05, the best induction time is 20 h, and the best IPTG concentration is 0.01 mmol/L. After optimization, the editing efficiency was improved to (30.35±0.75)%, which was 1.3-fold of that in initial condition. Finally, endogenous genomic loci of C. glutamicum were selected to assess if the optimized condition can improve genome editing in other loci. Editing efficiency of different loci in optimized condition were improved to 1.7-2.5 fold of that in original condition, indicating the effectiveness and versatility of the optimized condition. Our research will promote the better application of base editing technology in C. glutamicum.},
}

@article {pmid32072776,
year = {2020},
author = {Ouyang, J and Xue, S and Zhou, Q and Cui, H},
title = {[Research progress and applications of gene editing technology CRISPR/Cas in zebrafish].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {36},
number = {1},
pages = {1-12},
doi = {10.13345/j.cjb.190178},
pmid = {32072776},
issn = {1000-3061},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) are acquired immune system in bacteria and archaea. This system is used in site-directed gene editing. Recently, scientists discovered new CRISPR-associated (Cas) proteins, in which Cas12a-mediated gene editing can significantly reduce the off-target rate. In this article, we review CRISPR/Cas system's discovery of history, composition, classification, and working principle. The latest research progress of the CRISPR/Cas system, and its application in zebrafish are introduced.},
}

@article {pmid32071241,
year = {2020},
author = {Bradde, S and Nourmohammad, A and Goyal, S and Balasubramanian, V},
title = {The size of the immune repertoire of bacteria.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {},
number = {},
pages = {},
doi = {10.1073/pnas.1903666117},
pmid = {32071241},
issn = {1091-6490},
abstract = {Some bacteria and archaea possess an immune system, based on the CRISPR-Cas mechanism, that confers adaptive immunity against viruses. In such species, individual prokaryotes maintain cassettes of viral DNA elements called spacers as a memory of past infections. Typically, the cassettes contain several dozen expressed spacers. Given that bacteria can have very large genomes and since having more spacers should confer a better memory, it is puzzling that so little genetic space would be devoted by prokaryotes to their adaptive immune systems. Here, assuming that CRISPR functions as a long-term memory-based defense against a diverse landscape of viral species, we identify a fundamental tradeoff between the amount of immune memory and effectiveness of response to a given threat. This tradeoff implies an optimal size for the prokaryotic immune repertoire in the observational range.},
}

@article {pmid32071159,
year = {2020},
author = {Brooks, MR and Padilla-Vélez, L and Khan, TA and Qureshi, AA and Pieper, JB and Maddox, CW and Alam, MT},
title = {Prophage-Mediated Disruption of Genetic Competence in Staphylococcus pseudintermedius.},
journal = {mSystems},
volume = {5},
number = {1},
pages = {},
doi = {10.1128/mSystems.00684-19},
pmid = {32071159},
issn = {2379-5077},
abstract = {Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of soft tissue infections in dogs and occasionally infects humans. Hypervirulent multidrug-resistant (MDR) MRSP clones have emerged globally. The sequence types ST71 and ST68, the major epidemic clones of Europe and North America, respectively, have spread to other regions. The genetic factors underlying the success of these clones have not been investigated thoroughly. Here, we performed a comprehensive genomic analysis of 371 S. pseudintermedius isolates to dissect the differences between major clonal lineages. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, restriction-modification (RM), and CRISPR/Cas systems differs significantly among MRSP clones. The isolates with GyrA+GrlA mutations, conferring fluoroquinolone resistance, carry more of these genes than those without GyrA+GrlA mutations. ST71 and ST68 clones carry lineage-specific prophages with genes that are likely associated with their increased fitness and virulence. We have discovered that a prophage, SpST71A, is inserted within the comGA gene of the late competence operon comG in the ST71 lineage. A functional comG is essential for natural genetic competence, which is one of the major modes of horizontal gene transfer (HGT) in bacteria. The RM and CRISPR/Cas systems, both major genetic barriers to HGT, are also lineage specific. Clones harboring CRISPR/Cas or a prophage-disrupted comG exhibited less genetic diversity and lower rates of recombination than clones lacking these systems. After Listeria monocytogenes, this is the second example of prophage-mediated competence disruption reported in any bacteria. These findings are important for understanding the evolution and clonal expansion of MDR MRSP clones.IMPORTANCEStaphylococcus pseudintermedius is a bacterium responsible for clinically important infections in dogs and can infect humans. In this study, we performed genomic analysis of 371 S. pseudintermedius isolates to understand the evolution of antibiotic resistance and virulence in this organism. The analysis covered significant reported clones, including ST71 and ST68, the major epidemic clones of Europe and North America, respectively. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, and horizontal gene transfer differs among clones. ST71 and ST68 carry prophages with novel virulence and antibiotic resistance genes. Importantly, site-specific integration of a prophage, SpST71A, has led to the disruption of the genetic competence operon comG in ST71 clone. A functional comG is essential for the natural uptake of foreign DNA and thus plays an important role in the evolution of bacteria. This study provides insight into the emergence and evolution of antibiotic resistance and virulence in S. pseudintermedius, which may help in efforts to combat this pathogen.},
}

@article {pmid31738765,
year = {2019},
author = {Lutz, S and Brion, C and Kliebhan, M and Albert, FW},
title = {DNA variants affecting the expression of numerous genes in trans have diverse mechanisms of action and evolutionary histories.},
journal = {PLoS genetics},
volume = {15},
number = {11},
pages = {e1008375},
pmid = {31738765},
issn = {1553-7404},
support = {R35 GM124676/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA-Binding Proteins/*genetics ; *Evolution, Molecular ; Fatty Acids/genetics/metabolism ; Gene Expression Regulation, Fungal/genetics ; Green Fluorescent Proteins/genetics ; Lipid Metabolism/genetics ; Monosaccharide Transport Proteins/genetics ; Mutation, Missense/genetics ; Phenotype ; Regulatory Sequences, Nucleic Acid/*genetics ; Saccharomyces cerevisiae/genetics/metabolism ; Saccharomyces cerevisiae Proteins/*genetics ; Stearoyl-CoA Desaturase/*genetics ; Transcription Factors/*genetics ; },
abstract = {DNA variants that alter gene expression contribute to variation in many phenotypic traits. In particular, trans-acting variants, which are often located on different chromosomes from the genes they affect, are an important source of heritable gene expression variation. However, our knowledge about the identity and mechanism of causal trans-acting variants remains limited. Here, we developed a fine-mapping strategy called CRISPR-Swap and dissected three expression quantitative trait locus (eQTL) hotspots known to alter the expression of numerous genes in trans in the yeast Saccharomyces cerevisiae. Causal variants were identified by engineering recombinant alleles and quantifying the effects of these alleles on the expression of a green fluorescent protein-tagged gene affected by the given locus in trans. We validated the effect of each variant on the expression of multiple genes by RNA-sequencing. The three variants differed in their molecular mechanism, the type of genes they reside in, and their distribution in natural populations. While a missense leucine-to-serine variant at position 63 in the transcription factor Oaf1 (L63S) was almost exclusively present in the reference laboratory strain, the two other variants were frequent among S. cerevisiae isolates. A causal missense variant in the glucose receptor Rgt2 (V539I) occurred at a poorly conserved amino acid residue and its effect was strongly dependent on the concentration of glucose in the culture medium. A noncoding variant in the conserved fatty acid regulated (FAR) element of the OLE1 promoter influenced the expression of the fatty acid desaturase Ole1 in cis and, by modulating the level of this essential enzyme, other genes in trans. The OAF1 and OLE1 variants showed a non-additive genetic interaction, and affected cellular lipid metabolism. These results demonstrate that the molecular basis of trans-regulatory variation is diverse, highlighting the challenges in predicting which natural genetic variants affect gene expression.},
}

CRISPR-Cas Systems/genetics
DNA-Binding Proteins/*genetics
*Evolution, Molecular
Fatty Acids/genetics/metabolism
Gene Expression Regulation, Fungal/genetics
Green Fluorescent Proteins/genetics
Lipid Metabolism/genetics
Monosaccharide Transport Proteins/genetics
Mutation, Missense/genetics
Phenotype
Regulatory Sequences, Nucleic Acid/*genetics
Saccharomyces cerevisiae/genetics/metabolism
Saccharomyces cerevisiae Proteins/*genetics
Stearoyl-CoA Desaturase/*genetics
Transcription Factors/*genetics

@article {pmid31730647,
year = {2019},
author = {Snedeker, J and Gibbons, WJ and Paulding, DF and Abdelhamed, Z and Prows, DR and Stottmann, RW},
title = {Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants.},
journal = {PLoS genetics},
volume = {15},
number = {11},
pages = {e1008467},
pmid = {31730647},
issn = {1553-7404},
support = {R01 GM112744/GM/NIGMS NIH HHS/United States ; R35 GM131875/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptor Proteins, Signal Transducing/*genetics ; Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Chromosome Mapping ; Cilia/genetics ; Embryo, Mammalian ; Embryonic Development/*genetics ; Genotype ; Humans ; Mice ; Mice, Inbred C57BL ; Microcephaly/*genetics/physiopathology ; Prosencephalon/growth & development/metabolism ; Quantitative Trait Loci/*genetics ; Receptors, G-Protein-Coupled/*genetics ; Spina Bifida Cystica/genetics/physiopathology ; Synthetic Lethal Mutations/genetics ; },
abstract = {The primary cilium is a signaling center critical for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background and mutants on an FVB/NJ (FVB) background develop a forebrain significantly smaller than mutants on a C57BL/6J (B6) background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified an orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP that is polymorphic between the FVB and B6 strains in Gpr63 and creates a missense mutation predicted to be deleterious in the FVB protein. We used CRISPR-Cas9 genome editing to create two lines of FVB congenic mice: one with the B6 sequence of Gpr63 and the other with a deletion allele leading to a truncation of the GPR63 C-terminal tail. We then demonstrated that Gpr63 can localize to the cilium in vitro. These alleles affect ciliary localization of GPR63 in vitro and genetically interact with Ttc21baln/aln as Gpr63;Ttc21b double mutants show unique phenotypes including spina bifida aperta and earlier embryonic lethality. This validated Gpr63 as a modifier of multiple Ttc21b neural phenotypes and strongly supports Gpr63 as a causal gene (i.e., a quantitative trait gene, QTG) within the Moaq1 QTL.},
}

Adaptor Proteins, Signal Transducing/*genetics
Alleles
Animals
CRISPR-Cas Systems/genetics
Chromosome Mapping
Cilia/genetics
Embryo, Mammalian
Embryonic Development/*genetics
Genotype
Humans
Mice
Mice, Inbred C57BL
Microcephaly/*genetics/physiopathology
Prosencephalon/growth & development/metabolism
Quantitative Trait Loci/*genetics
Receptors, G-Protein-Coupled/*genetics
Spina Bifida Cystica/genetics/physiopathology
Synthetic Lethal Mutations/genetics

@article {pmid31695185,
year = {2019},
author = {Gorelik, A and Bartual, SG and Borodkin, VS and Varghese, J and Ferenbach, AT and van Aalten, DMF},
title = {Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation.},
journal = {Nature structural & molecular biology},
volume = {26},
number = {11},
pages = {1071-1077},
pmid = {31695185},
issn = {1545-9985},
support = {/WT_/Wellcome Trust/United Kingdom ; 105310/WT_/Wellcome Trust/United Kingdom ; 110061/WT_/Wellcome Trust/United Kingdom ; 200208/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Acetylglucosamine/analogs & derivatives/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Glycosylation ; HEK293 Cells ; Humans ; Mice ; Models, Molecular ; N-Acetylglucosaminyltransferases/chemistry/*metabolism ; Protein Processing, Post-Translational ; Substrate Specificity ; beta-N-Acetylhexosaminidases/chemistry/genetics/*metabolism ; },
abstract = {Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GlcNAc, catalyzed by O-GlcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GlcNAcase (OGA), precise functional dissection of site-specific O-GlcNAc modification in vivo is currently not possible without affecting the entire O-GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GlcNAc is a hydrolytically stable and accurate structural mimic of O-GlcNAc that can be encoded in mammalian systems with CRISPR-Cas9 in an otherwise unperturbed O-GlcNAcome. Using this approach, we target an elusive Ser 405 O-GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.},
}

Acetylglucosamine/analogs & derivatives/genetics/*metabolism
Animals
CRISPR-Cas Systems
Glycosylation
HEK293 Cells
Humans
Mice
Models, Molecular
N-Acetylglucosaminyltransferases/chemistry/*metabolism
Protein Processing, Post-Translational
Substrate Specificity
beta-N-Acetylhexosaminidases/chemistry/genetics/*metabolism

@article {pmid31694550,
year = {2019},
author = {Cram, D and Kulkarni, M and Buchwaldt, M and Rajagopalan, N and Bhowmik, P and Rozwadowski, K and Parkin, IAP and Sharpe, AG and Kagale, S},
title = {WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {474},
pmid = {31694550},
issn = {1471-2229},
mesh = {*CRISPR-Cas Systems ; Computational Biology/methods ; Databases, Genetic ; Gene Editing/*methods ; Gene Targeting ; Genome, Plant ; Internet ; *RNA, Guide ; Triticum/*genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement.

DESCRIPTION: We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184-191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR.

CONCLUSION: The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/ .},
}

*CRISPR-Cas Systems
Computational Biology/methods
Databases, Genetic
Gene Editing/*methods
Gene Targeting
Genome, Plant
Internet
*RNA, Guide
Triticum/*genetics

@article {pmid31668930,
year = {2019},
author = {Sun, W and Yang, J and Cheng, Z and Amrani, N and Liu, C and Wang, K and Ibraheim, R and Edraki, A and Huang, X and Wang, M and Wang, J and Liu, L and Sheng, G and Yang, Y and Lou, J and Sontheimer, EJ and Wang, Y},
title = {Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States.},
journal = {Molecular cell},
volume = {76},
number = {6},
pages = {938-952.e5},
doi = {10.1016/j.molcel.2019.09.025},
pmid = {31668930},
issn = {1097-4164},
support = {F31 DK120333/DK/NIDDK NIH HHS/United States ; F31 NS110328/NS/NINDS NIH HHS/United States ; R01 GM125797/GM/NIGMS NIH HHS/United States ; UG3 TR002668/TR/NCATS NIH HHS/United States ; },
mesh = {Bacteriophages/genetics/*metabolism ; Binding Sites ; CRISPR-Associated Protein 9/genetics/*metabolism/ultrastructure ; *CRISPR-Cas Systems ; Catalysis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/*metabolism/ultrastructure ; Escherichia coli/enzymology/genetics ; Neisseria meningitidis/*enzymology/genetics ; Protein Binding ; Protein Domains ; RNA, Guide/genetics/metabolism ; Structure-Activity Relationship ; Viral Proteins/genetics/*metabolism/ultrastructure ; },
abstract = {High-resolution Cas9 structures have yet to reveal catalytic conformations due to HNH nuclease domain positioning away from the cleavage site. Nme1Cas9 and Nme2Cas9 are compact nucleases for in vivo genome editing. Here, we report structures of meningococcal Cas9 homologs in complex with sgRNA, dsDNA, or the AcrIIC3 anti-CRISPR protein. DNA-bound structures represent an early step of target recognition, a later HNH pre-catalytic state, the HNH catalytic state, and a cleaved-target-DNA-bound state. In the HNH catalytic state of Nme1Cas9, the active site is seen poised at the scissile phosphodiester linkage of the target strand, providing a high-resolution view of the active conformation. The HNH active conformation activates the RuvC domain. Our structures explain how Nme1Cas9 and Nme2Cas9 read distinct PAM sequences and how AcrIIC3 inhibits Nme1Cas9 activity. These structures provide insights into Cas9 domain rearrangements, guide-target engagement, cleavage mechanism, and anti-CRISPR inhibition, facilitating the optimization of these genome-editing platforms.},
}

Bacteriophages/genetics/*metabolism
Binding Sites
CRISPR-Associated Protein 9/genetics/*metabolism/ultrastructure
*CRISPR-Cas Systems
Catalysis
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA/genetics/*metabolism/ultrastructure
Escherichia coli/enzymology/genetics
Neisseria meningitidis/*enzymology/genetics
Protein Binding
Protein Domains
RNA, Guide/genetics/metabolism
Structure-Activity Relationship
Viral Proteins/genetics/*metabolism/ultrastructure

@article {pmid31607545,
year = {2019},
author = {Freije, CA and Myhrvold, C and Boehm, CK and Lin, AE and Welch, NL and Carter, A and Metsky, HC and Luo, CY and Abudayyeh, OO and Gootenberg, JS and Yozwiak, NL and Zhang, F and Sabeti, PC},
title = {Programmable Inhibition and Detection of RNA Viruses Using Cas13.},
journal = {Molecular cell},
volume = {76},
number = {5},
pages = {826-837.e11},
doi = {10.1016/j.molcel.2019.09.013},
pmid = {31607545},
issn = {1097-4164},
mesh = {A549 Cells ; Animals ; CRISPR-Associated Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Chlorocebus aethiops ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Dogs ; Escherichia coli/enzymology/genetics ; Gene Targeting/*methods ; HEK293 Cells ; Humans ; Madin Darby Canine Kidney Cells ; *RNA Stability ; RNA Viruses/*enzymology/genetics ; RNA, Viral/genetics/*metabolism ; Vero Cells ; },
abstract = {The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development.},
}

A549 Cells
Animals
CRISPR-Associated Proteins/genetics/*metabolism
*CRISPR-Cas Systems
Chlorocebus aethiops
*Clustered Regularly Interspaced Short Palindromic Repeats
Dogs
Escherichia coli/enzymology/genetics
Gene Targeting/*methods
HEK293 Cells
Humans
Madin Darby Canine Kidney Cells
*RNA Stability
RNA Viruses/*enzymology/genetics
RNA, Viral/genetics/*metabolism
Vero Cells

@article {pmid31285607,
year = {2019},
author = {Zhu, X and Clarke, R and Puppala, AK and Chittori, S and Merk, A and Merrill, BJ and Simonović, M and Subramaniam, S},
title = {Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.},
journal = {Nature structural & molecular biology},
volume = {26},
number = {8},
pages = {679-685},
doi = {10.1038/s41594-019-0258-2},
pmid = {31285607},
issn = {1545-9985},
support = {R01 GM097042/GM/NIGMS NIH HHS/United States ; R01 HD081534/HD/NICHD NIH HHS/United States ; ZIA BC010826-12/ImNIH/Intramural NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/chemistry/metabolism/*ultrastructure ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/*metabolism/ultrastructure ; Macromolecular Substances/ultrastructure ; Models, Molecular ; Motion ; Protein Conformation ; Protein Domains ; RNA Editing ; RNA, Guide/metabolism ; Streptococcus pyogenes/enzymology ; },
abstract = {The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg2+. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9.},
}

CRISPR-Associated Protein 9/chemistry/metabolism/*ultrastructure
*CRISPR-Cas Systems
Cryoelectron Microscopy
DNA/*metabolism/ultrastructure
Macromolecular Substances/ultrastructure
Models, Molecular
Motion
Protein Conformation
Protein Domains
RNA Editing
RNA, Guide/metabolism
Streptococcus pyogenes/enzymology

@article {pmid31285603,
year = {2019},
author = {Taylor, DW},
title = {The final cut: Cas9 editing.},
journal = {Nature structural & molecular biology},
volume = {26},
number = {8},
pages = {669-670},
doi = {10.1038/s41594-019-0267-1},
pmid = {31285603},
issn = {1545-9985},
mesh = {CRISPR-Associated Protein 9/chemistry/metabolism/*ultrastructure ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; DNA/metabolism/ultrastructure ; Gene Editing/methods ; Macromolecular Substances/ultrastructure ; Models, Molecular ; Protein Conformation ; *RNA Editing ; Streptococcus pyogenes/enzymology ; },
}

CRISPR-Associated Protein 9/chemistry/metabolism/*ultrastructure
*CRISPR-Cas Systems
Cryoelectron Microscopy
DNA/metabolism/ultrastructure
Gene Editing/methods
Macromolecular Substances/ultrastructure
Models, Molecular
Protein Conformation
*RNA Editing
Streptococcus pyogenes/enzymology

@article {pmid31246169,
year = {2019},
author = {Tracey, WD},
title = {The taste of water.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
doi = {10.7554/eLife.48654},
pmid = {31246169},
issn = {2050-084X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Culicidae/genetics/*physiology ; *Eggs ; Epithelial Sodium Channels/*genetics ; Female ; Fresh Water/chemistry ; Genome, Insect/genetics ; Neurons/metabolism ; Taste/*genetics/physiology ; },
abstract = {Female mosquitos require a specific ion-channel protein to sense the presence of fresh water in which they can lay their eggs.},
}

Animals
CRISPR-Cas Systems/genetics
Culicidae/genetics/*physiology
*Eggs
Epithelial Sodium Channels/*genetics
Female
Fresh Water/chemistry
Genome, Insect/genetics
Neurons/metabolism
Taste/*genetics/physiology

@article {pmid31004744,
year = {2019},
author = {Couch, T and Murphy, Z and Getman, M and Kurita, R and Nakamura, Y and Steiner, LA},
title = {Human erythroblasts with c-Kit activating mutations have reduced cell culture costs and remain capable of terminal maturation.},
journal = {Experimental hematology},
volume = {74},
number = {},
pages = {19-24.e4},
doi = {10.1016/j.exphem.2019.04.001},
pmid = {31004744},
issn = {1873-2399},
mesh = {CRISPR-Cas Systems ; *Cell Culture Techniques/economics/methods ; *Cell Differentiation ; Cell Line, Transformed ; Erythroblasts/*enzymology ; Gene Editing ; Humans ; K562 Cells ; *Mutation ; *Proto-Oncogene Proteins c-kit/genetics/metabolism ; },
abstract = {A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.},
}

CRISPR-Cas Systems
*Cell Culture Techniques/economics/methods
*Cell Differentiation
Cell Line, Transformed
Erythroblasts/*enzymology
Gene Editing
Humans
K562 Cells
*Mutation
*Proto-Oncogene Proteins c-kit/genetics/metabolism

@article {pmid30840598,
year = {2019},
author = {Schmich, F and Kuipers, J and Merdes, G and Beerenwinkel, N},
title = {netprioR: a probabilistic model for integrative hit prioritisation of genetic screens.},
journal = {Statistical applications in genetics and molecular biology},
volume = {18},
number = {3},
pages = {},
doi = {10.1515/sagmb-2018-0033},
pmid = {30840598},
issn = {1544-6115},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Computational Biology/*statistics & numerical data ; Drosophila melanogaster/genetics ; Genetic Testing/*statistics & numerical data ; *Models, Statistical ; Phenotype ; Receptors, Notch/genetics ; },
abstract = {In the post-genomic era of big data in biology, computational approaches to integrate multiple heterogeneous data sets become increasingly important. Despite the availability of large amounts of omics data, the prioritisation of genes relevant for a specific functional pathway based on genetic screening experiments, remains a challenging task. Here, we introduce netprioR, a probabilistic generative model for semi-supervised integrative prioritisation of hit genes. The model integrates multiple network data sets representing gene-gene similarities and prior knowledge about gene functions from the literature with gene-based covariates, such as phenotypes measured in genetic perturbation screens, for example, by RNA interference or CRISPR/Cas9. We evaluate netprioR on simulated data and show that the model outperforms current state-of-the-art methods in many scenarios and is on par otherwise. In an application to real biological data, we integrate 22 network data sets, 1784 prior knowledge class labels and 3840 RNA interference phenotypes in order to prioritise novel regulators of Notch signalling in Drosophila melanogaster. The biological relevance of our predictions is evaluated using in silico and in vivo experiments. An efficient implementation of netprioR is available as an R package at http://bioconductor.org/packages/netprioR.},
}

Animals
CRISPR-Cas Systems/genetics
Computational Biology/*statistics & numerical data
Drosophila melanogaster/genetics
Genetic Testing/*statistics & numerical data
*Models, Statistical
Phenotype
Receptors, Notch/genetics

@article {pmid32061705,
year = {2020},
author = {Lee, J and Bayarsaikhan, D and Bayarsaikhan, G and Kim, JS and Schwarzbach, E and Lee, B},
title = {Recent advances in genome editing of stem cells for drug discovery and therapeutic application.},
journal = {Pharmacology & therapeutics},
volume = {},
number = {},
pages = {107501},
doi = {10.1016/j.pharmthera.2020.107501},
pmid = {32061705},
issn = {1879-016X},
abstract = {Genome engineering technologies right from viral vector-mediated to protein-based editing- which include zinc finger nucleases, TALENs, and CRISPR/Cas systems-have been improved significantly. These technologies have facilitated drug discovery and have resulted in the development of potential curative therapies for many intractable diseases. They can efficiently correct genetic errors; however, these technologies have limitations, such as off-target effects and possible safety issues, which need to be considered when employing these techniques in humans. Significant efforts have been made to overcome these limitations and to accelerate the clinical implementation of these technologies. In this review, we focus on the recent technological advancements in genome engineering and their applications in stem cells to enable efficient discovery of drugs and treatment of intractable diseases.},
}

@article {pmid32016291,
year = {2020},
author = {Hampton, T},
title = {DNA Prime Editing: A New CRISPR-Based Method to Correct Most Disease-Causing Mutations.},
journal = {JAMA},
volume = {323},
number = {5},
pages = {405-406},
doi = {10.1001/jama.2019.21827},
pmid = {32016291},
issn = {1538-3598},
mesh = {*CRISPR-Cas Systems ; *DNA ; Gene Editing/*methods ; Humans ; *Mutation ; },
}

*CRISPR-Cas Systems
*DNA
Gene Editing/*methods
Humans
*Mutation

@article {pmid31104397,
year = {2019},
author = {Narimani, M and Sharifi, M and Hakhamaneshi, MS and Roshani, D and Kazemi, M and Hejazi, SH and Jalili, A},
title = {BIRC5 Gene Disruption via CRISPR/Cas9n Platform Suppress Acute Myelocytic Leukemia Progression.},
journal = {Iranian biomedical journal},
volume = {23},
number = {6},
pages = {369-378},
pmid = {31104397},
issn = {2008-823X},
mesh = {Apoptosis ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; *Disease Progression ; *Gene Deletion ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Myeloid, Acute/*genetics/*pathology ; RNA, Guide/metabolism ; Survivin/*genetics ; },
abstract = {Background: Acute myelocytic leukemia (AML) is a clonal malignancy resulting from the accumulation of genetic abnormalities in the cells. Human baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), encodes survivin, is one of only a handful of genes that is differentially over-expressed in numerous malignant diseases including AML.

Methods: The BIRC5 was silenced permanently in two AML cell lines, HL‑60 and KG-1, via the CRISPR/Cas9n system. After transfection of CRISPR constructs, genomic DNA was extracted and amplified to assess mutation detection. To evaluate BIRC5 gene expression, quantitative real-time PCR was performed. Also, MTT cell viability and Annexin‑V/propidium iodide flowcytometric staining were performed, and the data were analyzed using the Kolmogorov-Smirnov, Levene's, and ANOVA tests.

Results: The results indicated that Cas9n and its sgRNAs successfully triggered site-specific cleavage and mutation in the BIRC5 gene locus. Moreover, suppression of BIRC5 resulted in the reduction of cell viability, and induction of apoptosis and necrosis in HL60 and KG1 suggested that the permanent suppression of BIRC5 remarkably dropped the gene expression and cells viability.

Conclusion: This study reinforces the idea that BIRC5 disruption via Cas9n:sgRNAs has favorable effects on the AML clinical outcome. It thereby can be a promising candidate in a variety of leukemia treatments.},
}

Apoptosis
Base Sequence
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Cell Line, Tumor
Cell Proliferation
Cell Survival
*Disease Progression
*Gene Deletion
Gene Expression Regulation, Leukemic
Humans
Leukemia, Myeloid, Acute/*genetics/*pathology
RNA, Guide/metabolism
Survivin/*genetics

@article {pmid30722048,
year = {2019},
author = {Botella, JR},
title = {Now for the hard ones: is there a limit on CRISPR genome editing in crops?.},
journal = {Journal of experimental botany},
volume = {70},
number = {3},
pages = {734-737},
doi = {10.1093/jxb/erz007},
pmid = {30722048},
issn = {1460-2431},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Fragaria ; *Gene Editing ; Mutagenesis ; },
}

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Fragaria
*Gene Editing
Mutagenesis

@article {pmid32058568,
year = {2020},
author = {Wu, M and Hu, N and Du, X and Wei, J},
title = {Application of CRISPR/Cas9 technology in sepsis research.},
journal = {Briefings in functional genomics},
volume = {},
number = {},
pages = {},
doi = {10.1093/bfgp/elz040},
pmid = {32058568},
issn = {2041-2657},
abstract = {CRISPR/Cas9, as a new genome-editing tool, offers new approaches to understand and treat diseases, which is being rapidly applied in various areas of biomedical research including sepsis field. The type II prokaryotic CRISPR/Cas system uses a single-guide RNA (sgRNA) to target the Cas9 nuclease to a specific genomic sequence, which is introduced into disease models for functional characterization and for testing of therapeutic strategies. This incredibly precise technology can be used for therapeutic research of gene-related diseases and to program any sequence in a target cell. Most importantly, the multifunctional capacity of this technology allows simultaneous editing of several genes. In this review, we focus on the basic principles, advantages and limitations of CRISPR/Cas9 and the use of the CRISPR/Cas9 system as a powerful tool in sepsis research and as a new strategy for the treatment of sepsis.},
}

@article {pmid32056963,
year = {2020},
author = {He, Q and Yu, D and Bao, M and Korensky, G and Chen, J and Shin, M and Kim, J and Park, M and Qin, P and Du, K},
title = {High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system.},
journal = {Biosensors & bioelectronics},
volume = {154},
number = {},
pages = {112068},
doi = {10.1016/j.bios.2020.112068},
pmid = {32056963},
issn = {1873-4235},
abstract = {Here we report the development of a high throughput, all-solution phase, and isothermal detection system for African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with a fluorescence-based point-of-care (POC) system for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 h. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 h of incubation, resulting in an improved detection limit of 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact and simple ASFV detection, intended for low resource settings.},
}

@article {pmid32054870,
year = {2020},
author = {Hirotsune, S and Kiyonari, H and Jin, M and Kumamoto, K and Yoshida, K and Shinohara, M and Watanabe, H and Wynshaw-Boris, A and Matsuzaki, F},
title = {Enhanced homologous recombination by the modulation of targeting vector ends.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {2518},
doi = {10.1038/s41598-020-58893-9},
pmid = {32054870},
issn = {2045-2322},
abstract = {The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.},
}

@article {pmid32052827,
year = {2020},
author = {Rossi, CC and Pereira, MF and Giambiagi-deMarval, M},
title = {Underrated Staphylococcus species and their role in antimicrobial resistance spreading.},
journal = {Genetics and molecular biology},
volume = {43},
number = {1 suppl 2},
pages = {e20190065},
doi = {10.1590/1678-4685-GMB-2019-0065},
pmid = {32052827},
issn = {1415-4757},
abstract = {The increasing threat of antimicrobial resistance has shed light on the interconnection between humans, animals, the environment, and their roles in the exchange and spreading of resistance genes. In this review, we present evidences that show that Staphylococcus species, usually referred to as harmless or opportunistic pathogens, represent a threat to human and animal health for acting as reservoirs of antimicrobial resistance genes. The capacity of genetic exchange between isolates of different sources and species of the Staphylococcus genus is discussed with emphasis on mobile genetic elements, the contribution of biofilm formation, and evidences obtained either experimentally or through genome analyses. We also discuss the involvement of CRISPR-Cas systems in the limitation of horizontal gene transfer and its suitability as a molecular clock to describe the history of genetic exchange between staphylococci.},
}

@article {pmid32052006,
year = {2020},
author = {Chen, X and Fan, S and Wen, C and Du, X},
title = {CRISPR/Cas9 for cancer treatment: technology, clinical applications and challenges.},
journal = {Briefings in functional genomics},
volume = {},
number = {},
pages = {},
doi = {10.1093/bfgp/elaa001},
pmid = {32052006},
issn = {2041-2657},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) is described as RNA mediated adaptive immune system defense, which is naturally found in bacteria and archaea. CRISPR-Cas9 has shown great promise for cancer treatment in cancer immunotherapy, manipulation of cancer genome and epigenome and elimination or inactivation of carcinogenic viral infections. However, many challenges remain to be addressed to increase its efficacy, including off-target effects, editing efficiency, fitness of edited cells, immune response and delivery methods. Here, we explain CRISPR-Cas classification and its general function mechanism for gene editing. Then, we summarize these preclinical CRISPR-Cas9-based therapeutic strategies against cancer. Moreover, the challenges and improvements of CRISPR-Cas9 clinical applications will be discussed.},
}

@article {pmid32051592,
year = {2020},
author = {Al-Shayeb, B and Sachdeva, R and Chen, LX and Ward, F and Munk, P and Devoto, A and Castelle, CJ and Olm, MR and Bouma-Gregson, K and Amano, Y and He, C and Méheust, R and Brooks, B and Thomas, A and Lavy, A and Matheus-Carnevali, P and Sun, C and Goltsman, DSA and Borton, MA and Sharrar, A and Jaffe, AL and Nelson, TC and Kantor, R and Keren, R and Lane, KR and Farag, IF and Lei, S and Finstad, K and Amundson, R and Anantharaman, K and Zhou, J and Probst, AJ and Power, ME and Tringe, SG and Li, WJ and Wrighton, K and Harrison, S and Morowitz, M and Relman, DA and Doudna, JA and Lehours, AC and Warren, L and Cate, JHD and Santini, JM and Banfield, JF},
title = {Clades of huge phages from across Earth's ecosystems.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-020-2007-4},
pmid = {32051592},
issn = {1476-4687},
abstract = {Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.},
}

@article {pmid32051236,
year = {2020},
author = {Spoto, M and Guan, C and Fleming, E and Oh, J},
title = {A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes.},
journal = {mSphere},
volume = {5},
number = {1},
pages = {},
doi = {10.1128/mSphere.00086-20},
pmid = {32051236},
issn = {2379-5042},
abstract = {The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful-focusing on CRISPRi as a potential application-as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species.IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.},
}

@article {pmid32050613,
year = {2020},
author = {Grigonyte, AM and Harrison, C and MacDonald, PR and Montero-Blay, A and Tridgett, M and Duncan, J and Sagona, AP and Constantinidou, C and Jaramillo, A and Millard, A},
title = {Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7.},
journal = {Viruses},
volume = {12},
number = {2},
pages = {},
doi = {10.3390/v12020193},
pmid = {32050613},
issn = {1999-4915},
support = {NE/N019881/1//Natural Environment Research Council/ ; MR/L015080/1/MRC_/Medical Research Council/United Kingdom ; },
abstract = {With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.},
}

@article {pmid32046217,
year = {2020},
author = {Burmistrz, M and Krakowski, K and Krawczyk-Balska, A},
title = {RNA-Targeting CRISPR-Cas Systems and Their Applications.},
journal = {International journal of molecular sciences},
volume = {21},
number = {3},
pages = {},
doi = {10.3390/ijms21031122},
pmid = {32046217},
issn = {1422-0067},
support = {2016/21/B/NZ6/00963//National Center of Science, Poland/ ; intramural grant DSM no. 501-D114-01-1140400//Ministry of Science and Higher Education through the Faculty of Biology, University of Warsaw/ ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems have revolutionized modern molecular biology. Numerous types of these systems have been discovered to date. Many CRISPR-Cas systems have been used as a backbone for the development of potent research tools, with Cas9 being the most widespread. While most of the utilized systems are DNA-targeting, recently more and more attention is being gained by those that target RNA. Their ability to specifically recognize a given RNA sequence in an easily programmable way makes them ideal candidates for developing new research tools. In this review we summarize current knowledge on CRISPR-Cas systems which have been shown to target RNA molecules, that is type III (Csm/Cmr), type VI (Cas13), and type II (Cas9). We also present a list of available technologies based on these systems.},
}

@article {pmid32044725,
year = {2020},
author = {Aquino-Jarquin, G},
title = {Novel Engineered Programmable Systems for ADAR-Mediated RNA Editing.},
journal = {Molecular therapy. Nucleic acids},
volume = {19},
number = {},
pages = {1065-1072},
doi = {10.1016/j.omtn.2019.12.042},
pmid = {32044725},
issn = {2162-2531},
abstract = {One of the most prevalent forms of post-transcriptional RNA modification is the conversion of adenosine-to-inosine (A-to-I), mediated by adenosine deaminase acting on RNA (ADAR) enzymes. The advent of the CRISPR/Cas systems inspires researchers to work actively in the engineering of programmable RNA-guided machines for basic research and biomedical applications. In this regard, CIRTS (CRISPR-Cas-Inspired RNA Targeting System), RESCUE (RNA Editing for Specific C to U Exchange), RESTORE (Recruiting Endogenous ADAR to Specific Transcripts for Oligonucleotide-mediated RNA Editing), and LEAPER (Leveraging Endogenous ADAR for Programmable Editing of RNA) are innovative RNA base-editing platforms that have recently been engineered to perform programmable base conversions on target RNAs mediated by ADAR enzymes in mammalian cells. Thus, these four currently characterized RNA-editing systems constitute novel molecular tools with compelling programmability, specificity, and efficiency that show us some creative ways to take advantage of the engineered deaminases for precise base editing. Moreover, the advanced engineering of these systems permits editing of full-length transcripts containing disease-causing point mutations without the loss of genomic information, providing an attractive alternative for in vivo research and in the therapeutic setting if the challenges encountered in off-target edits and delivery are appropriately addressed. Here, I present an analytical approach of the current status and rapid progress of the novel ADAR-mediated RNA-editing systems when highlighting the qualities of each new RNA-editing platform and how these RNA-targeting strategies could be used to recruit human ADARs on endogenous transcripts, not only for our understanding of RNA-modification-mediated regulation of gene expression but also for editing clinically relevant mutations in a programmable and straightforward manner.},
}

@article {pmid32038537,
year = {2019},
author = {Wimmer, F and Beisel, CL},
title = {CRISPR-Cas Systems and the Paradox of Self-Targeting Spacers.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {3078},
pmid = {32038537},
issn = {1664-302X},
abstract = {CRISPR-Cas immune systems in bacteria and archaea record prior infections as spacers within each system's CRISPR arrays. Spacers are normally derived from invasive genetic material and direct the immune system to complementary targets as part of future infections. However, not all spacers appear to be derived from foreign genetic material and instead can originate from the host genome. Their presence poses a paradox, as self-targeting spacers would be expected to induce an autoimmune response and cell death. In this review, we discuss the known frequency of self-targeting spacers in natural CRISPR-Cas systems, how these spacers can be incorporated into CRISPR arrays, and how the host can evade lethal attack. We also discuss how self-targeting spacers can become the basis for alternative functions performed by CRISPR-Cas systems that extend beyond adaptive immunity. Overall, the acquisition of genome-targeting spacers poses a substantial risk but can aid in the host's evolution and potentially lead to or support new functionalities.},
}

@article {pmid32037739,
year = {2020},
author = {Wang, J and Zhang, C and Feng, B},
title = {The rapidly advancing Class 2 CRISPR-Cas technologies: A customizable toolbox for molecular manipulations.},
journal = {Journal of cellular and molecular medicine},
volume = {},
number = {},
pages = {},
doi = {10.1111/jcmm.15039},
pmid = {32037739},
issn = {1582-4934},
support = {31771635//National Natural Science Foundation of China/ ; 14119518//Research Grants Council, University Grants Committee/ ; T13-605/18-W//Research Grants Council, University Grants Committee/ ; T13-402/17-N//Research Grants Council, University Grants Committee/ ; CTFHK 2018/02//Children's Thalassaemia Foundation in Hong Kong/ ; },
abstract = {The CRISPR-Cas technologies derived from bacterial and archaeal adaptive immune systems have emerged as a series of groundbreaking nucleic acid-guided gene editing tools, ultimately standing out among several engineered nucleases because of their high efficiency, sequence-specific targeting, ease of programming and versatility. Facilitated by the advancement across multiple disciplines such as bioinformatics, structural biology and high-throughput sequencing, the discoveries and engineering of various innovative CRISPR-Cas systems are rapidly expanding the CRISPR toolbox. This is revolutionizing not only genome editing but also various other types of nucleic acid-guided manipulations such as transcriptional control and genomic imaging. Meanwhile, the adaptation of various CRISPR strategies in multiple settings has realized numerous previously non-existing applications, ranging from the introduction of sophisticated approaches in basic research to impactful agricultural and therapeutic applications. Here, we summarize the recent advances of CRISPR technologies and strategies, as well as their impactful applications.},
}