@article {pmid31304778,
year = {2019},
author = {Krimsky, S},
title = {Breaking the Germline Barrier in a Moral Vacuum.},
journal = {Accountability in research},
volume = {},
number = {},
pages = {},
doi = {10.1080/08989621.2019.1644171},
pmid = {31304778},
issn = {1545-5815},
abstract = {In November 2018 a Chinese scientist claimed to have used CRISPR/Cas 9 technology to genetically modify two human embryos that were then gestated in one adult woman through an IVF pregnancy and brought to term. The twin girls are allegedly the first babies born with their prenatal genomes edited. Using both English language and Chinese supporting documents, this paper discusses the background of this human experiment, the social context of Chinese science, and the alleged ethical transgressions of its principal scientist.},
}

@article {pmid31303969,
year = {2019},
author = {Chen, T and Olsen, I},
title = {Porphyromonas gingivalis and its CRISPR-Cas system.},
journal = {Journal of oral microbiology},
volume = {11},
number = {1},
pages = {1638196},
doi = {10.1080/20002297.2019.1638196},
pmid = {31303969},
issn = {2000-2297},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated proteins (Cas) are immune systems in prokaryotes present in most Bacteria and Archaea. They provide adaptive immunity against foreign elements such as bacteriophages/viruses, plasmids and transposons. During immunization a small sequence of foreign DNA, a so-called spacer is integrated into the CRISPR locus in the host cell. Spacers are then transcribed into small RNA guides that direct cleavage of foreign DNA by Cas nucleases. Immunization through spacer acquisition is transferred vertically to the progeny. It is possible that this genetic immune system of bacteria participates in modulating the microbiome of 'chronic' periodontitis, in which Porphyromonas gingivalis has been identified as a keystone pathogen causing microbial dysbiosis. An in-depth review of our current knowledge on the CRISPR-Cas systems in P. gingivalis is given in this paper with the attempt to understand how this anaerobic bacterium may protect itself in the periodontal pocket where bacteriophages are abundant and even out-number bacteria.},
}

@article {pmid31086349,
year = {2019},
author = {Azvolinsky, A},
title = {Molecular scissors cut in on stem cells.},
journal = {Nature medicine},
volume = {25},
number = {6},
pages = {864-866},
doi = {10.1038/s41591-019-0467-6},
pmid = {31086349},
issn = {1546-170X},
mesh = {Anemia, Sickle Cell/genetics/therapy ; CRISPR-Cas Systems ; Carrier Proteins/genetics ; Clinical Trials as Topic ; Gene Editing/*methods ; Humans ; Nuclear Proteins/genetics ; Stem Cell Transplantation ; Stem Cells/*metabolism ; beta-Globins/genetics ; beta-Thalassemia/genetics/therapy ; },
}

Anemia, Sickle Cell/genetics/therapy
CRISPR-Cas Systems
Carrier Proteins/genetics
Clinical Trials as Topic
Gene Editing/*methods
Humans
Nuclear Proteins/genetics
Stem Cell Transplantation
Stem Cells/*metabolism
beta-Globins/genetics
beta-Thalassemia/genetics/therapy

@article {pmid30872121,
year = {2019},
author = {Rollins, MF and Chowdhury, S and Carter, J and Golden, SM and Miettinen, HM and Santiago-Frangos, A and Faith, D and Lawrence, CM and Lander, GC and Wiedenheft, B},
title = {Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry.},
journal = {Molecular cell},
volume = {74},
number = {1},
pages = {132-142.e5},
doi = {10.1016/j.molcel.2019.02.001},
pmid = {30872121},
issn = {1097-4164},
support = {R21 AI130670/AI/NIAID NIH HHS/United States ; R01 GM110270/GM/NIGMS NIH HHS/United States ; DP2 EB020402/EB/NIBIB NIH HHS/United States ; S10 OD021634/OD/NIH HHS/United States ; P20 GM103500/GM/NIGMS NIH HHS/United States ; R01 GM108888/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Proteins/chemistry/genetics/immunology/*metabolism ; CRISPR-Associated Proteins/chemistry/genetics/immunology/*metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cryoelectron Microscopy ; DNA, Bacterial/chemistry/genetics/*metabolism ; Models, Molecular ; *Molecular Mimicry ; Nucleic Acid Conformation ; Protein Conformation ; Pseudomonas aeruginosa/*enzymology/genetics/immunology ; RNA, Bacterial/chemistry/genetics/*metabolism ; RNA, Guide/chemistry/genetics/*metabolism ; Structure-Activity Relationship ; Viral Proteins/chemistry/genetics/immunology/*metabolism ; },
abstract = {Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.},
}

Bacterial Proteins/chemistry/genetics/immunology/*metabolism
CRISPR-Associated Proteins/chemistry/genetics/immunology/*metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Cryoelectron Microscopy
DNA, Bacterial/chemistry/genetics/*metabolism
Models, Molecular
*Molecular Mimicry
Nucleic Acid Conformation
Protein Conformation
Pseudomonas aeruginosa/*enzymology/genetics/immunology
RNA, Bacterial/chemistry/genetics/*metabolism
RNA, Guide/chemistry/genetics/*metabolism
Structure-Activity Relationship
Viral Proteins/chemistry/genetics/immunology/*metabolism

@article {pmid30716580,
year = {2019},
author = {King, MW and Munger, J},
title = {Editing the human cytomegalovirus genome with the CRISPR/Cas9 system.},
journal = {Virology},
volume = {529},
number = {},
pages = {186-194},
doi = {10.1016/j.virol.2019.01.021},
pmid = {30716580},
issn = {1096-0341},
support = {R01 AI081773/AI/NIAID NIH HHS/United States ; R01 AI127370/AI/NIAID NIH HHS/United States ; R25 GM064133/GM/NIGMS NIH HHS/United States ; R56 AI081773/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cytomegalovirus/*genetics ; Genetic Engineering/*methods ; Genome, Viral/*genetics ; Humans ; },
abstract = {Human Cytomegalovirus (HCMV) is an opportunistic pathogen that causes substantial disease in neonates and immunocompromised individuals. Reverse genetic analysis of the HCMV genome is a powerful tool to dissect the roles that various viral genes play during infection. However, genetic engineering of HCMV is hampered by both the large size of the HCMV genome and HCMV's slow replication cycle. Currently, most laboratories that genetically engineer HCMV employ Bacterial Artificial Chromosome (BAC) mediated recombineering, which is a relatively lengthy process. We explored an alternative method of producing recombinant HCMV using the CRISPR/Cas9 system. We employed both homologous recombination (HR) and Non-homologous end-joining (NHEJ)-based methods, and find that each approach is capable of efficiently mutating the HCMV genome, with optimal efficiencies of 42% and 81% respectively. Our results suggest that CRISPR-mediated genomic engineering of HCMV is competitive with BAC-mediated recombineering and provide a framework for using CRISPR/Cas9 for mutational analysis of the HCMV genome.},
}

*CRISPR-Cas Systems
Cytomegalovirus/*genetics
Genetic Engineering/*methods
Genome, Viral/*genetics
Humans

@article {pmid29954751,
year = {2018},
author = {Patil, A and Manzano, M and Gottwein, E},
title = {CK1α and IRF4 are essential and independent effectors of immunomodulatory drugs in primary effusion lymphoma.},
journal = {Blood},
volume = {132},
number = {6},
pages = {577-586},
pmid = {29954751},
issn = {1528-0020},
support = {R21 CA210904/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Casein Kinase Ialpha/genetics/*physiology ; Cell Line, Tumor ; Down-Regulation/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; Humans ; Ikaros Transcription Factor/physiology ; Immunologic Factors/*pharmacology/therapeutic use ; Interferon Regulatory Factors/biosynthesis/genetics/*physiology ; Lenalidomide/*pharmacology/therapeutic use ; Lymphoma, Primary Effusion/*drug therapy/genetics/metabolism ; Molecular Targeted Therapy ; Neoplasm Proteins/biosynthesis/genetics/*physiology ; RNA Interference ; RNA, Small Interfering/genetics ; Signal Transduction ; Thalidomide/*analogs & derivatives/pharmacology/therapeutic use ; Ubiquitin-Protein Ligases/physiology ; },
abstract = {Primary effusion lymphoma (PEL) is an aggressive cancer with few treatment options. The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide have recently been shown to kill PEL cell lines, and lenalidomide is in clinical trials against PEL. IMiDs bind to the CRL4CRBN E3 ubiquitin ligase complex, leading to the acquisition of the Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3), casein kinase 1 α (CK1α), and zinc finger protein 91 (ZFP91) as neosubstrates. IMiDs are effective against multiple myeloma because of degradation of IKZF1 and IKZF3 and the consequent loss of interferon regulatory factor 4 (IRF4) and MYC expression. Lenalidomide is also effective in chromosome 5q deletion-associated myelodysplastic syndrome as a result of degradation of CK1α. An essential IKZF1-IRF4-MYC axis has recently been proposed to underlie the toxicity of IMiDs in PEL. Here, we further investigate IMiD effectors in PEL cell lines, based on genome-wide CRISPR/Cas9 screens for essential human genes. These screens and extensive validation experiments show that, of the 4 neosubstrates, only CK1α is essential for the survival of PEL cell lines. In contrast, IKZF1 and IKZF3 are dispensable, individually or in combination. IRF4 was critical in all 8 PEL cell lines tested, and surprisingly, IMiDs triggered downregulation of IRF4 expression independently of both IKZF1 and IKZF3. Reexpression of CK1α and/or IRF4 partially rescued PEL cell lines from IMiD-mediated toxicity. In conclusion, IMiD toxicity in PEL cell lines is independent of IKZF1 and IKZF3 but proceeds through degradation of the neosubstrate CK1α and downregulation of IRF4.},
}

CRISPR-Cas Systems
Casein Kinase Ialpha/genetics/*physiology
Cell Line, Tumor
Down-Regulation/drug effects
Gene Expression Regulation, Neoplastic/drug effects
Gene Knockout Techniques
Humans
Ikaros Transcription Factor/physiology
Immunologic Factors/*pharmacology/therapeutic use
Interferon Regulatory Factors/biosynthesis/genetics/*physiology
Lenalidomide/*pharmacology/therapeutic use
Lymphoma, Primary Effusion/*drug therapy/genetics/metabolism
Molecular Targeted Therapy
Neoplasm Proteins/biosynthesis/genetics/*physiology
RNA Interference
RNA, Small Interfering/genetics
Signal Transduction
Thalidomide/*analogs & derivatives/pharmacology/therapeutic use
Ubiquitin-Protein Ligases/physiology

@article {pmid29954749,
year = {2018},
author = {Kahn, JD and Miller, PG and Silver, AJ and Sellar, RS and Bhatt, S and Gibson, C and McConkey, M and Adams, D and Mar, B and Mertins, P and Fereshetian, S and Krug, K and Zhu, H and Letai, A and Carr, SA and Doench, J and Jaiswal, S and Ebert, BL},
title = {PPM1D-truncating mutations confer resistance to chemotherapy and sensitivity to PPM1D inhibition in hematopoietic cells.},
journal = {Blood},
volume = {132},
number = {11},
pages = {1095-1105},
pmid = {29954749},
issn = {1528-0020},
support = {P01 CA066996/CA/NCI NIH HHS/United States ; P01 CA108631/CA/NCI NIH HHS/United States ; P50 CA206963/CA/NCI NIH HHS/United States ; R01 HL082945/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Base Sequence ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/drug effects/genetics ; Enzyme Inhibitors/*pharmacology ; *Hematologic Neoplasms/drug therapy/enzymology/genetics/pathology ; Hematopoietic Stem Cells/*enzymology/pathology ; Humans ; *Myeloproliferative Disorders/drug therapy/enzymology/genetics/pathology ; *Neoplasm Proteins/antagonists & inhibitors/genetics/metabolism ; Neoplastic Stem Cells/*enzymology/pathology ; *Protein Phosphatase 2C/antagonists & inhibitors/genetics/metabolism ; *Sequence Deletion ; },
abstract = {Truncating mutations in the terminal exon of protein phosphatase Mg2+/Mn2+ 1D (PPM1D) have been identified in clonal hematopoiesis and myeloid neoplasms, with a striking enrichment in patients previously exposed to chemotherapy. In this study, we demonstrate that truncating PPM1D mutations confer a chemoresistance phenotype, resulting in the selective expansion of PPM1D-mutant hematopoietic cells in the presence of chemotherapy in vitro and in vivo. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease mutational profiling of PPM1D in the presence of chemotherapy selected for the same exon 6 mutations identified in patient samples. These exon 6 mutations encode for a truncated protein that displays elevated expression and activity due to loss of a C-terminal degradation domain. Global phosphoproteomic profiling revealed altered phosphorylation of target proteins in the presence of the mutation, highlighting multiple pathways including the DNA damage response (DDR). In the presence of chemotherapy, PPM1D-mutant cells have an abrogated DDR resulting in altered cell cycle progression, decreased apoptosis, and reduced mitochondrial priming. We demonstrate that treatment with an allosteric, small molecule inhibitor of PPM1D reverts the phosphoproteomic, DDR, apoptotic, and mitochondrial priming changes observed in PPM1D-mutant cells. Finally, we show that the inhibitor preferentially kills PPM1D-mutant cells, sensitizes the cells to chemotherapy, and reverses the chemoresistance phenotype. These results provide an explanation for the enrichment of truncating PPM1D mutations in the blood of patients exposed to chemotherapy and in therapy-related myeloid neoplasms, and demonstrate that PPM1D can be a targeted in the prevention of clonal expansion of PPM1D-mutant cells and the treatment of PPM1D-mutant disease.},
}

*Base Sequence
CRISPR-Cas Systems
Cell Line, Tumor
*Drug Resistance, Neoplasm/drug effects/genetics
Enzyme Inhibitors/*pharmacology
*Hematologic Neoplasms/drug therapy/enzymology/genetics/pathology
Hematopoietic Stem Cells/*enzymology/pathology
Humans
*Myeloproliferative Disorders/drug therapy/enzymology/genetics/pathology
*Neoplasm Proteins/antagonists & inhibitors/genetics/metabolism
Neoplastic Stem Cells/*enzymology/pathology
*Protein Phosphatase 2C/antagonists & inhibitors/genetics/metabolism
*Sequence Deletion

@article {pmid29790585,
year = {2018},
author = {Leibman-Markus, M and Pizarro, L and Schuster, S and Lin, ZJD and Gershony, O and Bar, M and Coaker, G and Avni, A},
title = {The intracellular nucleotide-binding leucine-rich repeat receptor (SlNRC4a) enhances immune signalling elicited by extracellular perception.},
journal = {Plant, cell & environment},
volume = {41},
number = {10},
pages = {2313-2327},
doi = {10.1111/pce.13347},
pmid = {29790585},
issn = {1365-3040},
support = {R01 GM092772/GM/NIGMS NIH HHS/United States ; },
mesh = {Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Ethylenes/metabolism ; Gene Editing ; Immunoprecipitation ; Lycopersicon esculentum/immunology/physiology ; Mass Spectrometry ; Microscopy, Confocal ; NLR Proteins/metabolism/*physiology ; Plant Growth Regulators/metabolism ; *Plant Immunity ; Plant Proteins/metabolism/*physiology ; Plants, Genetically Modified ; Polymerase Chain Reaction ; Reactive Oxygen Species/metabolism ; Receptors, Pattern Recognition/metabolism/physiology ; Signal Transduction ; },
abstract = {Plant recognition and defence against pathogens employs a two-tiered perception system. Surface-localized pattern recognition receptors (PRRs) act to recognize microbial features, whereas intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) directly or indirectly recognize pathogen effectors inside host cells. Employing the tomato PRR LeEIX2/EIX model system, we explored the molecular mechanism of signalling pathways. We identified an NLR that can associate with LeEIX2, termed SlNRC4a (NB-LRR required for hypersensitive response-associated cell death-4). Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defence responses, whereas silencing SlNRC4 reduces plant immunity. Moreover, the coiled-coil domain of SlNRC4a is able to associate with LeEIX2 and is sufficient to enhance responses upon EIX perception. On the basis of these findings, we propose that SlNRC4a acts as a noncanonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perceptions.},
}

Blotting, Western
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Ethylenes/metabolism
Gene Editing
Immunoprecipitation
Lycopersicon esculentum/immunology/physiology
Mass Spectrometry
Microscopy, Confocal
NLR Proteins/metabolism/*physiology
Plant Growth Regulators/metabolism
*Plant Immunity
Plant Proteins/metabolism/*physiology
Plants, Genetically Modified
Polymerase Chain Reaction
Reactive Oxygen Species/metabolism
Receptors, Pattern Recognition/metabolism/physiology
Signal Transduction

@article {pmid29789357,
year = {2018},
author = {Li, C and Psatha, N and Sova, P and Gil, S and Wang, H and Kim, J and Kulkarni, C and Valensisi, C and Hawkins, RD and Stamatoyannopoulos, G and Lieber, A},
title = {Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing.},
journal = {Blood},
volume = {131},
number = {26},
pages = {2915-2928},
pmid = {29789357},
issn = {1528-0020},
support = {R01 HL130040/HL/NHLBI NIH HHS/United States ; R21 CA193077/CA/NCI NIH HHS/United States ; R21 CA204036/CA/NCI NIH HHS/United States ; R01 HL120888/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Erythrocytes/metabolism ; Female ; Gene Editing/*methods ; *Gene Expression ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*metabolism ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Promoter Regions, Genetic ; beta-Globins/*genetics ; gamma-Globins/*genetics ; },
abstract = {Disorders involving β-globin gene mutations, primarily β-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human β-globin locus-transgenic (β-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from β-YAC/human CD46-transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into β-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human β- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.},
}

Animals
*CRISPR-Cas Systems
Erythrocytes/metabolism
Female
Gene Editing/*methods
*Gene Expression
Hematopoietic Stem Cell Mobilization
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells/*metabolism
Humans
Mice
Mice, Inbred C57BL
Mice, Transgenic
Promoter Regions, Genetic
beta-Globins/*genetics
gamma-Globins/*genetics

@article {pmid29760161,
year = {2018},
author = {Luo, H and Wang, F and Zha, J and Li, H and Yan, B and Du, Q and Yang, F and Sobh, A and Vulpe, C and Drusbosky, L and Cogle, C and Chepelev, I and Xu, B and Nimer, SD and Licht, J and Qiu, Y and Chen, B and Xu, M and Huang, S},
title = {CTCF boundary remodels chromatin domain and drives aberrant HOX gene transcription in acute myeloid leukemia.},
journal = {Blood},
volume = {132},
number = {8},
pages = {837-848},
pmid = {29760161},
issn = {1528-0020},
support = {R01 CA166835/CA/NCI NIH HHS/United States ; R01 DK110108/DK/NIDDK NIH HHS/United States ; R01 CA204044/CA/NCI NIH HHS/United States ; R01 CA172408/CA/NCI NIH HHS/United States ; R01 HL112294/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CCCTC-Binding Factor/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Chromatin Assembly and Disassembly ; *Gene Expression Regulation, Leukemic ; Homeodomain Proteins/*biosynthesis/genetics ; Humans ; Leukemia, Myeloid, Acute/genetics/*metabolism/pathology ; Mice ; Mice, Inbred NOD ; Neoplasm Proteins/genetics/*metabolism ; *Transcription, Genetic ; },
abstract = {HOX gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant HOX gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal HOX gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the HOX gene loci. We discovered that the CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) is critical for establishing and maintaining aberrant HOXA9-HOXA13 gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active HOXA chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior HOXA genes. Consistent with the role of the CBS7/9 boundary in HOXA locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the HOXA locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.},
}

Animals
CCCTC-Binding Factor/genetics/*metabolism
CRISPR-Cas Systems
Cell Line, Tumor
*Chromatin Assembly and Disassembly
*Gene Expression Regulation, Leukemic
Homeodomain Proteins/*biosynthesis/genetics
Humans
Leukemia, Myeloid, Acute/genetics/*metabolism/pathology
Mice
Mice, Inbred NOD
Neoplasm Proteins/genetics/*metabolism
*Transcription, Genetic

@article {pmid29447390,
year = {2018},
author = {Zhou, Z and Wang, L and Ge, F and Gong, P and Wang, H and Wang, F and Chen, L and Liu, L},
title = {Pold3 is required for genomic stability and telomere integrity in embryonic stem cells and meiosis.},
journal = {Nucleic acids research},
volume = {46},
number = {7},
pages = {3468-3486},
pmid = {29447390},
issn = {1362-4962},
mesh = {Animals ; Ataxia Telangiectasia Mutated Proteins/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; DNA Damage/genetics ; DNA Polymerase III/*genetics ; DNA Repair/genetics ; DNA Replication/*genetics ; Embryonic Stem Cells/*metabolism ; Genomic Instability/*genetics ; Meiosis/genetics ; Mice, Knockout ; Telomere/*genetics ; Telomere-Binding Proteins/genetics ; Tumor Suppressor p53-Binding Protein 1/genetics ; },
abstract = {Embryonic stem cells (ESCs) and meiosis are featured by relatively higher frequent homologous recombination associated with DNA double strand breaks (DSB) repair. Here, we show that Pold3 plays important roles in DSB repair, telomere maintenance and genomic stability of both ESCs and spermatocytes in mice. By attempting to generate Pold3 deficient mice using CRISPR/Cas9 or transcription activator-like effector nucleases, we show that complete loss of Pold3 (Pold3-/-) resulted in early embryonic lethality at E6.5. Rapid DNA damage response and massive apoptosis occurred in both outgrowths of Pold3-null (Pold3-/-) blastocysts and Pold3 inducible knockout (iKO) ESCs. While Pold3-/- ESCs were not achievable, Pold3 iKO led to increased DNA damage response, telomere loss and chromosome breaks accompanied by extended S phase. Meanwhile, loss of Pold3 resulted in replicative stress, micronucleation and aneuploidy. Also, DNA repair was impaired in Pold3+/- or Pold3 knockdown ESCs. Moreover, Pold3 mediates DNA replication and repair by regulating 53BP1, RIF1, ATR and ATM pathways. Furthermore, spermatocytes of Pold3 haploinsufficient (Pold3+/-) mice with increasing age displayed impaired DSB repair, telomere shortening and loss, and chromosome breaks, like Pold3 iKO ESCs. These data suggest that Pold3 maintains telomere integrity and genomic stability of both ESCs and meiosis by suppressing replicative stress.},
}

Animals
Ataxia Telangiectasia Mutated Proteins/genetics
CRISPR-Cas Systems/genetics
DNA Breaks, Double-Stranded
DNA Damage/genetics
DNA Polymerase III/*genetics
DNA Repair/genetics
DNA Replication/*genetics
Embryonic Stem Cells/*metabolism
Genomic Instability/*genetics
Meiosis/genetics
Mice, Knockout
Telomere/*genetics
Telomere-Binding Proteins/genetics
Tumor Suppressor p53-Binding Protein 1/genetics

@article {pmid29337302,
year = {2018},
author = {Wang, Y and Ji, T and Nelson, AD and Glanowska, K and Murphy, GG and Jenkins, PM and Parent, JM},
title = {Critical roles of αII spectrin in brain development and epileptic encephalopathy.},
journal = {The Journal of clinical investigation},
volume = {128},
number = {2},
pages = {760-773},
pmid = {29337302},
issn = {1558-8238},
support = {K08 NS099379/NS/NINDS NIH HHS/United States ; R01 AG052934/AG/NIA NIH HHS/United States ; T32 GM007544/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Axons/pathology ; Brain/*embryology/pathology ; Brain Diseases/*pathology ; CRISPR-Cas Systems ; Carrier Proteins/genetics/physiology ; Dendrites/pathology ; Epilepsy/*pathology ; Female ; Gene Deletion ; Hippocampus/pathology ; Humans ; Male ; Mice ; Mice, Transgenic ; Microfilament Proteins/genetics/physiology ; Mutation ; Neurons/pathology ; Phenotype ; Prosencephalon/pathology ; Rats ; Spectrin/genetics/*physiology ; },
abstract = {The nonerythrocytic α-spectrin-1 (SPTAN1) gene encodes the cytoskeletal protein αII spectrin. Mutations in SPTAN1 cause early infantile epileptic encephalopathy type 5 (EIEE5); however, the role of αII spectrin in neurodevelopment and EIEE5 pathogenesis is unknown. Prior work suggests that αII spectrin is absent in the axon initial segment (AIS) and contributes to a diffusion barrier in the distal axon. Here, we have shown that αII spectrin is expressed ubiquitously in rodent and human somatodendritic and axonal domains. CRISPR-mediated deletion of Sptan1 in embryonic rat forebrain by in utero electroporation caused altered dendritic and axonal development, loss of the AIS, and decreased inhibitory innervation. Overexpression of human EIEE5 mutant SPTAN1 in embryonic rat forebrain and mouse hippocampal neurons led to similar developmental defects that were also observed in EIEE5 patient-derived neurons. Additionally, patient-derived neurons displayed aggregation of spectrin complexes. Taken together, these findings implicate αII spectrin in critical aspects of dendritic and axonal development and synaptogenesis, and support a dominant-negative mechanism of SPTAN1 mutations in EIEE5.},
}

Animals
Axons/pathology
Brain/*embryology/pathology
Brain Diseases/*pathology
CRISPR-Cas Systems
Carrier Proteins/genetics/physiology
Dendrites/pathology
Epilepsy/*pathology
Female
Gene Deletion
Hippocampus/pathology
Humans
Male
Mice
Mice, Transgenic
Microfilament Proteins/genetics/physiology
Mutation
Neurons/pathology
Phenotype
Prosencephalon/pathology
Rats
Spectrin/genetics/*physiology

@article {pmid29259316,
year = {2017},
author = {Ikeda, M and Matsuyama, S and Akagi, S and Ohkoshi, K and Nakamura, S and Minabe, S and Kimura, K and Hosoe, M},
title = {Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {17827},
pmid = {29259316},
issn = {2045-2322},
mesh = {Animals ; Blastocyst/metabolism ; CRISPR-Cas Systems/*genetics ; Cattle ; Cattle Diseases/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/genetics ; Endonucleases/genetics ; Gene Editing/methods ; Genome/*genetics ; Green Fluorescent Proteins/genetics ; Isoleucine-tRNA Ligase/genetics ; Japan ; Mutation/*genetics ; Nuclear Transfer Techniques ; RNA, Messenger/genetics ; Transposases/genetics ; },
abstract = {Isoleucyl-tRNA synthetase (IARS) syndrome is a recessive disease of Japanese Black cattle caused by a single nucleotide substitution. To repair the mutated IARS gene, we designed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create a double-strand break near the mutation site. CRISPR/Cas9 and donor DNA that contained a synonymous codon for the correct amino acid and an Aequorea coerulescens Green Fluorescent Protein (AcGFP) cassette with a piggyBac transposase recognition site at both ends were introduced into bovine fetal fibroblast (BFF) cells isolated from a homozygous mutant calf. Recombinant cells were enriched on the basis of expression of AcGFP, and two cell lines that contained the repaired allele were subcloned. We generated somatic cell nuclear transfer (SCNT) embryos from the repaired cells and transferred 22 blastocysts to recipient cows. In total, five viable fetuses were retrieved at Days 34 and 36. PiggyBac transposase mRNA was introduced into BFF cells isolated from cloned foetuses and AcGFP-negative cells were used for second round of cloning. We transferred nine SCNT embryos to recipient cows and retrieved two fetuses at Day 34. Fetal genomic DNA analysis showed correct repair of the IARS mutation without any additional DNA footprint.},
}

Animals
Blastocyst/metabolism
CRISPR-Cas Systems/*genetics
Cattle
Cattle Diseases/*genetics
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
DNA/genetics
Endonucleases/genetics
Gene Editing/methods
Genome/*genetics
Green Fluorescent Proteins/genetics
Isoleucine-tRNA Ligase/genetics
Japan
Mutation/*genetics
Nuclear Transfer Techniques
RNA, Messenger/genetics
Transposases/genetics

@article {pmid29255251,
year = {2017},
author = {le Sage, C and Lawo, S and Panicker, P and Scales, TME and Rahman, SA and Little, AS and McCarthy, NJ and Moore, JD and Cross, BCS},
title = {Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {17693},
pmid = {29255251},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics/physiology ; DNA Cleavage ; Drug Evaluation, Preclinical/methods ; Drug Resistance/*genetics ; Endonucleases/genetics ; Gene Editing/methods ; Gene Expression Regulation/genetics ; Gene Knockout Techniques/*methods ; Gene Regulatory Networks/*genetics ; Genome/genetics ; Humans ; Proto-Oncogene Proteins B-raf/antagonists & inhibitors ; Vemurafenib/pharmacology ; },
abstract = {Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.},
}

CRISPR-Cas Systems/genetics
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/genetics/physiology
DNA Cleavage
Drug Evaluation, Preclinical/methods
Drug Resistance/*genetics
Endonucleases/genetics
Gene Editing/methods
Gene Expression Regulation/genetics
Gene Knockout Techniques/*methods
Gene Regulatory Networks/*genetics
Genome/genetics
Humans
Proto-Oncogene Proteins B-raf/antagonists & inhibitors
Vemurafenib/pharmacology

@article {pmid29190429,
year = {2018},
author = {Kook, S and Qi, A and Wang, P and Meng, S and Gulleman, P and Young, LR and Guttentag, SH},
title = {Gene-edited MLE-15 Cells as a Model for the Hermansky-Pudlak Syndromes.},
journal = {American journal of respiratory cell and molecular biology},
volume = {58},
number = {5},
pages = {566-574},
doi = {10.1165/rcmb.2017-0324MA},
pmid = {29190429},
issn = {1535-4989},
support = {P01 HL092870/HL/NHLBI NIH HHS/United States ; P60 DK020593/DK/NIDDK NIH HHS/United States ; R01 GM108807/GM/NIGMS NIH HHS/United States ; P30 DK058404/DK/NIDDK NIH HHS/United States ; P30 EY008126/EY/NEI NIH HHS/United States ; P30 DK020593/DK/NIDDK NIH HHS/United States ; P30 CA068485/CA/NCI NIH HHS/United States ; R01 HL119503/HL/NHLBI NIH HHS/United States ; U24 DK059637/DK/NIDDK NIH HHS/United States ; },
mesh = {Alveolar Epithelial Cells/*metabolism/pathology ; Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Markers ; Genetic Predisposition to Disease ; Hermanski-Pudlak Syndrome/*genetics/metabolism/pathology ; Humans ; Mice, Inbred C57BL ; Mice, Transgenic ; *Mutation ; Phenotype ; },
abstract = {Defining the mechanisms of cellular pathogenesis in rare lung diseases such as Hermansky-Pudlak syndrome (HPS) is often complicated by loss of the differentiated phenotype of cultured primary alveolar type 2 (AT2) cells, as well as by a lack of durable cell lines that are faithful to both AT2-cell and rare disease phenotypes. We used CRISPR/Cas9 gene editing to generate a series of HPS-specific mutations in the MLE-15 cell line. The resulting MLE-15/HPS cell lines exhibit preservation of AT2 cellular functions, including formation of lamellar body-like organelles, complete processing of surfactant protein B, and known features of HPS specific to each trafficking complex, including loss of protein targeting to lamellar bodies. MLE-15/HPS1 and MLE-15/HPS2 (with a mutation in Ap3β1) express increased macrophage chemotactic protein-1, a well-described mediator of alveolitis in patients with HPS and in mouse models. We show that MLE-15/HPS9 and pallid AT2 cells (with a mutation in Bloc1s6) also express increased macrophage chemotactic protein-1, suggesting that mice and humans with BLOC-1 mutations may also be susceptible to alveolitis. In addition to providing a flexible platform to examine the role of HPS-specific mutations in trafficking AT2 cells, MLE-15/HPS cell lines provide a durable resource for high-throughput screening and studies of cellular pathophysiology that are likely to accelerate progress toward developing novel therapies for this rare lung disease.},
}

Alveolar Epithelial Cells/*metabolism/pathology
Animals
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
Cell Line
*Clustered Regularly Interspaced Short Palindromic Repeats
Disease Models, Animal
Gene Editing/*methods
Genetic Markers
Genetic Predisposition to Disease
Hermanski-Pudlak Syndrome/*genetics/metabolism/pathology
Humans
Mice, Inbred C57BL
Mice, Transgenic
*Mutation
Phenotype

@article {pmid28944962,
year = {2018},
author = {Leung, TH and Tang, HW and Siu, MK and Chan, DW and Chan, KK and Cheung, AN and Ngan, HY},
title = {Human papillomavirus E6 protein enriches the CD55(+) population in cervical cancer cells, promoting radioresistance and cancer aggressiveness.},
journal = {The Journal of pathology},
volume = {244},
number = {2},
pages = {151-163},
doi = {10.1002/path.4991},
pmid = {28944962},
issn = {1096-9896},
mesh = {Animals ; CD55 Antigens/genetics/*metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cell Self Renewal ; Female ; Gene Editing/methods ; Genetic Therapy/methods ; Host-Pathogen Interactions ; Humans ; Mice, Nude ; Oncogene Proteins, Viral/genetics/*metabolism ; Papillomaviridae/genetics/*metabolism ; Papillomavirus Infections/diagnosis/*virology ; Radiation Tolerance ; Repressor Proteins/genetics/*metabolism ; Signal Transduction ; Tumor Cells, Cultured ; Up-Regulation ; Uterine Cervical Neoplasms/genetics/metabolism/therapy/*virology ; Xenograft Model Antitumor Assays ; },
abstract = {Accumulating evidence indicates that the human papillomavirus (HPV) E6 protein plays a crucial role in the development of cervical cancer. Subpopulations of cells that reside within tumours are responsible for tumour resistance to cancer therapy and recurrence. However, the identity of such cells residing in cervical cancer and their relationship with the HPV-E6 protein have not been identified. Here, we isolated sphere-forming cells, which showed self-renewal ability, from primary cervical tumours. Gene expression profiling revealed that cluster of differentiation (CD) 55 was upregulated in primary cervical cancer sphere cells. Flow-cytometric analysis detected abundant CD55(+) populations among a panel of HPV-positive cervical cancer cell lines, whereas few CD55(+) cells were found in HPV-negative cervical cancer and normal cervical epithelial cell lines. The CD55(+) subpopulation isolated from the C33A cell line showed significant sphere-forming ability and enhanced tumourigenicity, cell migration, and radioresistance. In contrast, the suppression of CD55 in HPV-positive CaSki cells inhibited tumourigenicity both in vitro and in vivo, and sensitized cells to radiation treatment. In addition, ectopic expression of the HPV-E6 protein in HPV-negative cervical cancer cells dramatically enriched the CD55(+) subpopulation. CRISPR/Cas9 knockout of CD55 in an HPV-E6-overexpressing stable clone abolished the tumourigenic effects of the HPV-E6 protein. Taken together, our data suggest that HPV-E6 protein expression enriches the CD55(+) population, which contributes to tumourigenicity and radioresistance in cervical cancer cells. Targeting CD55 via CRISPR/Cas9 may represent a novel avenue for developing new strategies and effective therapies for the treatment of cervical cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.},
}

Animals
CD55 Antigens/genetics/*metabolism
CRISPR-Associated Protein 9/genetics/metabolism
CRISPR-Cas Systems
Cell Line, Tumor
Cell Movement
Cell Proliferation
Cell Self Renewal
Female
Gene Editing/methods
Genetic Therapy/methods
Host-Pathogen Interactions
Humans
Mice, Nude
Oncogene Proteins, Viral/genetics/*metabolism
Papillomaviridae/genetics/*metabolism
Papillomavirus Infections/diagnosis/*virology
Radiation Tolerance
Repressor Proteins/genetics/*metabolism
Signal Transduction
Tumor Cells, Cultured
Up-Regulation
Uterine Cervical Neoplasms/genetics/metabolism/therapy/*virology
Xenograft Model Antitumor Assays

@article {pmid31298604,
year = {2019},
author = {Guo, T and Zheng, F and Zeng, Z and Yang, Y and Li, Q and She, Q and Han, W},
title = {Cmr3 regulates the suppression on cyclic oligoadenylate synthesis by tag complementarity in a Type III-B CRISPR-Cas system.},
journal = {RNA biology},
volume = {},
number = {},
pages = {},
doi = {10.1080/15476286.2019.1642725},
pmid = {31298604},
issn = {1555-8584},
abstract = {Type III CRISPR-Cas systems code for a multi-subunit ribonucleoprotein (RNP) complex that mediates DNA cleavage and synthesizes cyclic oligoadenylate (cOA) second messenger to confer anti-viral immunity. Both immune activities are to be activated upon binding to target RNA transcripts by their complementarity to crRNA, and autoimmunity avoidance is determined by extended complementarity between the 5'-repeat tag of crRNA and 3'-flanking sequences of target transcripts (anti-tag). However, as to how the strategy could achieve stringent autoimmunity avoidance remained elusive. In this study, we systematically investigated how the complementarity of the crRNA 5'-tag and anti-tag (i.e., tag complementarity) could affect the interference activities (DNA cleavage activity and cOA synthesis activity) of Cmr-α, a type III-B system in Sulfolobus islandicus Rey15A. The results revealed an increasing suppression on both activities by increasing degrees of tag complementarity and a critical function of the 7th nucleotide of crRNA in avoiding autoimmunity. More importantly, mutagenesis of Cmr3α exerts either positive or negative effects on the cOA synthesis activity depending on the degrees of tag complementarity, suggesting that the subunit, coupling with the interaction between crRNA tag and anti-tag, function in facilitating immunity and avoiding autoimmunity in Type III-B systems.},
}

@article {pmid31294157,
year = {2017},
author = {Abedon, ST},
title = {Phage "delay" towards enhancing bacterial escape from biofilms: a more comprehensive way of viewing resistance to bacteriophages.},
journal = {AIMS microbiology},
volume = {3},
number = {2},
pages = {186-226},
doi = {10.3934/microbiol.2017.2.186},
pmid = {31294157},
issn = {2471-1888},
abstract = {In exploring bacterial resistance to bacteriophages, emphasis typically is placed on those mechanisms which completely prevent phage replication. Such resistance can be detected as extensive reductions in phage ability to form plaques, that is, reduced efficiency of plating. Mechanisms include restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Alternatively, phages may be reduced in their "vigor" when infecting certain bacterial hosts, that is, with phages displaying smaller burst sizes or extended latent periods rather than being outright inactivated. It is well known, as well, that most phages poorly infect bacteria that are less metabolically active. Extracellular polymers such as biofilm matrix material also may at least slow phage penetration to bacterial surfaces. Here I suggest that such "less-robust" mechanisms of resistance to bacteriophages could serve bacteria by slowing phage propagation within bacterial biofilms, that is, delaying phage impact on multiple bacteria rather than necessarily outright preventing such impact. Related bacteria, ones that are relatively near to infected bacteria, e.g., roughly 10+ µm away, consequently may be able to escape from biofilms with greater likelihood via standard dissemination-initiating mechanisms including erosion from biofilm surfaces or seeding dispersal/central hollowing. That is, given localized areas of phage infection, so long as phage spread can be reduced in rate from initial points of contact with susceptible bacteria, then bacterial survival may be enhanced due to bacteria metaphorically "running away" to more phage-free locations. Delay mechanisms-to the extent that they are less specific in terms of what phages are targeted-collectively could represent broader bacterial strategies of phage resistance versus outright phage killing, the latter especially as require specific, evolved molecular recognition of phage presence. The potential for phage delay should be taken into account when developing protocols of phage-mediated biocontrol of biofilm bacteria, e.g., as during phage therapy of chronic bacterial infections.},
}

@article {pmid31295471,
year = {2019},
author = {Gupta, D and Bhattacharjee, O and Mandal, D and Sen, MK and Dey, D and Dasgupta, A and Kazi, TA and Gupta, R and Sinharoy, S and Acharya, K and Chattopadhyay, D and Ravichandiran, V and Roy, S and Ghosh, D},
title = {CRISPR-Cas9 system: A new-fangled dawn in gene editing.},
journal = {Life sciences},
volume = {},
number = {},
pages = {116636},
doi = {10.1016/j.lfs.2019.116636},
pmid = {31295471},
issn = {1879-0631},
abstract = {Till date, only three techniques namely Zinc Finger Nuclease (ZFN), Transcription-Activator Like Effector Molecules (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9) are available for targeted genome editing. CRISPR-Cas system is very efficient, fast, easy and cheap technique for achieving knock-out gene in the cell. CRISPR-Cas9 system refurbishes the targeted genome editing approach into a more expedient and competent way, thus facilitating proficient genome editing through embattled double-strand breaks in approximately any organism and cell type. The off-target effects of CRISPR Cas system has been circumnavigated by using paired nickases. Moreover, CRISPR-Cas9 has been used effectively for numerous purposes, like knock-out of a gene, regulation of endogenous gene expression, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The execution of the CRISPR-Cas9 system has amplified the number of accessible scientific substitutes for studying gene function, thus enabling generation of CRISPR-based disease models. Even though many mechanistic questions are left behind to be answered and the system is not yet fool-proof i.e., a number of challenges are yet to be addressed, the employment of CRISPR-Cas9-based genome engineering technologies will increase our understanding to disease processes and their treatment in the near future. In this review we have discussed the history of CRISPR-Cas9, its mechanism for genome editing and its application in animal, plant and protozoan parasites. Additionally, the pros and cons of CRISPR-Cas9 and its potential in therapeutic application have also been detailed here.},
}

@article {pmid31293824,
year = {2019},
author = {Ashari, KS and Roslan, NS and Omar, AR and Bejo, MH and Ideris, A and Mat Isa, N},
title = {Genome sequencing and analysis of Salmonella enterica subsp. enterica serovar Stanley UPM 517: Insights on its virulence-associated elements and their potentials as vaccine candidates.},
journal = {PeerJ},
volume = {7},
number = {},
pages = {e6948},
doi = {10.7717/peerj.6948},
pmid = {31293824},
issn = {2167-8359},
abstract = {Salmonella enterica subsp. enterica serovar Stanley (S. Stanley) is a pathogen that contaminates food, and is related to Salmonella outbreaks in a variety of hosts such as humans and farm animals through products like dairy items and vegetables. Despite the fact that several vaccines of Salmonella strains had been constructed, none of them were developed according to serovar Stanley up to this day. This study presents results of genome sequencing and analysis on our S. Stanley UPM 517 strain taken from fecal swabs of 21-day-old healthy commercial chickens in Perak, Malaysia and used Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S. Typhimurium LT2) as a reference to be compared with. First, sequencing and assembling of the Salmonella Stanley UPM 517 genome into a contiguous form were done. The work was then continued with scaffolding and gap filling. Annotation and alignment of the draft genome was performed with S. Typhimurium LT2. The other elements of virulence estimated in this study included Salmonella pathogenicity islands, resistance genes, prophages, virulence factors, plasmid regions, restriction-modification sites and the CRISPR-Cas system. The S. Stanley UPM 517 draft genome had a length of 4,736,817 bp with 4,730 coding sequence and 58 RNAs. It was discovered via genomic analysis on this strain that there were antimicrobial resistance properties toward a wide variety of antibiotics. Tcf and ste, the two fimbrial virulence clusters related with human and broiler intestinal colonizations which were not found in S. Typhimurium LT2, were atypically discovered in the S. Stanley UPM 517 genome. These clusters are involved in the intestinal colonization of human and broilers, respectively. There were seven Salmonella pathogenicity islands (SPIs) within the draft genome, which contained the virulence factors associated with Salmonella infection (except SPI-14). Five intact prophage regions, mostly comprising of the protein encoding Gifsy-1, Fels-1, RE-2010 and SEN34 prophages, were also encoded in the draft genome. Also identified were Type I-III restriction-modification sites and the CRISPR-Cas system of the Type I-E subtype. As this strain exhibited resistance toward numerous antibiotics, we distinguished several genes that had the potential for removal in the construction of a possible vaccine candidate to restrain and lessen the pervasiveness of salmonellosis and to function as an alternative to antibiotics.},
}

@article {pmid31293420,
year = {2019},
author = {Lima, R and Del Fiol, FS and Balcão, VM},
title = {Prospects for the Use of New Technologies to Combat Multidrug-Resistant Bacteria.},
journal = {Frontiers in pharmacology},
volume = {10},
number = {},
pages = {692},
doi = {10.3389/fphar.2019.00692},
pmid = {31293420},
issn = {1663-9812},
abstract = {The increasing use of antibiotics is being driven by factors such as the aging of the population, increased occurrence of infections, and greater prevalence of chronic diseases that require antimicrobial treatment. The excessive and unnecessary use of antibiotics in humans has led to the emergence of bacteria resistant to the antibiotics currently available, as well as to the selective development of other microorganisms, hence contributing to the widespread dissemination of resistance genes at the environmental level. Due to this, attempts are being made to develop new techniques to combat resistant bacteria, among them the use of strictly lytic bacteriophage particles, CRISPR-Cas, and nanotechnology. The use of these technologies, alone or in combination, is promising for solving a problem that humanity faces today and that could lead to human extinction: the domination of pathogenic bacteria resistant to artificial drugs. This prospective paper discusses the potential of bacteriophage particles, CRISPR-Cas, and nanotechnology for use in combating human (bacterial) infections.},
}

@article {pmid31142266,
year = {2019},
author = {Léger, S and Costa, MBW and Tulpan, D},
title = {Pairwise visual comparison of small RNA secondary structures with base pair probabilities.},
journal = {BMC bioinformatics},
volume = {20},
number = {1},
pages = {293},
doi = {10.1186/s12859-019-2902-6},
pmid = {31142266},
issn = {1471-2105},
mesh = {Algorithms ; *Base Pairing ; CRISPR-Cas Systems/genetics ; Evolution, Molecular ; Humans ; *Nucleic Acid Conformation ; *Probability ; RNA/*chemistry ; RNA, Guide/genetics ; Virulence/genetics ; Yersinia/pathogenicity ; },
abstract = {BACKGROUND: Predicted RNA secondary structures are typically visualized using dot-plots for base pair binding probabilities and planar graphs for unique structures, such as the minimum free energy structure. These are however difficult to analyze simultaneously.

RESULTS: This work introduces a compact unified view of the most stable conformation of an RNA secondary structure and its base pair probabilities, which is called the Circular Secondary Structure Base Pairs Probabilities Plot (CS2BP2-Plot). Along with our design we provide access to a web server implementation of our solution that facilitates pairwise comparison of short RNA (and DNA) sequences up to 200 base pairs. The web server first calculates the minimum free energy secondary structure and the base pair probabilities for up to 10 RNA or DNA sequences using RNAfold and then provides a two panel comparative view that includes CS2BP2-Plots along with the traditional graph, planar and circular diagrams obtained with VARNA. The CS2BP2-Plots include highlighting of the nucleotide differences between two selected sequences using ClustalW local alignments. We also provide descriptive statistics, dot-bracket secondary structure representations and ClustalW local alignments for compared sequences.

CONCLUSIONS: Using circular diagrams and colour and weight-coded arcs, we demonstrate how a single image can replace the state-of-the-art dual representations (dot-plots and minimum free energy structures) for base-pair probabilities of RNA secondary structures while allowing efficient exploration and comparison of different RNA conformations via a web server front end. With that, we provide the community, especially the biologically oriented, with an intuitive tool for ncRNA visualization. Web-server: https://nrcmonsrv01.nrc.ca/cs2bp2plot.},
}

Algorithms
*Base Pairing
CRISPR-Cas Systems/genetics
Evolution, Molecular
Humans
*Nucleic Acid Conformation
*Probability
RNA/*chemistry
RNA, Guide/genetics
Virulence/genetics
Yersinia/pathogenicity

@article {pmid31065830,
year = {2019},
author = {Huang, JM and Chang, YT and Shih, MH and Lin, WC and Huang, FC},
title = {Identification and characterization of a secreted M28 aminopeptidase protein in Acanthamoeba.},
journal = {Parasitology research},
volume = {118},
number = {6},
pages = {1865-1874},
doi = {10.1007/s00436-019-06332-8},
pmid = {31065830},
issn = {1432-1955},
mesh = {Acanthamoeba/isolation & purification/*metabolism ; Amebiasis/parasitology ; Aminopeptidases/genetics/*immunology/*metabolism ; CRISPR-Cas Systems ; Complement C3/*metabolism ; Humans ; Lakes/parasitology ; Protozoan Proteins/genetics/*immunology/*metabolism ; Soil/parasitology ; },
abstract = {Acanthamoeba is a free-living pathogenic protozoan that is distributed in different environmental reservoirs, including lakes and soil. Pathogenic Acanthamoeba can cause severe human diseases, such as blinding keratitis and granulomatous encephalitis. Therefore, it is important to understand the pathogenic relationship between humans and Acanthamoeba. By comparison of systemic analysis results for Acanthamoeba isolates, we identified a novel secreted protein of Acanthamoeba, an M28 aminopeptidase (M28AP), which targets of the human innate immune defense. We investigated the molecular functions and characteristics of the M28AP protein by anti-M28 antibodies and a M28AP mutant strain generated by the CRISPR/Cas9 system. Human complement proteins such as C3b and iC3b were degraded by Acanthamoeba M28AP. We believe that M28AP is an important factor in human innate immunity. This study provides new insight for the development of more efficient medicines to treat Acanthamoeba infection.},
}

Acanthamoeba/isolation & purification/*metabolism
Amebiasis/parasitology
Aminopeptidases/genetics/*immunology/*metabolism
CRISPR-Cas Systems
Complement C3/*metabolism
Humans
Lakes/parasitology
Protozoan Proteins/genetics/*immunology/*metabolism
Soil/parasitology

@article {pmid31011186,
year = {2019},
author = {Mimitou, EP and Cheng, A and Montalbano, A and Hao, S and Stoeckius, M and Legut, M and Roush, T and Herrera, A and Papalexi, E and Ouyang, Z and Satija, R and Sanjana, NE and Koralov, SB and Smibert, P},
title = {Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells.},
journal = {Nature methods},
volume = {16},
number = {5},
pages = {409-412},
doi = {10.1038/s41592-019-0392-0},
pmid = {31011186},
issn = {1548-7105},
support = {R01 HL125816/HL/NHLBI NIH HHS/United States ; R21 HG009748/HG/NHGRI NIH HHS/United States ; R35 GM124998/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Profiling ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Leukocytes, Mononuclear/metabolism/pathology ; Lymphoma, T-Cell, Cutaneous/genetics/metabolism/pathology ; Mice ; NIH 3T3 Cells ; Proteins/*genetics ; RNA, Guide/genetics ; Sequence Analysis, RNA/*methods ; Single-Cell Analysis/*methods ; Skin Neoplasms/genetics/metabolism/pathology ; Transcriptome/*genetics ; Tumor Cells, Cultured ; },
abstract = {Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.},
}

Animals
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Expression Profiling
High-Throughput Nucleotide Sequencing/*methods
Humans
Leukocytes, Mononuclear/metabolism/pathology
Lymphoma, T-Cell, Cutaneous/genetics/metabolism/pathology
Mice
NIH 3T3 Cells
Proteins/*genetics
RNA, Guide/genetics
Sequence Analysis, RNA/*methods
Single-Cell Analysis/*methods
Skin Neoplasms/genetics/metabolism/pathology
Transcriptome/*genetics
Tumor Cells, Cultured

@article {pmid30962622,
year = {2019},
author = {Chow, RD and Wang, G and Ye, L and Codina, A and Kim, HR and Shen, L and Dong, MB and Errami, Y and Chen, S},
title = {In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens.},
journal = {Nature methods},
volume = {16},
number = {5},
pages = {405-408},
doi = {10.1038/s41592-019-0371-5},
pmid = {30962622},
issn = {1548-7105},
support = {T32 GM007223/GM/NIGMS NIH HHS/United States ; U54 CA209992/CA/NCI NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Mice ; Neoplasm Metastasis/*genetics ; Sequence Analysis, RNA ; },
abstract = {Systematic investigation of the genetic interactions that influence metastatic potential has been challenging. Here we developed massively parallel CRISPR-Cpf1/Cas12a crRNA array profiling (MCAP), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library of 11,934 arrays targeting 325 pairwise combinations of genes implicated in metastasis. By assessing the metastatic potential of the double knockouts in mice, we unveiled a quantitative landscape of genetic interactions that drive metastasis.},
}

Animals
Bacterial Proteins/*genetics
CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems/*genetics
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Endonucleases/*genetics
Gene Editing/*methods
Gene Knockout Techniques/*methods
Mice
Neoplasm Metastasis/*genetics
Sequence Analysis, RNA

@article {pmid30788819,
year = {2019},
author = {Sibbritt, T and Osteil, P and Fan, X and Sun, J and Salehin, N and Knowles, H and Shen, J and Tam, PPL},
title = {Gene Editing of Mouse Embryonic and Epiblast Stem Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1940},
number = {},
pages = {77-95},
doi = {10.1007/978-1-4939-9086-3_6},
pmid = {30788819},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Frameshift Mutation/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Germ Layers/*cytology ; Mice ; Mouse Embryonic Stem Cells/*cytology ; Plasmids/genetics ; RNA, Guide/genetics ; },
abstract = {Efficient and reliable methods for gene editing are critical for the generation of loss-of-gene function stem cells and genetically modified mice. Here, we outline the application of CRISPR-Cas9 technology for gene editing in mouse embryonic stem cells (mESCs) to generate knockout ESC chimeras for the fast-tracked analysis of gene function. Furthermore, we describe the application of gene editing directly to mouse epiblast stem cells (mEpiSCs) for modelling germ layer differentiation in vitro.},
}

Animals
CRISPR-Cas Systems/genetics
Cells, Cultured
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Frameshift Mutation/*genetics
Gene Editing/*methods
Gene Knockout Techniques/*methods
Germ Layers/*cytology
Mice
Mouse Embryonic Stem Cells/*cytology
Plasmids/genetics
RNA, Guide/genetics

@article {pmid30575812,
year = {2019},
author = {Burgess, DJ},
title = {Genome editing for disease locus dissection.},
journal = {Nature reviews. Genetics},
volume = {20},
number = {2},
pages = {67},
doi = {10.1038/s41576-018-0091-1},
pmid = {30575812},
issn = {1471-0064},
mesh = {CRISPR-Cas Systems ; *Cardiovascular Diseases ; *Gene Editing ; Haplotypes ; Humans ; Risk Factors ; },
}

CRISPR-Cas Systems
*Cardiovascular Diseases
*Gene Editing
Haplotypes
Humans
Risk Factors

@article {pmid30446729,
year = {2019},
author = {Cloney, R},
title = {The oracle of inDelphi predicts Cas9 repair outcomes.},
journal = {Nature reviews. Genetics},
volume = {20},
number = {1},
pages = {4-5},
doi = {10.1038/s41576-018-0077-z},
pmid = {30446729},
issn = {1471-0064},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; RNA, Guide ; },
}

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

@article {pmid30358158,
year = {2019},
author = {Maguire, JA and Cardenas-Diaz, FL and Gadue, P and French, DL},
title = {Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.},
journal = {Current protocols in stem cell biology},
volume = {48},
number = {1},
pages = {e64},
doi = {10.1002/cpsc.64},
pmid = {30358158},
issn = {1938-8969},
support = {U01 HL099656/HL/NHLBI NIH HHS/United States ; U01 HL134696/HL/NHLBI NIH HHS/United States ; },
mesh = {Alleles ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Targeting/*methods ; Genome, Human ; Humans ; Mutation ; *Pluripotent Stem Cells ; },
abstract = {Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele. To overcome this obstacle, we describe the use of two oligonucleotides, one expressing the sequence change, with the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodology to make this protocol efficient with small numbers of cells and to limit the amount of labor-intensive clone passaging. This protocol provides a streamlined and technically simple approach for generating valuable tools to model human disease in stem cells. © 2018 by John Wiley & Sons, Inc.},
}

Alleles
CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Gene Editing/*methods
Gene Targeting/*methods
Genome, Human
Humans
Mutation
*Pluripotent Stem Cells

@article {pmid29727166,
year = {2018},
author = {Yang, Z and Wang, H and Wang, Y and Ren, Y and Wei, D},
title = {Manufacturing Multienzymatic Complex Reactors In Vivo by Self-Assembly To Improve the Biosynthesis of Itaconic Acid in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {7},
number = {5},
pages = {1244-1250},
doi = {10.1021/acssynbio.8b00086},
pmid = {29727166},
issn = {2161-5063},
mesh = {Aconitate Hydratase/genetics/metabolism ; CRISPR-Cas Systems ; Carboxy-Lyases/genetics/metabolism ; Citrate (si)-Synthase/genetics/metabolism ; Escherichia coli/genetics/*metabolism ; Fluorescence ; Metabolic Engineering/methods ; Microorganisms, Genetically-Modified ; Microscopy, Atomic Force ; Microscopy, Electron, Scanning ; Multiprotein Complexes/chemistry/genetics/metabolism ; Protein Engineering/*methods ; Recombinant Proteins/genetics/metabolism ; Succinates/*metabolism ; },
abstract = {The self-assembly of multienzyme into bioreactors is of extensive interest to spatially regulate valuable reactions. Despite the important progresses achieved, methods to precisely manufacture multienzymatic complex reactors (MECRs) are still poorly proposed both in vivo and in vitro, particularly for more than three biocatalytically relevant enzymes. Here, we developed a sequential self-assembly system to form multitude MECRs involving three enzymes in the itaconic acid (IA) pathway with two pairs of protein-peptide interactions. The MECRs were identified as nanoscale particle-like structures when self-assembled in vitro and produced higher IA production than the unassembled and linearly assembled systems when applied in vivo coupling with CRISPR-Cas9 based metabolic engineering. This work provides novel insights into the construction of multifarious multienzyme complex into bioreactors by the self-assembly strategy for multistep cascades to sequentially control metabolic fluxes inside cells.},
}

Aconitate Hydratase/genetics/metabolism
CRISPR-Cas Systems
Carboxy-Lyases/genetics/metabolism
Citrate (si)-Synthase/genetics/metabolism
Escherichia coli/genetics/*metabolism
Fluorescence
Metabolic Engineering/methods
Microorganisms, Genetically-Modified
Microscopy, Atomic Force
Microscopy, Electron, Scanning
Multiprotein Complexes/chemistry/genetics/metabolism
Protein Engineering/*methods
Recombinant Proteins/genetics/metabolism
Succinates/*metabolism

@article {pmid29709170,
year = {2018},
author = {Agrawal, DK and Tang, X and Westbrook, A and Marshall, R and Maxwell, CS and Lucks, J and Noireaux, V and Beisel, CL and Dunlop, MJ and Franco, E},
title = {Mathematical Modeling of RNA-Based Architectures for Closed Loop Control of Gene Expression.},
journal = {ACS synthetic biology},
volume = {7},
number = {5},
pages = {1219-1228},
doi = {10.1021/acssynbio.8b00040},
pmid = {29709170},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems ; Computational Biology/*methods ; Feedback, Physiological/*physiology ; *Gene Expression Regulation ; *Models, Theoretical ; RNA/*chemistry ; Synthetic Biology/methods ; },
abstract = {Feedback allows biological systems to control gene expression precisely and reliably, even in the presence of uncertainty, by sensing and processing environmental changes. Taking inspiration from natural architectures, synthetic biologists have engineered feedback loops to tune the dynamics and improve the robustness and predictability of gene expression. However, experimental implementations of biomolecular control systems are still far from satisfying performance specifications typically achieved by electrical or mechanical control systems. To address this gap, we present mathematical models of biomolecular controllers that enable reference tracking, disturbance rejection, and tuning of the temporal response of gene expression. These controllers employ RNA transcriptional regulators to achieve closed loop control where feedback is introduced via molecular sequestration. Sensitivity analysis of the models allows us to identify which parameters influence the transient and steady state response of a target gene expression process, as well as which biologically plausible parameter values enable perfect reference tracking. We quantify performance using typical control theory metrics to characterize response properties and provide clear selection guidelines for practical applications. Our results indicate that RNA regulators are well-suited for building robust and precise feedback controllers for gene expression. Additionally, our approach illustrates several quantitative methods useful for assessing the performance of biomolecular feedback control systems.},
}

CRISPR-Cas Systems
Computational Biology/*methods
Feedback, Physiological/*physiology
*Gene Expression Regulation
*Models, Theoretical
RNA/*chemistry
Synthetic Biology/methods

@article {pmid29704747,
year = {2018},
author = {Li, L and Hu, S and Chen, X},
title = {Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities.},
journal = {Biomaterials},
volume = {171},
number = {},
pages = {207-218},
pmid = {29704747},
issn = {1878-5905},
support = {Z99 EB999999//NULL/International ; ZIA EB000073-07//NULL/International ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Disease/genetics ; *Gene Editing ; *Gene Transfer Techniques ; Viruses/*metabolism ; },
abstract = {In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed.},
}

CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Disease/genetics
*Gene Editing
*Gene Transfer Techniques
Viruses/*metabolism

@article {pmid29683658,
year = {2018},
author = {Wang, W and Zheng, W and Hu, F and He, X and Wu, D and Zhang, W and Liu, H and Ma, X},
title = {Enhanced Biosynthesis Performance of Heterologous Proteins in CHO-K1 Cells Using CRISPR-Cas9.},
journal = {ACS synthetic biology},
volume = {7},
number = {5},
pages = {1259-1268},
doi = {10.1021/acssynbio.7b00375},
pmid = {29683658},
issn = {2161-5063},
mesh = {Animals ; Apoptosis/drug effects/genetics ; CHO Cells ; *CRISPR-Cas Systems ; Cricetulus ; DNA End-Joining Repair ; Endoplasmic Reticulum/genetics/metabolism ; Glutathione/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; Luciferases/genetics/metabolism ; Oxidoreductases Acting on Sulfur Group Donors/genetics/metabolism ; Plasmids ; Protein Engineering/*methods ; Recombinant Proteins/*genetics/metabolism ; Staurosporine/pharmacology ; Survivin/genetics/metabolism ; },
abstract = {Chinese hamster ovary (CHO) cells are the famous expression system for industrial production of recombinant proteins, such as therapeutic antibodies. However, there still remain bottlenecks in protein quality and weakness in expression efficiency because of the intrinsic genetic properties of the cell. Here we have enhanced biosynthesis performance of heterologous proteins in CHO-K1 cells using CRISPR-Cas9 by editing the genome precisely with two genes for improving ER microenvironment and reinforcing antiapoptotic ability. A linear donor plasmid harboring eGFP-HsQSOX1b and Survivin genes was knocked in specific locus in CHO-K1 genome by the CRISPR-Cas9 RNA guided nucleases via NHEJ with efficiencies of up to 3.85% in the CHO-K1 cell pools following FACS, and the hQSOX1 and hSurvivin genes were integrated into expected genome locus successfully. Compared with control, the antiapoptotic viability of edited CHO-K1 cells was increased by 6.40 times, and the yield has been raised by 5.55 times with GLuc as model protein. The possible molecular mechanisms and pathways of remarkable antiapoptotic ability and protein biosynthesis in modified CHO-K1 cells have been elucidated reasonably. In conclusion, the novel ideas and reliable techniques for obtaining foreign proteins more efficiently in engineered animal cells were very valuable to meet large clinical needs.},
}

Animals
Apoptosis/drug effects/genetics
CHO Cells
*CRISPR-Cas Systems
Cricetulus
DNA End-Joining Repair
Endoplasmic Reticulum/genetics/metabolism
Glutathione/metabolism
Green Fluorescent Proteins/genetics/metabolism
Luciferases/genetics/metabolism
Oxidoreductases Acting on Sulfur Group Donors/genetics/metabolism
Plasmids
Protein Engineering/*methods
Recombinant Proteins/*genetics/metabolism
Staurosporine/pharmacology
Survivin/genetics/metabolism

@article {pmid29668266,
year = {2018},
author = {Gao, W and Yin, J and Bao, L and Wang, Q and Hou, S and Yue, Y and Yao, W and Gao, X},
title = {Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State.},
journal = {ACS synthetic biology},
volume = {7},
number = {5},
pages = {1291-1302},
doi = {10.1021/acssynbio.7b00400},
pmid = {29668266},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems ; Escherichia coli/*genetics/growth & development ; Extracellular Matrix/genetics ; Fluorescence ; Gene Deletion ; Gene Expression Profiling/methods ; Genetic Engineering/*methods ; Interferon-alpha/genetics/metabolism ; Microorganisms, Genetically-Modified ; Molecular Weight ; Mutation ; Recombinant Proteins/genetics/metabolism ; },
abstract = {Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.},
}

*CRISPR-Cas Systems
Escherichia coli/*genetics/growth & development
Extracellular Matrix/genetics
Fluorescence
Gene Deletion
Gene Expression Profiling/methods
Genetic Engineering/*methods
Interferon-alpha/genetics/metabolism
Microorganisms, Genetically-Modified
Molecular Weight
Mutation
Recombinant Proteins/genetics/metabolism

@article {pmid29634237,
year = {2018},
author = {Liu, Y and Wei, WP and Ye, BC},
title = {High GC Content Cas9-Mediated Genome-Editing and Biosynthetic Gene Cluster Activation in Saccharopolyspora erythraea.},
journal = {ACS synthetic biology},
volume = {7},
number = {5},
pages = {1338-1348},
doi = {10.1021/acssynbio.7b00448},
pmid = {29634237},
issn = {2161-5063},
mesh = {*Base Composition ; *CRISPR-Cas Systems ; Erythromycin/*biosynthesis ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Gene Knock-In Techniques ; Genetic Vectors ; Genome, Bacterial ; Microorganisms, Genetically-Modified ; Multigene Family ; Plasmids ; Promoter Regions, Genetic ; RNA, Guide ; Saccharopolyspora/*genetics ; Temperature ; },
abstract = {The overexpression of bacterial secondary metabolite biosynthetic enzymes is the basis for industrial overproducing strains. Genome editing tools can be used to further improve gene expression and yield. Saccharopolyspora erythraea produces erythromycin, which has extensive clinical applications. In this study, the CRISPR-Cas9 system was used to edit genes in the S. erythraea genome. A temperature-sensitive plasmid containing the PermE promoter, to drive Cas9 expression, and the Pj23119 and PkasO promoters, to drive sgRNAs, was designed. Erythromycin esterase, encoded by S. erythraea SACE_1765, inactivates erythromycin by hydrolyzing the macrolactone ring. Sequencing and qRT-PCR confirmed that reporter genes were successfully inserted into the SACE_1765 gene. Deletion of SACE_1765 in a high-producing strain resulted in a 12.7% increase in erythromycin levels. Subsequent PermE- egfp knock-in at the SACE_0712 locus resulted in an 80.3% increase in erythromycin production compared with that of wild type. Further investigation showed that PermE promoter knock-in activated the erythromycin biosynthetic gene clusters at the SACE_0712 locus. Additionally, deletion of indA (SACE_1229) using dual sgRNA targeting without markers increased the editing efficiency to 65%. In summary, we have successfully applied Cas9-based genome editing to a bacterial strain, S. erythraea, with a high GC content. This system has potential application for both genome-editing and biosynthetic gene cluster activation in Actinobacteria.},
}

*Base Composition
*CRISPR-Cas Systems
Erythromycin/*biosynthesis
Gene Editing/*methods
Gene Expression Regulation, Bacterial
Gene Knock-In Techniques
Genetic Vectors
Genome, Bacterial
Microorganisms, Genetically-Modified
Multigene Family
Plasmids
Promoter Regions, Genetic
RNA, Guide
Saccharopolyspora/*genetics
Temperature

@article {pmid29527014,
year = {2018},
author = {Wrighton, KH},
title = {Genetic engineering: Expanding the reach of Cas9.},
journal = {Nature reviews. Genetics},
volume = {19},
number = {5},
pages = {250-251},
pmid = {29527014},
issn = {1471-0064},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Genetic Engineering ; },
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA
*Genetic Engineering

@article {pmid29249815,
year = {2018},
author = {Burgess, DJ},
title = {Translational genetics: CRISPR therapies - making the grade not the cut.},
journal = {Nature reviews. Genetics},
volume = {19},
number = {2},
pages = {63},
pmid = {29249815},
issn = {1471-0064},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Epigenomics ; Proteomics ; Transcriptional Activation ; },
}

*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Epigenomics
Proteomics
Transcriptional Activation

@article {pmid29192199,
year = {2017},
author = {Zhao, D and Feng, X and Zhu, X and Wu, T and Zhang, X and Bi, C},
title = {CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {16624},
pmid = {29192199},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Gene Deletion ; *Gene Editing/methods ; Gene Targeting ; Genetic Loci ; Plasmids/genetics ; *RNA, Guide ; },
abstract = {The CRISPR/Cas9 system is a powerful, revolutionary tool for genome editing. However, it is not without limitations. There are PAM-free and CRISPR-tolerant regions that cannot be modified by the standard CRISPR/Cas9 system, and off-target activity impedes its broader applications. To avoid these drawbacks, we developed a very simple CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique which does not require the construction of a plasmid to express a specific gRNA. Instead, a universal N20 sequence with a very high targeting efficiency is inserted into the E. coli chromosome by homologous recombination, which in turn undergoes a double-stranded break by CRISPR/Cas9 and induces an intra-chromosomal recombination event to accomplish the editing process. This technique was shown to be able to edit PAM-free and CRISPR-tolerant regions with no off-target effects in Escherichia coli. When applied to multi-locus editing, CAGO was able to modify one locus in two days with a near 100% editing efficiency. Furthermore, modified CAGO was used to edit large regions of up to 100 kbp with at least 75% efficiency. Finally, genome editing by CAGO only requires a transformation procedure and the construction of a linear donor DNA cassette, which was further simplified by applying a modular design strategy. Although the technique was established in E. coli, it should be applicable to other organisms with only minor modifications.},
}

*CRISPR-Cas Systems
Gene Deletion
*Gene Editing/methods
Gene Targeting
Genetic Loci
Plasmids/genetics
*RNA, Guide

@article {pmid28974517,
year = {2017},
author = {Musunuru, K and Lagor, WR and Miano, JM},
title = {What Do We Really Think About Human Germline Genome Editing, and What Does It Mean for Medicine?.},
journal = {Circulation. Cardiovascular genetics},
volume = {10},
number = {5},
pages = {},
pmid = {28974517},
issn = {1942-3268},
support = {R01 HL112793/HL/NHLBI NIH HHS/United States ; R01 HL117907/HL/NHLBI NIH HHS/United States ; R01 HL126875/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Female ; *Gene Editing/ethics/trends ; Genome, Human ; *Germ-Line Mutation ; Humans ; Infant, Newborn ; Male ; Pregnancy ; Public Opinion ; Societies, Medical ; Surveys and Questionnaires ; United States ; },
}

CRISPR-Cas Systems
Female
*Gene Editing/ethics/trends
Genome, Human
*Germ-Line Mutation
Humans
Infant, Newborn
Male
Pregnancy
Public Opinion
Societies, Medical
Surveys and Questionnaires
United States

@article {pmid28768752,
year = {2017},
author = {Hennessy, EJ},
title = {Cardiovascular Disease and Long Noncoding RNAs: Tools for Unraveling the Mystery Lnc-ing RNA and Phenotype.},
journal = {Circulation. Cardiovascular genetics},
volume = {10},
number = {4},
pages = {e001556},
doi = {10.1161/CIRCGENETICS.117.001556},
pmid = {28768752},
issn = {1942-3268},
mesh = {Animals ; CRISPR-Cas Systems ; Cardiovascular Diseases/*genetics/therapy ; Epigenesis, Genetic ; Gene Editing ; Humans ; Models, Genetic ; Nucleic Acid Conformation ; Phenotype ; Polymorphism, Single Nucleotide ; RNA, Long Noncoding/chemistry/*genetics/metabolism ; },
}

Animals
CRISPR-Cas Systems
Cardiovascular Diseases/*genetics/therapy
Epigenesis, Genetic
Gene Editing
Humans
Models, Genetic
Nucleic Acid Conformation
Phenotype
Polymorphism, Single Nucleotide
RNA, Long Noncoding/chemistry/*genetics/metabolism

@article {pmid28096529,
year = {2017},
author = {Burgess, DJ},
title = {Genetic screens: Combining CRISPR perturbations and RNA-seq.},
journal = {Nature reviews. Genetics},
volume = {18},
number = {2},
pages = {67},
pmid = {28096529},
issn = {1471-0064},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Testing ; *RNA ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Testing
*RNA

@article {pmid28029162,
year = {2017},
author = {Cloney, R},
title = {Genetic engineering: A genome-editing off switch.},
journal = {Nature reviews. Genetics},
volume = {18},
number = {2},
pages = {68-69},
pmid = {28029162},
issn = {1471-0064},
mesh = {*Bacteriophages ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering ; Genome ; },
}

*Bacteriophages
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Engineering
Genome

@article {pmid27982043,
year = {2017},
author = {McMahon, MA and Cleveland, DW},
title = {Gene therapy: Gene-editing therapy for neurological disease.},
journal = {Nature reviews. Neurology},
volume = {13},
number = {1},
pages = {7-9},
pmid = {27982043},
issn = {1759-4766},
mesh = {Alleles ; *CRISPR-Cas Systems ; Genetic Therapy ; Humans ; Huntington Disease/*genetics ; Mutation ; },
}

Alleles
*CRISPR-Cas Systems
Genetic Therapy
Humans
Huntington Disease/*genetics
Mutation

@article {pmid31291570,
year = {2019},
author = {Steinecke, A and Kurabayashi, N and Hayano, Y and Ishino, Y and Taniguchi, H},
title = {In Vivo Single-Cell Genotyping of Mouse Cortical Neurons Transfected with CRISPR/Cas9.},
journal = {Cell reports},
volume = {28},
number = {2},
pages = {325-331.e4},
doi = {10.1016/j.celrep.2019.06.038},
pmid = {31291570},
issn = {2211-1247},
abstract = {CRISPR/Cas-based technologies have revolutionized genetic approaches to addressing a wide range of neurobiological questions. The ability of CRISPR/Cas to introduce mutations into target genes allows us to perform in vivo loss-of-function experiments without generating genetically engineered mice. However, the lack of a reliable method to determine genotypes of individual CRISPR/Cas-transfected cells has made it impossible to unambiguously identify the genetic cause of their phenotypes in vivo. Here, we report a strategy for single-cell genotyping in CRISPR/Cas-transfected neurons that were phenotypically characterized in vivo. We show that re-sectioning of cortical slices and subsequent laser microdissection allow us to isolate individual CRISPR/Cas-transfected neurons. Sequencing of PCR products containing a CRISPR/Cas-targeted genomic region in single reference neurons provided genotypes that completely correspond with those deduced from their target protein expression and phenotypes. Thus, our study establishes a powerful strategy to determine the causality between genotypes and phenotypes in CRISPR/Cas-transfected neurons.},
}

@article {pmid31290648,
year = {2019},
author = {Jin, M and Garreau de Loubresse, N and Kim, Y and Kim, J and Yin, P},
title = {Programmable CRISPR-Cas Repression, Activation, and Computation with Sequence-Independent Targets and Triggers.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.9b00141},
pmid = {31290648},
issn = {2161-5063},
abstract = {The programmability of CRISPR-derived Cas9 as a sequence-specific DNA-targeting protein has made it a powerful tool for genomic manipulation in biological research and translational applications. Cas9 activity can be programmably engineered to respond to nucleic acids, but these efforts have focused primarily on single-input control of Cas9, and until recently, they were limited by sequence dependence between parts of the guide RNA and the sequence to be detected. Here, we not only design and present DNA- and RNA-sensing conditional guide RNA (cgRNA) that have no such sequence constraints, but also demonstrate a complete set of logical computations using these designs on DNA and RNA sequence inputs, including AND, OR, NAND, and NOR. The development of sequence-independent nucleic acid-sensing CRISPR-Cas9 systems with multi-input logic computation capabilities could lead to improved genome engineering and regulation as well as the construction of synthetic circuits with broader functionality.},
}

@article {pmid31288753,
year = {2019},
author = {Lam, TJ and Ye, Y},
title = {Long reads reveal the diversification and dynamics of CRISPR reservoir in microbiomes.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {567},
doi = {10.1186/s12864-019-5922-8},
pmid = {31288753},
issn = {1471-2164},
support = {1R01AI143254//National Institute of Allergy and Infectious Diseases/ ; 1R01AI108888//National Institute of Allergy and Infectious Diseases/ ; },
abstract = {BACKGROUND: Sequencing of microbiomes has accelerated the characterization of the diversity of CRISPR-Cas immune systems. However, the utilization of next generation short read sequences for the characterization of CRISPR-Cas dynamics remains limited due to the repetitive nature of CRISPR arrays. CRISPR arrays are comprised of short spacer segments (derived from invaders' genomes) interspaced between flanking repeat sequences. The repetitive structure of CRISPR arrays poses a computational challenge for the accurate assembly of CRISPR arrays from short reads. In this paper we evaluate the use of long read sequences for the analysis of CRISPR-Cas system dynamics in microbiomes.

RESULTS: We analyzed a dataset of Illumina's TruSeq Synthetic Long-Reads (SLR) derived from a gut microbiome. We showed that long reads captured CRISPR spacers at a high degree of redundancy, which highlights the spacer conservation of spacer sharing CRISPR variants, enabling the study of CRISPR array dynamics in ways difficult to achieve though short read sequences. We introduce compressed spacer graphs, a visual abstraction of spacer sharing CRISPR arrays, to provide a simplified view of complex organizational structures present within CRISPR array dynamics. Utilizing compressed spacer graphs, several key defining characteristics of CRISPR-Cas system dynamics were observed including spacer acquisition and loss events, conservation of the trailer end spacers, and CRISPR arrays' directionality (transcription orientation). Other result highlights include the observation of intense array contraction and expansion events, and reconstruction of a full-length genome for a potential invader (Faecalibacterium phage) based on identified spacers.

CONCLUSION: We demonstrate in an in silico system that long reads provide the necessary context for characterizing the organization of CRISPR arrays in a microbiome, and reveal dynamic and evolutionary features of CRISPR-Cas systems in a microbial population.},
}

@article {pmid31273110,
year = {2019},
author = {Hou, Z and Zhang, Y},
title = {Inserting DNA with CRISPR.},
journal = {Science (New York, N.Y.)},
volume = {365},
number = {6448},
pages = {25-26},
doi = {10.1126/science.aay2056},
pmid = {31273110},
issn = {1095-9203},
support = {R00 GM117268/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; RNA ; *Transposases ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA
RNA
*Transposases

@article {pmid31110297,
year = {2019},
author = {Pei, W and Wang, X and Rössler, J and Feyerabend, TB and Höfer, T and Rodewald, HR},
title = {Using Cre-recombinase-driven Polylox barcoding for in vivo fate mapping in mice.},
journal = {Nature protocols},
volume = {14},
number = {6},
pages = {1820-1840},
doi = {10.1038/s41596-019-0163-5},
pmid = {31110297},
issn = {1750-2799},
mesh = {Animals ; CRISPR-Cas Systems ; *Cell Lineage ; DNA/genetics/metabolism ; DNA Barcoding, Taxonomic/methods ; Female ; *Genes, Reporter ; Genotyping Techniques/methods ; Integrases/genetics ; Male ; Mice ; Mice, Transgenic ; Polymerase Chain Reaction/methods ; Recombination, Genetic ; Sequence Analysis, DNA/*methods ; },
abstract = {Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox. In Polylox, random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice (Rosa26Polylox reporter mice). Here, we provide a step-by-step protocol for the use of Polylox, including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR-Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2-3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis.},
}

Animals
CRISPR-Cas Systems
*Cell Lineage
DNA/genetics/metabolism
DNA Barcoding, Taxonomic/methods
Female
*Genes, Reporter
Genotyping Techniques/methods
Integrases/genetics
Male
Mice
Mice, Transgenic
Polymerase Chain Reaction/methods
Recombination, Genetic
Sequence Analysis, DNA/*methods

@article {pmid30911135,
year = {2019},
author = {Wu, Y and Zeng, J and Roscoe, BP and Liu, P and Yao, Q and Lazzarotto, CR and Clement, K and Cole, MA and Luk, K and Baricordi, C and Shen, AH and Ren, C and Esrick, EB and Manis, JP and Dorfman, DM and Williams, DA and Biffi, A and Brugnara, C and Biasco, L and Brendel, C and Pinello, L and Tsai, SQ and Wolfe, SA and Bauer, DE},
title = {Highly efficient therapeutic gene editing of human hematopoietic stem cells.},
journal = {Nature medicine},
volume = {25},
number = {5},
pages = {776-783},
doi = {10.1038/s41591-019-0401-y},
pmid = {30911135},
issn = {1546-170X},
support = {2013137/DDCF/Doris Duke Charitable Foundation/United States ; K08 DK093705/DK/NIDDK NIH HHS/United States ; R03 DK109232/DK/NIDDK NIH HHS/United States ; DP2 HL137300/HL/NHLBI NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; P01 HL032262/HL/NHLBI NIH HHS/United States ; U01 HL117720/HL/NHLBI NIH HHS/United States ; R01 AI117839/AI/NIAID NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; R01 HL137848/HL/NHLBI NIH HHS/United States ; 2015134/DDCF/Doris Duke Charitable Foundation/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Anemia, Sickle Cell/blood/genetics/therapy ; Base Sequence ; CRISPR-Cas Systems ; Carrier Proteins/genetics ; Enhancer Elements, Genetic ; Erythroid Precursor Cells/metabolism ; Fetal Hemoglobin/biosynthesis/genetics ; Gene Editing/*methods ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*metabolism ; Humans ; INDEL Mutation ; Nuclear Proteins/genetics ; RNA, Guide/genetics ; beta-Thalassemia/blood/genetics/therapy ; gamma-Globins/biosynthesis/genetics ; },
abstract = {Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.},
}

Amino Acid Sequence
Anemia, Sickle Cell/blood/genetics/therapy
Base Sequence
CRISPR-Cas Systems
Carrier Proteins/genetics
Enhancer Elements, Genetic
Erythroid Precursor Cells/metabolism
Fetal Hemoglobin/biosynthesis/genetics
Gene Editing/*methods
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells/*metabolism
Humans
INDEL Mutation
Nuclear Proteins/genetics
RNA, Guide/genetics
beta-Thalassemia/blood/genetics/therapy
gamma-Globins/biosynthesis/genetics

@article {pmid30794383,
year = {2019},
author = {Rui, Y and Wilson, DR and Sanders, K and Green, JJ},
title = {Reducible Branched Ester-Amine Quadpolymers (rBEAQs) Codelivering Plasmid DNA and RNA Oligonucleotides Enable CRISPR/Cas9 Genome Editing.},
journal = {ACS applied materials & interfaces},
volume = {11},
number = {11},
pages = {10472-10480},
doi = {10.1021/acsami.8b20206},
pmid = {30794383},
issn = {1944-8252},
mesh = {Amines/chemistry ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Survival/drug effects ; DNA/chemistry/*metabolism ; Esters/chemistry ; Gene Editing/*methods ; Humans ; Nanoparticles/chemistry/toxicity ; Plasmids/genetics/metabolism ; Polymers/chemical synthesis/*chemistry ; RNA, Small Interfering/chemistry/*metabolism ; Transfection ; },
abstract = {Functional codelivery of plasmid DNA and RNA oligonucleotides in the same nanoparticle system is challenging due to differences in their physical properties as well as their intracellular locations of function. In this study, we synthesized a series of reducible branched ester-amine quadpolymers (rBEAQs) and investigated their ability to coencapsulate and deliver DNA plasmids and RNA oligos. The rBEAQs are designed to leverage polymer branching, reducibility, and hydrophobicity to successfully cocomplex DNA and RNA in nanoparticles at low polymer to nucleic acid w/w ratios and enable high delivery efficiency. We validate the synthesis of this new class of biodegradable polymers, characterize the self-assembled nanoparticles that these polymers form with diverse nucleic acids, and demonstrate that the nanoparticles enable safe, effective, and efficient DNA-siRNA codelivery as well as nonviral CRISPR-mediated gene editing utilizing Cas9 DNA and sgRNA codelivery.},
}

Amines/chemistry
CRISPR-Cas Systems/*genetics
Cell Line
Cell Survival/drug effects
DNA/chemistry/*metabolism
Esters/chemistry
Gene Editing/*methods
Humans
Nanoparticles/chemistry/toxicity
Plasmids/genetics/metabolism
Polymers/chemical synthesis/*chemistry
RNA, Small Interfering/chemistry/*metabolism
Transfection

@article {pmid30016959,
year = {2018},
author = {Montaño, A and Forero-Castro, M and Hernández-Rivas, JM and García-Tuñón, I and Benito, R},
title = {Targeted genome editing in acute lymphoblastic leukemia: a review.},
journal = {BMC biotechnology},
volume = {18},
number = {1},
pages = {45},
pmid = {30016959},
issn = {1472-6750},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; *Gene Editing ; *Genetic Therapy ; Humans ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*genetics/*therapy ; },
abstract = {BACKGROUND: Genome editing technologies offers new opportunities for tackling diseases such as acute lymphoblastic leukemia (ALL) that have been beyond the reach of previous therapies.

RESULTS: We show how the recent availability of genome-editing tools such as CRISPR-Cas9 are an important means of advancing functional studies of ALL through the incorporation, elimination and modification of somatic mutations and fusion genes in cell lines and mouse models. These tools not only broaden the understanding of the involvement of various genetic alterations in the pathogenesis of the disease but also identify new therapeutic targets for future clinical trials.

CONCLUSIONS: New approaches including CRISPR-Cas9 are crucial for functional studies of genetic aberrations driving cancer progression, and that may be responsible for treatment resistance and relapses. By using this approach, diseases can be more faithfully reproduced and new therapeutic targets and approaches found.},
}

Animals
CRISPR-Cas Systems/*genetics
Disease Models, Animal
*Gene Editing
*Genetic Therapy
Humans
Precursor Cell Lymphoblastic Leukemia-Lymphoma/*genetics/*therapy

@article {pmid29875470,
year = {2018},
author = {Bleijenberg, A and Dekker, E},
title = {Reverse-engineering the serrated neoplasia pathway using CRISPR-Cas9.},
journal = {Nature reviews. Gastroenterology & hepatology},
volume = {15},
number = {9},
pages = {522-524},
doi = {10.1038/s41575-018-0035-4},
pmid = {29875470},
issn = {1759-5053},
mesh = {*CRISPR-Cas Systems ; Carcinogenesis ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Organoids ; },
}

*CRISPR-Cas Systems
Carcinogenesis
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
*Organoids

@article {pmid29663646,
year = {2018},
author = {Paulo, JA and Gygi, SP},
title = {Isobaric Tag-Based Protein Profiling of a Nicotine-Treated Alpha7 Nicotinic Receptor-Null Human Haploid Cell Line.},
journal = {Proteomics},
volume = {18},
number = {11},
pages = {e1700475},
pmid = {29663646},
issn = {1615-9861},
support = {K01 DK098285/DK/NIDDK NIH HHS/United States ; R01 GM067945/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Gene Ontology ; *Haploidy ; Humans ; Isotope Labeling ; Nicotine/*pharmacology ; Nicotinic Agonists/*pharmacology ; Proteome/analysis/drug effects/*metabolism ; Tandem Mass Spectrometry/*methods ; alpha7 Nicotinic Acetylcholine Receptor/antagonists & inhibitors/genetics/*metabolism ; },
abstract = {Nicotinic acetylcholine receptors (nAChR), the primary cell surface targets of nicotine, have implications in various neurological disorders. Here we investigate the proteome-wide effects of nicotine on human haploid cell lines (wildtype HAP1 and α7KO-HAP1) to address differences in nicotine-induced protein abundance profiles between these cell lines. We performed an SPS-MS3-based TMT10-plex experiment arranged in a 2-3-2-3 design with two replicates of the untreated samples and three of the treated samples for each cell line. We quantified 8775 proteins across all ten samples, of which several hundred differed significantly in abundance. Comparing α7KO-HAP1 and HAP1wt cell lines to each other revealed significant protein abundance alterations; however, we also measured differences resulting from nicotine treatment in both cell lines. Among proteins with increased abundance levels due to nicotine treatment included those previously identified: APP, APLP2, and ITM2B. The magnitude of these changes was greater in HAP1wt compared to the α7KO-HAP1 cell line, implying a potential role for the α7 nAChR in HAP1 cells. Moreover, the data revealed that membrane proteins and proteins commonly associated with neurons were predominant among those with altered abundance. This study, which is the first TMT-based proteome profiling of HAP1 cells, defines further the effects of nicotine on non-neuronal cellular proteomes.},
}

CRISPR-Cas Systems
Cells, Cultured
Gene Ontology
*Haploidy
Humans
Isotope Labeling
Nicotine/*pharmacology
Nicotinic Agonists/*pharmacology
Proteome/analysis/drug effects/*metabolism
Tandem Mass Spectrometry/*methods
alpha7 Nicotinic Acetylcholine Receptor/antagonists & inhibitors/genetics/*metabolism

@article {pmid29259215,
year = {2017},
author = {Phan, QV and Contzen, J and Seemann, P and Gossen, M},
title = {Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {17771},
pmid = {29259215},
issn = {2045-2322},
mesh = {Animals ; CHO Cells ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Chromosomes/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cricetulus ; DNA Nucleotidyltransferases/*genetics ; HEK293 Cells ; Humans ; Mutagenesis, Insertional/*genetics ; Recombination, Genetic/*genetics ; Transgenes/genetics ; },
abstract = {Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent protein patterns identified the recombination status of individual cells. In derivatives of a CHO master cell line the expression of the introduced transgene of interest could be dramatically increased almost 20-fold by subsequent deletion of the fluorescent protein gene that provided the initial isolation principle. The same master cell line was employed in a comparative analysis using CRISPR/Cas9 for transgene integration in identical loci. Even though the overall targeting efficacy was comparable, multi-loci targeting was considerably more effective for Cas9-mediated transgene insertion when compared to RMCE. While Cas9 is inherently more flexible, our results also alert to the risk of aberrant recombination events around the cut site. Together, this study points at the individual strengths in performance of both systems and provides guidance for their appropriate use.},
}

Animals
CHO Cells
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/genetics
Cell Line
Chromosomes/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cricetulus
DNA Nucleotidyltransferases/*genetics
HEK293 Cells
Humans
Mutagenesis, Insertional/*genetics
Recombination, Genetic/*genetics
Transgenes/genetics

@article {pmid29192200,
year = {2017},
author = {Jacobs, EZ and Warrier, S and Volders, PJ and D'haene, E and Van Lombergen, E and Vantomme, L and Van der Jeught, M and Heindryckx, B and Menten, B and Vergult, S},
title = {CRISPR/Cas9-mediated genome editing in naïve human embryonic stem cells.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {16650},
pmid = {29192200},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; High-Throughput Nucleotide Sequencing ; Human Embryonic Stem Cells/*metabolism ; Humans ; Neural Stem Cells/cytology/metabolism ; Workflow ; },
abstract = {The combination of genome-edited human embryonic stem cells (hESCs) and subsequent neural differentiation is a powerful tool to study neurodevelopmental disorders. Since the naïve state of pluripotency has favourable characteristics for efficient genome-editing, we optimized a workflow for the CRISPR/Cas9 system in these naïve stem cells. Editing efficiencies of respectively 1.3-8.4% and 3.8-19% were generated with the Cas9 nuclease and the D10A Cas9 nickase mutant. Next to this, wildtype and genome-edited naïve hESCs were successfully differentiated to neural progenitor cells. As a proof-of-principle of our workflow, two monoclonal genome-edited naïve hESCs colonies were obtained for TUNA, a long non-coding RNA involved in pluripotency and neural differentiation. In these genome-edited hESCs, an effect was seen on expression of TUNA, although not on neural differentiation potential. In conclusion, we optimized a genome-editing workflow in naïve hESCs that can be used to study candidate genes involved in neural differentiation and/or functioning.},
}

*CRISPR-Cas Systems
Cell Differentiation/genetics
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
High-Throughput Nucleotide Sequencing
Human Embryonic Stem Cells/*metabolism
Humans
Neural Stem Cells/cytology/metabolism
Workflow

@article {pmid31284938,
year = {2019},
author = {Goulin, EH and Galdeano, DM and Granato, LM and Matsumura, EE and Dalio, RJD and Machado, MA},
title = {RNA interference and CRISPR: Promising approaches to better understand and control citrus pathogens.},
journal = {Microbiological research},
volume = {226},
number = {},
pages = {1-9},
doi = {10.1016/j.micres.2019.03.006},
pmid = {31284938},
issn = {1618-0623},
abstract = {Citrus crops have great economic importance worldwide. However, citrus production faces many diseases caused by different pathogens, such as bacteria, oomycetes, fungi and viruses. To overcome important plant diseases in general, new technologies have been developed and applied to crop protection, including RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. RNAi has been demonstrated to be a powerful tool for application in plant defence mechanisms against different pathogens as well as their respective vectors, and CRISPR/Cas system has become widely used in gene editing or reprogramming or knocking out any chosen DNA/RNA sequence. In this article, we provide an overview of the use of RNAi and CRISPR/Cas technologies in management strategies to control several plants diseases, and we discuss how these strategies can be potentially used against citrus pathogens.},
}

@article {pmid31222014,
year = {2019},
author = {Bell, CC and Fennell, KA and Chan, YC and Rambow, F and Yeung, MM and Vassiliadis, D and Lara, L and Yeh, P and Martelotto, LG and Rogiers, A and Kremer, BE and Barbash, O and Mohammad, HP and Johanson, TM and Burr, ML and Dhar, A and Karpinich, N and Tian, L and Tyler, DS and MacPherson, L and Shi, J and Pinnawala, N and Yew Fong, C and Papenfuss, AT and Grimmond, SM and Dawson, SJ and Allan, RS and Kruger, RG and Vakoc, CR and Goode, DL and Naik, SH and Gilan, O and Lam, EYN and Marine, JC and Prinjha, RK and Dawson, MA},
title = {Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2723},
doi = {10.1038/s41467-019-10652-9},
pmid = {31222014},
issn = {2041-1723},
support = {55008729//Howard Hughes Medical Institute (HHMI)/International ; 1085015//Department of Health | National Health and Medical Research Council (NHMRC)/International ; 1106444//Department of Health | National Health and Medical Research Council (NHMRC)/International ; 1128984//Department of Health | National Health and Medical Research Council (NHMRC)/International ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; Bone Marrow/pathology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*drug effects ; Epigenesis, Genetic/drug effects ; Female ; Gene Expression Regulation, Leukemic/*drug effects ; HEK293 Cells ; Humans ; Kaplan-Meier Estimate ; Leukemia, Myeloid, Acute/*drug therapy/genetics/mortality/pathology ; Mice ; Mice, Inbred C57BL ; Sequence Analysis, RNA ; Single-Cell Analysis ; Trans-Activators/*antagonists & inhibitors/genetics/metabolism ; Transcription, Genetic/drug effects ; Treatment Outcome ; Xenograft Model Antitumor Assays ; },
abstract = {Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.},
}

Animals
Antineoplastic Agents/*pharmacology/therapeutic use
Bone Marrow/pathology
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Drug Resistance, Neoplasm/*drug effects
Epigenesis, Genetic/drug effects
Female
Gene Expression Regulation, Leukemic/*drug effects
HEK293 Cells
Humans
Kaplan-Meier Estimate
Leukemia, Myeloid, Acute/*drug therapy/genetics/mortality/pathology
Mice
Mice, Inbred C57BL
Sequence Analysis, RNA
Single-Cell Analysis
Trans-Activators/*antagonists & inhibitors/genetics/metabolism
Transcription, Genetic/drug effects
Treatment Outcome
Xenograft Model Antitumor Assays

@article {pmid31197154,
year = {2019},
author = {Naseri, G and Behrend, J and Rieper, L and Mueller-Roeber, B},
title = {COMPASS for rapid combinatorial optimization of biochemical pathways based on artificial transcription factors.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2615},
doi = {10.1038/s41467-019-10224-x},
pmid = {31197154},
issn = {2041-1723},
support = {031A172//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/International ; 031B0223//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/International ; },
mesh = {Arabidopsis Proteins/genetics/metabolism ; Biosensing Techniques/methods ; Biosynthetic Pathways/*genetics ; CRISPR-Cas Systems/genetics ; Cloning, Molecular/methods ; Flavanones/biosynthesis ; Homologous Recombination/genetics ; Metabolic Engineering/*methods ; Norisoprenoids/biosynthesis ; Promoter Regions, Genetic/genetics ; Saccharomyces cerevisiae/genetics/*metabolism ; Saccharomyces cerevisiae Proteins/*genetics/metabolism ; Synthetic Biology/methods ; Transcription Factors/genetics/*metabolism ; beta Carotene/biosynthesis ; },
abstract = {Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing β-carotene and co-producing β-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.},
}

Arabidopsis Proteins/genetics/metabolism
Biosensing Techniques/methods
Biosynthetic Pathways/*genetics
CRISPR-Cas Systems/genetics
Cloning, Molecular/methods
Flavanones/biosynthesis
Homologous Recombination/genetics
Metabolic Engineering/*methods
Norisoprenoids/biosynthesis
Promoter Regions, Genetic/genetics
Saccharomyces cerevisiae/genetics/*metabolism
Saccharomyces cerevisiae Proteins/*genetics/metabolism
Synthetic Biology/methods
Transcription Factors/genetics/*metabolism
beta Carotene/biosynthesis

@article {pmid31197149,
year = {2019},
author = {Hojo, MA and Masuda, K and Hojo, H and Nagahata, Y and Yasuda, K and Ohara, D and Takeuchi, Y and Hirota, K and Suzuki, Y and Kawamoto, H and Kawaoka, S},
title = {Identification of a genomic enhancer that enforces proper apoptosis induction in thymic negative selection.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2603},
doi = {10.1038/s41467-019-10525-1},
pmid = {31197149},
issn = {2041-1723},
support = {15H01478//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; 18K15409//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; 18H04810: S.K//MEXT | Japan Society for the Promotion of Science (JSPS)/International ; na//Uehara Memorial Foundation/International ; na//Japan Foundation for Applied Enzymology/International ; JPMJER1303//MEXT | Japan Science and Technology Agency (JST)/International ; },
mesh = {Animals ; Apoptosis/genetics/immunology ; Autoimmunity/*genetics/immunology ; Bcl-2-Like Protein 11/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Enhancer Elements, Genetic/genetics/*immunology ; Female ; Gene Expression Regulation/immunology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Models, Animal ; Receptors, Antigen, T-Cell/immunology/*metabolism ; Signal Transduction/genetics/immunology ; Thymocytes/*physiology ; Thymus Gland/cytology/immunology ; },
abstract = {During thymic negative selection, autoreactive thymocytes carrying T cell receptor (TCR) with overtly strong affinity to self-MHC/self-peptide are removed by Bim-dependent apoptosis, but how Bim is specifically regulated to link TCR activation and apoptosis induction is unclear. Here we identify a murine T cell-specific genomic enhancer EBAB (Bub1-Acoxl-Bim), whose deletion leads to accumulation of thymocytes expressing high affinity TCRs. Consistently, EBAB knockout mice have defective negative selection and fail to delete autoreactive thymocytes in various settings, with this defect accompanied by reduced Bim expression and apoptosis induction. By contrast, EBAB is dispensable for maintaining peripheral T cell homeostasis via Bim-dependent pathways. Our data thus implicate EBAB as an important, developmental stage-specific regulator of Bim expression and apoptosis induction to enforce thymic negative selection and suppress autoimmunity. Our study unravels a part of genomic enhancer codes that underlie complex and context-dependent gene regulation in TCR signaling.},
}

Animals
Apoptosis/genetics/immunology
Autoimmunity/*genetics/immunology
Bcl-2-Like Protein 11/*genetics/metabolism
CRISPR-Cas Systems/genetics
Enhancer Elements, Genetic/genetics/*immunology
Female
Gene Expression Regulation/immunology
Male
Mice
Mice, Inbred C57BL
Mice, Knockout
Models, Animal
Receptors, Antigen, T-Cell/immunology/*metabolism
Signal Transduction/genetics/immunology
Thymocytes/*physiology
Thymus Gland/cytology/immunology

@article {pmid31133747,
year = {2019},
author = {Chen, Z and Zhang, Y},
title = {Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development.},
journal = {Nature genetics},
volume = {51},
number = {6},
pages = {947-951},
doi = {10.1038/s41588-019-0418-7},
pmid = {31133747},
issn = {1546-1718},
support = {R01 HD092465/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Embryonic Development/*genetics ; Gene Expression Regulation, Developmental ; Gene Targeting ; *Genetic Association Studies/methods ; *Genome ; Homeodomain Proteins/*genetics ; Mice ; Mice, Knockout ; Zygote/*metabolism ; },
abstract = {How maternal factors in oocytes trigger zygotic genome activation (ZGA) is a long-standing question in developmental biology. Recent studies in 2-cell-like embryonic stem cells (2C-like cells) suggest that transcription factors of the DUX family are key regulators of ZGA in placental mammals1,2. To characterize the role of DUX in ZGA, we generated Dux cluster knockout (KO) mouse lines. Unexpectedly, we found that both Dux zygotic KO (Z-KO) and maternal and zygotic KO (MZ-KO) embryos can survive to adulthood despite showing reduced developmental potential. Furthermore, transcriptome profiling of the MZ-KO embryos revealed that loss of DUX has minimal effects on ZGA and most DUX targets in 2C-like cells are normally activated in MZ-KO embryos. Thus, contrary to the key function of DUX in inducing 2C-like cells, our data indicate that DUX has only a minor role in ZGA and that loss of DUX is compatible with mouse development.},
}

Animals
CRISPR-Cas Systems
Embryonic Development/*genetics
Gene Expression Regulation, Developmental
Gene Targeting
*Genetic Association Studies/methods
*Genome
Homeodomain Proteins/*genetics
Mice
Mice, Knockout
Zygote/*metabolism

@article {pmid30899116,
year = {2019},
author = {Soriano, V},
title = {Gene Editing for HIV Cure at the Edge.},
journal = {AIDS reviews},
volume = {21},
number = {1},
pages = {50a-51},
pmid = {30899116},
issn = {1698-6997},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; HIV Infections/*therapy ; HIV-1/*genetics ; Humans ; },
}

CRISPR-Cas Systems
Gene Editing/*methods
HIV Infections/*therapy
HIV-1/*genetics
Humans

@article {pmid30885208,
year = {2019},
author = {Yuan, T and Zhong, Y and Wang, Y and Zhang, T and Lu, R and Zhou, M and Lu, Y and Yan, K and Chen, Y and Hu, Z and Liang, J and Fan, J and Cheng, Y},
title = {Generation of hyperlipidemic rabbit models using multiple sgRNAs targeted CRISPR/Cas9 gene editing system.},
journal = {Lipids in health and disease},
volume = {18},
number = {1},
pages = {69},
doi = {10.1186/s12944-019-1013-8},
pmid = {30885208},
issn = {1476-511X},
support = {No.81570392//National Natural Science Foundation of China/ ; 81770457//National Natural Science Foundation of China/ ; No.2016YFE0126000//National Key Research and Development Program of China/ ; 15H04718//Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Technology, Japan/ ; },
mesh = {Animals ; Animals, Genetically Modified ; Apolipoproteins E/genetics ; Atherosclerosis/etiology/genetics ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Female ; Gene Editing/*methods ; Gene Knockout Techniques ; Hyperlipidemias/*genetics ; Lipids/blood/genetics ; Lipoproteins/blood/genetics ; Pregnancy ; RNA, Guide ; Rabbits ; Receptors, LDL/genetics ; },
abstract = {OBJECTIVE: To generate novel rabbit models with a large-fragment deletion of either LDL receptor (LDLR) and/or apolipoprotein (apoE) genes for the study of hyperlipidemic and atherosclerosis.

METHODS: CRISPR/Cas9 system directed by a multiple sgRNAs system was used in rabbit embryos to edit their LDLR and apoE genes. The LDLR and apoE genes of founder rabbits were sequenced, and their plasma lipids and lipoprotein profiles on a normal chow diet were analyzed, western blotting was also performed to evaluate the expression of apolipoprotein. Sudan IV and HE staining of aortic were performed to confirm the formation of atherosclerosis.

RESULTS: Six knockout (KO) rabbits by injection of both LDLR and apoE sgRNAs were obtained, including four LDLR KO rabbits and two LDLR/apoE double- KO rabbits. Sequence analysis of these KO rabbits revealed that they contained multiple mutations including indels, deletions, and substitutions, as well as two rabbit lines containing biallelic large fragment deletion in the LDLR region. Analysis of their plasma lipids and lipoprotein profiles of these rabbits fed on a normal chow diet revealed that all of these KO rabbits exhibited remarkable hyperlipidemia with total cholesterol levels increased by up to 10-fold over those of wild-type rabbits. Pathological examinations of two founder rabbits showed that KO rabbits developed prominent aortic and coronary atherosclerosis.

CONCLUSION: Large fragment deletions can be achieved in rabbits using Cas9 mRNA and multiple sgRNAs. LDLR KO along with LDLR/apoE double KO rabbits should provide a novel means for translational investigations of human hyperlipidemia and atherosclerosis.},
}

Animals
Animals, Genetically Modified
Apolipoproteins E/genetics
Atherosclerosis/etiology/genetics
*CRISPR-Cas Systems
*Disease Models, Animal
Female
Gene Editing/*methods
Gene Knockout Techniques
Hyperlipidemias/*genetics
Lipids/blood/genetics
Lipoproteins/blood/genetics
Pregnancy
RNA, Guide
Rabbits
Receptors, LDL/genetics

@article {pmid30828060,
year = {2019},
author = {Çiçek, YA and Luther, DC and Kretzmann, JA and Rotello, VM},
title = {Advances in CRISPR/Cas9 Technology for in Vivo Translation.},
journal = {Biological & pharmaceutical bulletin},
volume = {42},
number = {3},
pages = {304-311},
doi = {10.1248/bpb.b18-00811},
pmid = {30828060},
issn = {1347-5215},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy/*methods ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized therapeutic gene editing by providing researchers with a new method to study and cure diseases previously considered untreatable. While the full range and power of CRISPR technology for therapeutics is being elucidated through in vitro studies, translation to in vivo studies is slow. To date there is no totally effective delivery strategy to carry CRISPR components to the target site in vivo. The complexity of in vivo delivery is furthered by the number of potential delivery methods, the different forms in which CRISPR can be delivered as a therapeutic, and the disease target and tissue type in question. There are major challenges and limitations to delivery strategies, and it is imperative that future directions are guided by well-conducted studies that consider the full effect these variables have on the eventual outcome. In this review we will discuss the advances of the latest in vivo CRISPR/Cas9 delivery strategies and highlight the challenges yet to be overcome.},
}

CRISPR-Cas Systems
*Gene Editing
Genetic Therapy/*methods
Humans

@article {pmid30704461,
year = {2019},
author = {Malzahn, AA and Tang, X and Lee, K and Ren, Q and Sretenovic, S and Zhang, Y and Chen, H and Kang, M and Bao, Y and Zheng, X and Deng, K and Zhang, T and Salcedo, V and Wang, K and Zhang, Y and Qi, Y},
title = {Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis.},
journal = {BMC biology},
volume = {17},
number = {1},
pages = {9},
doi = {10.1186/s12915-019-0629-5},
pmid = {30704461},
issn = {1741-7007},
mesh = {Arabidopsis/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Genome, Plant ; Oryza/*genetics ; Plants, Genetically Modified/*genetics ; Temperature ; Zea mays/*genetics ; },
abstract = {BACKGROUND: CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing.

RESULTS: We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis.

CONCLUSION: Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.},
}

Arabidopsis/*genetics
*CRISPR-Cas Systems
*Gene Editing
Genome, Plant
Oryza/*genetics
Plants, Genetically Modified/*genetics
Temperature
Zea mays/*genetics

@article {pmid30542997,
year = {2019},
author = {Slipek, NJ and Varshney, J and Largaespada, DA},
title = {CRISPR/Cas9-Based Positive Screens for Cancer-Related Traits.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1907},
number = {},
pages = {137-144},
doi = {10.1007/978-1-4939-8967-6_11},
pmid = {30542997},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Genome, Human ; Genomics/*methods ; Humans ; Neoplasm Proteins/antagonists & inhibitors/*genetics ; Neoplasms/*genetics/pathology ; *Quantitative Trait Loci ; },
abstract = {Since the advent of large-scale, detailed descriptive cancer genomics studies at the beginning of the century, such as The Cancer Genome Atlas (TCGA), labs around the world have been working to make this data useful. Data like these can be made more useful by comparison with complementary functional genomic data. One new example is the application of CRISPR/Cas9-based library screening for cancer-related traits in cell lines. Such screens can reveal genome-wide suppressors of tumorigenesis and metastasis. Here we describe the use of widely available lentiviral libraries for such screens in cultured cell lines.},
}

*CRISPR-Cas Systems
Genome, Human
Genomics/*methods
Humans
Neoplasm Proteins/antagonists & inhibitors/*genetics
Neoplasms/*genetics/pathology
*Quantitative Trait Loci

@article {pmid30542996,
year = {2019},
author = {Adelmann, CH and Wang, T and Sabatini, DM and Lander, ES},
title = {Genome-Wide CRISPR/Cas9 Screening for Identification of Cancer Genes in Cell Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1907},
number = {},
pages = {125-136},
doi = {10.1007/978-1-4939-8967-6_10},
pmid = {30542996},
issn = {1940-6029},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Knockout Techniques ; *Genome, Human ; Genome-Wide Association Study/*methods ; Genomics/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; Neoplasm Proteins/antagonists & inhibitors/*genetics ; Neoplasms/diagnosis/*genetics ; Phenotype ; Tumor Cells, Cultured ; },
abstract = {In this protocol, pooled sgRNA libraries targeting thousands of genes are computationally designed, generated using microarray-based synthesis techniques, and packaged into lentiviral particles. Target cells of interest are transduced with the lentiviral sgRNA pools to generate a collection of knockout mutants-via Cas9-mediated genomic cleavage-and screened for a phenotype of interest. The relative abundance of each mutant in the population can be monitored over time through high-throughput sequencing of the integrated sgRNA expression cassettes. Using this technique, we outline strategies for the identification of cancer driver genes and genes mediating drug response.},
}

*CRISPR-Cas Systems
Gene Knockout Techniques
*Genome, Human
Genome-Wide Association Study/*methods
Genomics/*methods
High-Throughput Nucleotide Sequencing/*methods
Humans
Neoplasm Proteins/antagonists & inhibitors/*genetics
Neoplasms/diagnosis/*genetics
Phenotype
Tumor Cells, Cultured

@article {pmid30289463,
year = {2018},
author = {Zhang, S and Voigt, CA},
title = {Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design.},
journal = {Nucleic acids research},
volume = {46},
number = {20},
pages = {11115-11125},
pmid = {30289463},
issn = {1362-4962},
mesh = {Amino Acid Motifs ; Binding Sites ; *CRISPR-Cas Systems ; Escherichia coli/*genetics ; *Gene Expression Regulation, Bacterial ; Gene Regulatory Networks ; Metabolic Engineering ; Mutation ; Promoter Regions, Genetic ; RNA, Guide/*metabolism ; Synthetic Biology ; },
abstract = {Large synthetic genetic circuits require the simultaneous expression of many regulators. Deactivated Cas9 (dCas9) can serve as a repressor by having a small guide RNA (sgRNA) direct it to bind a promoter. The programmability and specificity of RNA:DNA basepairing simplifies the generation of many orthogonal sgRNAs that, in theory, could serve as a large set of regulators in a circuit. However, dCas9 is toxic in many bacteria, thus limiting how high it can be expressed, and low concentrations are quickly sequestered by multiple sgRNAs. Here, we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF). Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter. The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9. Further, PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF). A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates; however, the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold. This work introduces a non-toxic variant of dCas9, critical for its use in applications in metabolic engineering and synthetic biology, and exposes a limitation in the number of regulators that can be used in one cell when they rely on a shared resource.},
}

Amino Acid Motifs
Binding Sites
*CRISPR-Cas Systems
Escherichia coli/*genetics
*Gene Expression Regulation, Bacterial
Gene Regulatory Networks
Metabolic Engineering
Mutation
Promoter Regions, Genetic
RNA, Guide/*metabolism
Synthetic Biology

@article {pmid30184135,
year = {2018},
author = {van den Berg, J and G Manjón, A and Kielbassa, K and Feringa, FM and Freire, R and Medema, RH},
title = {A limited number of double-strand DNA breaks is sufficient to delay cell cycle progression.},
journal = {Nucleic acids research},
volume = {46},
number = {19},
pages = {10132-10144},
pmid = {30184135},
issn = {1362-4962},
mesh = {Aneuploidy ; CRISPR-Cas Systems ; Cell Cycle/*genetics ; Cell Cycle Checkpoints/*genetics ; Cell Line ; *DNA Breaks, Double-Stranded ; DNA Damage ; *DNA Repair ; Genome, Human/genetics ; Humans ; Mitosis/genetics ; },
abstract = {DNA damaging agents cause a variety of lesions, of which DNA double-strand breaks (DSBs) are the most genotoxic. Unbiased approaches aimed at investigating the relationship between the number of DSBs and outcome of the DNA damage response have been challenging due to the random nature in which damage is induced by classical DNA damaging agents. Here, we describe a CRISPR/Cas9-based system that permits us to efficiently introduce DSBs at defined sites in the genome. Using this system, we show that a guide RNA targeting only a single site in the human genome can trigger a checkpoint response that is potent enough to delay cell cycle progression. Abrogation of this checkpoint leads to DNA breaks in mitosis which gives rise to aneuploid progeny.},
}

Aneuploidy
CRISPR-Cas Systems
Cell Cycle/*genetics
Cell Cycle Checkpoints/*genetics
Cell Line
*DNA Breaks, Double-Stranded
DNA Damage
*DNA Repair
Genome, Human/genetics
Humans
Mitosis/genetics

@article {pmid30044923,
year = {2019},
author = {Cai, C and Sang, C and Du, J and Jia, H and Tu, J and Wan, Q and Bao, B and Xie, S and Huang, Y and Li, A and Li, J and Yang, K and Wang, S and Lu, Q},
title = {Knockout of tnni1b in zebrafish causes defects in atrioventricular valve development via the inhibition of the myocardial wnt signaling pathway.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {1},
pages = {696-710},
doi = {10.1096/fj.201800481RR},
pmid = {30044923},
issn = {1530-6860},
mesh = {Animals ; Animals, Genetically Modified/embryology/genetics/metabolism ; Atrioventricular Node/metabolism/*pathology ; CRISPR-Cas Systems ; Cells, Cultured ; Embryo, Nonmammalian/metabolism/*pathology ; Gene Expression Regulation, Developmental ; Heart Valves/embryology/metabolism/*pathology ; Myocardium/metabolism/*pathology ; Organogenesis ; Rats ; Troponin I/*antagonists & inhibitors/genetics/metabolism ; *Wnt Signaling Pathway ; Zebrafish/*embryology/genetics/metabolism ; Zebrafish Proteins/*antagonists & inhibitors/genetics/metabolism ; },
abstract = {The proper development of atrioventricular (AV) valves is critical for heart morphogenesis and for the formation of the cardiac conduction system. Defects in AV valve development are the most common type of congenital heart defect. Cardiac troponin I (ctnni), a structural and regulatory protein involved in cardiac muscle contraction, is a subunit of the troponin complex, but the functions and molecular mechanisms of ctnni during early heart development remain unclear. We created a knockout zebrafish model in which troponin I type 1b (tnni1b) (Tnni-HC, heart and craniofacial) was deleted using the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein system. In the homozygous mutant, the embryos showed severe pericardial edema, malformation of the heart tube, reduction of heart rate without contraction and with almost no blood flow, heart cavity congestion, and lack of an endocardial ring or valve leaflet, resulting in 88.8 ± 6.0% lethality at 7 d postfertilization. Deletion of tnni1b caused the abnormal expression of several markers involved in AV valve development, including bmp4, cspg2, has2, notch1b, spp1, and Alcam. Myocardial re-expression of tnni1b in mutants partially rescued the pericardial edema phenotype and AV canal (AVC) developmental defects. We further showed that tnni1b knockout in zebrafish and ctnni knockdown in rat h9c2 myocardial cells inhibited cardiac wnt signaling and that myocardial reactivation of wnt signaling partially rescued the abnormal expression of AVC markers caused by the tnni1b deletion. Taken together, our data suggest that tnni1b plays a vital role in zebrafish AV valve development by regulating the myocardial wnt signaling pathway.-Cai, C., Sang, C., Du, J., Jia, H., Tu, J., Wan, Q., Bao, B., Xie, S., Huang, Y., Li, A., Li, J., Yang, K., Wang, S., Lu, Q. Knockout of tnni1b in zebrafish causes defects in atrioventricular valve development via the inhibition of myocardial wnt signaling pathway.},
}

Animals
Animals, Genetically Modified/embryology/genetics/metabolism
Atrioventricular Node/metabolism/*pathology
CRISPR-Cas Systems
Cells, Cultured
Embryo, Nonmammalian/metabolism/*pathology
Gene Expression Regulation, Developmental
Heart Valves/embryology/metabolism/*pathology
Myocardium/metabolism/*pathology
Organogenesis
Rats
Troponin I/*antagonists & inhibitors/genetics/metabolism
*Wnt Signaling Pathway
Zebrafish/*embryology/genetics/metabolism
Zebrafish Proteins/*antagonists & inhibitors/genetics/metabolism

@article {pmid30032296,
year = {2018},
author = {Clements, KE and Thakar, T and Nicolae, CM and Liang, X and Wang, HG and Moldovan, GL},
title = {Loss of E2F7 confers resistance to poly-ADP-ribose polymerase (PARP) inhibitors in BRCA2-deficient cells.},
journal = {Nucleic acids research},
volume = {46},
number = {17},
pages = {8898-8907},
pmid = {30032296},
issn = {1362-4962},
support = {R01 ES026184/ES/NIEHS NIH HHS/United States ; },
mesh = {Antineoplastic Agents/*pharmacology ; BRCA2 Protein/*deficiency ; CRISPR-Cas Systems ; Cell Line, Tumor ; DNA Replication/drug effects/physiology ; DNA, Neoplasm/genetics/metabolism ; Drug Resistance, Neoplasm/*physiology ; E2F7 Transcription Factor/deficiency/*physiology ; Enzyme Inhibitors/*pharmacology ; Gene Knockout Techniques ; Genes, BRCA2 ; Humans ; Neoplasm Proteins/deficiency/*physiology ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Poly(ADP-ribose) Polymerases ; Rad51 Recombinase/biosynthesis/genetics ; Recombinational DNA Repair/drug effects/physiology ; },
abstract = {BRCA proteins are essential for homologous recombination (HR) DNA repair, and their germline or somatic inactivation is frequently observed in human tumors. Understanding the molecular mechanisms underlying the response of BRCA-deficient tumors to chemotherapy is paramount for developing improved personalized cancer therapies. While PARP inhibitors have been recently approved for treatment of BRCA-mutant breast and ovarian cancers, not all patients respond to this therapy, and resistance to these novel drugs remains a major clinical problem. Several mechanisms of chemoresistance in BRCA2-deficient cells have been identified. Rather than restoring normal recombination, these mechanisms result in stabilization of stalled replication forks, which can be subjected to degradation in BRCA2-mutated cells. Here, we show that the transcriptional repressor E2F7 modulates the chemosensitivity of BRCA2-deficient cells. We found that BRCA2-deficient cells are less sensitive to PARP inhibitor and cisplatin treatment after E2F7 depletion. Moreover, we show that the mechanism underlying this activity involves increased expression of RAD51, a target for E2F7-mediated transcriptional repression, which enhances both HR DNA repair, and replication fork stability in BRCA2-deficient cells. Our work describes a new mechanism of therapy resistance in BRCA2-deficient cells, and identifies E2F7 as a putative biomarker for tumor response to PARP inhibitor therapy.},
}

Antineoplastic Agents/*pharmacology
BRCA2 Protein/*deficiency
CRISPR-Cas Systems
Cell Line, Tumor
DNA Replication/drug effects/physiology
DNA, Neoplasm/genetics/metabolism
Drug Resistance, Neoplasm/*physiology
E2F7 Transcription Factor/deficiency/*physiology
Enzyme Inhibitors/*pharmacology
Gene Knockout Techniques
Genes, BRCA2
Humans
Neoplasm Proteins/deficiency/*physiology
Phthalazines/pharmacology
Piperazines/pharmacology
Poly(ADP-ribose) Polymerases
Rad51 Recombinase/biosynthesis/genetics
Recombinational DNA Repair/drug effects/physiology

@article {pmid30032250,
year = {2018},
author = {Schleicher, EM and Galvan, AM and Imamura-Kawasawa, Y and Moldovan, GL and Nicolae, CM},
title = {PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress.},
journal = {Nucleic acids research},
volume = {46},
number = {17},
pages = {8908-8916},
pmid = {30032250},
issn = {1362-4962},
support = {R01 ES026184/ES/NIEHS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis/*genetics ; Cell Division ; Cell Line, Transformed ; DNA Damage ; DNA Replication ; Female ; Gene Knockout Techniques ; HeLa Cells ; Heterografts ; Humans ; Mice ; Mice, Nude ; Neoplasm Proteins/deficiency/*physiology ; Poly(ADP-ribose) Polymerases/deficiency/*physiology ; Proto-Oncogene Proteins/deficiency/*physiology ; Retinal Pigment Epithelium/cytology ; Up-Regulation ; },
abstract = {During carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and difficult to replicate genomic regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein proliferating cell nuclear antigen and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP10 is overexpressed in a large proportion of human tumors. To understand the role of PARP10 in cellular transformation, we inactivated PARP10 in HeLa cancer cells by CRISPR/Cas9-mediated gene knockout, and overexpressed it in non-transformed RPE-1 cells. We found that PARP10 promotes cellular proliferation, and its overexpression alleviates cellular sensitivity to replication stress and fosters the restart of stalled replication forks. Importantly, mouse xenograft studies showed that loss of PARP10 reduces the tumorigenesis activity of HeLa cells, while its overexpression results in tumor formation by non-transformed RPE-1 cells. Our findings indicate that PARP10 promotes cellular transformation, potentially by alleviating replication stress and suggest that targeting PARP10 may represent a novel therapeutic approach.},
}

Animals
CRISPR-Cas Systems
Carcinogenesis/*genetics
Cell Division
Cell Line, Transformed
DNA Damage
DNA Replication
Female
Gene Knockout Techniques
HeLa Cells
Heterografts
Humans
Mice
Mice, Nude
Neoplasm Proteins/deficiency/*physiology
Poly(ADP-ribose) Polymerases/deficiency/*physiology
Proto-Oncogene Proteins/deficiency/*physiology
Retinal Pigment Epithelium/cytology
Up-Regulation

@article {pmid29992232,
year = {2018},
author = {Du, J and Kirk, B and Zeng, J and Ma, J and Wang, Q},
title = {Three classes of response elements for human PRC2 and MLL1/2-Trithorax complexes.},
journal = {Nucleic acids research},
volume = {46},
number = {17},
pages = {8848-8864},
pmid = {29992232},
issn = {1362-4962},
support = {R01 AI067839/AI/NIAID NIH HHS/United States ; R01 GM067801/GM/NIGMS NIH HHS/United States ; R01 GM116280/GM/NIGMS NIH HHS/United States ; R01 GM127628/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Chromatin Immunoprecipitation ; CpG Islands ; DNA-Binding Proteins/*genetics ; Epigenetic Repression/*genetics ; Gene Knockout Techniques ; Genes, Reporter ; HEK293 Cells ; HeLa Cells ; Histone Code/genetics ; Histone-Lysine N-Methyltransferase/*genetics ; Humans ; K562 Cells ; Multiprotein Complexes/*genetics ; Mutagenesis, Insertional ; Myeloid-Lymphoid Leukemia Protein/*genetics ; Neoplasm Proteins/*genetics ; Polycomb Repressive Complex 2/*genetics ; Polymerase Chain Reaction ; RNA Interference ; RNA, Small Interfering/genetics ; Response Elements/*genetics ; YY1 Transcription Factor/genetics ; },
abstract = {Polycomb group (PcG) and Trithorax group (TrxG) proteins are essential for maintaining epigenetic memory in both embryonic stem cells and differentiated cells. To date, how they are localized to hundreds of specific target genes within a vertebrate genome had remained elusive. Here, by focusing on short cis-acting DNA elements of single functions, we discovered three classes of response elements in human genome: Polycomb response elements (PREs), Trithorax response elements (TREs) and Polycomb/Trithorax response elements (P/TREs). In particular, the four PREs (PRE14, 29, 39 and 48) are the first set of, to our knowledge, bona fide vertebrate PREs ever discovered, while many previously reported Drosophila or vertebrate PREs are likely P/TREs. We further demonstrated that YY1 and CpG islands are specifically enriched in the four TREs (PRE30, 41, 44 and 55), but not in the PREs. The three classes of response elements as unraveled in this study should guide further global investigation and open new doors for a deeper understanding of PcG and TrxG mechanisms in vertebrates.},
}

CRISPR-Cas Systems
Chromatin Immunoprecipitation
CpG Islands
DNA-Binding Proteins/*genetics
Epigenetic Repression/*genetics
Gene Knockout Techniques
Genes, Reporter
HEK293 Cells
HeLa Cells
Histone Code/genetics
Histone-Lysine N-Methyltransferase/*genetics
Humans
K562 Cells
Multiprotein Complexes/*genetics
Mutagenesis, Insertional
Myeloid-Lymphoid Leukemia Protein/*genetics
Neoplasm Proteins/*genetics
Polycomb Repressive Complex 2/*genetics
Polymerase Chain Reaction
RNA Interference
RNA, Small Interfering/genetics
Response Elements/*genetics
YY1 Transcription Factor/genetics

@article {pmid29979630,
year = {2019},
author = {Schoenauer, R and Larpin, Y and Babiychuk, EB and Drücker, P and Babiychuk, VS and Avota, E and Schneider-Schaulies, S and Schumacher, F and Kleuser, B and Köffel, R and Draeger, A},
title = {Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {1},
pages = {275-285},
doi = {10.1096/fj.201800033R},
pmid = {29979630},
issn = {1530-6860},
mesh = {Bacterial Proteins/pharmacology ; Bacterial Toxins/*pharmacology ; Biological Transport ; CRISPR-Cas Systems ; Calcium/metabolism ; Cell Membrane/chemistry/drug effects/*metabolism ; Cell Survival ; Cell-Derived Microparticles/chemistry/drug effects/metabolism ; Heat-Shock Proteins/*pharmacology ; Hemolysin Proteins/*pharmacology ; Humans ; Sphingomyelin Phosphodiesterase/*antagonists & inhibitors/genetics/metabolism ; Streptolysins/*pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism ; },
abstract = {Bacterial pore-forming toxins compromise plasmalemmal integrity, leading to Ca2+ influx, leakage of the cytoplasm, and cell death. Such lesions can be repaired by microvesicular shedding or by the endocytic uptake of the injured membrane sites. Cells have at their disposal an entire toolbox of repair proteins for the identification and elimination of membrane lesions. Sphingomyelinases catalyze the breakdown of sphingomyelin into ceramide and phosphocholine. Sphingomyelin is predominantly localized in the outer leaflet, where it is hydrolyzed by acid sphingomyelinase (ASM) after lysosomal fusion with the plasma membrane. The magnesium-dependent neutral sphingomyelinase (NSM)-2 is found at the inner leaflet of the plasmalemma. Because either sphingomyelinase has been ascribed a role in the cellular stress response, we investigated their role in plasma membrane repair and cellular survival after treatment with the pore-forming toxins listeriolysin O (LLO) or pneumolysin (PLY). Jurkat T cells, in which ASM or NSM-2 was down-regulated [ASM knockdown (KD) or NSM-2 KD cells], showed inverse reactions to toxin-induced membrane damage: ASM KD cells displayed reduced toxin resistance, decreased viability, and defects in membrane repair. In contrast, the down-regulation of NSM-2 led to an increase in viability and enhanced plasmalemmal repair. Yet, in addition to the increased plasmalemmal repair, the enhanced toxin resistance of NSM-2 KD cells also appeared to be dependent on the activation of p38/MAPK, which was constitutively activated, whereas in ASM KD cells, the p38/MAPK activation was constitutively blunted.-Schoenauer, R., Larpin, Y., Babiychuk, E. B., Drücker, P., Babiychuk, V. S., Avota, E., Schneider-Schaulies, S., Schumacher, F., Kleuser, B., Köffel, R., Draeger, A. Down-regulation of acid sphingomyelinase and neutral sphingomyelinase-2 inversely determines the cellular resistance to plasmalemmal injury by pore-forming toxins.},
}

Bacterial Proteins/pharmacology
Bacterial Toxins/*pharmacology
Biological Transport
CRISPR-Cas Systems
Calcium/metabolism
Cell Membrane/chemistry/drug effects/*metabolism
Cell Survival
Cell-Derived Microparticles/chemistry/drug effects/metabolism
Heat-Shock Proteins/*pharmacology
Hemolysin Proteins/*pharmacology
Humans
Sphingomyelin Phosphodiesterase/*antagonists & inhibitors/genetics/metabolism
Streptolysins/*pharmacology
p38 Mitogen-Activated Protein Kinases/metabolism

@article {pmid29944446,
year = {2019},
author = {Robert, A and Tian, P and Adam, SA and Kittisopikul, M and Jaqaman, K and Goldman, RD and Gelfand, VI},
title = {Kinesin-dependent transport of keratin filaments: a unified mechanism for intermediate filament transport.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {1},
pages = {388-399},
doi = {10.1096/fj.201800604R},
pmid = {29944446},
issn = {1530-6860},
support = {P01 GM096971/GM/NIGMS NIH HHS/United States ; R01 GM052111/GM/NIGMS NIH HHS/United States ; R35 GM119619/GM/NIGMS NIH HHS/United States ; },
mesh = {Actin Cytoskeleton/metabolism ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; Intermediate Filaments/*metabolism ; Keratin-18/*metabolism ; Keratin-8/*metabolism ; Kinesin/antagonists & inhibitors/*physiology ; Mice ; Mice, Knockout ; Microscopy, Fluorescence ; Microtubules/*metabolism ; Retinal Pigment Epithelium/*metabolism ; Vimentin/*metabolism ; },
abstract = {Keratin intermediate filaments (IFs) are the major cytoskeletal component in epithelial cells. The dynamics of keratin IFs have been described to depend mostly on the actin cytoskeleton, but the rapid transport of fully polymerized keratin filaments has not been reported. In this work, we used a combination of photoconversion experiments and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 genome editing to study the role of microtubules and microtubule motors in keratin filament transport. We found that long keratin filaments, like other types of IFs, are transported along microtubules by kinesin-1. Our data revealed that keratin and vimentin are nonconventional kinesin-1 cargoes because their transport did not require kinesin light chains, which are a typical adapter for kinesin-dependent cargo transport. Furthermore, we found that the same domain of the kinesin heavy chain tail is involved in keratin and vimentin IF transport, strongly suggesting that multiple types of IFs move along microtubules using an identical mechanism.-Robert, A., Tian, P., Adam, S. A., Kittisopikul, M., Jaqaman, K., Goldman, R. D., Gelfand, V. I. Kinesin-dependent transport of keratin filaments: a unified mechanism for intermediate filament transport.},
}

Actin Cytoskeleton/metabolism
Animals
CRISPR-Cas Systems
Cells, Cultured
Intermediate Filaments/*metabolism
Keratin-18/*metabolism
Keratin-8/*metabolism
Kinesin/antagonists & inhibitors/*physiology
Mice
Mice, Knockout
Microscopy, Fluorescence
Microtubules/*metabolism
Retinal Pigment Epithelium/*metabolism
Vimentin/*metabolism

@article {pmid29929184,
year = {2018},
author = {Mikkelsen, JG},
title = {Viral delivery of genome-modifying proteins for cellular reprogramming.},
journal = {Current opinion in genetics & development},
volume = {52},
number = {},
pages = {92-99},
doi = {10.1016/j.gde.2018.06.005},
pmid = {29929184},
issn = {1879-0380},
mesh = {CRISPR-Cas Systems/*genetics ; Cellular Reprogramming/*genetics ; *Gene Editing ; Genetic Vectors ; Genome ; Humans ; Viruses/*genetics ; },
abstract = {Following the successful development of virus-based gene vehicles for genetic therapies, exploitation of viruses as carriers of genetic tools for cellular reprogramming and genome editing should be right up the street. However, whereas persistent, potentially life-long gene expression is the main goal of conventional genetic therapies, tools and bits for genome engineering should ideally be short-lived and active only for a limited time. Although viral vector systems have already been adapted for potent genome editing both in vitro and in vivo, regulatable gene expression systems or self-limiting expression circuits need to be implemented limiting exposure of chromatin to genome-modifying enzymes. As an alternative approach, emerging virus-based protein delivery technologies support direct protein delivery, providing a short, robust boost of enzymatic activity in transduced cells. Is this potentially the perfect way of shipping loads of cargo to many recipients and still maintain short-term activity?},
}

CRISPR-Cas Systems/*genetics
Cellular Reprogramming/*genetics
*Gene Editing
Genetic Vectors
Genome
Humans
Viruses/*genetics

@article {pmid29913329,
year = {2018},
author = {Shukla, A and Huangfu, D},
title = {Decoding the noncoding genome via large-scale CRISPR screens.},
journal = {Current opinion in genetics & development},
volume = {52},
number = {},
pages = {70-76},
pmid = {29913329},
issn = {1879-0380},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 DK096239/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Epigenomics/*trends ; Gene Editing/trends ; Gene Expression Regulation, Developmental/genetics ; Genome, Human/*genetics ; Genomics/trends ; Humans ; RNA Interference ; },
abstract = {Large portions of the human genome harbor functional noncoding elements, which can regulate a variety of biological processes and have important implications for disease risk and therapeutic outcomes. However, assigning specific functions to noncoding sequences remains a major challenge. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas) systems have emerged as a powerful approach for targeted genome and epigenome perturbation. CRISPR systems are now harnessed for high-throughput screening of the noncoding genome to uncover functional regulatory elements and to define their precise functions with superior speed. Here, we summarize the various tools developed for such screens in mammalian systems and discuss screening methods and technical considerations. We further highlight screens that are already transforming our understanding of gene regulation and disease mechanisms, consider the impact of such discoveries on the development of new therapeutics, and provide our viewpoint on the challenges for future development of the field.},
}

Animals
CRISPR-Cas Systems/*genetics
Epigenomics/*trends
Gene Editing/trends
Gene Expression Regulation, Developmental/genetics
Genome, Human/*genetics
Genomics/trends
Humans
RNA Interference

@article {pmid29879620,
year = {2018},
author = {Urnov, FD},
title = {Ctrl-Alt-inDel: genome editing to reprogram a cell in the clinic.},
journal = {Current opinion in genetics & development},
volume = {52},
number = {},
pages = {48-56},
doi = {10.1016/j.gde.2018.05.005},
pmid = {29879620},
issn = {1879-0380},
mesh = {CRISPR-Cas Systems/*genetics ; Cellular Reprogramming/*genetics ; *Gene Editing ; Genetic Engineering/trends ; Genetic Therapy/trends ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/cytology ; Humans ; },
abstract = {Genome editing with engineered nucleases (zinc finger, TAL effector, or CRISPR/Cas9-based) enables `write' access to regulatory programs executed by primary human cells. A decade of its clinical development, along with a reduction of conventional gene therapy to medical and commercial practice, has made cell reprogramming via editing a viable clinical modality. Reviewed here are the first examples of this to enter the clinic: ex vivo edited T cells for infectious disease and cancer, and hematopoietic stem/progenitor cells for the hemoglobinopathies. Three ongoing developments will ensure that the range of edited and reprogrammed cells to enter the clinic, and the scope of target indications, will grow markedly in the next five years: our ability to identify disease-relevant targets in noncoding regulatory DNA, which is uniquely suited for editing-based cell program control; recent reduction to clinical practice of in vivo editing; and progress in engineering and manufacture of differentiated cells from pluripotent progenitors.},
}

CRISPR-Cas Systems/*genetics
Cellular Reprogramming/*genetics
*Gene Editing
Genetic Engineering/trends
Genetic Therapy/trends
*Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells/cytology
Humans

@article {pmid29664675,
year = {2018},
author = {Byrne, FL and Olzomer, EM and Brink, R and Hoehn, KL},
title = {Knockout of glucose transporter GLUT6 has minimal effects on whole body metabolic physiology in mice.},
journal = {American journal of physiology. Endocrinology and metabolism},
volume = {315},
number = {2},
pages = {E286-E293},
doi = {10.1152/ajpendo.00082.2018},
pmid = {29664675},
issn = {1522-1555},
mesh = {Adiposity/genetics ; Animals ; Blood Glucose/metabolism ; Body Weight/genetics ; CRISPR-Cas Systems ; Diet ; Energy Metabolism/genetics ; Female ; Genotype ; Glucose/metabolism ; Glucose Tolerance Test ; Homeostasis/genetics ; Insulin/blood ; Male ; Metabolism/*genetics/*physiology ; Mice ; Mice, Knockout ; },
abstract = {Glucose transporter 6 (GLUT6) is a member of the facilitative glucose transporter family. GLUT6 is upregulated in several cancers but is not widely expressed in normal tissues. Previous studies have shown that GLUT6 knockdown kills endometrial cancer cells that express elevated levels of the protein. However, whether GLUT6 represents a viable anticancer drug target is unclear because the role of GLUT6 in normal metabolic physiology is unknown. Herein we generated GLUT6 knockout mice to determine how loss of GLUT6 affected whole body glucose homeostasis and metabolic physiology. We found that the mouse GLUT6 (Slc2a6) gene expression pattern was similar to humans with mRNA found primarily in brain and spleen. CRISPR-Cas9-mediated deletion of Slc2a6 did not alter mouse development, growth, or whole body glucose metabolism in male or female mice fed either a chow diet or Western diet. GLUT6 deletion did not impact glucose tolerance or blood glucose and insulin levels in male or female mice fed either diet. However, compared with wild-type littermate controls, GLUT6 null female mice had a relatively minor decrease in fat accumulation when fed Western diet and had a lower respiratory exchange ratio when fed chow diet. Collectively, these data show that GLUT6 is not a major regulator of whole body metabolic physiology; therefore, GLUT6 inhibition may have minimal adverse effects if targeted for cancer therapy.},
}

Adiposity/genetics
Animals
Blood Glucose/metabolism
Body Weight/genetics
CRISPR-Cas Systems
Diet
Energy Metabolism/genetics
Female
Genotype
Glucose/metabolism
Glucose Tolerance Test
Homeostasis/genetics
Insulin/blood
Male
Metabolism/*genetics/*physiology
Mice
Mice, Knockout

@article {pmid29631357,
year = {2018},
author = {Adeosun, SO and Gordon, DM and Weeks, MF and Moore, KH and Hall, JE and Hinds, TD and Stec, DE},
title = {Loss of biliverdin reductase-A promotes lipid accumulation and lipotoxicity in mouse proximal tubule cells.},
journal = {American journal of physiology. Renal physiology},
volume = {315},
number = {2},
pages = {F323-F331},
pmid = {29631357},
issn = {1522-1466},
support = {K01 HL125445/HL/NHLBI NIH HHS/United States ; P01 HL051971/HL/NHLBI NIH HHS/United States ; P20 GM104357/GM/NIGMS NIH HHS/United States ; U54 GM115428/GM/NIGMS NIH HHS/United States ; R01 HL088421/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Apoptosis/*drug effects ; CD36 Antigens/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Energy Metabolism/drug effects ; Gene Deletion ; Gene Editing/methods ; Kidney Tubules, Proximal/*drug effects/enzymology/pathology ; L-Lactate Dehydrogenase/metabolism ; Lipocalin-2/metabolism ; Lipogenesis/*drug effects ; Mice ; Mitochondria/drug effects/enzymology/pathology ; Oxidoreductases Acting on CH-CH Group Donors/*deficiency/genetics ; Palmitic Acid/metabolism/*toxicity ; Phosphorylation ; Triglycerides/metabolism ; bcl-Associated Death Protein/metabolism ; },
abstract = {Obesity and increased lipid availability have been implicated in the development and progression of chronic kidney disease. One of the major sites of renal lipid accumulation is in the proximal tubule cells of the kidney, suggesting that these cells may be susceptible to lipotoxicity. We previously demonstrated that loss of hepatic biliverdin reductase A (BVRA) causes fat accumulation in livers of mice on a high-fat diet. To determine the role of BVRA in mouse proximal tubule cells, we generated a CRISPR targeting BVRA for a knockout in mouse proximal tubule cells (BVRA KO). The BVRA KO cells had significantly less metabolic potential and mitochondrial respiration, which was exacerbated by treatment with palmitic acid, a saturated fatty acid. The BVRA KO cells also showed increased intracellular triglycerides which were associated with higher fatty acid uptake gene cluster of differentiation 36 as well as increased de novo lipogenesis as measured by higher neutral lipids. Additionally, neutrophil gelatinase-associated lipocalin 1 expression, annexin-V FITC staining, and lactate dehydrogenase assays all demonstrated that BVRA KO cells are more sensitive to palmitic acid-induced lipotoxicity than wild-type cells. Phosphorylation of BAD which plays a role in cell survival pathways, was significantly reduced in palmitic acid-treated BVRA KO cells. These data demonstrate the protective role of BVRA in proximal tubule cells against saturated fatty acid-induced lipotoxicity and suggest that activating BVRA could provide a benefit in protecting from obesity-induced kidney injury.},
}

Animals
Apoptosis/*drug effects
CD36 Antigens/metabolism
CRISPR-Cas Systems
Cells, Cultured
Energy Metabolism/drug effects
Gene Deletion
Gene Editing/methods
Kidney Tubules, Proximal/*drug effects/enzymology/pathology
L-Lactate Dehydrogenase/metabolism
Lipocalin-2/metabolism
Lipogenesis/*drug effects
Mice
Mitochondria/drug effects/enzymology/pathology
Oxidoreductases Acting on CH-CH Group Donors/*deficiency/genetics
Palmitic Acid/metabolism/*toxicity
Phosphorylation
Triglycerides/metabolism
bcl-Associated Death Protein/metabolism

@article {pmid29278415,
year = {2018},
author = {Alonso-Gutierrez, J and Koma, D and Hu, Q and Yang, Y and Chan, LJG and Petzold, CJ and Adams, PD and Vickers, CE and Nielsen, LK and Keasling, JD and Lee, TS},
title = {Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway.},
journal = {Biotechnology and bioengineering},
volume = {115},
number = {4},
pages = {1000-1013},
doi = {10.1002/bit.26530},
pmid = {29278415},
issn = {1097-0290},
mesh = {Biofuels/microbiology ; CRISPR-Cas Systems/*genetics ; Chromosomes, Bacterial/*genetics ; Escherichia coli/*genetics ; Genome, Bacterial/genetics ; Industrial Microbiology ; Metabolic Engineering ; Mevalonic Acid/*metabolism ; Microorganisms, Genetically-Modified/genetics ; Operon/genetics ; Sesquiterpenes/*metabolism ; Sucrose/metabolism ; },
abstract = {Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.},
}

Biofuels/microbiology
CRISPR-Cas Systems/*genetics
Chromosomes, Bacterial/*genetics
Escherichia coli/*genetics
Genome, Bacterial/genetics
Industrial Microbiology
Metabolic Engineering
Mevalonic Acid/*metabolism
Microorganisms, Genetically-Modified/genetics
Operon/genetics
Sesquiterpenes/*metabolism
Sucrose/metabolism

@article {pmid29259295,
year = {2017},
author = {Kanda, N and Ichikawa, M and Ono, A and Toyoda, A and Fujiyama, A and Abe, J and Tsuchikane, Y and Nishiyama, T and Sekimoto, H},
title = {CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {17873},
pmid = {29259295},
issn = {2045-2322},
mesh = {Amino Acid Sequence ; Arabidopsis Proteins/*genetics ; Biological Phenomena/genetics ; CRISPR-Cas Systems/genetics/*physiology ; Carrier Proteins/*genetics ; Closterium/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/physiology ; Gene Knockout Techniques/methods ; Protoplasts/physiology ; Reproduction/genetics/*physiology ; Sex Attractants/genetics/*physiology ; },
abstract = {Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt+) and mating-type minus (mt-). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt+ and mt- cells, respectively. PR-IP mediates the release of protoplasts from mt- cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt- cells of the C. psl. complex. When the knockout mt- cells were mixed with wild-type mt+ cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.},
}

Amino Acid Sequence
Arabidopsis Proteins/*genetics
Biological Phenomena/genetics
CRISPR-Cas Systems/genetics/*physiology
Carrier Proteins/*genetics
Closterium/genetics/*physiology
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics/physiology
Gene Knockout Techniques/methods
Protoplasts/physiology
Reproduction/genetics/*physiology
Sex Attractants/genetics/*physiology

@article {pmid29215041,
year = {2017},
author = {Ehrke-Schulz, E and Schiwon, M and Leitner, T and Dávid, S and Bergmann, T and Liu, J and Ehrhardt, A},
title = {CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {17113},
pmid = {29215041},
issn = {2045-2322},
mesh = {Adenoviridae/*genetics ; *CRISPR-Cas Systems ; Cells, Cultured ; DNA-Binding Proteins/genetics/metabolism ; Dystrophin/genetics/metabolism ; Gene Editing/*methods ; *Gene Transfer Techniques ; Genetic Vectors/*genetics ; HeLa Cells ; Humans ; Oncogene Proteins, Viral/genetics/metabolism ; Receptors, CCR5/genetics/metabolism ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.},
}

Adenoviridae/*genetics
*CRISPR-Cas Systems
Cells, Cultured
DNA-Binding Proteins/genetics/metabolism
Dystrophin/genetics/metabolism
Gene Editing/*methods
*Gene Transfer Techniques
Genetic Vectors/*genetics
HeLa Cells
Humans
Oncogene Proteins, Viral/genetics/metabolism
Receptors, CCR5/genetics/metabolism

@article {pmid29170458,
year = {2017},
author = {Christie, KA and Courtney, DG and DeDionisio, LA and Shern, CC and De Majumdar, S and Mairs, LC and Nesbit, MA and Moore, CBT},
title = {Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {16174},
pmid = {29170458},
issn = {2045-2322},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Corneal Dystrophies, Hereditary/genetics/therapy ; DNA End-Joining Repair/genetics ; Gene Editing/*methods ; Mice ; Mice, Mutant Strains ; Mutation/genetics ; Streptococcus pyogenes/enzymology ; },
abstract = {CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at "off-target" sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.},
}

Alleles
Animals
CRISPR-Cas Systems/genetics/physiology
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Corneal Dystrophies, Hereditary/genetics/therapy
DNA End-Joining Repair/genetics
Gene Editing/*methods
Mice
Mice, Mutant Strains
Mutation/genetics
Streptococcus pyogenes/enzymology

@article {pmid29162881,
year = {2017},
author = {Feng, X and Peng, C and Chen, Y and Liu, X and Feng, X and He, Y},
title = {Discrimination of CRISPR/Cas9-induced mutants of rice seeds using near-infrared hyperspectral imaging.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {15934},
pmid = {29162881},
issn = {2045-2322},
mesh = {Biomass ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Imaging, Three-Dimensional ; Mutation/*genetics ; Oryza/*genetics ; Principal Component Analysis ; Seeds/*genetics ; *Spectroscopy, Near-Infrared ; Support Vector Machine ; },
abstract = {Identifying individuals with target mutant phenotypes is a significant procedure in mutant exploitation for implementing genome editing technology in a crop breeding programme. In the present study, a rapid and non-invasive method was proposed to identify CRISPR/Cas9-induced rice mutants from their acceptor lines (huaidao-1 and nanjing46) using hyperspectral imaging in the near-infrared (NIR) range (874.41-1733.91 nm) combined with chemometric analysis. The hyperspectral imaging data were analysed using principal component analysis (PCA) for exploratory purposes, and a support vector machine (SVM) and an extreme learning machine (ELM) were applied to build discrimination models for classification. Meanwhile, PCA loadings and a successive projections algorithm (SPA) were used for extracting optimal spectral wavelengths. The SVM-SPA model achieved best performance, with classification accuracies of 93% and 92.75% being observed for calibration and prediction sets for huaidao-1 and 91.25% and 89.50% for nanjing46, respectively. Furthermore, the classification of mutant seeds was visualized on prediction maps by predicting the features of each pixel on individual hyperspectral images based on the SPA-SVM model. The above results indicated that NIR hyperspectral imaging together with chemometric data analysis could be a reliable tool for identifying CRISPR/Cas9-induced rice mutants, which would help to accelerate selection and crop breeding processes.},
}

Biomass
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Imaging, Three-Dimensional
Mutation/*genetics
Oryza/*genetics
Principal Component Analysis
Seeds/*genetics
*Spectroscopy, Near-Infrared
Support Vector Machine

@article {pmid29145633,
year = {2018},
author = {Zeng, Y and Cui, Y and Zhang, Y and Zhang, Y and Liang, M and Chen, H and Lan, J and Song, G and Lou, J},
title = {The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex.},
journal = {Nucleic acids research},
volume = {46},
number = {1},
pages = {350-361},
pmid = {29145633},
issn = {1362-4962},
mesh = {Bacterial Proteins/chemistry/genetics/*metabolism ; Base Sequence ; CRISPR-Associated Protein 9/chemistry/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA/chemistry/genetics/metabolism ; *DNA Cleavage ; Gene Editing/*methods ; Models, Molecular ; Nucleic Acid Conformation ; Protein Binding ; Protein Domains ; RNA, Guide/genetics/*metabolism ; Sequence Homology, Nucleic Acid ; Streptococcus pyogenes/enzymology/genetics ; },
abstract = {CRISPR-Cas9 system has been widely used for efficient genome editing. Although the structures of Cas9 protein in complex with single-guided RNA (sgRNA) and target DNA have been resolved, the molecular details about the formation of Cas9 endonuclease R-loop structure remain elusive. Here we examine the DNA cleavage activities of Streptococcus pyogenes Cas9 (SpyCas9) and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique. Our results show that Cas9-sgRNA complex divides the target DNA into several distinct domains: protospacer adjacent motif, linker, Seed, Middle and Tail. After seed pairing, the Cas9 transiently retains a semi-active conformation and induces the cleavage of either target or non-target strand. smFRET studies demonstrate that an intermediate state exists in prior to the formation of the fully stable R-loop complex. Kinetics analysis of this new intermediate state indicates that the lifetime of this state increases when the base-pairing length of guide-DNA hybrid duplex increases and reaches the maximum at the size of 18 bp. These data provide new insights into the process of R-loop formation and reveal the source of off-targeting in CRISPR/Cas9 system.},
}

Bacterial Proteins/chemistry/genetics/*metabolism
Base Sequence
CRISPR-Associated Protein 9/chemistry/genetics/*metabolism
*CRISPR-Cas Systems
DNA/chemistry/genetics/metabolism
*DNA Cleavage
Gene Editing/*methods
Models, Molecular
Nucleic Acid Conformation
Protein Binding
Protein Domains
RNA, Guide/genetics/*metabolism
Sequence Homology, Nucleic Acid
Streptococcus pyogenes/enzymology/genetics

@article {pmid29118424,
year = {2017},
author = {Coggins, NB and Stultz, J and O'Geen, H and Carvajal-Carmona, LG and Segal, DJ},
title = {Methods for Scarless, Selection-Free Generation of Human Cells and Allele-Specific Functional Analysis of Disease-Associated SNPs and Variants of Uncertain Significance.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {15044},
pmid = {29118424},
issn = {2045-2322},
support = {K12 CA138464/CA/NCI NIH HHS/United States ; R21 CA199631/CA/NCI NIH HHS/United States ; R21 CA204563/CA/NCI NIH HHS/United States ; T32 GM007377/GM/NIGMS NIH HHS/United States ; },
mesh = {Alleles ; Base Sequence ; CRISPR-Cas Systems ; Colorectal Neoplasms/genetics/metabolism/pathology ; Fanconi Anemia Complementation Group N Protein/genetics/metabolism ; Gene Editing/*methods ; Genetic Predisposition to Disease/*genetics ; Genome-Wide Association Study/*methods ; Genotype ; HCT116 Cells ; HEK293 Cells ; Humans ; Polymerase Chain Reaction/*methods ; *Polymorphism, Single Nucleotide ; Sequence Homology, Nucleic Acid ; Transcription Factor 7-Like 2 Protein/genetics/metabolism ; },
abstract = {With the continued emergence of risk loci from Genome-Wide Association studies and variants of uncertain significance identified from patient sequencing, better methods are required to translate these human genetic findings into improvements in public health. Here we combine CRISPR/Cas9 gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) genotyping technology to create scarless isogenic cell models of cancer variants in ~1 month. We successfully modeled two novel variants previously identified by our lab in the PALB2 gene in HEK239 cells, resulting in isogenic cells representing all three genotypes for both variants. We also modeled a known functional risk SNP of colorectal cancer, rs6983267, in HCT-116 cells. Cells with extremely low levels of gene editing could still be identified and isolated using this approach. We also introduce a novel molecular assay, ChIPnQASO (Chromatin Immunoprecipitation and Quantitative Allele-Specific Occupation), which uses the same technology to reveal allele-specific function of these variants at the DNA-protein interaction level. We demonstrated preferential binding of the transcription factor TCF7L2 to the rs6983267 risk allele over the non-risk. Our pipeline provides a platform for functional variant discovery and validation that is accessible and broadly applicable for the progression of efforts towards precision medicine.},
}

Alleles
Base Sequence
CRISPR-Cas Systems
Colorectal Neoplasms/genetics/metabolism/pathology
Fanconi Anemia Complementation Group N Protein/genetics/metabolism
Gene Editing/*methods
Genetic Predisposition to Disease/*genetics
Genome-Wide Association Study/*methods
Genotype
HCT116 Cells
HEK293 Cells
Humans
Polymerase Chain Reaction/*methods
*Polymorphism, Single Nucleotide
Sequence Homology, Nucleic Acid
Transcription Factor 7-Like 2 Protein/genetics/metabolism

@article {pmid28191783,
year = {2017},
author = {Hong, SN and Dunn, JC and Stelzner, M and Martín, MG},
title = {Concise Review: The Potential Use of Intestinal Stem Cells to Treat Patients with Intestinal Failure.},
journal = {Stem cells translational medicine},
volume = {6},
number = {2},
pages = {666-676},
pmid = {28191783},
issn = {2157-6564},
support = {U01 DK085535/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Lineage ; Cell Proliferation ; Gene Editing/*methods ; Gene Expression Regulation, Developmental ; Humans ; Intestines/pathology/physiopathology/*transplantation ; Recovery of Function ; *Regeneration ; Risk Factors ; Short Bowel Syndrome/genetics/pathology/physiopathology/*surgery ; Signal Transduction ; Stem Cell Transplantation/adverse effects/*methods ; *Stem Cells/metabolism/pathology ; Treatment Outcome ; },
abstract = {Intestinal failure is a rare life-threatening condition that results in the inability to maintain normal growth and hydration status by enteral nutrition alone. Although parenteral nutrition and whole organ allogeneic transplantation have improved the survival of these patients, current therapies are associated with a high risk for morbidity and mortality. Development of methods to propagate adult human intestinal stem cells (ISCs) and pluripotent stem cells raises the possibility of using stem cell-based therapy for patients with monogenic and polygenic forms of intestinal failure. Organoids have demonstrated the capacity to proliferate indefinitely and differentiate into the various cellular lineages of the gut. Genome-editing techniques, including the overexpression of the corrected form of the defective gene, or the use of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 to selectively correct the monogenic disease-causing variant within the stem cell, make autologous ISC transplantation a feasible approach. However, numerous techniques still need to be further optimized, including more robust ex vivo ISC expansion, native ISC ablation, and engraftment protocols. Large-animal models can to be used to develop such techniques and protocols and to establish the safety of autologous ISC transplantation because outcomes in such models can be extrapolated more readily to humans. Stem Cells Translational Medicine 2017;6:666-676.},
}

Animals
CRISPR-Cas Systems
Cell Differentiation
Cell Lineage
Cell Proliferation
Gene Editing/*methods
Gene Expression Regulation, Developmental
Humans
Intestines/pathology/physiopathology/*transplantation
Recovery of Function
*Regeneration
Risk Factors
Short Bowel Syndrome/genetics/pathology/physiopathology/*surgery
Signal Transduction
Stem Cell Transplantation/adverse effects/*methods
*Stem Cells/metabolism/pathology
Treatment Outcome

@article {pmid31280424,
year = {2019},
author = {Mitsui, R and Yamada, R and Ogino, H},
title = {CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals.},
journal = {World journal of microbiology & biotechnology},
volume = {35},
number = {7},
pages = {111},
doi = {10.1007/s11274-019-2688-8},
pmid = {31280424},
issn = {1573-0972},
support = {JP18K14069//the Japan Society for the Promotion of Science KAKENHI/ ; JP18KK0413//the Japan Society for the Promotion of Science KAKENHI/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.},
}

@article {pmid31279547,
year = {2019},
author = {Selle, K and Andersen, JM and Barrangou, R},
title = {Short communication: Transcriptional response to a large genomic island deletion in the dairy starter culture Streptococcus thermophilus.},
journal = {Journal of dairy science},
volume = {},
number = {},
pages = {},
doi = {10.3168/jds.2019-16397},
pmid = {31279547},
issn = {1525-3198},
abstract = {Streptococcus thermophilus is a lactic acid bacterium widely used in the syntrophic fermentation of milk into yogurt and cheese. Streptococcus thermophilus has adapted to ferment milk primarily through reductive genome evolution but also through acquisition of genes conferring proto-cooperation with Lactobacillus bulgaricus and efficient metabolism of milk macronutrients. Genomic analysis of Strep. thermophilus strains suggests that mobile genetic elements have contributed to genomic evolution through horizontal gene transfer and genomic plasticity. We previously used the endogenous type II CRISPR-Cas [clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated sequences (Cas)] system in Strep. thermophilus to isolate derivatives lacking the chromosomal mobile genetic element and expandable island that display decreased fitness under routine culturing conditions. Of note, the Lac operon and Leloir pathway genes were deleted in the largest expendable genomic island (102 kbp), rendering the strain incapable of acidifying milk. However, the removal of other open reading frames in the same island had unclear effects on the fitness and regulatory networks of Strep. thermophilus. To uncover the physiological basis for the observed phenotypic changes and underlying regulatory networks affected by deletion of the 102-kbp genomic island in Strep. thermophilus, we analyzed the transcriptome of the mutant that lacked ~5% of its genome. In addition to the loss of transcripts encoded by the deleted material, we detected a total of 56 genes that were differentially expressed, primarily encompassing 10 select operons. Several predicted metabolic pathways were affected, including amino acid and purine metabolism, oligopeptide transport, and iron transport. Collectively, these results suggest that deletion of a 102-kb genomic island in Strep. thermophilus influences compensatory transcription of starvation stress response genes and metabolic pathways involved in important niche-related adaptation.},
}

@article {pmid31279087,
year = {2019},
author = {Mion, S and Plener, L and Rémy, B and Daudé, D and Chabrière, É},
title = {Lactonase SsoPox modulates CRISPR-Cas expression in Gram-negative proteobacteria using AHL-based Quorum Sensing systems.},
journal = {Research in microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.resmic.2019.06.004},
pmid = {31279087},
issn = {1769-7123},
abstract = {Quorum sensing (QS) is a molecular communication system that bacteria use to harmonize the regulation of genes in a cell density-dependent manner. In proteobacteria, QS is involved, among others, in virulence, biofilm formation or CRISPR-Cas gene regulation. Here, we report for the first time the effect of a QS-interfering enzyme to alter the regulation of CRISPR-Cas systems in model and clinical strains of Pseudomonas aeruginosa, as well as in the marine bacterium Chromobacterium violaceum CV12472. The expression of CRISPR-Cas genes decreased in most cases suggesting that enzymatic disruption of QS is promising for modulating phage-bacteria interactions.},
}

@article {pmid31278272,
year = {2019},
author = {Liu, TY and Liu, JJ and Aditham, AJ and Nogales, E and Doudna, JA},
title = {Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3001},
doi = {10.1038/s41467-019-10780-2},
pmid = {31278272},
issn = {2041-1723},
support = {P01GM051487//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; F32GM122334//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
abstract = {Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.},
}

@article {pmid31277669,
year = {2019},
author = {Shabbir, MAB and Shabbir, MZ and Wu, Q and Mahmood, S and Sajid, A and Maan, MK and Ahmed, S and Naveed, U and Hao, H and Yuan, Z},
title = {CRISPR-cas system: biological function in microbes and its use to treat antimicrobial resistant pathogens.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {18},
number = {1},
pages = {21},
doi = {10.1186/s12941-019-0317-x},
pmid = {31277669},
issn = {1476-0711},
abstract = {The development of antibiotic resistance in bacteria is a major public health threat. Infection rates of resistant pathogens continue to rise against nearly all antimicrobials, which has led to development of different strategies to combat the antimicrobial resistance. In this review, we discuss how the newly popular CRISPR-cas system has been applied to combat antibiotic resistance in both extracellular and intracellular pathogens. We also review a recently developed method in which nano-size CRISPR complex was used without any phage to target the mecA gene. However, there is still challenge to practice these methods in field against emerging antimicrobial resistant pathogens.},
}

@article {pmid31276437,
year = {2019},
author = {Toro, N and Martínez-Abarca, F and Mestre, MR and González-Delgado, A},
title = {Multiple origins of reverse transcriptases linked to CRISPR-Cas systems.},
journal = {RNA biology},
volume = {},
number = {},
pages = {},
doi = {10.1080/15476286.2019.1639310},
pmid = {31276437},
issn = {1555-8584},
abstract = {Prokaryotic genomes harbor a plethora of uncharacterized reverse transcriptases (RTs). RTs phylogenetically related to those encoded by group II introns have been found associated with type III CRISPR-Cas systems, adjacent or fused at the C-terminus to Cas1. It is thought that these RTs may have a relevant function in the CRISPR immune response mediating spacer acquisition from RNA molecules. The origin and relationships of these RTs and the ways in which the various protein domains evolved remain matters of debate. We carried out a large survey of annotated RTs in databases (198,760 sequences), and constructed a large dataset of unique representative sequences (9,141). The combined phylogenetic reconstruction and identification of the RTs and their various protein domains in the vicinity of CRISPR adaptation and effector modules revealed three different origins for these RTs, consistent with their emergence on multiple occasions: a larger group that have evolved from group II intron RTs, and two minor lineages that may have arisen more recently from retron/retron-like sequences and Abi-P2 RTs, the latter associated with type I-C systems. We also identified a particular group of RTs associated with CRISPR-cas loci in clade 12, fused C-terminally to an archaeo-eukaryotic primase (AEP), a protein domain (AE-Prim_S_like) forming a particular family within the AEP proper clade. Together, these data provide new insight into the evolution of CRISPR-Cas/RT systems.},
}

@article {pmid31088931,
year = {2019},
author = {Gruffaz, M and Yuan, H and Meng, W and Liu, H and Bae, S and Kim, JS and Lu, C and Huang, Y and Gao, SJ},
title = {CRISPR-Cas9 Screening of Kaposi's Sarcoma-Associated Herpesvirus-Transformed Cells Identifies XPO1 as a Vulnerable Target of Cancer Cells.},
journal = {mBio},
volume = {10},
number = {3},
pages = {},
doi = {10.1128/mBio.00866-19},
pmid = {31088931},
issn = {2150-7511},
support = {R01 CA197153/CA/NCI NIH HHS/United States ; R01 CA096512/CA/NCI NIH HHS/United States ; R01 CA124332/CA/NCI NIH HHS/United States ; R01 CA213275/CA/NCI NIH HHS/United States ; R01 DE025465/DE/NIDCR NIH HHS/United States ; R01 CA177377/CA/NCI NIH HHS/United States ; R01 CA132637/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Cycle Checkpoints ; Cell Proliferation/*genetics ; Cell Transformation, Neoplastic/*genetics ; Early Detection of Cancer ; Genes, p53 ; Herpesvirus 8, Human/*pathogenicity ; Humans ; Karyopherins/antagonists & inhibitors/*genetics ; Leukemia, Promyelocytic, Acute ; Liver Neoplasms ; Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors/*genetics ; Sequestosome-1 Protein/genetics ; Stomach Neoplasms ; Tumor Cells, Cultured ; },
abstract = {The abnormal proliferation of cancer cells is driven by deregulated oncogenes or tumor suppressors, among which the cancer-vulnerable genes are attractive therapeutic targets. Targeting mislocalization of oncogenes and tumor suppressors resulting from aberrant nuclear export is effective for inhibiting growth transformation of cancer cells. We performed a clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) screening in a unique model of matched primary and oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed cells and identified genes that were growth promoting and growth suppressive for both types of cells, among which exportin XPO1 was demonstrated to be critical for the survival of transformed cells. Using XPO1 inhibitor KPT-8602 and by small interfering RNA (siRNA) knockdown, we confirmed the essential role of XPO1 in cell proliferation and growth transformation of KSHV-transformed cells and in cell lines of other cancers, including gastric cancer and liver cancer. XPO1 inhibition induced cell cycle arrest through p53 activation, but the mechanisms of p53 activation differed among the different types of cancer cells. p53 activation depended on the formation of promyelocytic leukemia (PML) nuclear bodies in gastric cancer and liver cancer cells. Mechanistically, XPO1 inhibition induced relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. Taken the data together, we have identified novel growth-promoting and growth-suppressive genes of primary and cancer cells and have demonstrated that XPO1 is a vulnerable target of cancer cells. XPO1 inhibition induces cell arrest through a novel PML- and p62-dependent mechanism of p53 activation in some types of cancer cells.IMPORTANCE Using a model of oncogenic virus KSHV-driven cellular transformation of primary cells, we have performed a genome-wide CRISPR-Cas9 screening to identify vulnerable genes of cancer cells. This screening is unique in that this virus-induced oncogenesis model does not depend on any cellular genetic alterations and has matched primary and KSHV-transformed cells, which are not available for similar screenings in other types of cancer. We have identified genes that are both growth promoting and growth suppressive in primary and transformed cells, some of which could represent novel proto-oncogenes and tumor suppressors. In particular, we have demonstrated that the exportin XPO1 is a critical factor for the survival of transformed cells. Using a XPO1 inhibitor (KPT-8602) and siRNA-mediated knockdown, we have confirmed the essential role of XPO1 in cell proliferation and in growth transformation of KSHV-transformed cells, as well as of gastric and liver cancer cells. XPO1 inhibition induces cell cycle arrest by activating p53, but the mechanisms of p53 activation differed among different types of cancer cells. p53 activation is dependent on the formation of PML nuclear bodies in gastric and liver cancer cells. Mechanistically, XPO1 inhibition induces relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. These results illustrate that XPO1 is a vulnerable target of cancer cells and reveal a novel mechanism for blocking cancer cell proliferation by XPO1 inhibition as well as a novel PML- and p62-mediated mechanism of p53 activation in some types of cancer cells.},
}

CRISPR-Cas Systems
Cell Cycle Checkpoints
Cell Proliferation/*genetics
Cell Transformation, Neoplastic/*genetics
Early Detection of Cancer
Genes, p53
Herpesvirus 8, Human/*pathogenicity
Humans
Karyopherins/antagonists & inhibitors/*genetics
Leukemia, Promyelocytic, Acute
Liver Neoplasms
Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors/*genetics
Sequestosome-1 Protein/genetics
Stomach Neoplasms
Tumor Cells, Cultured

@article {pmid30887099,
year = {2019},
author = {Xiang, X and Luo, L and Nodzyński, M and Li, C and Han, P and Dou, H and Petersen, TS and Liang, X and Pan, X and Qu, K and Yang, L and Dang, Y and Liu, X and Bolund, L and Zhang, X and Tong, G and Xing, Y and Luo, Y and Lin, L},
title = {LION: a simple and rapid method to achieve CRISPR gene editing.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {76},
number = {13},
pages = {2633-2645},
doi = {10.1007/s00018-019-03064-x},
pmid = {30887099},
issn = {1420-9071},
support = {SZSM201612074//Sanming Project of Medicine in Shenzhen/ ; DFF-1337-00128//Teknologi og Produktion, Det Frie Forskningsråd/ ; DFF-1335-00763A//Teknologi og Produktion, Det Frie Forskningsråd/ ; 8048-00072A//Teknologi og Produktion, Det Frie Forskningsråd/ ; AU-iCRISPR//Sundhedsvidenskabelige Fakultet, Aarhus Universitet/ ; R219-2016-1375//Lundbeckfonden (DK)/ ; },
mesh = {Breast/*metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; Female ; Gene Editing/*methods ; Gene Targeting ; Genetic Vectors/*administration & dosage/genetics ; HEK293 Cells ; Humans ; Ovarian Neoplasms/*genetics ; *RNA, Guide ; Uterine Cervical Neoplasms/*genetics ; },
abstract = {The RNA-guided CRISPR-Cas9 technology has paved the way for rapid and cost-effective gene editing. However, there is still a great need for effective methods for rapid generation and validation of CRISPR/Cas9 gRNAs. Previously, we have demonstrated that highly efficient generation of multiplexed CRISPR guide RNA (gRNA) expression array can be achieved with Golden Gate Assembly (GGA). Here, we present an optimized and rapid method for generation and validation in less than 1 day of CRISPR gene targeting vectors. The method (LION) is based on ligation of double-stranded gRNA oligos into CRISPR vectors with GGA followed by nucleic acid purification. Using a dual-fluorescent reporter vector (C-Check), T7E1 assay, TIDE assay and a traffic light reporter assay, we proved that the LION-based generation of CRISPR vectors are functionally active, and equivalent to CRISPR plasmids generated by traditional methods. We also tested the activity of LION CRISPR vectors in different human cell types. The LION method presented here advances the rapid functional validation and application of CRISPR system for gene editing and simplified the CRISPR gene-editing procedures.},
}

Breast/*metabolism
*CRISPR-Cas Systems
Cells, Cultured
Female
Gene Editing/*methods
Gene Targeting
Genetic Vectors/*administration & dosage/genetics
HEK293 Cells
Humans
Ovarian Neoplasms/*genetics
*RNA, Guide
Uterine Cervical Neoplasms/*genetics

@article {pmid29596744,
year = {2018},
author = {Li, SY and Liu, JK and Zhao, GP and Wang, J},
title = {CADS: CRISPR/Cas12a-Assisted DNA Steganography for Securing the Storage and Transfer of DNA-Encoded Information.},
journal = {ACS synthetic biology},
volume = {7},
number = {4},
pages = {1174-1178},
doi = {10.1021/acssynbio.8b00074},
pmid = {29596744},
issn = {2161-5063},
mesh = {CRISPR-Cas Systems/*genetics ; Computer Security ; *DNA ; DNA Primers ; DNA, Single-Stranded ; Escherichia coli/genetics ; HEK293 Cells ; Humans ; Information Storage and Retrieval/*methods ; Polymerase Chain Reaction ; RNA ; },
abstract = {Because DNA has the merit of high capacity and complexity, DNA steganography, which conceals DNA-encoded messages, is very promising in information storage. The classical DNA steganography method hides DNA with a "secret message" in a mount of junk DNA, and the message can be extracted by polymerase chain reaction (PCR) using specific primers (key), followed by DNA sequencing and sequence decoding. As leakage of the primer information may result in message insecurity, new methods are needed to better secure the DNA information. Here, we develop a pre-key by either mixing specific primers (real key) with nonspecific primers (fake key) or linking a real key with 3'-end redundant sequences. Then, the single-stranded DNA (ssDNA) trans cleavage activity of CRISPR/Cas12a is employed to cut a fake key or remove the 3'-end redundant sequences, generating a real key for further information extraction. Therefore, with the Cas12a-assisted DNA steganography method, both storage and transfer of DNA-encoding data can be better protected.},
}

CRISPR-Cas Systems/*genetics
Computer Security
*DNA
DNA Primers
DNA, Single-Stranded
Escherichia coli/genetics
HEK293 Cells
Humans
Information Storage and Retrieval/*methods
Polymerase Chain Reaction
RNA