@article {pmid31934359,
year = {2020},
author = {Li, H and Yang, Y and Hong, W and Huang, M and Wu, M and Zhao, X},
title = {Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects.},
journal = {Signal transduction and targeted therapy},
volume = {5},
number = {},
pages = {1},
doi = {10.1038/s41392-019-0089-y},
pmid = {31934359},
issn = {2059-3635},
abstract = {Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.},
}

@article {pmid31932164,
year = {2020},
author = {Lau, RK and Ye, Q and Birkholz, EA and Berg, KR and Patel, L and Mathews, IT and Watrous, JD and Ego, K and Whiteley, AT and Lowey, B and Mekalanos, JJ and Kranzusch, PJ and Jain, M and Pogliano, J and Corbett, KD},
title = {Structure and Mechanism of a Cyclic Trinucleotide-Activated Bacterial Endonuclease Mediating Bacteriophage Immunity.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2019.12.010},
pmid = {31932164},
issn = {1097-4164},
abstract = {Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.},
}

@article {pmid31615983,
year = {2019},
author = {Wei, L and Lee, D and Law, CT and Zhang, MS and Shen, J and Chin, DW and Zhang, A and Tsang, FH and Wong, CL and Ng, IO and Wong, CC and Wong, CM},
title = {Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4681},
pmid = {31615983},
issn = {2041-1723},
mesh = {Antineoplastic Agents/*therapeutic use ; Apoptosis ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*drug therapy/genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/*genetics ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Humans ; Liver Neoplasms/*drug therapy/genetics ; Phenylurea Compounds/therapeutic use ; Phosphoglycerate Dehydrogenase/antagonists & inhibitors/*genetics ; Pyridines/therapeutic use ; Quinolines/therapeutic use ; Reactive Oxygen Species/metabolism ; Sorafenib/*therapeutic use ; },
abstract = {Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common. By using genome-wide CRISPR/Cas9 library screening, we identify phosphoglycerate dehydrogenase (PHGDH), the first committed enzyme in the serine synthesis pathway (SSP), as a critical driver for Sorafenib resistance. Sorafenib treatment activates SSP by inducing PHGDH expression. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that inactivation of PHGDH paralyzes the SSP and reduce the production of αKG, serine, and NADPH. Concomitantly, inactivation of PHGDH elevates ROS level and induces HCC apoptosis upon Sorafenib treatment. More strikingly, treatment of PHGDH inhibitor NCT-503 works synergistically with Sorafenib to abolish HCC growth in vivo. Similar findings are also obtained in other FDA-approved tyrosine kinase inhibitors (TKIs), including Regorafenib or Lenvatinib. In summary, our results demonstrate that targeting PHGDH is an effective approach to overcome TKI drug resistance in HCC.},
}

Antineoplastic Agents/*therapeutic use
Apoptosis
CRISPR-Cas Systems
Carcinoma, Hepatocellular/*drug therapy/genetics
Cell Line, Tumor
Drug Resistance, Neoplasm/*genetics
Gene Knock-In Techniques
Gene Knockout Techniques
Humans
Liver Neoplasms/*drug therapy/genetics
Phenylurea Compounds/therapeutic use
Phosphoglycerate Dehydrogenase/antagonists & inhibitors/*genetics
Pyridines/therapeutic use
Quinolines/therapeutic use
Reactive Oxygen Species/metabolism
Sorafenib/*therapeutic use

@article {pmid31404948,
year = {2019},
author = {Wu, TM and Huang, JZ and Oung, HM and Hsu, YT and Tsai, YC and Hong, CY},
title = {H2O2-Based Method for Rapid Detection of Transgene-Free Rice Plants from Segregating CRISPR/Cas9 Genome-Edited Progenies.},
journal = {International journal of molecular sciences},
volume = {20},
number = {16},
pages = {},
doi = {10.3390/ijms20163885},
pmid = {31404948},
issn = {1422-0067},
support = {MOST 105-2628-B-002 -036 -MY3//Ministry of Science and Technology, Taiwan/ ; },
mesh = {CRISPR-Cas Systems ; Gene Editing ; *Genome, Plant ; Hydrogen Peroxide/*analysis ; Oryza/*genetics ; Phosphotransferases (Alcohol Group Acceptor)/genetics ; Plant Proteins/genetics ; Plants, Genetically Modified/*genetics ; Transgenes ; },
abstract = {Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3'-diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.},
}

CRISPR-Cas Systems
Gene Editing
*Genome, Plant
Hydrogen Peroxide/*analysis
Oryza/*genetics
Phosphotransferases (Alcohol Group Acceptor)/genetics
Plant Proteins/genetics
Plants, Genetically Modified/*genetics
Transgenes

@article {pmid31359433,
year = {2019},
author = {Liu, D and Awazu, A and Sakuma, T and Yamamoto, T and Sakamoto, N},
title = {Establishment of knockout adult sea urchins by using a CRISPR-Cas9 system.},
journal = {Development, growth & differentiation},
volume = {61},
number = {6},
pages = {378-388},
doi = {10.1111/dgd.12624},
pmid = {31359433},
issn = {1440-169X},
support = {JP17K07241//JSPS KAKENHI/ ; },
mesh = {Animals ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Gene Editing/methods ; Gene Knockout Techniques/methods ; Sea Urchins/*embryology/*genetics ; },
abstract = {Sea urchins are used as a model organism for research on developmental biology and gene regulatory networks during early development. Gene knockdown by microinjection of morpholino antisense oligonucleotide (MASO) has been used to analyze gene function in early sea urchin embryos. However, as the effect of MASO is not long lasting, it is impossible to perturb genes expressed during late development by MASO. Recent advances in genome editing technologies have enabled gene modification in various organisms. We previously reported genome editing in the sea urchin Hemicentrotus pulcherrimus using zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN); however, the efficiencies of these technologies were not satisfactory. Here, we applied clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology to knock out the Pks1 gene in H. pulcherrimus. When sgRNAs targeting Pks1, which is required for the biosynthesis of larval pigment, were microinjected into fertilized eggs with SpCas9 mRNA, high-efficiency mutagenesis was achieved within 24 hr post fertilization and SpCas9/sgRNA-injected pluteus larvae had an albino phenotype. One of the sgRNAs yielded 100% mutagenesis efficiency, and no off-target effect was detected. In addition, the albino phenotype was maintained in juvenile sea urchins after metamorphosis, and the knockout sea urchins survived for at least one year and grew to albino adult sea urchins. These findings suggest that knockout adult sea urchins were successfully established and the CRISPR-Cas9 system is a feasible method for analyzing gene functions from late developmental to adult stage.},
}

Animals
*CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Gene Editing/methods
Gene Knockout Techniques/methods
Sea Urchins/*embryology/*genetics

@article {pmid31278987,
year = {2019},
author = {Wang, Y and Chen, X and Liu, Z and Xu, J and Li, X and Bi, H and Andongma, AA and Niu, C and Huang, Y},
title = {Mutation of doublesex induces sex-specific sterility of the diamondback moth Plutella xylostella.},
journal = {Insect biochemistry and molecular biology},
volume = {112},
number = {},
pages = {103180},
doi = {10.1016/j.ibmb.2019.103180},
pmid = {31278987},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Cas Systems ; Female ; Gene Expression Regulation, Developmental ; Genitalia/abnormalities ; Infertility/genetics ; Male ; Moths/*genetics/growth & development ; Mutation ; Phylogeny ; Sequence Alignment ; Sex Differentiation/*genetics ; },
abstract = {DOUBLESEX (DSX): the downstream gene in the insect sex determination pathway, plays a critical role in sexual differentiation and development. The functions of dsx have been characterized in several model insect species. However, the molecular mechanism and functions of sex determination of dsx in Plutella xylostella, an agricultural pest, are still unknown. In present study, we identified a male-specific and three female-specific Pxdsx transcripts in P. xylostella. Phylogenetic analyses and multiple sequence alignment revealed that Pxdsx is highly conserved in lepidopterans. The CRISPR/Cas9 technology was used to induce mutations in the male-specific isoform, the female-specific isoform, and common regions of Pxdsx. Disruptions of Pxdsx sex-specific isoforms caused sex-specific defects in external genitals and partial sexual reversal. In addition, we found that female specific transcripts were detected in PxdsxM male mutants and male-specific transcripts were detected in PxdsxF female mutants. Mutations also caused changes in expression of several sex-biased genes and induced sex-specific sterility. This study demonstrates that Pxdsx plays a key role in sex determination of P. xylostella and suggests novel genetic control approaches for the management of P. xylostella.},
}

Animals
CRISPR-Cas Systems
Female
Gene Expression Regulation, Developmental
Genitalia/abnormalities
Infertility/genetics
Male
Moths/*genetics/growth & development
Mutation
Phylogeny
Sequence Alignment
Sex Differentiation/*genetics

@article {pmid31248723,
year = {2019},
author = {Segal, DJ},
title = {The Right Tools for the Right Job: CRISPR-pass Could Offer Safe Gene Correction for Many Disorders.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {8},
pages = {1346-1347},
doi = {10.1016/j.ymthe.2019.06.008},
pmid = {31248723},
issn = {1525-0024},
mesh = {Adenine ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Codon, Nonsense ; Gene Editing ; },
}

Adenine
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Codon, Nonsense
Gene Editing

@article {pmid30569609,
year = {2019},
author = {Parry, G and Harrison, CJ},
title = {GARNet gene editing workshop.},
journal = {The New phytologist},
volume = {221},
number = {2},
pages = {640-643},
doi = {10.1111/nph.15573},
pmid = {30569609},
issn = {1469-8137},
support = {//New Phytologist Trust/International ; //Bristol Centre for Agricultural Innovation/International ; BB/M004376/1//UKRI BBSRC- funded GARNet2020/International ; },
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Endonucleases ; *Gene Editing/legislation & jurisprudence ; Legislation as Topic ; Plants/*genetics ; Plants, Genetically Modified ; },
}

Base Sequence
CRISPR-Cas Systems/genetics
Endonucleases
*Gene Editing/legislation & jurisprudence
Legislation as Topic
Plants/*genetics
Plants, Genetically Modified

@article {pmid30375858,
year = {2019},
author = {White, J and Dhingra, SK and Deng, X and El Mazouni, F and Lee, MCS and Afanador, GA and Lawong, A and Tomchick, DR and Ng, CL and Bath, J and Rathod, PK and Fidock, DA and Phillips, MA},
title = {Identification and Mechanistic Understanding of Dihydroorotate Dehydrogenase Point Mutations in Plasmodium falciparum that Confer in Vitro Resistance to the Clinical Candidate DSM265.},
journal = {ACS infectious diseases},
volume = {5},
number = {1},
pages = {90-101},
doi = {10.1021/acsinfecdis.8b00211},
pmid = {30375858},
issn = {2373-8227},
support = {R01 AI090141/AI/NIAID NIH HHS/United States ; R01 AI093380/AI/NIAID NIH HHS/United States ; R01 AI103058/AI/NIAID NIH HHS/United States ; R01 AI103947/AI/NIAID NIH HHS/United States ; },
mesh = {Amino Acid Sequence ; Animals ; Antimalarials/*pharmacology ; Binding Sites ; CRISPR-Cas Systems ; Crystallography, X-Ray ; Drug Resistance/*genetics ; Gene Editing ; Humans ; Malaria, Falciparum ; Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors/*genetics ; Plasmodium falciparum/*drug effects/enzymology/*genetics ; *Point Mutation ; Pyrimidines/*pharmacology ; Triazoles/*pharmacology ; },
abstract = {Malaria is one of the most challenging human infectious diseases, and both prevention and control have been hindered by the development of Plasmodium falciparum resistance to existing therapies. Several new compounds with novel mechanisms are in clinical development for the treatment of malaria, including DSM265, an inhibitor of Plasmodium dihydroorotate dehydrogenase. To explore the mechanisms by which resistance might develop to DSM265 in the field, we selected for DSM265-resistant P. falciparum parasites in vitro. Any of five different amino acid changes led to reduced efficacy on the parasite and to decreased DSM265 binding to P. falciparum DHODH. The DSM265-resistant parasites retained full sensitivity to atovaquone. All but one of the observed mutations were in the DSM265 binding site, and the remaining C276F was in the adjacent flavin cofactor site. The C276F mutation was previously identified in a recrudescent parasite during a Phase IIa clinical study. We confirmed that this mutation (and the related C276Y) accounted for the full level of observed DSM265 resistance by regenerating the mutation using CRISPR/Cas9 genome editing. X-ray structure analysis of the C276F mutant enzyme showed that conformational changes of nearby residues were required to accommodate the larger F276 residue, which in turn led to a restriction in the size of the DSM265 binding pocket. These findings underscore the importance of developing DSM265 as part of a combination therapy with other agents for successful use against malaria.},
}

Amino Acid Sequence
Animals
Antimalarials/*pharmacology
Binding Sites
CRISPR-Cas Systems
Crystallography, X-Ray
Drug Resistance/*genetics
Gene Editing
Humans
Malaria, Falciparum
Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors/*genetics
Plasmodium falciparum/*drug effects/enzymology/*genetics
*Point Mutation
Pyrimidines/*pharmacology
Triazoles/*pharmacology

@article {pmid30317650,
year = {2019},
author = {Liang, W and van Wersch, S and Tong, M and Li, X},
title = {TIR-NB-LRR immune receptor SOC3 pairs with truncated TIR-NB protein CHS1 or TN2 to monitor the homeostasis of E3 ligase SAUL1.},
journal = {The New phytologist},
volume = {221},
number = {4},
pages = {2054-2066},
doi = {10.1111/nph.15534},
pmid = {30317650},
issn = {1469-8137},
support = {9975971//NSF Plant Genome Program Award/International ; //China Scholarship Council (CSC)/International ; //NSERC-Discovery program/International ; //CFI/International ; },
mesh = {Arabidopsis/genetics ; Arabidopsis Proteins/genetics/*metabolism ; Autoimmunity ; Base Sequence ; CRISPR-Cas Systems/genetics ; Gene Deletion ; Gene Expression Regulation, Plant ; *Homeostasis ; NLR Proteins/genetics/metabolism ; Protein Binding ; Receptors, Cell Surface/*metabolism ; Receptors, Immunologic/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; },
abstract = {Intracellular nucleotide binding (NB) and leucine-rich repeat (NLR) proteins function as immune receptors to recognize effectors from pathogens. They often guard host proteins that are the direct targets of those effectors. Recent findings have revealed that a typical NLR sometimes cooperates with another atypical NLR for effector recognition. Here, by using the CRISPR/Cas9 gene editing method, knockout analysis and biochemical assays, we uncovered differential pairings of typical Toll Interleukin1 receptor (TIR) type NLR (TNL) receptor SOC3 with atypical truncated TIR-NB (TN) proteins CHS1 or TN2 to guard the homeostasis of the E3 ligase SAUL1. Overaccumulation of SAUL1 is monitored by the SOC3-TN2 pair, while SAUL1's disappearance is guarded by the SOC3-CHS1 pair. SOC3 forms a head-to-head genomic arrangement with CHS1 and TN2, indicative of transcriptional co-regulation. Such intricate cooperative interactions can probably enlarge the recognition spectrum and increase the functional flexibility of NLRs, which can partly explain the overwhelming occurrence of NLR gene clustering in higher plants.},
}

Arabidopsis/genetics
Arabidopsis Proteins/genetics/*metabolism
Autoimmunity
Base Sequence
CRISPR-Cas Systems/genetics
Gene Deletion
Gene Expression Regulation, Plant
*Homeostasis
NLR Proteins/genetics/metabolism
Protein Binding
Receptors, Cell Surface/*metabolism
Receptors, Immunologic/genetics/metabolism
Ubiquitin-Protein Ligases/genetics/metabolism

@article {pmid30242813,
year = {2018},
author = {Hildebrandt, CC and Marron, JM},
title = {Justice in CRISPR/Cas9 Research and Clinical Applications.},
journal = {AMA journal of ethics},
volume = {20},
number = {9},
pages = {E826-833},
doi = {10.1001/amajethics.2018.826},
pmid = {30242813},
issn = {2376-6980},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Databases, Genetic/ethics ; Ethics, Clinical ; Ethics, Research ; Ethnic Groups ; Gene Editing/*ethics ; Genetic Therapy/*ethics ; *Health Equity ; *Healthcare Disparities ; Humans ; Minority Groups ; *Social Justice ; },
abstract = {CRISPR/Cas9 is a rapidly developing gene editing technology that will soon have many clinical applications. As with many other new technologies, somatic gene editing with CRISPR/Cas9 raises concerns about equitable access to therapies by historically disenfranchised racial and ethnic minorities. We describe justice concerns related to CRISPR/Cas9, including its potential impact on historically mistreated populations through underrepresentation of minorities in genomic databases and the potential for disparate access to somatic gene therapies when they become clinically available. We then describe ongoing work that aims to address these justice concerns. We conclude by highlighting important considerations to ensure equitable access to therapies going forward, including enhancing diversity in genomic sequencing efforts, improving education and transparency, and building partnerships with underserved and socially disenfranchised communities.},
}

*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Databases, Genetic/ethics
Ethics, Clinical
Ethics, Research
Ethnic Groups
Gene Editing/*ethics
Genetic Therapy/*ethics
*Health Equity
*Healthcare Disparities
Humans
Minority Groups
*Social Justice

@article {pmid31927331,
year = {2019},
author = {Weng, Y and Huang, Q and Li, C and Yang, Y and Wang, X and Yu, J and Huang, Y and Liang, XJ},
title = {Improved Nucleic Acid Therapy with Advanced Nanoscale Biotechnology.},
journal = {Molecular therapy. Nucleic acids},
volume = {19},
number = {},
pages = {581-601},
doi = {10.1016/j.omtn.2019.12.004},
pmid = {31927331},
issn = {2162-2531},
abstract = {Due to a series of systemic and intracellular obstacles in nucleic acid (NA) therapy, including fast degradation in blood, renal clearance, poor cellular uptake, and inefficient endosomal escape, NAs may need delivery methods to transport to the cell nucleus or cytosol to be effective. Advanced nanoscale biotechnology-associated strategies, such as controlling the particle size, charge, drug loading, response to environmental signals, or other physical/chemical properties of delivery carriers, have provided great help for the in vivo and in vitro delivery of NA therapeutics. In this review, we introduce the characteristics of different NA modalities and illustrate how advanced nanoscale biotechnology assists NA therapy. The specific features and challenges of various nanocarriers in clinical and preclinical studies are summarized and discussed. With the help of advanced nanoscale biotechnology, some of the major barriers to the development of NA therapy will eventually be overcome in the near future.},
}

@article {pmid31926765,
year = {2020},
author = {Wang, R and Angenent, GC and Seymour, G and de Maagd, RA},
title = {Revisiting the Role of Master Regulators in Tomato Ripening.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2019.11.005},
pmid = {31926765},
issn = {1878-4372},
abstract = {The study of transcriptional regulation of tomato ripening has been led by spontaneous mutations in transcription factor (TF) genes that completely inhibit normal ripening, suggesting that they are 'master regulators'. Studies using CRISPR/Cas9 mutagenesis to produce knockouts of the underlying genes indicate a different picture, suggesting that the regulation is more robust than previously thought. This requires us to revisit our model of the regulation of ripening and replace it with one involving a network of partially redundant components. At the same time, the fast rise of CRISPR/Cas mutagenesis, resulting in unexpectedly weak phenotypes, compared with knockdown technology, suggests that compensatory mechanisms may obscure protein functions. This emphasises the need for assessment of these mechanisms in plants and for the careful design of mutagenesis experiments.},
}

@article {pmid31564075,
year = {2019},
author = {Vo, KTX and Lee, SK and Halane, MK and Song, MY and Hoang, TV and Kim, CY and Park, SY and Jeon, J and Kim, ST and Sohn, KH and Jeon, JS},
title = {Pi5 and Pii Paired NLRs Are Functionally Exchangeable and Confer Similar Disease Resistance Specificity.},
journal = {Molecules and cells},
volume = {42},
number = {9},
pages = {637-645},
pmid = {31564075},
issn = {0219-1032},
mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; DNA, Complementary/genetics ; Disease Resistance/*genetics ; Gene Expression Regulation, Plant ; *Genes, Plant ; Magnaporthe/*physiology ; Mutation/genetics ; NLR Proteins/*metabolism ; Oryza/genetics/*microbiology ; Plant Diseases/*microbiology ; Plants, Genetically Modified ; },
abstract = {Effector-triggered immunity (ETI) is an effective layer of plant defense initiated upon recognition of avirulence (Avr) effectors from pathogens by cognate plant disease resistance (R) proteins. In rice, a large number of R genes have been characterized from various cultivars and have greatly contributed to breeding programs to improve resistance against the rice blast pathogen Magnaporthe oryzae. The extreme diversity of R gene repertoires is thought to be a result of co-evolutionary history between rice and its pathogens including M. oryzae. Here we show that Pii is an allele of Pi5 by DNA sequence characterization and complementation analysis. Pii-1 and Pii-2 cDNAs were cloned by reverse transcription polymerase chain reaction from the Pii -carrying cultivar Fujisaka5 . The complementation test in susceptible rice cultivar Dongjin demonstrated that the rice blast resistance mediated by Pii , similar to Pi5 , requires the presence of two nucleotide-binding leucine-rich repeat genes, Pii-1 and Pii-2 . Consistent with our hypothesis that Pi5 and Pii are functionally indistinguishable, the replacement of Pii-1 by Pi5-1 and Pii-2 by Pi5-2 , respectively, does not change the level of disease resistance to M. oryzae carrying AVR-Pii. Surprisingly, Exo70F3, required for Pii-mediated resistance, is dispensable for Pi5-mediated resistance. Based on our results, despite similarities observed between Pi5 and Pii, we hypothesize that Pi5 and Pii pairs require partially distinct mechanisms to function.},
}

Base Sequence
CRISPR-Cas Systems/genetics
DNA, Complementary/genetics
Disease Resistance/*genetics
Gene Expression Regulation, Plant
*Genes, Plant
Magnaporthe/*physiology
Mutation/genetics
NLR Proteins/*metabolism
Oryza/genetics/*microbiology
Plant Diseases/*microbiology
Plants, Genetically Modified

@article {pmid31331377,
year = {2019},
author = {Tong, Z and Sathe, A and Ebner, B and Qi, P and Veltkamp, C and Gschwend, JE and Holm, PS and Nawroth, R},
title = {Functional genomics identifies predictive markers and clinically actionable resistance mechanisms to CDK4/6 inhibition in bladder cancer.},
journal = {Journal of experimental & clinical cancer research : CR},
volume = {38},
number = {1},
pages = {322},
doi = {10.1186/s13046-019-1322-9},
pmid = {31331377},
issn = {1756-9966},
mesh = {Animals ; Apoptosis/drug effects ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chickens ; Chorioallantoic Membrane/drug effects ; Cyclin-Dependent Kinase 4/antagonists & inhibitors/genetics ; Cyclin-Dependent Kinase 6/antagonists & inhibitors/genetics ; Drug Resistance, Neoplasm/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; *Genomics ; Heterografts ; Humans ; Mice ; Piperazines/*pharmacology ; Protein Kinase Inhibitors/*pharmacology ; Pyridines/*pharmacology ; Urinary Bladder Neoplasms/*drug therapy/genetics/pathology ; },
abstract = {BACKGROUND: CDK4/6 inhibitors are a promising treatment strategy in tumor therapy but are hampered by resistance mechanisms. This study was performed to reveal predictive markers, mechanisms of resistance and to develop rational combination therapies for a personalized therapy approach in bladder cancer.

METHODS: A genome-scale CRISPR-dCas9 activation screen for resistance to the CDK4/6 inhibitor Palbociclib was performed in the bladder cancer derived cell line T24. sgRNA counts were analyzed using next generation sequencing and MAGeCK-VISPR. Significantly enriched sgRNAs were cloned and validated on a molecular and functional level for mediating resistance to Palbociclib treatment. Analysis was done in vitro and in vivo in the chorioallantois membrane model of the chicken embryo. Comparison of screen hits to signaling pathways and clinically relevant molecular alterations was performed using DAVID, Reactome, DGIdb and cBioPortal.

RESULTS: In the screen, 1024 sgRNAs encoding for 995 genes were significantly enriched indicative of mediators of resistance. 8 random sgRNAs were validated, revealing partial rescue to Palbociclib treatment. Within this gene panel, members of Receptor-Tyrosine Kinases, PI3K-Akt, Ras/MAPK, JAK/STAT or Wnt signaling pathways were identified. Combination of Palbociclib with inhibitors against these signaling pathways revealed beneficial effects in vitro and in in vivo xenografts.

CONCLUSIONS: Identification of potential predictive markers, resistance mechanisms and rational combination therapies could be achieved by applying a CRISPR-dCas9 screening approach in bladder cancer.},
}

Animals
Apoptosis/drug effects
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Cell Proliferation/drug effects
Chickens
Chorioallantoic Membrane/drug effects
Cyclin-Dependent Kinase 4/antagonists & inhibitors/genetics
Cyclin-Dependent Kinase 6/antagonists & inhibitors/genetics
Drug Resistance, Neoplasm/drug effects
Gene Expression Regulation, Neoplastic/drug effects
*Genomics
Heterografts
Humans
Mice
Piperazines/*pharmacology
Protein Kinase Inhibitors/*pharmacology
Pyridines/*pharmacology
Urinary Bladder Neoplasms/*drug therapy/genetics/pathology

@article {pmid31323920,
year = {2019},
author = {Milenkovic, VM and Slim, D and Bader, S and Koch, V and Heinl, ES and Alvarez-Carbonell, D and Nothdurfter, C and Rupprecht, R and Wetzel, CH},
title = {CRISPR-Cas9 Mediated TSPO Gene Knockout alters Respiration and Cellular Metabolism in Human Primary Microglia Cells.},
journal = {International journal of molecular sciences},
volume = {20},
number = {13},
pages = {},
doi = {10.3390/ijms20133359},
pmid = {31323920},
issn = {1422-0067},
support = {WE2298//Deutsche Forschungsgemeinschaft/ ; },
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Calcium/metabolism ; Cell Line ; Cells, Cultured ; Humans ; Membrane Potential, Mitochondrial/physiology ; Microglia/*metabolism ; Oxidative Phosphorylation ; Receptors, GABA/deficiency/genetics/*metabolism ; Steroids/metabolism ; },
abstract = {The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. It has been implicated in the regulation of various cellular processes including oxidative stress, proliferation, apoptosis, and steroid hormone biosynthesis. Since the expression of TSPO in activated microglia is upregulated in various neuroinflammatory and neurodegenerative disorders, we set out to examine the role of TSPO in an immortalized human microglia C20 cell line. To this end, we performed a dual approach and used (i) lentiviral shRNA silencing to reduce TSPO expression, and (ii) the CRISPR/Cas9 technology to generate complete TSPO knockout microglia cell lines. Functional characterization of control and TSPO knockdown as well as knockout cells, revealed only low de novo steroidogenesis in C20 cells, which was not dependent on the level of TSPO expression or influenced by the treatment with TSPO-specific ligands. In contrast to TSPO knockdown C20 cells, which did not show altered mitochondrial function, the TSPO deficient knockout cells displayed a significantly decreased mitochondrial membrane potential and cytosolic Ca2+ levels, as well as reduced respiratory function. Performing the rescue experiment by lentiviral overexpression of TSPO in knockout cells, increased oxygen consumption and restored respiratory function. Our study provides further evidence for a significant role of TSPO in cellular and mitochondrial metabolism and demonstrates that different phenotypes of mitochondrial function are dependent on the level of TSPO expression.},
}

CRISPR-Cas Systems/genetics/*physiology
Calcium/metabolism
Cell Line
Cells, Cultured
Humans
Membrane Potential, Mitochondrial/physiology
Microglia/*metabolism
Oxidative Phosphorylation
Receptors, GABA/deficiency/genetics/*metabolism
Steroids/metabolism

@article {pmid31248401,
year = {2019},
author = {Wilson, LOW and Hetzel, S and Pockrandt, C and Reinert, K and Bauer, DC},
title = {VARSCOT: variant-aware detection and scoring enables sensitive and personalized off-target detection for CRISPR-Cas9.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {40},
doi = {10.1186/s12896-019-0535-5},
pmid = {31248401},
issn = {1472-6750},
mesh = {*CRISPR-Cas Systems ; Computational Biology/*methods ; Gene Editing/*methods/standards ; Gene Targeting/*methods/standards ; Genomics/*methods/standards ; Internet ; Reproducibility of Results ; *Software ; },
abstract = {BACKGROUND: Natural variations in a genome can drastically alter the CRISPR-Cas9 off-target landscape by creating or removing sites. Despite the resulting potential side-effects from such unaccounted for sites, current off-target detection pipelines are not equipped to include variant information. To address this, we developed VARiant-aware detection and SCoring of Off-Targets (VARSCOT).

RESULTS: VARSCOT identifies only 0.6% of off-targets to be common between 4 individual genomes and the reference, with an average of 82% of off-targets unique to an individual. VARSCOT is the most sensitive detection method for off-targets, finding 40 to 70% more experimentally verified off-targets compared to other popular software tools and its machine learning model allows for CRISPR-Cas9 concentration aware off-target activity scoring.

CONCLUSIONS: VARSCOT allows researchers to take genomic variation into account when designing individual or population-wide targeting strategies. VARSCOT is available from https://github.com/BauerLab/VARSCOT .},
}

*CRISPR-Cas Systems
Computational Biology/*methods
Gene Editing/*methods/standards
Gene Targeting/*methods/standards
Genomics/*methods/standards
Internet
Reproducibility of Results
*Software

@article {pmid31208390,
year = {2019},
author = {Petersen, BL and Möller, SR and Mravec, J and Jørgensen, B and Christensen, M and Liu, Y and Wandall, HH and Bennett, EP and Yang, Z},
title = {Improved CRISPR/Cas9 gene editing by fluorescence activated cell sorting of green fluorescence protein tagged protoplasts.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {36},
doi = {10.1186/s12896-019-0530-x},
pmid = {31208390},
issn = {1472-6750},
mesh = {*CRISPR-Cas Systems ; Flow Cytometry ; Fluorescence ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics/*metabolism ; Microscopy, Fluorescence ; Mutation ; Plant Leaves/cytology/genetics/*metabolism ; Plants, Genetically Modified ; Protoplasts/cytology/*metabolism ; Tobacco/cytology/genetics/*metabolism ; },
abstract = {BACKGROUND: CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand.

RESULTS: In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3-5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations.

CONCLUSIONS: FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.},
}

*CRISPR-Cas Systems
Flow Cytometry
Fluorescence
Gene Editing/*methods
Green Fluorescent Proteins/genetics/*metabolism
Microscopy, Fluorescence
Mutation
Plant Leaves/cytology/genetics/*metabolism
Plants, Genetically Modified
Protoplasts/cytology/*metabolism
Tobacco/cytology/genetics/*metabolism

@article {pmid31152927,
year = {2019},
author = {J, B and Das, A},
title = {An edible fungi Pleurotus ostreatus inhibits adipogenesis via suppressing expression of PPAR γ and C/EBP α in 3T3-L1 cells: In vitro validation of gene knock out of RNAs in PPAR γ using CRISPR spcas9.},
journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie},
volume = {116},
number = {},
pages = {109030},
doi = {10.1016/j.biopha.2019.109030},
pmid = {31152927},
issn = {1950-6007},
mesh = {3T3-L1 Cells ; *Adipogenesis ; Animals ; Base Sequence ; CCAAT-Enhancer-Binding Protein-alpha/*metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Differentiation ; *Gene Knockout Techniques ; Humans ; Mice ; PPAR gamma/*metabolism ; Pleurotus/*chemistry ; RNA/*metabolism ; },
abstract = {OBJECTIVE: Obesity is now well recognized as a disorder, one that is essentially preventable through changes in lifestyle. Obesity is also a main concern associated with expanded morbidity and mortality from many noncommunicable illnesses (NCDs). The study aimed to determine the antiobesity effect of Pleurotus ostreatus (PO) and its bioactive anthraquinone (AQ). The overall promoter genes CEBPα (CCAAT enhancer binding protein α) and PPARγ (Peroxisome proliferator activated receptor γ) in controlling the homeostasis of glucose was analysed using 3T3-L1 cell line. Finally, an insilico study was carried out using CRISPR software to identify the RNA's involved in adipogenesis especially of the control gene PPARγ.

MATERIALS AND METHODS: Preliminary screening of the edible fungi and their bio actives led to the marvellous discovery of side effect free agonists for treating obesity (adipogenesis). An edible fungi Pleurotus ostreatus (PO) were analysed in a screening platform with different series of tests for adipocyte differentiation, triglyceride analysis, lipolysis determination, glucose uptake assay, cytotoxicity assay and lipase activity followed by specific gene expression analysis. The gene knockout mechanism was also elucidated by CRISPR spcas 9 tool.

RESULTS: The antiadipogenic (antiobesity) activity of DMSO extract of PO were found to stimulate the insulin dependent uptake of glucose. The extract also decreased the levels of triglycerides and glycerol accumulation in differentiated adipocyte cells. The binding FABP4 (Fatty acid binding protein) and transport protein FATP1 (Fatty acid transport protein) along with the fat breaking LPL (lipoprotein lipase) was found to be inhibited after the PO treatment at varying concentration (0-300 μg/ml). CRISPR spcas9 genome editing software was used as an insilico approach in validating the efficiency of mouse embryonic and human adipogenic cell line (3T3-L1). These tool analysed and found 4 RNAs gene knock out possibilities in PPARγ and their efficiency for further treating obesity.

CONCLUSION: These novel finding contribute to the confirmation that edible fungi PO and it's bioactive AQ is an adequate supplement for constraining the lipid and triglycerides in differentiated mature adipocytes by reversing the fat deposition. Thereby, forbidding the enzymes linked with fat absorption. Besides, the CRISPR tool identified gene knock out possibilities of control gene PPARγ, will pave a way in further research for treating obesity.},
}

3T3-L1 Cells
*Adipogenesis
Animals
Base Sequence
CCAAT-Enhancer-Binding Protein-alpha/*metabolism
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Cell Differentiation
*Gene Knockout Techniques
Humans
Mice
PPAR gamma/*metabolism
Pleurotus/*chemistry
RNA/*metabolism

@article {pmid31130364,
year = {2019},
author = {Jia, R and Bonifacino, JS},
title = {Lysosome Positioning Influences mTORC2 and AKT Signaling.},
journal = {Molecular cell},
volume = {75},
number = {1},
pages = {26-38.e3},
doi = {10.1016/j.molcel.2019.05.009},
pmid = {31130364},
issn = {1097-4164},
mesh = {ADP-Ribosylation Factors/deficiency/genetics ; CRISPR-Cas Systems ; Cell Nucleus/*metabolism/ultrastructure ; Culture Media, Serum-Free ; Endosomes/metabolism/ultrastructure ; Gene Editing ; Gene Expression Regulation ; HEK293 Cells ; HeLa Cells ; Humans ; Kinesin/deficiency/genetics ; Lysosomes/*metabolism/ultrastructure ; MEF2 Transcription Factors/deficiency/genetics ; Mechanistic Target of Rapamycin Complex 1/*genetics/metabolism ; Mechanistic Target of Rapamycin Complex 2/*genetics/metabolism ; Proto-Oncogene Proteins c-akt/*genetics/metabolism ; Signal Transduction ; },
abstract = {Growth factor signaling is initiated at the plasma membrane and propagated through the cytoplasm for eventual relay to intracellular organelles such as lysosomes. The serine/threonine kinase mTOR participates in growth factor signaling as a component of two multi-subunit complexes, mTORC1 and mTORC2. mTORC1 associates with lysosomes, and its activity depends on the positioning of lysosomes within the cytoplasm, although there is no consensus regarding the exact effect of perinuclear versus peripheral distribution. mTORC2 and its substrate kinase AKT have a widespread distribution, but they are thought to act mainly at the plasma membrane. Using cell lines with knockout of components of the lysosome-positioning machinery, we show that perinuclear clustering of lysosomes delays reactivation of not only mTORC1, but also mTORC2 and AKT upon serum replenishment. These experiments demonstrate the existence of pools of mTORC2 and AKT that are sensitive to lysosome positioning.},
}

ADP-Ribosylation Factors/deficiency/genetics
CRISPR-Cas Systems
Cell Nucleus/*metabolism/ultrastructure
Culture Media, Serum-Free
Endosomes/metabolism/ultrastructure
Gene Editing
Gene Expression Regulation
HEK293 Cells
HeLa Cells
Humans
Kinesin/deficiency/genetics
Lysosomes/*metabolism/ultrastructure
MEF2 Transcription Factors/deficiency/genetics
Mechanistic Target of Rapamycin Complex 1/*genetics/metabolism
Mechanistic Target of Rapamycin Complex 2/*genetics/metabolism
Proto-Oncogene Proteins c-akt/*genetics/metabolism
Signal Transduction

@article {pmid31060546,
year = {2019},
author = {Carey, K and Ryu, J and Uh, K and Lengi, AJ and Clark-Deener, S and Corl, BA and Lee, K},
title = {Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryos.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {25},
doi = {10.1186/s12896-019-0517-7},
pmid = {31060546},
issn = {1472-6750},
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Embryo, Mammalian/embryology/*metabolism ; Gene Editing/*methods ; Gene Targeting/*methods ; Mutation ; Sequence Analysis, DNA ; Swine ; },
abstract = {BACKGROUND: The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined.

RESULTS: First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5' side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5' side, indicating unpredictability of the events.

CONCLUSIONS: The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.},
}

Animals
Base Sequence
*CRISPR-Cas Systems
Embryo, Mammalian/embryology/*metabolism
Gene Editing/*methods
Gene Targeting/*methods
Mutation
Sequence Analysis, DNA
Swine

@article {pmid31035982,
year = {2019},
author = {Yuan, M and Zhu, J and Gong, L and He, L and Lee, C and Han, S and Chen, C and He, G},
title = {Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {24},
doi = {10.1186/s12896-019-0516-8},
pmid = {31035982},
issn = {1472-6750},
mesh = {Arachis/enzymology/*genetics ; Base Sequence ; *CRISPR-Cas Systems ; Fatty Acid Desaturases/*genetics/metabolism ; Gene Editing/*methods ; Linoleic Acid/metabolism ; *Mutagenesis ; Oleic Acid/metabolism ; Plant Breeding/methods ; Plant Proteins/*genetics/metabolism ; Plants, Genetically Modified ; Seeds/enzymology/genetics ; },
abstract = {BACKGROUND: Increasing the content of oleic acid in peanut seeds is one of the major goals in peanut breeding due to consumer and industry benefits, such as anti-oxidation and long shelf-life. Homeologous ahFAD2A and ahFAD2B genes encode fatty acid desaturases, which are the key enzymes for converting oleic acid to linoleic acid that oxidizes readily. To date, all high oleic acid peanut varieties result from natural mutations occurred in both genes. A method to induce mutations in the genes of other elite cultivars could speed introgression of this valuable trait. The gene-editing approach utilizing CRISPR/Cas9 technology was employed to induce de novo mutations in the ahFAD2 genes using peanut protoplasts and hairy root cultures as models.

RESULTS: The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441_442insA and G451T in ahFAD2B. The G448A and 441_442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content.

CONCLUSIONS: Overall, these results showed that mutations were, for the first time, induced by CRISPR-based gene editing approach in peanut. This research demonstrated the potential application of gene editing for mutagenesis in peanut and suggested that CRISPR/Cas9 technology may be useful in the peanut breeding programs.},
}

Arachis/enzymology/*genetics
Base Sequence
*CRISPR-Cas Systems
Fatty Acid Desaturases/*genetics/metabolism
Gene Editing/*methods
Linoleic Acid/metabolism
*Mutagenesis
Oleic Acid/metabolism
Plant Breeding/methods
Plant Proteins/*genetics/metabolism
Plants, Genetically Modified
Seeds/enzymology/genetics

@article {pmid31005419,
year = {2019},
author = {Seruggia, D and Oti, M and Tripathi, P and Canver, MC and LeBlanc, L and Di Giammartino, DC and Bullen, MJ and Nefzger, CM and Sun, YBY and Farouni, R and Polo, JM and Pinello, L and Apostolou, E and Kim, J and Orkin, SH and Das, PP},
title = {TAF5L and TAF6L Maintain Self-Renewal of Embryonic Stem Cells via the MYC Regulatory Network.},
journal = {Molecular cell},
volume = {74},
number = {6},
pages = {1148-1163.e7},
doi = {10.1016/j.molcel.2019.03.025},
pmid = {31005419},
issn = {1097-4164},
support = {DP2 DA043813/DA/NIDA NIH HHS/United States ; U01 HL100001/HL/NHLBI NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; R01 GM112722/GM/NIGMS NIH HHS/United States ; F30 DK103359/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle/genetics ; Cell Proliferation ; Cellular Reprogramming ; Embryo, Mammalian ; Embryonic Stem Cells/cytology/*metabolism ; Epigenesis, Genetic ; Fibroblasts/cytology/metabolism ; Gene Editing ; Gene Expression Regulation ; *Gene Regulatory Networks ; HEK293 Cells ; Histones/genetics/metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Mice ; Primary Cell Culture ; Protein Isoforms/genetics/metabolism ; Proto-Oncogene Proteins c-myc/*genetics/metabolism ; Signal Transduction ; TATA-Binding Protein Associated Factors/*genetics/metabolism ; },
abstract = {Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.},
}

Animals
CRISPR-Cas Systems
Cell Cycle/genetics
Cell Proliferation
Cellular Reprogramming
Embryo, Mammalian
Embryonic Stem Cells/cytology/*metabolism
Epigenesis, Genetic
Fibroblasts/cytology/metabolism
Gene Editing
Gene Expression Regulation
*Gene Regulatory Networks
HEK293 Cells
Histones/genetics/metabolism
Humans
Induced Pluripotent Stem Cells/cytology/*metabolism
Mice
Primary Cell Culture
Protein Isoforms/genetics/metabolism
Proto-Oncogene Proteins c-myc/*genetics/metabolism
Signal Transduction
TATA-Binding Protein Associated Factors/*genetics/metabolism

@article {pmid30951658,
year = {2019},
author = {Trounson, A and Boyd, NR and Boyd, RL},
title = {Toward a Universal Solution: Editing Compatibility into Pluripotent Stem Cells.},
journal = {Cell stem cell},
volume = {24},
number = {4},
pages = {508-510},
doi = {10.1016/j.stem.2019.03.003},
pmid = {30951658},
issn = {1875-9777},
mesh = {CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Induced Pluripotent Stem Cells ; *Pluripotent Stem Cells ; },
abstract = {In this issue of Cell Stem Cell, Xu et al. (2019) demonstrate that editing iPSCs' major histocompatibility antigens may potentially provide a small set of universally compatible stem cell lines for therapies. However, these modifications may result in patient minor histocompatibility responses and deficiencies in their T cell response repertoire to infection and cancer.},
}

CRISPR-Cas Systems
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
*Induced Pluripotent Stem Cells
*Pluripotent Stem Cells

@article {pmid30817134,
year = {2019},
author = {Neukranz, Y and Kotter, A and Beilschmidt, L and Marelja, Z and Helm, M and Gräf, R and Leimkühler, S},
title = {Analysis of the Cellular Roles of MOCS3 Identifies a MOCS3-Independent Localization of NFS1 at the Tips of the Centrosome.},
journal = {Biochemistry},
volume = {58},
number = {13},
pages = {1786-1798},
doi = {10.1021/acs.biochem.8b01160},
pmid = {30817134},
issn = {1520-4995},
mesh = {Aconitate Hydratase/metabolism ; CRISPR-Cas Systems ; Carbon-Sulfur Lyases/analysis/*metabolism ; Centrosome/*metabolism/ultrastructure ; Coenzymes/metabolism ; HEK293 Cells ; HeLa Cells ; Humans ; Isocitrate Dehydrogenase/metabolism ; Metalloproteins/metabolism ; Nucleotidyltransferases/analysis/genetics/*metabolism ; Pteridines/metabolism ; RNA, Transfer/metabolism ; Sulfite Oxidase/metabolism ; Sulfurtransferases/analysis/genetics/*metabolism ; },
abstract = {The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the l-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm5s2U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm5s2U thio-modified tRNAs were not detectable. Because the l-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.},
}

Aconitate Hydratase/metabolism
CRISPR-Cas Systems
Carbon-Sulfur Lyases/analysis/*metabolism
Centrosome/*metabolism/ultrastructure
Coenzymes/metabolism
HEK293 Cells
HeLa Cells
Humans
Isocitrate Dehydrogenase/metabolism
Metalloproteins/metabolism
Nucleotidyltransferases/analysis/genetics/*metabolism
Pteridines/metabolism
RNA, Transfer/metabolism
Sulfite Oxidase/metabolism
Sulfurtransferases/analysis/genetics/*metabolism

@article {pmid30745836,
year = {2019},
author = {Chen, J and Wang, H and Bai, J and Liu, W and Liu, X and Yu, D and Feng, T and Sun, Z and Zhang, L and Ma, L and Hu, Y and Zou, Y and Tan, T and Zhong, J and Hu, M and Bai, X and Pan, D and Xing, Y and Zhao, Y and Tian, K and Hu, X and Li, N},
title = {Generation of Pigs Resistant to Highly Pathogenic-Porcine Reproductive and Respiratory Syndrome Virus through Gene Editing of CD163.},
journal = {International journal of biological sciences},
volume = {15},
number = {2},
pages = {481-492},
doi = {10.7150/ijbs.25862},
pmid = {30745836},
issn = {1449-2288},
mesh = {Animals ; Antigens, CD/*genetics ; Antigens, Differentiation, Myelomonocytic/*genetics ; CRISPR-Cas Systems/genetics ; Exons/genetics ; Gene Editing/*methods ; Porcine respiratory and reproductive syndrome virus/*genetics ; Receptors, Cell Surface/*genetics ; Swine ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease and the most economically important disease of the swine industry worldwide. Highly pathogenic-PRRS virus (HP-PRRSV) is a variant of PRRSV, which caused high morbidity and mortality. Scavenger receptor CD163, which contains nine scavenger receptor cysteine-rich (SRCR) domains, is a key entry mediator for PRRSV. A previous study demonstrated that SRCR domain 5 (SRCR5), encoded by exon 7, was essential for PRRSV infection in vitro. Here, we substituted exon 7 of porcine CD163 with the corresponding exon of human CD163-like 1 (hCD163L1) using a CRISPR/Cas9 system combined with a donor vector. In CD163Mut/Mut pigs, modifying CD163 gene had no adverse effects on hemoglobin-haptoglobin (Hb-Hp) complex clearance or erythroblast growth. In vitro infection experiments showed that the CD163 mutant strongly inhibited HP-PRRSV replication by inhibiting virus uncoating and genome release. Compared to wild-type (WT) pigs in vivo, HP-PRRSV-infected CD163Mut/Mut pigs showed a substantially decreased viral load in blood and relief from PRRSV-induced fever. While all WT pigs were dead, there of four CD163Mut/Mut pigs survived and recovered at the termination of the experiment. Our data demonstrated that modifying CD163 remarkably inhibited PRRSV replication and protected pigs from HP-PRRSV infection, thus establishing a good foundation for breeding PRRSV-resistant pigs via gene editing technology.},
}

Animals
Antigens, CD/*genetics
Antigens, Differentiation, Myelomonocytic/*genetics
CRISPR-Cas Systems/genetics
Exons/genetics
Gene Editing/*methods
Porcine respiratory and reproductive syndrome virus/*genetics
Receptors, Cell Surface/*genetics
Swine

@article {pmid31925391,
year = {2020},
author = {Jia, N and Xie, W and de la Cruz, MJ and Eng, ET and Patel, DJ},
title = {Structure-function insights into the initial step of DNA integration by a CRISPR-Cas-Transposon complex.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41422-019-0272-2},
pmid = {31925391},
issn = {1748-7838},
}

@article {pmid31925273,
year = {2019},
author = {Sansbury, BM and Hewes, AM and Kmiec, EB},
title = {Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair.},
journal = {Communications biology},
volume = {2},
number = {1},
pages = {458},
doi = {10.1038/s42003-019-0705-y},
pmid = {31925273},
issn = {2399-3642},
support = {1558//BIRD Foundation (Israel-U.S. Binational Industrial Research and Development Foundation)/ ; },
abstract = {As CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.},
}

@article {pmid31925225,
year = {2019},
author = {Young, JK and Gasior, SL and Jones, S and Wang, L and Navarro, P and Vickroy, B and Barrangou, R},
title = {The repurposing of type I-E CRISPR-Cascade for gene activation in plants.},
journal = {Communications biology},
volume = {2},
number = {1},
pages = {383},
doi = {10.1038/s42003-019-0637-6},
pmid = {31925225},
issn = {2399-3642},
abstract = {CRISPR-Cas systems are robust and facile tools for manipulating the genome, epigenome and transcriptome of eukaryotic organisms. Most groups use class 2 effectors, such as Cas9 and Cas12a, however, other CRISPR-Cas systems may provide unique opportunities for genome engineering. Indeed, the multi-subunit composition of class 1 systems offers to expand the number of domains and functionalities that may be recruited to a genomic target. Here we report DNA targeting in Zea mays using a class 1 type I-E CRISPR-Cas system from S. thermophilus. First, we engineer its Cascade complex to modulate gene expression by tethering a plant transcriptional activation domain to 3 different subunits. Next, using an immunofluorescent assay, we confirm Cascade cellular complex formation and observe enhanced gene activation when multiple subunits tagged with the transcriptional activator are combined. Finally, we examine Cascade mediated gene activation at chromosomal DNA targets by reprogramming Zea mays cells to change color.},
}

@article {pmid31924979,
year = {2019},
author = {Qiao, J and Li, W and Lin, S and Sun, W and Ma, L and Liu, Y},
title = {Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins.},
journal = {Communications biology},
volume = {2},
number = {1},
pages = {161},
doi = {10.1038/s42003-019-0402-x},
pmid = {31924979},
issn = {2399-3642},
abstract = {CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas9 and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. The prepared Cas RNPs show remarkable stability in the absence of RNase inhibitors, as well as profound gene-editing efficiency in vitro and in vivo. Our method is convenient, cost-effective, and can be used to prepare other CRISPR/Cas RNPs.},
}

@article {pmid31922192,
year = {2020},
author = {Hidalgo-Cantabrana, C and Barrangou, R},
title = {Characterization and applications of Type I CRISPR-Cas systems.},
journal = {Biochemical Society transactions},
volume = {},
number = {},
pages = {},
doi = {10.1042/BST20190119},
pmid = {31922192},
issn = {1470-8752},
abstract = {CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.},
}

@article {pmid31921246,
year = {2019},
author = {Wesseler, J and Politiek, H and Zilberman, D},
title = {The Economics of Regulating New Plant Breeding Technologies - Implications for the Bioeconomy Illustrated by a Survey Among Dutch Plant Breeders.},
journal = {Frontiers in plant science},
volume = {10},
number = {},
pages = {1597},
doi = {10.3389/fpls.2019.01597},
pmid = {31921246},
issn = {1664-462X},
abstract = {New plant breeding technologies (NPBTs) are increasingly used for developing new plants with novel traits. The science tells us that those plants in general are as safe as than those once developed using "conventional" plant breeding methods. The knowledge about the induced changes and properties of the new plants by using NPBTs is more precise. This should lead to the conclusion that plants developed using NPBTs should not be regulated differently than those developed using "conventional" plant breeding methods. This contribution discusses the economics of regulating new plant breeding technologies. We first develop the theoretical model and elaborate on the different regulatory approaches being used and compare their advantages and disadvantages. Then we provide a perspectives on EU regulation around mutagenesis-based New Plant Breeding Techniques (NPBT), formed by new insights from a survey among Dutch plant breeding companies. The survey measures the attitude of breeding companies towards the ruling of the EU Court of Justice that subjected the use of CRISPR-Cas in the development of new plant varieties under the general EU regulations around GMOs. The results show that plant breeders experience a financial barrier because of the ruling, with perceived negative impact on competitiveness and investments in CRISPR-Cas as a result. The degree of negative impact differs however significantly among seed-sectors and company sizes. One of the most striking results was the relative optimism of companies in the sector about more lenient legislation in the next five years, despite the stated negative effects.},
}

@article {pmid31921024,
year = {2019},
author = {Watanabe, S and Cui, B and Kiga, K and Aiba, Y and Tan, XE and Sato'o, Y and Kawauchi, M and Boonsiri, T and Thitiananpakorn, K and Taki, Y and Li, FY and Azam, AH and Nakada, Y and Sasahara, T and Cui, L},
title = {Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {2838},
doi = {10.3389/fmicb.2019.02838},
pmid = {31921024},
issn = {1664-302X},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.},
}

@article {pmid31919672,
year = {2020},
author = {Zhao, Z and Zhang, RA and Fu, GY and Zhang, R and Nie, YF and Sun, C and Wu, M},
title = {The Complete Genome of Emcibacter congregatus ZYLT, a Marine Bacterium Encoding a CRISPR-Cas 9 Immune System.},
journal = {Current microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00284-019-01867-6},
pmid = {31919672},
issn = {1432-0991},
support = {2017FY100300//Science & Technology Basic Resources Investigation Program of China/ ; 2018M642382//China Postdoctoral Science Foundation/ ; LQ19C010005//Natural Science Foundation of Zhejiang Province/ ; },
abstract = {Emcibacter congregatus ZYLT was isolated from a sediment sample cultured in situ in a coast located in the East China Sea. The genome of E. congregatus ZYLT was sequenced and assembled into one single circular chromosome with the size of 4,189,011 bp and G+C content of 52.6%. Genomic annotation showed that E. congregatus ZYLT had an intact Type II-C CRISPR-Cas system consists of three cas genes (cas 9, cas 1, and cas 2), 34 direct repeat sequences with the length of 36 bp, and 33 spacers. The predicted Cas 9 protein was smaller than most of existing genome editing tools. This structure might have potential in developing new gene editing system and uncovering the regulatory mechanisms of CRISPR-Cas system. Besides, the comparison between E. congregatus ZYLT and its relative species living in neritic environments unraveled some common traits of the defective strategies of these bacteria to face inshore challenges including the motility, multidrug resistance, and universal efflux pumps.},
}

@article {pmid31919495,
year = {2020},
author = {Kukhtar, D and Rubio-Peña, K and Serrat, X and Cerón, J},
title = {Mimicking of splicing-related retinitis pigmentosa mutations in C. elegans allow drug screens and identification of disease modifiers.},
journal = {Human molecular genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/hmg/ddz315},
pmid = {31919495},
issn = {1460-2083},
abstract = {CRISPR/Cas and the high conservation of the spliceosome components facilitate the mimicking of human pathological mutations in splicing factors of model organisms. The degenerative retinal disease retinitis pigmentosa (RP) is caused by mutations in distinct types of genes, including missense mutations in splicing factors that provoke RP in an autosomal dominant form (s-adRP). Using CRISPR in Caenorhabditis elegans, we generated mutant strains to mimic s-adRP mutations reported in PRPF8 and SNRNP200. Whereas these inherited mutations are present in heterozygosis in patients, C. elegans allows the maintenance of these mutations as homozygotes, which is advantageous for genetic and drug screens. We found that snrp-200(cer23[V676 L]) and prp-8(cer14[H2302del]) display pleiotropic phenotypes, including reduced fertility. However, snrp-200(cer24[S1080 L]) and prp-8(cer22[R2303G]) are weak alleles suitable for RNAi screens for identifying genetic interactions, which could uncover potential disease modifiers. We screened a collection of RNAi clones for splicing-related genes and identified three splicing factors: isy-1/ISY1, cyn-15/PPWD1, and mog-2/SNRPA1 whose partial inactivation may modify the course of the disease. Interestingly, these three genes act as modifiers of prp-8(cer22) but not of snrp-200(cer24). Finally, a screen of the strong allele prp-8(cer14) with FDA-approved drugs did not identify molecules capable of alleviating the temperature-sensitive sterility. Instead, we detected drugs, such as dequalinium chloride, which exacerbated the phenotype, and therefore, are potentially harmful to s-adRP patients since they may accelerate the progression of the disease.},
}

@article {pmid31911695,
year = {2020},
author = {Ledford, H},
title = {Quest to use CRISPR against disease gains ground.},
journal = {Nature},
volume = {577},
number = {7789},
pages = {156},
doi = {10.1038/d41586-019-03919-0},
pmid = {31911695},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *HIV Infections ; Humans ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Stem Cells ; },
}

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*HIV Infections
Humans
*Precursor Cell Lymphoblastic Leukemia-Lymphoma
Stem Cells

@article {pmid31106480,
year = {2019},
author = {Bai, X and Zeng, T and Ni, XY and Su, HA and Huang, J and Ye, GY and Lu, YY and Qi, YX},
title = {CRISPR/Cas9-mediated knockout of the eye pigmentation gene white leads to alterations in colour of head spots in the oriental fruit fly, Bactrocera dorsalis.},
journal = {Insect molecular biology},
volume = {28},
number = {6},
pages = {837-849},
doi = {10.1111/imb.12592},
pmid = {31106480},
issn = {1365-2583},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Compound Eye, Arthropod/*physiology ; Female ; Gene Knockout Techniques ; Insect Proteins/chemistry/*genetics/metabolism ; Male ; Phylogeny ; Pigmentation/*genetics ; Sequence Alignment ; Tephritidae/genetics/*physiology ; },
abstract = {The intensely studied white gene is widely used as a genetic marker in Drosophila melanogaster. Here, we cloned and characterized the white gene in an important pest of the fruit industry, Bactrocera dorsalis, to understand its functional role in pigmentation. We obtained BdWhite knockout strains, based on the wild-type strain, using the CRISPR/Cas9 genome editing system, and found that mutants lost pigmentation in the compound eye and their black head spots. We then examined differences in the expression levels of genes associated with melanin pigmentation between mutants and the wild-type strain using quantitative reverse transcription PCR. We found that transcription levels of the Bd-yellow1 were lower in the head of mutants than in the wild-type strain, and there were no significant differences in expression of the other six genes between mutants and the wild type. Since yellow is critical for melanin biosynthesis (Heinze et al., Scientific Reports. 2017;7:4582), the lower levels of expression of Bd-yellow1 in mutants led to reduced dark pigmentation in head spots. Our results provide the first evidence, to our knowledge, that white may play a functional role in cuticle pigmentation by affecting the expression of yellow.},
}

Amino Acid Sequence
Animals
Base Sequence
*CRISPR-Cas Systems
Compound Eye, Arthropod/*physiology
Female
Gene Knockout Techniques
Insect Proteins/chemistry/*genetics/metabolism
Male
Phylogeny
Pigmentation/*genetics
Sequence Alignment
Tephritidae/genetics/*physiology

@article {pmid31022512,
year = {2019},
author = {Liu, Y and Ma, S and Chang, J and Zhang, T and Chen, X and Liang, Y and Xia, Q},
title = {Programmable targeted epigenetic editing using CRISPR system in Bombyx mori.},
journal = {Insect biochemistry and molecular biology},
volume = {110},
number = {},
pages = {105-111},
doi = {10.1016/j.ibmb.2019.04.013},
pmid = {31022512},
issn = {1879-0240},
mesh = {Animals ; Bombyx/*genetics ; *CRISPR-Cas Systems ; *DNA Methylation ; *Epigenesis, Genetic ; Gene Editing/*methods ; },
abstract = {DNA methylation has been proven to play roles in regulating gene expression, cell fate, disease determination, and chromatin architecture organization in mammals and plants, and is a significant component of epigenetic modification. Compared to mammals or plants, the status and function of DNA methylation are poorly understood in insects, which is partially due to the lack of efficient manipulation tools. In this study, we show that fusion protein of catalytically inactive Cas9 (dCas9) with TET1 can efficiently demethylate genomic DNA of silkworm Bombyx mori, in a programmable target region specific manner. We first developed an all-in-one vector to maximize the targeting efficiency of dCas9-TET1. Then we selected 3 endogenous genes that were previously found to harbor methylated DNA, and designed gRNAs within the methylated region. Co-transfection of dCas9-TET1 and gRNA successfully erased methylation marks near the targeting region, with efficiencies from about 17.50% to 40.00%. Furthermore, targeted demethylation on gene body resulted in increased mRNA transcription level. Unlike the previously widely used decitabine, a methylation inhibitor, dCas9-TET1 is more effective and specific, and has no unwanted impact on whole-genome methylation. DCas9-TET1 provides a powerful tool for investigating the functional significance of DNA methylation in a locus-specific manner, and for exploring the unknown links between methylation and development in insects.},
}

Animals
Bombyx/*genetics
*CRISPR-Cas Systems
*DNA Methylation
*Epigenesis, Genetic
Gene Editing/*methods

@article {pmid31015023,
year = {2019},
author = {Le Trionnaire, G and Tanguy, S and Hudaverdian, S and Gleonnec, F and Richard, G and Cayrol, B and Monsion, B and Pichon, E and Deshoux, M and Webster, C and Uzest, M and Herpin, A and Tagu, D},
title = {An integrated protocol for targeted mutagenesis with CRISPR-Cas9 system in the pea aphid.},
journal = {Insect biochemistry and molecular biology},
volume = {110},
number = {},
pages = {34-44},
doi = {10.1016/j.ibmb.2019.04.016},
pmid = {31015023},
issn = {1879-0240},
mesh = {Animals ; Aphids/*genetics ; *CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Male ; *Mutagenesis ; },
abstract = {CRISPR-Cas9 technology is a very efficient functional analysis tool and has been developed in several insects to edit their genome through injection of eggs with guide RNAs targeting coding sequences of genes of interest. However, its implementation in aphids is more challenging. Aphids are major pests of crops worldwide that alternate during their life cycle between clonality and sexual reproduction. The production of eggs after mating of sexual individuals is a single yearly event and is necessarily triggered by a photoperiod decrease. Fertilized eggs then experience an obligate 3-month diapause period before hatching as new clonal colonies. Taking into consideration these particularities, we developed in the pea aphid Acyrthosiphon pisum a step-by-step protocol of targeted mutagenesis based on the microinjection within fertilized eggs of CRISPR-Cas9 components designed for the editing of a cuticular protein gene (stylin-01). This protocol includes the following steps: i) the photoperiod-triggered induction of sexual morphs (2 months), ii) the mating and egg collection step (2 weeks), iii) egg microinjection and melanization, iv) the 3-month obligate diapause, v) the hatching of new lineages from injected eggs (2 weeks) and vi) the maintenance of stable lineages (2 weeks). Overall, this 7-month long procedure was applied to three different crosses in order to estimate the impact of the choice of the genetic combination on egg production dynamics by females as well as hatching rates after diapause. Mutation rates within eggs before diapause were estimated at 70-80%. The hatching rate of injected eggs following diapause ranged from 1 to 11% depending on the cross and finally a total of 17 stable lineages were obtained and maintained clonally. Out of these, 6 lineages were mutated at the defined sgRNAs target sites within stylin-01 coding sequence, either at the two alleles (2 lineages) or at one allele (4 lineages). The final germline transmission rate of the mutations was thus around 35%. Our protocol of an efficient targeted mutagenesis opens the avenue for functional studies through genome editing in aphids.},
}

Animals
Aphids/*genetics
*CRISPR-Cas Systems
Female
Gene Editing/*methods
Male
*Mutagenesis

@article {pmid30894699,
year = {2019},
author = {Fyfe, I},
title = {Alzheimer disease mice improve with CRISPR-Cas9 gene editing.},
journal = {Nature reviews. Neurology},
volume = {15},
number = {5},
pages = {247},
doi = {10.1038/s41582-019-0172-6},
pmid = {30894699},
issn = {1759-4766},
mesh = {*Alzheimer Disease ; Animals ; CRISPR-Cas Systems ; *Gene Editing ; Mice ; },
}

*Alzheimer Disease
Animals
CRISPR-Cas Systems
*Gene Editing
Mice

@article {pmid30700588,
year = {2019},
author = {Crivello, P and Ahci, M and Maaßen, F and Wossidlo, N and Arrieta-Bolaños, E and Heinold, A and Lange, V and Falkenburg, JHF and Horn, PA and Fleischhauer, K and Heinrichs, S},
title = {Multiple Knockout of Classical HLA Class II β-Chains by CRISPR/Cas9 Genome Editing Driven by a Single Guide RNA.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {202},
number = {6},
pages = {1895-1903},
doi = {10.4049/jimmunol.1800257},
pmid = {30700588},
issn = {1550-6606},
mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; *HLA-DR beta-Chains/genetics ; Humans ; *RNA, Guide ; },
abstract = {Comprehensive knockout of HLA class II (HLA-II) β-chain genes is complicated by their high polymorphism. In this study, we developed CRISPR/Cas9 genome editing to simultaneously target HLA-DRB, -DQB1, and -DPB1 through a single guide RNA recognizing a conserved region in exon 2. Abrogation of HLA-II surface expression was achieved in five different HLA-typed, human EBV-transformed B lymphoblastoid cell lines (BLCLs). Next-generation sequencing-based detection confirmed specific genomic insertion/deletion mutations with 99.5% penetrance in sorted cells for all three loci. No alterations were observed in HLA-I genes, the HLA-II peptide editor HLA-DMB, or its antagonist HLA-DOB, showing high on-target specificity. Transfection of full-length HLA-DPB1 mRNA into knockout BLCLs fully restored HLA-DP surface expression and recognition by alloreactive human CD4 T cells. The possibility to generate single HLA-II-expressing BLCLs by one-shot genome editing opens unprecedented opportunities for mechanistically dissecting the interaction of individual HLA variants with the immune system.},
}

*CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Cell Line, Tumor
Gene Editing/*methods
Gene Knockout Techniques/*methods
*HLA-DR beta-Chains/genetics
Humans
*RNA, Guide

@article {pmid31917244,
year = {2020},
author = {Gui, S and Nji Tizi Taning, C and Wei, D and Smagghe, G},
title = {First report on CRISPR/Cas9-targeted mutagenesis in the Colorado potato beetle,Leptinotarsa decemlineata.},
journal = {Journal of insect physiology},
volume = {},
number = {},
pages = {104013},
doi = {10.1016/j.jinsphys.2020.104013},
pmid = {31917244},
issn = {1879-1611},
abstract = {Leptinotarsa decemlineata (Say), commonly known as the Colorado potato beetle (CPB), is an agricultural important pest for potatoes and other solanaceous plants. The CRISPR/Cas system is an efficient genome editing technology, which could be exploited to study the biology of CPB and possibly also lead to the development of better environmentally friendly pest management strategies. However, the use of CRISPR/Cas9 has been limited to only a few model insects. Here, for the first time, a CRISPR/Cas9 protocol for mutagenesis studies in CPB was developed. A gene with a clear phenotype such as the vestigial gene (vest), known to be involved in wing development in other insect species, was selected as a good indicator for the knockout study. First, vest was functionally characterized in CPB by using RNAi technology for knockdown studies. Once the expected deformed wing phenotypes were observed, a CRISPR/Cas9 work flow was established for mutagenesis in CPB. By co-injecting the Cas9 protein and a vest-guide RNA into 539 CPB eggs of <1 h old, sixty-two successfully developed to adults, among which mutation in the vest loci was confirmed in 5 of the 18 wingless CPBs (29% phenotypic mutation efficiency). The mutation in vest resulted in a clear phenotype in the CPBs, which developed to adulthood with no hindwing and elytron formed. Altogether, this study provides for the first time a useful methodology involving the use of the CRISPR/Cas9 system for mutagenesis studies in one of the most important pest insects.},
}

@article {pmid31916673,
year = {2020},
author = {Wang, M and Xu, Z and Gosavi, G and Ren, B and Cao, Y and Kuang, Y and Zhou, C and Spetz, C and Yan, F and Zhou, X and Zhou, H},
title = {Targeted base editing in rice with CRISPR/ScCas9 system.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13330},
pmid = {31916673},
issn = {1467-7652},
abstract = {The CRISPR/Cas system has rapidly become the preferred tool for genome engineering in various organisms due to high efficiency, specificity, simplicity and versatility. Currently, CRISPR/Cas-mediated base editing, a novel genome editing strategy that enables irreversible nucleotide changes at target loci without double-stranded DNA cleavage or any donor template, has been widely adopted for generating gain-of-function germplasms in functional genomics research and crop genetic improvement (Hua et al., 2019; Ren et al., 2018; Yan et al., 2018).},
}

@article {pmid31907146,
year = {2019},
author = {Chen, G and Cheng, D and Chen, B},
title = {[Development of CRISPR technology and its application in bone and cartilage tissue engineering].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {39},
number = {12},
pages = {1515-1520},
doi = {10.12122/j.issn.1673-4254.2019.12.19},
pmid = {31907146},
issn = {1673-4254},
mesh = {CRISPR-Cas Systems ; Cartilage ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide ; *Tissue Engineering ; },
abstract = {The CRISPR/Cas9 system, consisting of Cas9 nuclease and single guide RNA (sgRNA), is an emerging gene editing technology that can perform gene reprogramming operations such as deletion, insertion, and point mutation on DNA sequences targeted by sgRNA. In addition, CRISPR/dCas9 (a mutant that loses Cas9 nuclease activity) still retains the ability of sgRNA to target DNA. The fusion of dCas9 protein with transcriptional activator (CRISPRa) can activate the expression of the target gene, and fusion transcriptional repressors (CRISPRi) can also be used to suppress target gene expression. Efficient delivery of the CRISPR/Cas9 system is one of the main problems limiting its wide clinical application. Viral vectors are widely used to efficiently deliver CRISPR/Cas9 elements, but non-viral vector research is more attractive in terms of safety, simplicity, and flexibility. In this review, we summarize the principles and research advances of CRISPR technology, including CRISPR/ Cas9 delivery vectors, delivery methods, and obstacles to the delivery, and review the progress of CRISPR-based research in bone and cartilage tissue engineering. Finally, the challenges and future applications of CRISPR technology in bone and cartilage tissue engineering are discussed.},
}

CRISPR-Cas Systems
Cartilage
*Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Guide
*Tissue Engineering

@article {pmid31394891,
year = {2019},
author = {Butt, H and Piatek, A and Li, L and S N Reddy, A and M Mahfouz, M},
title = {Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice.},
journal = {Genes},
volume = {10},
number = {8},
pages = {},
doi = {10.3390/genes10080596},
pmid = {31394891},
issn = {2073-4425},
mesh = {Alternative Splicing ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Mutagenesis ; Oryza/*genetics ; Plant Proteins/*genetics/metabolism ; Serine-Arginine Splicing Factors/*genetics/metabolism ; },
abstract = {Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses.},
}

Alternative Splicing
*CRISPR-Cas Systems
Gene Editing/*methods
Mutagenesis
Oryza/*genetics
Plant Proteins/*genetics/metabolism
Serine-Arginine Splicing Factors/*genetics/metabolism

@article {pmid31003997,
year = {2019},
author = {Shin, TH and Baek, EJ and Corat, MAF and Chen, S and Metais, JY and AlJanahi, AA and Zhou, Y and Donahue, RE and Yu, KR and Dunbar, CE},
title = {CRISPR/Cas9 PIG-A gene editing in nonhuman primate model demonstrates no intrinsic clonal expansion of PNH HSPCs.},
journal = {Blood},
volume = {133},
number = {23},
pages = {2542-2545},
doi = {10.1182/blood.2019000800},
pmid = {31003997},
issn = {1528-0020},
mesh = {Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Gene Editing/methods ; *Hematopoietic Stem Cells ; Hemoglobinuria, Paroxysmal/*genetics ; Macaca mulatta ; Membrane Proteins/*genetics ; },
}

Animals
CRISPR-Cas Systems
*Disease Models, Animal
Gene Editing/methods
*Hematopoietic Stem Cells
Hemoglobinuria, Paroxysmal/*genetics
Macaca mulatta
Membrane Proteins/*genetics

@article {pmid30987342,
year = {2019},
author = {Pálinkás, HL and Rácz, GA and Gál, Z and Hoffmann, OI and Tihanyi, G and Róna, G and Gócza, E and Hiripi, L and Vértessy, BG},
title = {CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality.},
journal = {Biomolecules},
volume = {9},
number = {4},
pages = {},
doi = {10.3390/biom9040136},
pmid = {30987342},
issn = {2218-273X},
mesh = {Animals ; Blastocyst/metabolism/pathology ; CRISPR-Cas Systems ; Cells, Cultured ; Embryonic Development/*genetics ; *Gene Deletion ; Heterozygote ; Homozygote ; Mice ; Mice, Knockout ; Pyrophosphatases/*genetics/metabolism ; },
abstract = {Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2'-deoxyuridine 5'-triphosphate (dUTP) into 2'-deoxyuridine 5'-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/- animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut -/- embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice.},
}

Animals
Blastocyst/metabolism/pathology
CRISPR-Cas Systems
Cells, Cultured
Embryonic Development/*genetics
*Gene Deletion
Heterozygote
Homozygote
Mice
Mice, Knockout
Pyrophosphatases/*genetics/metabolism

@article {pmid30975639,
year = {2019},
author = {Wang, L and Yang, Y and Breton, CA and White, J and Zhang, J and Che, Y and Saveliev, A and McMenamin, D and He, Z and Latshaw, C and Li, M and Wilson, JM},
title = {CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX-knockout mice.},
journal = {Blood},
volume = {133},
number = {26},
pages = {2745-2752},
doi = {10.1182/blood.2019000790},
pmid = {30975639},
issn = {1528-0020},
mesh = {Animals ; Animals, Newborn ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Factor IX/*genetics ; Gene Targeting/*methods ; Genetic Therapy/methods ; Genetic Vectors ; Hemophilia B/*genetics ; Hemostasis/*genetics ; Humans ; Mice ; Mice, Knockout ; },
abstract = {Many genetic diseases, including hemophilia, require long-term therapeutic effects. Despite the initial success of liver-directed adeno-associated virus (AAV) gene therapy for hemophilia in clinical trials, long-term sustained therapeutic effects have yet to be seen. One explanation for the gradual decline of efficacy over time is that the nonintegrating AAV vector genome could be lost during cell division during hepatocyte turnover, albeit at a slow pace in adults. Readministering the same vector is challenging as a result of the AAV-neutralizing antibodies elicited by the initial treatment. Here, we investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed gene targeting for sustained treatment of hemophilia B. We developed a donor vector containing a promoterless partial human factor IX (FIX) complementary DNA carrying the hyperactive FIX Padua mutation. A single injection of dual AAV vectors in newborn and adult FIX-knockout (FIX-KO) mice led to stable expression of FIX at or above the normal levels for 8 months. Eight weeks after the vector treatment, we subjected a subgroup of newborn and adult treated FIX-KO mice to a two-thirds partial hepatectomy; all of these animals survived the procedure without any complications or interventions. FIX levels persisted at similar levels for 24 weeks after partial hepatectomy, indicating stable genomic targeting. Our results lend support for the use of a CRISPR/Cas9 approach to achieve lifelong expression of therapeutic proteins.},
}

Animals
Animals, Newborn
*CRISPR-Cas Systems
Dependovirus/genetics
Factor IX/*genetics
Gene Targeting/*methods
Genetic Therapy/methods
Genetic Vectors
Hemophilia B/*genetics
Hemostasis/*genetics
Humans
Mice
Mice, Knockout

@article {pmid30831220,
year = {2019},
author = {Xu, J and Yu, Y and Chen, K and Huang, Y},
title = {Intersex regulates female external genital and imaginal disc development in the silkworm.},
journal = {Insect biochemistry and molecular biology},
volume = {108},
number = {},
pages = {1-8},
doi = {10.1016/j.ibmb.2019.02.003},
pmid = {30831220},
issn = {1879-0240},
mesh = {Animals ; Bombyx/*genetics/*growth & development ; CRISPR-Cas Systems ; Female ; Gene Expression Regulation, Developmental ; Genitalia, Female/growth & development ; Imaginal Discs/*growth & development ; Male ; Mutation ; Signal Transduction ; },
abstract = {As a component of the mediator complex, the intersex (ix) gene product is involved in the sex determination pathway of the Drosophila melanogaster. IX functions together with the female-specific product of doublesex (dsx) at the bottom of the hierarchy to implement female sexual differentiation. Here we analyzed the functions of the ix gene in the model lepidopteran insect Bombyx mori. We found that Bmix is expressed in many tissues and is highly expressed in early pupal stages. We used the transgene-based CRISPR/Cas9 system to generate mutants of the Bmix gene. The Bmix female mutants were sterile and had irregular external genitalia, whereas in the mutant males external genitalia were normal. Mutants of both sexes had normal gonad development and normal splicing of the Bmdsx pre-mRNA, suggesting that Bmix functions independently of Bmdsx. Interestingly, both male and female mutants had defective development of the imaginal disc including wing, antenna, and leg. RNA-seq and gene expression analyses indicated that genes involved in WNT, Hippo, and Hedgehog signaling pathways and wing development genes Bmawd and Bmfng were up-regulated or down-regulated in the Bmix mutants compared with wild-type animals. Our data provide insights into the multiple functions of Bmix in female external genital and imaginal disc development in the silkworm.},
}

Animals
Bombyx/*genetics/*growth & development
CRISPR-Cas Systems
Female
Gene Expression Regulation, Developmental
Genitalia, Female/growth & development
Imaginal Discs/*growth & development
Male
Mutation
Signal Transduction

@article {pmid30747044,
year = {2019},
author = {Dyikanov, DT and Vasiluev, PA and Rysenkova, KD and Aleksandrushkina, NA and Tyurin-Kuzmin, PA and Kulebyakin, KY and Rubtsov, YP and Shmakova, AA and Evseeva, MN and Balatskiy, AV and Semina, EV and Rostovtseva, AI and Makarevich, PI and Karagyaur, MN},
title = {Optimization of CRISPR/Cas9 Technology to Knock Out Genes of Interest in Aneuploid Cell Lines.},
journal = {Tissue engineering. Part C, Methods},
volume = {25},
number = {3},
pages = {168-175},
doi = {10.1089/ten.TEC.2018.0365},
pmid = {30747044},
issn = {1937-3392},
mesh = {*Aneuploidy ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques/*standards ; Genes/*genetics ; HeLa Cells ; Hep G2 Cells ; Humans ; Mice ; NIH 3T3 Cells ; },
abstract = {IMPACT STATEMENT: Cell lines represent convenient models to elucidate specific causes of multigenetic and pluricausal diseases, to test breakthrough regenerative technologies. Most commonly used cell lines surpass diploid cells in their accessibility for delivery of large DNA molecules and genome editing, but the main obstacles for obtaining cell models with knockout-targeted protein from aneuploid cells are multiple allele copies and karyotype/phenotype heterogeneity. In the study, we report an original approach to CRISPR-/Cas9-mediated genome modification of aneuploid cell cultures to create functional cell models, achieving highly efficient targeted protein knockout and avoiding "clonal effect" (for the first time to our knowledge).},
}

*Aneuploidy
Animals
*CRISPR-Cas Systems
*Gene Editing
Gene Knockout Techniques/*standards
Genes/*genetics
HeLa Cells
Hep G2 Cells
Humans
Mice
NIH 3T3 Cells

@article {pmid30731491,
year = {2019},
author = {Kong, X and Ma, L and Chen, E and Shaw, CA and Edelstein, LC},
title = {Identification of the Regulatory Elements and Target Genes of Megakaryopoietic Transcription Factor MEF2C.},
journal = {Thrombosis and haemostasis},
volume = {119},
number = {5},
pages = {716-725},
pmid = {30731491},
issn = {2567-689X},
support = {R01 HL128234/HL/NHLBI NIH HHS/United States ; },
mesh = {Blood Platelets/*physiology ; Bone Marrow/*physiology ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Chromatin Immunoprecipitation ; Gene Expression Regulation ; Gene Ontology ; Humans ; Induced Pluripotent Stem Cells/*physiology ; MEF2 Transcription Factors/*genetics ; Megakaryocytes/*physiology ; RNA, Small Interfering/genetics ; Regulatory Elements, Transcriptional/*genetics ; Thrombopoiesis/*genetics ; },
abstract = {Megakaryopoiesis produces specialized haematopoietic stem cells in the bone marrow that give rise to megakaryocytes which ultimately produce platelets. Defects in megakaryopoiesis can result in altered platelet counts and physiology, leading to dysfunctional haemostasis and thrombosis. Additionally, dysregulated megakaryopoiesis is also associated with myeloid pathologies. Transcription factors play critical roles in cell differentiation by regulating the temporal and spatial patterns of gene expression which ultimately decide cell fate. Several transcription factors have been described as regulating megakaryopoiesis including myocyte enhancer factor 2C (MEF2C); however, the genes regulated by MEF2C that influence megakaryopoiesis have not been reported. Using chromatin immunoprecipitation-sequencing and Gene Ontology data we identified five candidate genes that are bound by MEF2C and regulate megakaryopoiesis: MOV10, AGO3, HDAC1, RBBP5 and WASF2. To study expression of these genes, we silenced MEF2C gene expression in the Meg01 megakaryocytic cell line and in induced pluripotent stem cells by CRISPR/Cas9 editing. We also knocked down MEF2C expression in cord blood-derived haematopoietic stem cells by siRNA. We found that absent or reduced MEF2C expression resulted in defects in megakaryocytic differentiation and reduced levels of the candidate target genes. Luciferase assays confirmed that genomic sequences within the target genes are regulated by MEF2C levels. Finally, we demonstrate that small deletions linked to a platelet count-associated single nucleotide polymorphism alter transcriptional activity, suggesting a mechanism by which genetic variation in MEF2C alters platelet production. These data help elucidate the mechanism behind MEF2C regulation of megakaryopoiesis and genetic variation driving platelet production.},
}

Blood Platelets/*physiology
Bone Marrow/*physiology
CRISPR-Cas Systems
Cell Differentiation
Cell Line
Chromatin Immunoprecipitation
Gene Expression Regulation
Gene Ontology
Humans
Induced Pluripotent Stem Cells/*physiology
MEF2 Transcription Factors/*genetics
Megakaryocytes/*physiology
RNA, Small Interfering/genetics
Regulatory Elements, Transcriptional/*genetics
Thrombopoiesis/*genetics

@article {pmid30622144,
year = {2019},
author = {Nasri, M and Mir, P and Dannenmann, B and Amend, D and Skroblyn, T and Xu, Y and Schulze-Osthoff, K and Klimiankou, M and Welte, K and Skokowa, J},
title = {Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response.},
journal = {Blood advances},
volume = {3},
number = {1},
pages = {63-71},
doi = {10.1182/bloodadvances.2017015511},
pmid = {30622144},
issn = {2473-9537},
mesh = {Antigens, Differentiation/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; DNA Damage ; Gene Editing/methods ; *Gene Knockout Techniques ; Gene Targeting/methods ; Hematopoietic Stem Cells/*metabolism ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Macromolecular Substances/metabolism ; Protein Binding ; RNA, Guide/genetics ; Ribonucleoproteins/*metabolism ; Stress, Physiological/*genetics/radiation effects ; },
abstract = {CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9-gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible β (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect.},
}

Antigens, Differentiation/*genetics/metabolism
*CRISPR-Cas Systems
Cell Line
DNA Damage
Gene Editing/methods
*Gene Knockout Techniques
Gene Targeting/methods
Hematopoietic Stem Cells/*metabolism
Humans
Induced Pluripotent Stem Cells/*metabolism
Macromolecular Substances/metabolism
Protein Binding
RNA, Guide/genetics
Ribonucleoproteins/*metabolism
Stress, Physiological/*genetics/radiation effects

@article {pmid31914418,
year = {2019},
author = {Ramachandran, A and Summerville, L and Learn, BA and DeBell, L and Bailey, S},
title = {Processing and integration of functionally oriented prespacers in the E. coli CRISPR system depends on bacterial host exonucleases.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA119.012196},
pmid = {31914418},
issn = {1083-351X},
abstract = {CRISPR/Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3' overhangs via specific recognition of a protospacer adjacent motif (PAM), but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing and quantitative PCR assays, we show here that the bacterial 3'-5' exonucleases DnaQ or ExoT can trim long 3' overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3'-5' exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.},
}

@article {pmid31913359,
year = {2020},
author = {Wang, B and Xu, W and Yang, H},
title = {Structural basis of a Tn7-like transposase recruitment and DNA loading to CRISPR-Cas surveillance complex.},
journal = {Cell research},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41422-020-0274-0},
pmid = {31913359},
issn = {1748-7838},
support = {31971135//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
}

@article {pmid31913120,
year = {2020},
author = {Hickman, AB and Kailasan, S and Genzor, P and Haase, AD and Dyda, F},
title = {Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
doi = {10.7554/eLife.50004},
pmid = {31913120},
issn = {2050-084X},
support = {Intramural Program/DK/NIDDK NIH HHS/United States ; },
abstract = {Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanosarcina mazei casposase can integrate varied forms of the casposon end in vitro, and recapitulates several properties of CRISPR-Cas integrases including site-specificity. The X-ray structure of the casposase bound to DNA representing the product of integration reveals a tetramer with target DNA bound snugly between two dimers in which single-stranded casposon end binding resembles that of spacer 3'-overhangs. The differences between transposase and CRISPR-Cas integrase are largely architectural, and it appears that evolutionary change involved changes in protein-protein interactions to favor Cas2 binding over tetramerization; this in turn led to preferred integration of single spacers over two transposon ends.},
}

@article {pmid31911609,
year = {2020},
author = {Munck, C and Sheth, RU and Freedberg, DE and Wang, HH},
title = {Recording mobile DNA in the gut microbiota using an Escherichia coli CRISPR-Cas spacer acquisition platform.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {95},
doi = {10.1038/s41467-019-14012-5},
pmid = {31911609},
issn = {2041-1723},
abstract = {The flow of genetic material between bacteria is central to the adaptation and evolution of bacterial genomes. However, our knowledge about DNA transfer within complex microbiomes is lacking, with most studies of horizontal gene transfer (HGT) relying on bioinformatic analyses of genetic elements maintained on evolutionary timescales or experimental measurements of phenotypically trackable markers. Here, we utilize the CRISPR-Cas spacer acquisition process to detect DNA acquisition events from complex microbiota in real-time and at nucleotide resolution. In this system, an E. coli recording strain is exposed to a microbial sample and spacers are acquired from transferred plasmids and permanently stored in genomic CRISPR arrays. Sequencing and analysis of acquired spacers enables identification of the transferred plasmids. This approach allowed us to identify individual mobile elements without relying on phenotypic markers or post-transfer replication. We found that HGT into the recording strain in human clinical fecal samples can be extensive and is driven by different plasmid types, with the IncX type being the most actively transferred.},
}

@article {pmid31911131,
year = {2019},
author = {Li, J and Hong, S and Chen, W and Zuo, E and Yang, H},
title = {Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2019.11.002},
pmid = {31911131},
issn = {1673-8527},
abstract = {CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors. Currently, poor efficiency and off-target problems have impeded the application of CRISPR systems. The on-target efficiency has been improved in several advanced versions of CRISPR systems, whereas the off-target detection still remains a key challenge. Here, we outline the different versions of CRISPR systems and off-target detection strategies, discuss the merits and limitations of off-target detection methods, and provide potential implications for further gene editing research.},
}

@article {pmid31910089,
year = {2020},
author = {Tatineni, S and Stewart, LR and Sanfaçon, H and Wang, X and Navas-Castillo, J and Hajimorad, MR},
title = {Fundamental Aspects of Plant Viruses-An Overview on Focus Issue Articles.},
journal = {Phytopathology},
volume = {110},
number = {1},
pages = {6-9},
doi = {10.1094/PHYTO-10-19-0404-FI},
pmid = {31910089},
issn = {0031-949X},
abstract = {Given the importance of and rapid research progress in plant virology in recent years, this Focus Issue broadly emphasizes advances in fundamental aspects of virus infection cycles and epidemiology. This Focus Issue comprises three review articles and 18 research articles. The research articles cover broad research areas on the identification of novel viruses, the development of detection methods, reverse genetics systems and functional genomics for plant viruses, vector and seed transmission studies, viral population studies, virus-virus interactions and their effect on vector transmission, and management strategies of viral diseases. The three review articles discuss recent developments in application of prokaryotic clustered regularly interspaced short palindromic repeats/CRISPR-associated genes (CRISPR/Cas) technology for plant virus resistance, mixed viral infections and their role in disease synergism and cross-protection, and viral transmission by whiteflies. The following briefly summarizes the articles appearing in this Focus Issue.},
}

@article {pmid31906943,
year = {2020},
author = {Yeo, WL and Heng, E and Tan, LL and Lim, YW and Ching, KC and Tsai, DJ and Jhang, YW and Lauderdale, TL and Shia, KS and Zhao, H and Ang, EL and Zhang, MM and Lim, YH and Wong, FT},
title = {Biosynthetic engineering of the antifungal, anti-MRSA auroramycin.},
journal = {Microbial cell factories},
volume = {19},
number = {1},
pages = {3},
doi = {10.1186/s12934-019-1274-y},
pmid = {31906943},
issn = {1475-2859},
support = {NRF2013-THE001-094//National Research Foundation Singapore/ ; NRF2013-THE001-094//National Research Foundation Singapore/ ; NRF2013-THE001-094//National Research Foundation Singapore/ ; Visiting Investigator Program//Agency for Science, Technology and Research, ASTAR, Singapore/ ; 1535j00137//ASTAR Visiting Investigator Program/ ; },
abstract = {Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.},
}

@article {pmid31853027,
year = {2020},
author = {Neff, EP},
title = {CRISPR takes genetic screens forward.},
journal = {Lab animal},
volume = {49},
number = {1},
pages = {13-16},
doi = {10.1038/s41684-019-0445-0},
pmid = {31853027},
issn = {1548-4475},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Early Detection of Cancer ; Humans ; Immunotherapy ; *Neoplasms ; Protein Tyrosine Phosphatase, Non-Receptor Type 2 ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Early Detection of Cancer
Humans
Immunotherapy
*Neoplasms
Protein Tyrosine Phosphatase, Non-Receptor Type 2

@article {pmid31614382,
year = {2019},
author = {Zhou, Y and Yang, H and Shi, J and Zhang, M and Yang, S and Wang, N and Sun, R and Wang, Z and Fei, J},
title = {Fate Tracing of Isl1+Cells in Adult Mouse Hearts under Physiological and Exercise Conditions.},
journal = {International journal of sports medicine},
volume = {40},
number = {14},
pages = {921-930},
doi = {10.1055/a-0961-1458},
pmid = {31614382},
issn = {1439-3964},
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Proliferation/physiology ; Chromosome Mapping ; Heart/embryology/growth & development/*physiology ; LIM-Homeodomain Proteins/*genetics ; Male ; Mice, Inbred C57BL ; Myocardium/*cytology ; Myocytes, Cardiac/*cytology ; *Physical Conditioning, Animal ; Stem Cells/*physiology ; Transcription Factors/*genetics ; },
abstract = {Myocardial damage due to dysfunctional myocardium has been increasing, and the prognosis of pharmacological and device-based therapies remain poor. Isl1-expressing cells were thought to be progenitor cells for cardiomyocyte proliferation after specific stimuli. However, the true origin of the proliferating myocardiac cells and the role of Isl1 in adult mammals remain unresolved. In this study, Isl1-CreERT2 knock-in mouse model was constructed using CRISPR/Cas9 technology. Using tamoxifen-inducible Isl1-CreERT/Rosa26R-LacZ system, Isl1+cells and their progeny were permanently marked by lacZ-expression. X-gal staining, immunostaining, and quantitative PCR were then used to reveal the fate of Isl1+cells under physiological and exercise conditions in mouse hearts from embryonic stage to adulthood. Isl1+cells were found to localize to the sinoatrial node, atrioventricular node, cardiac ganglia, aortic arch, and pulmonary roots in adult mice heart. However, they did not act as cardiac progenitor cells under physiological and exercise conditions. Although Isl1+cells showed progenitor cell properties in early mouse embryos (E7.5), this ability was lost by E9.5. Furthermore, although the proliferation and regeneration of heart cell was observed in response to exercise, the cells associated were not Isl1 positive.},
}

Animals
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Cell Proliferation/physiology
Chromosome Mapping
Heart/embryology/growth & development/*physiology
LIM-Homeodomain Proteins/*genetics
Male
Mice, Inbred C57BL
Myocardium/*cytology
Myocytes, Cardiac/*cytology
*Physical Conditioning, Animal
Stem Cells/*physiology
Transcription Factors/*genetics

@article {pmid31481656,
year = {2019},
author = {Young, J and Dominicus, C and Wagener, J and Butterworth, S and Ye, X and Kelly, G and Ordan, M and Saunders, B and Instrell, R and Howell, M and Stewart, A and Treeck, M},
title = {A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3963},
doi = {10.1038/s41467-019-11855-w},
pmid = {31481656},
issn = {2041-1723},
support = {FC001189/CRUK_/Cancer Research UK/United Kingdom ; FC001189/WT_/Wellcome Trust/United Kingdom ; FC001189/MRC_/Medical Research Council/United Kingdom ; R01 AI123457/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques/methods ; Gene Library ; Genome, Protozoan ; Humans ; Mice, Inbred C57BL ; RNA, Guide ; Toxoplasma/*genetics/pathogenicity ; Toxoplasmosis/genetics/parasitology/pathology ; Virulence Factors/*genetics ; },
abstract = {Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1.},
}

Animals
*CRISPR-Cas Systems
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Knockout Techniques/methods
Gene Library
Genome, Protozoan
Humans
Mice, Inbred C57BL
RNA, Guide
Toxoplasma/*genetics/pathogenicity
Toxoplasmosis/genetics/parasitology/pathology
Virulence Factors/*genetics

@article {pmid31465441,
year = {2019},
author = {Fleiss, A and O'Donnell, S and Fournier, T and Lu, W and Agier, N and Delmas, S and Schacherer, J and Fischer, G},
title = {Reshuffling yeast chromosomes with CRISPR/Cas9.},
journal = {PLoS genetics},
volume = {15},
number = {8},
pages = {e1008332},
doi = {10.1371/journal.pgen.1008332},
pmid = {31465441},
issn = {1553-7404},
mesh = {Anion Transport Proteins/genetics ; *CRISPR-Cas Systems ; Chromosomes, Fungal/*genetics ; DNA Shuffling/*methods ; Gene Editing/*methods ; Genome, Fungal/genetics ; Promoter Regions, Genetic/genetics ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/genetics ; Translocation, Genetic ; },
abstract = {Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions.},
}

Anion Transport Proteins/genetics
*CRISPR-Cas Systems
Chromosomes, Fungal/*genetics
DNA Shuffling/*methods
Gene Editing/*methods
Genome, Fungal/genetics
Promoter Regions, Genetic/genetics
Saccharomyces cerevisiae/*genetics
Saccharomyces cerevisiae Proteins/genetics
Translocation, Genetic

@article {pmid31405997,
year = {2019},
author = {Nance, J and Frøkjær-Jensen, C},
title = {The Caenorhabditis elegans Transgenic Toolbox.},
journal = {Genetics},
volume = {212},
number = {4},
pages = {959-990},
doi = {10.1534/genetics.119.301506},
pmid = {31405997},
issn = {1943-2631},
support = {R21 HD088779/HD/NICHD NIH HHS/United States ; R35 GM118081/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; *Genetic Techniques ; },
abstract = {The power of any genetic model organism is derived, in part, from the ease with which gene expression can be manipulated. The short generation time and invariant developmental lineage have made Caenorhabditis elegans very useful for understanding, e.g., developmental programs, basic cell biology, neurobiology, and aging. Over the last decade, the C. elegans transgenic toolbox has expanded considerably, with the addition of a variety of methods to control expression and modify genes with unprecedented resolution. Here, we provide a comprehensive overview of transgenic methods in C. elegans, with an emphasis on recent advances in transposon-mediated transgenesis, CRISPR/Cas9 gene editing, conditional gene and protein inactivation, and bipartite systems for temporal and spatial control of expression.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems
Caenorhabditis elegans/*genetics
*Genetic Techniques

@article {pmid31386652,
year = {2019},
author = {Bittermann, E and Abdelhamed, Z and Liegel, RP and Menke, C and Timms, A and Beier, DR and Stottmann, RW},
title = {Differential requirements of tubulin genes in mammalian forebrain development.},
journal = {PLoS genetics},
volume = {15},
number = {8},
pages = {e1008243},
doi = {10.1371/journal.pgen.1008243},
pmid = {31386652},
issn = {1553-7404},
support = {R01 NS085023/NS/NINDS NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo, Mammalian ; Ethylnitrosourea/toxicity ; Female ; Gene Deletion ; Gene Editing ; Gene Expression Regulation, Developmental ; Humans ; Male ; Malformations of Cortical Development/*genetics/mortality/pathology ; Mice ; Mice, Transgenic ; Microtubules/genetics ; Models, Animal ; Mutagenesis/drug effects ; Neurogenesis/*genetics ; Sensorimotor Cortex/abnormalities/*embryology ; Species Specificity ; Tubulin/*genetics/metabolism ; },
abstract = {Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases.},
}

Alleles
Animals
CRISPR-Cas Systems
Disease Models, Animal
Embryo, Mammalian
Ethylnitrosourea/toxicity
Female
Gene Deletion
Gene Editing
Gene Expression Regulation, Developmental
Humans
Male
Malformations of Cortical Development/*genetics/mortality/pathology
Mice
Mice, Transgenic
Microtubules/genetics
Models, Animal
Mutagenesis/drug effects
Neurogenesis/*genetics
Sensorimotor Cortex/abnormalities/*embryology
Species Specificity
Tubulin/*genetics/metabolism

@article {pmid31315144,
year = {2019},
author = {Afolabi, LO and Adeshakin, AO and Sani, MM and Bi, J and Wan, X},
title = {Genetic reprogramming for NK cell cancer immunotherapy with CRISPR/Cas9.},
journal = {Immunology},
volume = {158},
number = {2},
pages = {63-69},
doi = {10.1111/imm.13094},
pmid = {31315144},
issn = {1365-2567},
mesh = {Adaptive Immunity ; CRISPR-Associated Protein 9/genetics/immunology ; CRISPR-Cas Systems/*immunology ; Cellular Reprogramming/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/immunology ; Cytotoxicity, Immunologic ; Drug Delivery Systems/*methods ; Gene Editing/methods ; *Gene Transfer Techniques ; Humans ; Immunotherapy/methods ; Killer Cells, Natural/*immunology/metabolism ; Metal Nanoparticles/administration & dosage ; Neoplasms/genetics/immunology/pathology/*therapy ; Plasmids/chemistry/immunology ; RNA, Guide/genetics/immunology ; Receptors, Chimeric Antigen/*genetics/immunology ; Tumor Microenvironment/genetics/immunology ; },
abstract = {Natural killer cells are potent cytotoxic lymphocytes specialized in recognizing and eliminating transformed cells, and in orchestrating adaptive anti-tumour immunity. However, NK cells are usually functionally exhausted in the tumour microenvironment. Strategies such as checkpoint blockades are under investigation to overcome NK cell exhaustion in order to boost anti-tumour immunity. The discovery and development of the CRISPR/Cas9 technology offer a flexible and efficient gene-editing capability in modulating various pathways that mediate NK cell exhaustion, and in arming NK cells with novel chimeric antigen receptors to specifically target tumour cells. Despite the high efficiency in its gene-editing capability, difficulty in the delivery of the CRISPR/Cas9 system remains a major bottleneck for its therapeutic applications, particularly for NK cells. The current review discusses feasible approaches to deliver the CRISPR/Cas9 systems, as well as potential strategies in gene-editing for NK cell immunotherapy for cancers.},
}

Adaptive Immunity
CRISPR-Associated Protein 9/genetics/immunology
CRISPR-Cas Systems/*immunology
Cellular Reprogramming/immunology
Clustered Regularly Interspaced Short Palindromic Repeats/immunology
Cytotoxicity, Immunologic
Drug Delivery Systems/*methods
Gene Editing/methods
*Gene Transfer Techniques
Humans
Immunotherapy/methods
Killer Cells, Natural/*immunology/metabolism
Metal Nanoparticles/administration & dosage
Neoplasms/genetics/immunology/pathology/*therapy
Plasmids/chemistry/immunology
RNA, Guide/genetics/immunology
Receptors, Chimeric Antigen/*genetics/immunology
Tumor Microenvironment/genetics/immunology

@article {pmid31172498,
year = {2019},
author = {Sweeney, CL and Merling, RK and De Ravin, SS and Choi, U and Malech, HL},
title = {Gene Editing in Chronic Granulomatous Disease.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1982},
number = {},
pages = {623-665},
doi = {10.1007/978-1-4939-9424-3_36},
pmid = {31172498},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cell Differentiation/genetics/immunology ; Cell Line ; Cells, Cultured ; Cloning, Molecular ; *Gene Editing/methods ; Gene Order ; Gene Targeting ; Genetic Vectors ; Granulomatous Disease, Chronic/*genetics/metabolism ; Hematopoietic Stem Cells/cytology/metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Neutrophils/cytology/immunology/metabolism ; RNA, Guide ; Reactive Oxygen Species/metabolism ; },
abstract = {Chronic granulomatous disease (CGD) is an immune deficiency characterized by defects in the production of microbicidal reactive oxygen species (ROS) by the phagocytic oxidase (phox) enzyme complex in neutrophils. We have previously described targeted gene editing strategies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases for gene targeting with homology-directed repair in CGD patient stem cells to achieve functional restoration of expression of phox genes and NADPH oxidase activity in differentiated neutrophils. In this chapter, we describe detailed protocols for targeted gene editing in human-induced pluripotent stem cells and hematopoietic stem cells and for subsequent differentiation of these stem cells into mature neutrophils, as well as assays to characterize neutrophil identity and function including flow cytometry analysis of neutrophil surface markers, intracellular staining for phox proteins, and analysis of ROS generation.},
}

CRISPR-Cas Systems
Cell Differentiation/genetics/immunology
Cell Line
Cells, Cultured
Cloning, Molecular
*Gene Editing/methods
Gene Order
Gene Targeting
Genetic Vectors
Granulomatous Disease, Chronic/*genetics/metabolism
Hematopoietic Stem Cells/cytology/metabolism
Humans
Induced Pluripotent Stem Cells/cytology/metabolism
Neutrophils/cytology/immunology/metabolism
RNA, Guide
Reactive Oxygen Species/metabolism

@article {pmid30998719,
year = {2019},
author = {Stevens, RC and Steele, JL and Glover, WR and Sanchez-Garcia, JF and Simpson, SD and O'Rourke, D and Ramsdell, JS and MacManes, MD and Thomas, WK and Shuber, AP},
title = {A novel CRISPR/Cas9 associated technology for sequence-specific nucleic acid enrichment.},
journal = {PloS one},
volume = {14},
number = {4},
pages = {e0215441},
doi = {10.1371/journal.pone.0215441},
pmid = {30998719},
issn = {1932-6203},
support = {R35 GM128843/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/*genetics ; *Gene Editing ; High-Throughput Nucleotide Sequencing ; *Polymerase Chain Reaction ; RNA, Guide/*genetics ; Sequence Analysis, DNA ; },
abstract = {Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5' and 3' termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.},
}

*CRISPR-Cas Systems
DNA/*genetics
*Gene Editing
High-Throughput Nucleotide Sequencing
*Polymerase Chain Reaction
RNA, Guide/*genetics
Sequence Analysis, DNA

@article {pmid30986258,
year = {2019},
author = {Tormanen, K and Ton, C and Waring, BM and Wang, K and Sütterlin, C},
title = {Function of Golgi-centrosome proximity in RPE-1 cells.},
journal = {PloS one},
volume = {14},
number = {4},
pages = {e0215215},
doi = {10.1371/journal.pone.0215215},
pmid = {30986258},
issn = {1932-6203},
support = {R01 GM089913/GM/NIGMS NIH HHS/United States ; T32 GM008620/GM/NIGMS NIH HHS/United States ; },
mesh = {A Kinase Anchor Proteins/genetics/metabolism ; Autoantigens/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Centrosome/*metabolism ; Cytoskeletal Proteins/genetics/metabolism ; Epithelial Cells/cytology/*metabolism ; Gene Deletion ; Golgi Apparatus/genetics/*metabolism ; Humans ; Membrane Proteins/genetics/metabolism ; Retinal Pigment Epithelium/cytology/*metabolism ; },
abstract = {The close physical proximity between the Golgi and the centrosome is a unique feature of mammalian cells that has baffled scientists for years. Several knockdown and overexpression studies have linked the spatial relationship between these two organelles to the control of directional protein transport, directional migration, ciliogenesis and mitotic entry. However, most of these conditions have not only separated these two organelles, but also caused extensive fragmentation of the Golgi, making it difficult to dissect the specific contribution of Golgi-centrosome proximity. In this study, we present our results with stable retinal pigment epithelial (RPE-1) cell lines in which GM130 was knocked out using a CRISPR/Cas9 approach. While Golgi and centrosome organization appeared mostly intact in cells lacking GM130, there was a clear separation of these organelles from each other. We show that GM130 may control Golgi-centrosome proximity by anchoring AKAP450 to the Golgi. We also provide evidence that the physical proximity between these two organelles is dispensable for protein transport, cell migration, and ciliogenesis. These results suggest that Golgi-centrosome proximity per se is not necessary for the normal function of RPE-1 cells.},
}

A Kinase Anchor Proteins/genetics/metabolism
Autoantigens/genetics/metabolism
CRISPR-Cas Systems
Cell Line
Centrosome/*metabolism
Cytoskeletal Proteins/genetics/metabolism
Epithelial Cells/cytology/*metabolism
Gene Deletion
Golgi Apparatus/genetics/*metabolism
Humans
Membrane Proteins/genetics/metabolism
Retinal Pigment Epithelium/cytology/*metabolism

@article {pmid30941642,
year = {2019},
author = {Kumar, R and Kaur, A and Pandey, A and Mamrutha, HM and Singh, GP},
title = {CRISPR-based genome editing in wheat: a comprehensive review and future prospects.},
journal = {Molecular biology reports},
volume = {46},
number = {3},
pages = {3557-3569},
doi = {10.1007/s11033-019-04761-3},
pmid = {30941642},
issn = {1573-4978},
support = {1006474//Indian Council of Agricultural Research/ ; },
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genes, Plant/genetics ; Genetic Engineering/methods ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; Poaceae/genetics ; Triticum/*genetics ; },
abstract = {CRISPR technology has vividly increased its applications in last five years for genome editing in a wide range of organisms from bacteria to plants. It is mostly applied in the field of mammalian research. This emerging versatile tool can be utilized in crop improvement by targeting various traits to increase economic value and adaptability of the crop species under changing climate. In plants, Arabidopsis and rice are the most studied plant species in genome editing through CRISPR technology. Wheat is lagging behind in the utilization of CRISPR based genome modifications. The hexaploid, large genome size and the recalcitrant nature in terms of tissue culture are the major obstacles for CRISPR utilization in wheat. Recently, the IWGSC released the high quality of reference genome for wheat which will greatly accelerate the application of CRISPR-based genome engineering in wheat and helps to resolve the global issue of food security in coming decades. The exogenous DNA-free improved mutants with CRISPR technology having desired traits will increase the productivity under biotic and abiotic stress conditions. To address complex traits involving multigene, recently developed multiplex genome editing toolkits can be used. This is a first review of its kind in which the practical utilization and updates on CRISPR validation in wheat along with its future prospects for use of this technology in wheat improvement are comprehensively discussed. Thus, the compiled information will immensely benefit the researchers for utilization of CRISPR system in wheat improvement across the globe.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing/*methods
Genes, Plant/genetics
Genetic Engineering/methods
Genome, Plant/genetics
Plants, Genetically Modified/genetics
Poaceae/genetics
Triticum/*genetics

@article {pmid30730211,
year = {2019},
author = {Wang, Z and Zhang, Y and Lee, YW and Ivanova, NB},
title = {Combining CRISPR/Cas9-mediated knockout with genetic complementation for in-depth mechanistic studies in human ES cells.},
journal = {BioTechniques},
volume = {66},
number = {1},
pages = {23-27},
doi = {10.2144/btn-2018-0115},
pmid = {30730211},
issn = {1940-9818},
support = {R01 GM107092/NIGMS NIH HHS/National Institute of General Medical Sciences/United States ; },
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Doxycycline/pharmacology ; Gene Expression Regulation ; Gene Knockdown Techniques/*methods ; Genetic Complementation Test ; Human Embryonic Stem Cells/cytology/drug effects/*physiology ; Humans ; Polycomb Repressive Complex 1/genetics ; Proto-Oncogene Proteins/genetics ; RNA, Small Interfering/genetics ; Repressor Proteins/genetics ; SOXF Transcription Factors/genetics ; Transcription Activator-Like Effector Nucleases ; Transgenes ; },
abstract = {Gene regulatory networks that control pluripotency of human embryonic stem cells (hESCs) are of considerable interest for regenerative medicine. RNAi and CRISPR/Cas9 technologies have allowed the identification of hESC regulators on a genome-wide scale. However, these technologies are ill-suited for mechanistic studies because knockdown/knockout clones of essential genes do not grow in culture. We have developed a genetic rescue strategy that combines CRISPR/Cas9-mediated knockout with TALEN-mediated integration of a doxycycline-inducible rescue transgene into a constitutive AASV1 locus. The resulting rescue clones are stable in culture, allow modulation of the rescue transgene dosage by titration of doxycycline in the media and can be combined with various molecular assays, thus providing mechanistic insights into gene function in a variety of cellular contexts.},
}

*CRISPR-Cas Systems
Cells, Cultured
Doxycycline/pharmacology
Gene Expression Regulation
Gene Knockdown Techniques/*methods
Genetic Complementation Test
Human Embryonic Stem Cells/cytology/drug effects/*physiology
Humans
Polycomb Repressive Complex 1/genetics
Proto-Oncogene Proteins/genetics
RNA, Small Interfering/genetics
Repressor Proteins/genetics
SOXF Transcription Factors/genetics
Transcription Activator-Like Effector Nucleases
Transgenes

@article {pmid31901636,
year = {2019},
author = {Broeders, M and Herrero-Hernandez, P and Ernst, MPT and van der Ploeg, AT and Pijnappel, WWMP},
title = {Sharpening the Molecular Scissors: Advances in Gene-Editing Technology.},
journal = {iScience},
volume = {23},
number = {1},
pages = {100789},
doi = {10.1016/j.isci.2019.100789},
pmid = {31901636},
issn = {2589-0042},
abstract = {The ability to precisely modify human genes has been made possible by the development of tools such as meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas. These now make it possible to generate targeted deletions, insertions, gene knock outs, and point variants; to modulate gene expression by targeting transcription factors or epigenetic machineries to DNA; or to target and modify RNA. Endogenous repair mechanisms are used to make the modifications required in DNA; they include non-homologous end joining, homology-directed repair, homology-independent targeted integration, microhomology-mediated end joining, base-excision repair, and mismatch repair. Off-target effects can be monitored using in silico prediction and sequencing and minimized using Cas proteins with higher accuracy, such as high-fidelity Cas9, enhanced-specificity Cas9, and hyperaccurate Cas9. Alternatives to Cas9 have been identified, including Cpf1, Cas12a, Cas12b, and smaller Cas9 orthologs such as CjCas9. Delivery of gene-editing components is performed ex vivo using standard techniques or in vivo using AAV, lipid nanoparticles, or cell-penetrating peptides. Clinical development of gene-editing technology is progressing in several fields, including immunotherapy in cancer treatment, antiviral therapy for HIV infection, and treatment of genetic disorders such as β-thalassemia, sickle cell disease, lysosomal storage disorders, and retinal dystrophy. Here we review these technological advances and the challenges to their clinical implementation.},
}

@article {pmid31901522,
year = {2019},
author = {Chevallereau, A and Meaden, S and Fradet, O and Landsberger, M and Maestri, A and Biswas, A and Gandon, S and van Houte, S and Westra, ER},
title = {Exploitation of the Cooperative Behaviors of Anti-CRISPR Phages.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2019.12.004},
pmid = {31901522},
issn = {1934-6069},
abstract = {Bacteriophages encoding anti-CRISPR proteins (Acrs) must cooperate to overcome phage resistance mediated by the bacterial immune system CRISPR-Cas, where the first phage blocks CRISPR-Cas immunity in order to allow a second Acr phage to successfully replicate. However, in nature, bacteria are frequently not pre-immunized, and phage populations are often not clonal, exhibiting variations in Acr presence and strength. We explored how interactions between Acr phages and initially sensitive bacteria evolve, both in the presence and absence of competing phages lacking Acrs. We find that Acr phages benefit "Acr-negative" phages by limiting the evolution of CRISPR-based resistance and helping Acr-negative phages to replicate on resistant host sub-populations. These benefits depend on the strength of CRISPR-Cas inhibitors and result in strong Acrs providing smaller fitness advantages than weaker ones when Acr phages compete with Acr-negative phages. These results indicate that different Acr types shape the evolutionary dynamics and social interactions of phage populations in natural communities.},
}

@article {pmid31541098,
year = {2019},
author = {Taghbalout, A and Du, M and Jillette, N and Rosikiewicz, W and Rath, A and Heinen, CD and Li, S and Cheng, AW},
title = {Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4296},
doi = {10.1038/s41467-019-12339-7},
pmid = {31541098},
issn = {2041-1723},
support = {P30 CA034196/CA/NCI NIH HHS/United States ; R01 CA115783/CA/NCI NIH HHS/United States ; R01 HG009900/HG/NHGRI NIH HHS/United States ; },
mesh = {5-Methylcytosine/metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism ; Cell Line, Tumor ; Cell Proliferation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *DNA Demethylation ; DNA Glycosylases/metabolism ; DNA Methylation ; *DNA Repair ; DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism ; Gene Editing ; HEK293 Cells ; Humans ; Mixed Function Oxygenases/genetics ; Oxidation-Reduction ; Promoter Regions, Genetic ; Proto-Oncogene Proteins/genetics/metabolism ; RNA, Guide/*metabolism ; Sequence Analysis, RNA ; },
abstract = {Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.},
}

5-Methylcytosine/metabolism
CRISPR-Cas Systems
Cell Cycle Proteins/metabolism
Cell Line, Tumor
Cell Proliferation
*Clustered Regularly Interspaced Short Palindromic Repeats
*DNA Demethylation
DNA Glycosylases/metabolism
DNA Methylation
*DNA Repair
DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism
Gene Editing
HEK293 Cells
Humans
Mixed Function Oxygenases/genetics
Oxidation-Reduction
Promoter Regions, Genetic
Proto-Oncogene Proteins/genetics/metabolism
RNA, Guide/*metabolism
Sequence Analysis, RNA

@article {pmid31537810,
year = {2019},
author = {Wang, D and Zhang, C and Wang, B and Li, B and Wang, Q and Liu, D and Wang, H and Zhou, Y and Shi, L and Lan, F and Wang, Y},
title = {Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4284},
doi = {10.1038/s41467-019-12281-8},
pmid = {31537810},
issn = {2041-1723},
mesh = {Animals ; Base Sequence/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Deep Learning ; Endonucleases/metabolism ; Gene Editing/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; INDEL Mutation/genetics ; Internet ; Mice ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; },
abstract = {Highly specific Cas9 nucleases derived from SpCas9 are valuable tools for genome editing, but their wide applications are hampered by a lack of knowledge governing guide RNA (gRNA) activity. Here, we perform a genome-scale screen to measure gRNA activity for two highly specific SpCas9 variants (eSpCas9(1.1) and SpCas9-HF1) and wild-type SpCas9 (WT-SpCas9) in human cells, and obtain indel rates of over 50,000 gRNAs for each nuclease, covering ~20,000 genes. We evaluate the contribution of 1,031 features to gRNA activity and develope models for activity prediction. Our data reveals that a combination of RNN with important biological features outperforms other models for activity prediction. We further demonstrate that our model outperforms other popular gRNA design tools. Finally, we develop an online design tool DeepHF for the three Cas9 nucleases. The database, as well as the designer tool, is freely accessible via a web server, http://www.DeepHF.com/ .},
}

Animals
Base Sequence/genetics
CRISPR-Cas Systems/*genetics
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
*Deep Learning
Endonucleases/metabolism
Gene Editing/*methods
HEK293 Cells
HeLa Cells
Humans
INDEL Mutation/genetics
Internet
Mice
Promoter Regions, Genetic/genetics
RNA, Guide/*genetics

@article {pmid31537809,
year = {2019},
author = {Feng, W and Simpson, DA and Carvajal-Garcia, J and Price, BA and Kumar, RJ and Mose, LE and Wood, RD and Rashid, N and Purvis, JE and Parker, JS and Ramsden, DA and Gupta, GP},
title = {Genetic determinants of cellular addiction to DNA polymerase theta.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4286},
doi = {10.1038/s41467-019-12234-1},
pmid = {31537809},
issn = {2041-1723},
support = {P30 CA016086/CA/NCI NIH HHS/United States ; R01 CA222092/CA/NCI NIH HHS/United States ; },
mesh = {Aminoquinolines/toxicity ; Animals ; Breast Neoplasms/*genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/*genetics ; DNA-Directed DNA Polymerase/*genetics/metabolism ; HEK293 Cells ; Humans ; Mice ; Mitomycin/toxicity ; Picolinic Acids/toxicity ; },
abstract = {Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.},
}

Aminoquinolines/toxicity
Animals
Breast Neoplasms/*genetics
CRISPR-Cas Systems/genetics
Cell Line
DNA Breaks, Double-Stranded
DNA End-Joining Repair/*genetics
DNA-Directed DNA Polymerase/*genetics/metabolism
HEK293 Cells
Humans
Mice
Mitomycin/toxicity
Picolinic Acids/toxicity

@article {pmid31523835,
year = {2019},
author = {Sun, J and Nagel, R and Zaal, EA and Ugalde, AP and Han, R and Proost, N and Song, JY and Pataskar, A and Burylo, A and Fu, H and Poelarends, GJ and van de Ven, M and van Tellingen, O and Berkers, CR and Agami, R},
title = {SLC1A3 contributes to L-asparaginase resistance in solid tumors.},
journal = {The EMBO journal},
volume = {38},
number = {21},
pages = {e102147},
pmid = {31523835},
issn = {1460-2075},
support = {201503250056//China Scholarship Council/International ; KWF 0315/2016//Dutch Cancer Society/International ; ERC-PoC 665317/ERC_/European Research Council/International ; },
mesh = {Animals ; Antineoplastic Agents/pharmacology ; Apoptosis ; Asparaginase/*pharmacology ; CRISPR-Cas Systems ; Cell Proliferation ; Drug Resistance, Neoplasm/*genetics ; Excitatory Amino Acid Transporter 1/antagonists & inhibitors/genetics/*metabolism ; Humans ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasms/*drug therapy/enzymology/pathology ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.},
}

Animals
Antineoplastic Agents/pharmacology
Apoptosis
Asparaginase/*pharmacology
CRISPR-Cas Systems
Cell Proliferation
Drug Resistance, Neoplasm/*genetics
Excitatory Amino Acid Transporter 1/antagonists & inhibitors/genetics/*metabolism
Humans
Mice
Mice, Inbred NOD
Mice, SCID
Neoplasms/*drug therapy/enzymology/pathology
Tumor Cells, Cultured
Xenograft Model Antitumor Assays

@article {pmid31493470,
year = {2019},
author = {Filippova, J and Matveeva, A and Zhuravlev, E and Stepanov, G},
title = {Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems.},
journal = {Biochimie},
volume = {167},
number = {},
pages = {49-60},
doi = {10.1016/j.biochi.2019.09.003},
pmid = {31493470},
issn = {1638-6183},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Fungi/genetics ; Gene Editing/*methods ; Gene Expression ; Humans ; Plants/genetics ; *RNA, Guide/chemical synthesis/chemistry/genetics ; Transfection ; },
abstract = {Genome-editing technologies, in particular, CRISPR systems, are widely used for targeted regulation of gene expression and obtaining modified human and animal cell lines, plants, fungi, and animals with preassigned features. Despite being well described and easy to perform, the most common methods for construction and delivery of CRISPR/Cas9-containing plasmid systems possess significant disadvantages, mostly associated with effects of the presence of exogenous DNA within the cell. Transfection with active ribonucleoprotein complexes of Cas9 with single-guide RNAs (sgRNAs) represents one of the most promising options because of faster production of sgRNAs, the ability of a researcher to control the amount of sgRNA delivered into the cell, and consequently, fewer off-target mutations. Artificial-RNA synthesis strategies allow for the introduction of various modified components, such as backbone alterations, native structural motifs, and labels for visualization. Modifications of RNA can increase its resistance to hydrolysis, alter the thermodynamic stability of RNA-protein and RNA-DNA complexes, and reduce the immunogenic and cytotoxic effects. This review describes various approaches to improving synthetic guide RNA function through nucleotide modification.},
}

Animals
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/*genetics
Cell Line
Fungi/genetics
Gene Editing/*methods
Gene Expression
Humans
Plants/genetics
*RNA, Guide/chemical synthesis/chemistry/genetics
Transfection

@article {pmid31286885,
year = {2019},
author = {Reimer, M and Pulakanti, K and Shi, L and Abel, A and Liang, M and Malarkannan, S and Rao, S},
title = {Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression.},
journal = {BMC developmental biology},
volume = {19},
number = {1},
pages = {16},
doi = {10.1186/s12861-019-0196-6},
pmid = {31286885},
issn = {1471-213X},
support = {R01 CA204231/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; DNA Methylation/*genetics ; DNA-Binding Proteins/*genetics ; Embryonic Stem Cells/*cytology ; Gene Editing ; Gene Expression Regulation, Developmental/genetics ; Mice ; Mice, Knockout ; Proto-Oncogene Proteins/*genetics ; },
abstract = {BACKGROUND: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments.

RESULTS: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs.

CONCLUSIONS: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Differentiation/genetics
DNA Methylation/*genetics
DNA-Binding Proteins/*genetics
Embryonic Stem Cells/*cytology
Gene Editing
Gene Expression Regulation, Developmental/genetics
Mice
Mice, Knockout
Proto-Oncogene Proteins/*genetics

@article {pmid31262711,
year = {2019},
author = {Aripaka, K and Gudey, SK and Zang, G and Schmidt, A and Åhrling, SS and Österman, L and Bergh, A and von Hofsten, J and Landström, M},
title = {TRAF6 function as a novel co-regulator of Wnt3a target genes in prostate cancer.},
journal = {EBioMedicine},
volume = {45},
number = {},
pages = {192-207},
doi = {10.1016/j.ebiom.2019.06.046},
pmid = {31262711},
issn = {2352-3964},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Inflammation/*genetics/pathology ; Low Density Lipoprotein Receptor-Related Protein-5/genetics ; Male ; Prostatic Neoplasms/*genetics/pathology ; TNF Receptor-Associated Factor 6/*genetics ; Wnt Signaling Pathway/genetics ; Wnt3A Protein/*genetics ; Zebrafish ; Zebrafish Proteins/*genetics ; beta Catenin/genetics ; },
abstract = {BACKGROUND: Tumour necrosis factor receptor associated factor 6 (TRAF6) promotes inflammation in response to various cytokines. Aberrant Wnt3a signals promotes cancer progression through accumulation of β-Catenin. Here we investigated a potential role for TRAF6 in Wnt signaling.

METHODS: TRAF6 expression was silenced by siRNA in human prostate cancer (PC3U) and human colorectal SW480 cells and by CRISPR/Cas9 in zebrafish. Several biochemical methods and analyses of mutant phenotype in zebrafish were used to analyse the function of TRAF6 in Wnt signaling.

FINDINGS: Wnt3a-treatment promoted binding of TRAF6 to the Wnt co-receptors LRP5/LRP6 in PC3U and LNCaP cells in vitro. TRAF6 positively regulated mRNA expression of β-Catenin and subsequent activation of Wnt target genes in PC3U cells. Wnt3a-induced invasion of PC3U and SW480 cells were significantly reduced when TRAF6 was silenced by siRNA. Database analysis revealed a correlation between TRAF6 mRNA and Wnt target genes in patients with prostate cancer, and high expression of LRP5, TRAF6 and c-Myc correlated with poor prognosis. By using CRISPR/Cas9 to silence TRAF6 in zebrafish, we confirm TRAF6 as a key molecule in Wnt3a signaling for expression of Wnt target genes.

INTERPRETATION: We identify TRAF6 as an important component in Wnt3a signaling to promote activation of Wnt target genes, a finding important for understanding mechanisms driving prostate cancer progression. FUND: KAW 2012.0090, CAN 2017/544, Swedish Medical Research Council (2016-02513), Prostatacancerförbundet, Konung Gustaf V:s Frimurarestiftelse and Cancerforskningsfonden Norrland. The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
Humans
Inflammation/*genetics/pathology
Low Density Lipoprotein Receptor-Related Protein-5/genetics
Male
Prostatic Neoplasms/*genetics/pathology
TNF Receptor-Associated Factor 6/*genetics
Wnt Signaling Pathway/genetics
Wnt3A Protein/*genetics
Zebrafish
Zebrafish Proteins/*genetics
beta Catenin/genetics

@article {pmid31147716,
year = {2019},
author = {Su, G and Guo, D and Chen, J and Liu, M and Zheng, J and Wang, W and Zhao, X and Yin, Q and Zhang, L and Zhao, Z and Shi, J and Lu, W},
title = {A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.},
journal = {Nucleic acids research},
volume = {47},
number = {13},
pages = {6737-6752},
doi = {10.1093/nar/gkz482},
pmid = {31147716},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation/drug effects ; Cells, Cultured ; Chromatin/genetics/metabolism ; Embryonic Stem Cells/drug effects/*metabolism ; Endoderm/metabolism ; *Enhancer Elements, Genetic/genetics ; Gene Editing ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Gene Ontology ; Homeodomain Proteins/*biosynthesis/genetics ; Intracellular Signaling Peptides and Proteins/biosynthesis/genetics ; Mice ; Promoter Regions, Genetic ; RNA, Small Interfering/genetics ; Recombinant Proteins/metabolism ; Transcription Factors/*biosynthesis/genetics ; Tretinoin/*pharmacology ; },
abstract = {Retinoic acid (RA) induces rapid differentiation of embryonic stem cells (ESCs), partly by activating expression of the transcription factor Hoxa1, which regulates downstream target genes that promote ESCs differentiation. However, mechanisms of RA-induced Hoxa1 expression and ESCs early differentiation remain largely unknown. Here, we identify a distal enhancer interacting with the Hoxa1 locus through a long-range chromatin loop. Enhancer deletion significantly inhibited expression of RA-induced Hoxa1 and endoderm master control genes such as Gata4 and Gata6. Transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in Hoxa1 enhancer knockout cells, suggesting a requirement for the enhancer. Restoration of Hoxa1 expression partly rescued expression levels of ∼40% of genes whose expression changed following enhancer deletion, and ∼18% of promoters of those rescued genes were directly bound by Hoxa1. Our data show that a distal enhancer maintains Hoxa1 expression through long-range chromatin loop and that Hoxa1 directly regulates downstream target genes expression and then orchestrates RA-induced early differentiation of ESCs. This discovery reveals mechanisms of a novel enhancer regulating RA-induced Hoxa genes expression and early ESCs differentiation.},
}

Animals
CRISPR-Cas Systems
Cell Differentiation/drug effects
Cells, Cultured
Chromatin/genetics/metabolism
Embryonic Stem Cells/drug effects/*metabolism
Endoderm/metabolism
*Enhancer Elements, Genetic/genetics
Gene Editing
Gene Expression Regulation, Developmental
Gene Knockdown Techniques
Gene Ontology
Homeodomain Proteins/*biosynthesis/genetics
Intracellular Signaling Peptides and Proteins/biosynthesis/genetics
Mice
Promoter Regions, Genetic
RNA, Small Interfering/genetics
Recombinant Proteins/metabolism
Transcription Factors/*biosynthesis/genetics
Tretinoin/*pharmacology

@article {pmid30997496,
year = {2019},
author = {Truong, VA and Hsu, MN and Kieu Nguyen, NT and Lin, MW and Shen, CC and Lin, CY and Hu, YC},
title = {CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.},
journal = {Nucleic acids research},
volume = {47},
number = {13},
pages = {e74},
doi = {10.1093/nar/gkz267},
pmid = {30997496},
issn = {1362-4962},
mesh = {Adipogenesis ; Adult Stem Cells/*transplantation ; Animals ; Baculoviridae ; Bone Marrow Transplantation ; Bone Regeneration/*genetics ; CHO Cells ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Chondrogenesis/*genetics ; Cricetulus ; Gene Editing/*methods ; Luminescent Proteins ; *Mesenchymal Stem Cell Transplantation ; PPAR gamma/genetics ; Parietal Bone/injuries/*physiology ; RNA, Guide ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; SOX9 Transcription Factor/genetics ; *Tissue Scaffolds ; *Transcriptional Activation ; Wound Healing/*genetics ; },
abstract = {Calvarial bone healing remains difficult but may be improved by stimulating chondrogenesis of implanted stem cells. To simultaneously promote chondrogenesis and repress adipogenesis of stem cells, we built a CRISPRai system that comprised inactive Cas9 (dCas9), two fusion proteins as activation/repression complexes and two single guide RNA (sgRNA) as scaffolds for recruiting activator (sgRNAa) or inhibitor (sgRNAi). By plasmid transfection and co-expression in CHO cells, we validated that dCas9 coordinated with sgRNAa to recruit the activator for mCherry activation and also orchestrated with sgRNAi to recruit the repressor for d2EGFP inhibition, without cross interference. After changing the sgRNA sequence to target endogenous Sox9/PPAR-γ, we packaged the entire CRISPRai system into an all-in-one baculovirus for efficient delivery into rat bone marrow-derived mesenchymal stem cells (rBMSC) and verified simultaneous Sox9 activation and PPAR-γ repression. The activation/inhibition effects were further enhanced/prolonged by using the Cre/loxP-based hybrid baculovirus. The CRISPRai system delivered by the hybrid baculovirus stimulated chondrogenesis and repressed adipogenesis of rBMSC in 2D culture and promoted the formation of engineered cartilage in 3D culture. Importantly, implantation of the rBMSC engineered by the CRISPRai improved calvarial bone healing. This study paves a new avenue to translate the CRISPRai technology to regenerative medicine.},
}

Adipogenesis
Adult Stem Cells/*transplantation
Animals
Baculoviridae
Bone Marrow Transplantation
Bone Regeneration/*genetics
CHO Cells
CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Chondrogenesis/*genetics
Cricetulus
Gene Editing/*methods
Luminescent Proteins
*Mesenchymal Stem Cell Transplantation
PPAR gamma/genetics
Parietal Bone/injuries/*physiology
RNA, Guide
Rats, Sprague-Dawley
Recombinant Fusion Proteins
SOX9 Transcription Factor/genetics
*Tissue Scaffolds
*Transcriptional Activation
Wound Healing/*genetics

@article {pmid30978004,
year = {2019},
author = {Tovell, H and Testa, A and Maniaci, C and Zhou, H and Prescott, AR and Macartney, T and Ciulli, A and Alessi, DR},
title = {Rapid and Reversible Knockdown of Endogenously Tagged Endosomal Proteins via an Optimized HaloPROTAC Degrader.},
journal = {ACS chemical biology},
volume = {14},
number = {5},
pages = {882-892},
pmid = {30978004},
issn = {1554-8937},
support = {MC_UU_12016/2/MRC_/Medical Research Council/United Kingdom ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Class III Phosphatidylinositol 3-Kinases/genetics ; Endosomes/*metabolism ; Gene Editing/*methods ; Gene Knock-In Techniques ; HEK293 Cells ; Humans ; Protein-Serine-Threonine Kinases/genetics ; Proteins/*metabolism ; },
abstract = {Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC50 of 3-10 nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min) and complete (Dmax of ∼95% at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function.},
}

CRISPR-Cas Systems
Class III Phosphatidylinositol 3-Kinases/genetics
Endosomes/*metabolism
Gene Editing/*methods
Gene Knock-In Techniques
HEK293 Cells
Humans
Protein-Serine-Threonine Kinases/genetics
Proteins/*metabolism

@article {pmid30933966,
year = {2019},
author = {Shoemaker, CJ and Huang, TQ and Weir, NR and Polyakov, NJ and Schultz, SW and Denic, V},
title = {CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor.},
journal = {PLoS biology},
volume = {17},
number = {4},
pages = {e2007044},
doi = {10.1371/journal.pbio.2007044},
pmid = {30933966},
issn = {1545-7885},
support = {K99 GM117218/GM/NIGMS NIH HHS/United States ; R00 GM117218/GM/NIGMS NIH HHS/United States ; R35 GM127136/GM/NIGMS NIH HHS/United States ; P20 GM113132/GM/NIGMS NIH HHS/United States ; },
mesh = {Autophagy/*genetics/*physiology ; CRISPR-Cas Systems ; Endoplasmic Reticulum/metabolism ; Humans ; K562 Cells ; Membrane Proteins/*genetics/metabolism/physiology ; Protein Transport ; },
abstract = {The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.},
}

Autophagy/*genetics/*physiology
CRISPR-Cas Systems
Endoplasmic Reticulum/metabolism
Humans
K562 Cells
Membrane Proteins/*genetics/metabolism/physiology
Protein Transport

@article {pmid30914406,
year = {2019},
author = {Mulas, C and Kalkan, T and von Meyenn, F and Leitch, HG and Nichols, J and Smith, A},
title = {Defined conditions for propagation and manipulation of mouse embryonic stem cells.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {6},
pages = {},
doi = {10.1242/dev.173146},
pmid = {30914406},
issn = {1477-9129},
support = {/WT_/Wellcome Trust/United Kingdom ; G1100526/1/MRC_/Medical Research Council/United Kingdom ; 091484/Z/10/Z/WT_/Wellcome Trust/United Kingdom ; BB/G015678/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Cycle ; Cell Differentiation/*genetics ; Coculture Techniques ; Culture Media/chemistry ; Embryo, Mammalian/*cytology ; Embryonic Stem Cells/*cytology ; Humans ; Karyotyping ; Mice ; Mice, Inbred C57BL ; Mouse Embryonic Stem Cells/*cytology ; Neurons/cytology ; RNA, Small Interfering/genetics ; Signal Transduction ; },
abstract = {The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.},
}

Animals
CRISPR-Cas Systems
Cell Culture Techniques
Cell Cycle
Cell Differentiation/*genetics
Coculture Techniques
Culture Media/chemistry
Embryo, Mammalian/*cytology
Embryonic Stem Cells/*cytology
Humans
Karyotyping
Mice
Mice, Inbred C57BL
Mouse Embryonic Stem Cells/*cytology
Neurons/cytology
RNA, Small Interfering/genetics
Signal Transduction

@article {pmid30817136,
year = {2019},
author = {Auxillos, JY and Garcia-Ruiz, E and Jones, S and Li, T and Jiang, S and Dai, J and Cai, Y},
title = {Multiplex Genome Engineering for Optimizing Bioproduction in Saccharomyces cerevisiae.},
journal = {Biochemistry},
volume = {58},
number = {11},
pages = {1492-1500},
doi = {10.1021/acs.biochem.8b01086},
pmid = {30817136},
issn = {1520-4995},
support = {BB/P02114X/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Genetic Engineering/methods ; Metabolic Engineering/methods ; Protein Engineering/*methods ; Saccharomyces cerevisiae/genetics/metabolism ; Saccharomyces cerevisiae Proteins/*biosynthesis/genetics/metabolism ; Synthetic Biology/*methods ; },
abstract = {The field of synthetic biology is already beginning to realize its potential, with a wealth of examples showcasing the successful genetic engineering of microorganisms for the production of valuable compounds. The chassis Saccharomyces cerevisiae has been engineered to function as a microfactory for producing many of these economically and medically relevant compounds. However, strain construction and optimization to produce industrially relevant titers necessitate a wealth of underpinning biological knowledge alongside large investments of capital and time. Over the past decade, advances in DNA synthesis and editing tools have enabled multiplex genome engineering of yeast, permitting access to more complex modifications that could not have been easily generated in the past. These genome engineering efforts often result in large populations of strains with genetic diversity that can pose a significant challenge to screen individually via traditional methods such as mass spectrometry. The large number of samples generated would necessitate screening methods capable of analyzing all of the strains generated to maximize the explored genetic space. In this Perspective, we focus on recent innovations in multiplex genome engineering of S. cerevisiae, together with biosensors and high-throughput screening tools, such as droplet microfluidics, and their applications in accelerating chassis optimization.},
}

CRISPR-Cas Systems
Genetic Engineering/methods
Metabolic Engineering/methods
Protein Engineering/*methods
Saccharomyces cerevisiae/genetics/metabolism
Saccharomyces cerevisiae Proteins/*biosynthesis/genetics/metabolism
Synthetic Biology/*methods

@article {pmid30796047,
year = {2019},
author = {Gultekin, Y and Steller, H},
title = {Axin proteolysis by Iduna is required for the regulation of stem cell proliferation and intestinal homeostasis in Drosophila.},
journal = {Development (Cambridge, England)},
volume = {146},
number = {6},
pages = {},
doi = {10.1242/dev.169284},
pmid = {30796047},
issn = {1477-9129},
support = {R01 GM060124/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Axin Protein/*metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Crosses, Genetic ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/*metabolism ; Female ; Gene Expression Regulation, Developmental ; Green Fluorescent Proteins/metabolism ; Homeostasis ; Intestines/*physiology ; Male ; Models, Genetic ; Mutation ; RNA Interference ; Recombinant Proteins/metabolism ; Signal Transduction ; Stem Cells/*cytology ; Tankyrases/metabolism ; Ubiquitin-Protein Ligases/*metabolism ; Wnt1 Protein/*metabolism ; },
abstract = {Self-renewal of intestinal stem cells is controlled by Wingless/Wnt-β catenin signaling in both Drosophila and mammals. As Axin is a rate-limiting factor in Wingless signaling, its regulation is essential. Iduna is an evolutionarily conserved ubiquitin E3 ligase that has been identified as a crucial regulator for degradation of ADP-ribosylated Axin and, thus, of Wnt/β-catenin signaling. However, its physiological significance remains to be demonstrated. Here, we generated loss-of-function mutants of Iduna to investigate its physiological role in Drosophila Genetic depletion of Iduna causes the accumulation of both Tankyrase and Axin. Increase of Axin protein in enterocytes non-autonomously enhanced stem cell divisions in the Drosophila midgut. Enterocytes secreted Unpaired proteins and thereby stimulated the activity of the JAK-STAT pathway in intestinal stem cells. A decrease in Axin gene expression suppressed the over-proliferation of stem cells and restored their numbers to normal levels in Iduna mutants. These findings suggest that Iduna-mediated regulation of Axin proteolysis is essential for tissue homeostasis in the Drosophila midgut.},
}

Animals
Axin Protein/*metabolism
CRISPR-Cas Systems
Cell Proliferation
Crosses, Genetic
Drosophila Proteins/*metabolism
Drosophila melanogaster/*metabolism
Female
Gene Expression Regulation, Developmental
Green Fluorescent Proteins/metabolism
Homeostasis
Intestines/*physiology
Male
Models, Genetic
Mutation
RNA Interference
Recombinant Proteins/metabolism
Signal Transduction
Stem Cells/*cytology
Tankyrases/metabolism
Ubiquitin-Protein Ligases/*metabolism
Wnt1 Protein/*metabolism

@article {pmid30618248,
year = {2019},
author = {Liu, Z and Zhang, Y and Nielsen, J},
title = {Synthetic Biology of Yeast.},
journal = {Biochemistry},
volume = {58},
number = {11},
pages = {1511-1520},
doi = {10.1021/acs.biochem.8b01236},
pmid = {30618248},
issn = {1520-4995},
mesh = {CRISPR-Cas Systems ; Gene Regulatory Networks/genetics ; Genetic Engineering/methods ; Metabolic Engineering/methods ; Promoter Regions, Genetic/genetics ; Protein Engineering/methods/trends ; Saccharomyces cerevisiae/genetics/metabolism ; Saccharomyces cerevisiae Proteins/*biosynthesis/genetics/metabolism ; Synthetic Biology/*methods/*trends ; },
abstract = {With the rapid development of DNA synthesis and next-generation sequencing, synthetic biology that aims to standardize, modularize, and innovate cellular functions, has achieved vast progress. Here we review key advances in synthetic biology of the yeast Saccharomyces cerevisiae, which serves as an important eukaryal model organism and widely applied cell factory. This covers the development of new building blocks, i.e., promoters, terminators and enzymes, pathway engineering, tools developments, and gene circuits utilization. We will also summarize impacts of synthetic biology on both basic and applied biology, and end with further directions for advancing synthetic biology in yeast.},
}

CRISPR-Cas Systems
Gene Regulatory Networks/genetics
Genetic Engineering/methods
Metabolic Engineering/methods
Promoter Regions, Genetic/genetics
Protein Engineering/methods/trends
Saccharomyces cerevisiae/genetics/metabolism
Saccharomyces cerevisiae Proteins/*biosynthesis/genetics/metabolism
Synthetic Biology/*methods/*trends

@article {pmid30573670,
year = {2019},
author = {Globyte, V and Lee, SH and Bae, T and Kim, JS and Joo, C},
title = {CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion.},
journal = {The EMBO journal},
volume = {38},
number = {4},
pages = {},
doi = {10.15252/embj.201899466},
pmid = {30573670},
issn = {1460-2075},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; DNA/*genetics/metabolism ; *Gene Editing ; Genetic Engineering/*methods ; *Nucleotide Motifs ; Sequence Homology ; Streptococcus pyogenes/*genetics/metabolism ; Substrate Specificity ; },
abstract = {The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) nuclease has been widely applied in genetic engineering. Despite its importance in genome editing, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. In particular, because of the lack of a method with high spatio-temporal resolution, transient interactions between Cas9 and DNA could not be reliably investigated. It therefore remains controversial how Cas9 searches for protospacer adjacent motif (PAM) sequences. We have developed single-molecule Förster resonance energy transfer (smFRET) assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the PAM sequence weakly, yet probing neighboring sequences via facilitated diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with three-dimensional diffusion and thus is involved in PAM finding and consequently on-target binding. Our results imply that the neighboring sequences can be very important when choosing a target in genetic engineering applications.},
}

Base Sequence
*CRISPR-Cas Systems
DNA/*genetics/metabolism
*Gene Editing
Genetic Engineering/*methods
*Nucleotide Motifs
Sequence Homology
Streptococcus pyogenes/*genetics/metabolism
Substrate Specificity

@article {pmid30573669,
year = {2019},
author = {Al-Raawi, D and Jones, R and Wijesinghe, S and Halsall, J and Petric, M and Roberts, S and Hotchin, NA and Kanhere, A},
title = {A novel form of JARID2 is required for differentiation in lineage-committed cells.},
journal = {The EMBO journal},
volume = {38},
number = {3},
pages = {},
doi = {10.15252/embj.201798449},
pmid = {30573669},
issn = {1460-2075},
mesh = {CRISPR-Cas Systems ; *Cell Differentiation ; *Cell Lineage ; Embryonic Stem Cells/*cytology/metabolism ; HEK293 Cells ; Humans ; Keratinocytes/*cytology/metabolism ; Polycomb Repressive Complex 2/antagonists & inhibitors/genetics/*metabolism ; Protein Binding ; Protein Isoforms ; },
abstract = {Polycomb repressive complex-2 (PRC2) is a group of proteins that play an important role during development and in cell differentiation. PRC2 is a histone-modifying complex that catalyses methylation of lysine 27 of histone H3 (H3K27me3) at differentiation genes leading to their transcriptional repression. JARID2 is a co-factor of PRC2 and is important for targeting PRC2 to chromatin. Here, we show that, unlike in embryonic stem cells, in lineage-committed human cells, including human epidermal keratinocytes, JARID2 predominantly exists as a novel low molecular weight form, which lacks the N-terminal PRC2-interacting domain (ΔN-JARID2). We show that ΔN-JARID2 is a cleaved product of full-length JARID2 spanning the C-terminal conserved jumonji domains. JARID2 knockout in keratinocytes results in up-regulation of cell cycle genes and repression of many epidermal differentiation genes. Surprisingly, repression of epidermal differentiation genes in JARID2-null keratinocytes can be rescued by expression of ΔN-JARID2 suggesting that, in contrast to PRC2, ΔN-JARID2 promotes activation of differentiation genes. We propose that a switch from expression of full-length JARID2 to ΔN-JARID2 is important for the up-regulation differentiation genes.},
}

CRISPR-Cas Systems
*Cell Differentiation
*Cell Lineage
Embryonic Stem Cells/*cytology/metabolism
HEK293 Cells
Humans
Keratinocytes/*cytology/metabolism
Polycomb Repressive Complex 2/antagonists & inhibitors/genetics/*metabolism
Protein Binding
Protein Isoforms

@article {pmid30478227,
year = {2019},
author = {Katayama, T and Nakamura, H and Zhang, Y and Pascal, A and Fujii, W and Maruyama, JI},
title = {Forced Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene Deletion/Integration in the Industrial Filamentous Fungus Aspergillus oryzae.},
journal = {Applied and environmental microbiology},
volume = {85},
number = {3},
pages = {},
doi = {10.1128/AEM.01896-18},
pmid = {30478227},
issn = {1098-5336},
mesh = {Aspergillus oryzae/*genetics/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; Gene Editing ; Genetic Engineering ; Genome, Fungal ; Industrial Microbiology ; Mutagenesis ; Plasmids/genetics/metabolism ; },
abstract = {Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established. We have previously developed a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9-based mutagenesis technique for the industrial filamentous fungus Aspergillus oryzae, enabling mutation efficiency of 10 to 20%. Here, we improved the CRISPR/Cas9 approach by including an AMA1-based autonomously replicating plasmid harboring the drug resistance marker ptrA By using the improved mutagenesis technique, we successfully modified A. oryzae wild and industrial strains, with a mutation efficiency of 50 to 100%. Conditional expression of the Aoace2 gene from the AMA1-based plasmid severely inhibited fungal growth. This enabled forced recycling of the plasmid, allowing repeated genome editing. Further, double mutant strains were successfully obtained with high efficiency by expressing two guide RNA molecules from the genome-editing plasmid. Cotransformation of fungal cells with the genome-editing plasmid together with a circular donor DNA enabled marker-free multiplex gene deletion/integration in A. oryzae The presented repeatable marker-free genetic engineering approach for mutagenesis and gene deletion/integration will allow for efficient modification of multiple genes in industrial fungal strains, increasing their applicability.IMPORTANCE Multiple gene modifications of specific fungal strains are required for achieving industrial-scale production of enzymes and secondary metabolites. In the present study, we developed an efficient multiple genetic engineering technique for the filamentous fungus Aspergillus oryzae The approach is based on a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and recycling of an AMA1-based autonomous replicating plasmid. Because the plasmid harbors a drug resistance marker (ptrA), the approach does not require the construction of auxotrophic industrial strains prior to genome editing and allows for forced recycling of the gene-editing plasmid. The established plasmid-recycling technique involves an Aoace2-conditional expression cassette, whose induction severely impairs fungal growth. We used the developed genetic engineering techniques for highly efficient marker-free multiple gene deletion/integration in A. oryzae The genome-editing approaches established in the present study, which enable unlimited repeatable genetic engineering, will facilitate multiple gene modification of industrially important fungal strains.},
}

Aspergillus oryzae/*genetics/metabolism
CRISPR-Cas Systems
Gene Deletion
Gene Editing
Genetic Engineering
Genome, Fungal
Industrial Microbiology
Mutagenesis
Plasmids/genetics/metabolism

@article {pmid31900288,
year = {2020},
author = {Agudelo, D and Carter, S and Velimirovic, M and Duringer, A and Rivest, JF and Levesque, S and Loehr, J and Mouchiroud, M and Cyr, D and Waters, PJ and Laplante, M and Moineau, S and Goulet, A and Doyon, Y},
title = {Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9.},
journal = {Genome research},
volume = {},
number = {},
pages = {},
doi = {10.1101/gr.255414.119},
pmid = {31900288},
issn = {1549-5469},
abstract = {Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.},
}

@article {pmid31896785,
year = {2020},
author = {Broniewski, JM and Meaden, S and Paterson, S and Buckling, A and Westra, ER},
title = {The effect of phage genetic diversity on bacterial resistance evolution.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41396-019-0577-7},
pmid = {31896785},
issn = {1751-7370},
abstract = {CRISPR-Cas adaptive immune systems are found in bacteria and archaea and provide defence against phage by inserting phage-derived sequences into CRISPR loci on the host genome to provide sequence specific immunological memory against re-infection. Under laboratory conditions the bacterium Pseudomonas aeruginosa readily evolves the high levels of CRISPR-based immunity against clonal populations of its phage DMS3vir, which in turn causes rapid extinction of the phage. However, in nature phage populations are likely to be more genetically diverse, which could theoretically impact the frequency at which CRISPR-based immunity evolves which in turn can alter phage persistence over time. Here we experimentally test these ideas and found that a smaller proportion of infected bacterial populations evolved CRISPR-based immunity against more genetically diverse phage populations, with the majority of the population evolving a sm preventing phage adsorption and providing generalised defence against a broader range of phage genotypes. However, those cells that do evolve CRISPR-based immunity in response to infection with more genetically diverse phage acquire greater numbers of CRISPR memory sequences in order to resist a wider range of phage genotypes. Despite differences in bacterial resistance evolution, the rates of phage extinction were similar in the context of clonal and diverse phage infections suggesting selection for CRISPR-based immunity or sm-based resistance plays a relatively minor role in the ecological dynamics in this study. Collectively, these data help to understand the drivers of CRISPR-based immunity and their consequences for bacteria-phage coexistence, and, more broadly, when generalised defences will be favoured over more specific defences.},
}

@article {pmid31534175,
year = {2019},
author = {Bernkopf, DB and Brückner, M and Hadjihannas, MV and Behrens, J},
title = {An aggregon in conductin/axin2 regulates Wnt/β-catenin signaling and holds potential for cancer therapy.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4251},
doi = {10.1038/s41467-019-12203-8},
pmid = {31534175},
issn = {2041-1723},
mesh = {Axin Protein/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms/genetics/*pathology ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Wnt Proteins/metabolism ; Wnt Signaling Pathway/*genetics/physiology ; beta Catenin/*metabolism ; },
abstract = {The paralogous scaffold proteins axin and conductin/axin2 are key factors in the negative regulation of the Wnt pathway transcription factor β-catenin, thereby representing interesting targets for signaling regulation. Polymerization of axin proteins is essential for their activity in suppressing Wnt/β-catenin signaling. Notably, conductin shows less polymerization and lower activity than axin. By domain swapping between axin and conductin we here identify an aggregation site in the conductin RGS domain which prevents conductin polymerization. Induction of conductin polymerization by point mutations of this aggregon results in enhanced inhibition of Wnt/β-catenin signaling. Importantly, we identify a short peptide which induces conductin polymerization via masking the aggregon, thereby enhancing β-catenin degradation, inhibiting β-catenin-dependent transcription and repressing growth of colorectal cancer cells. Our study reveals a mechanism for regulating signaling pathways via the polymerization status of scaffold proteins and suggests a strategy for targeted colorectal cancer therapy.},
}

Axin Protein/genetics/*metabolism
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Cell Proliferation
Colorectal Neoplasms/genetics/*pathology
Gene Knockout Techniques
HEK293 Cells
Humans
Wnt Proteins/metabolism
Wnt Signaling Pathway/*genetics/physiology
beta Catenin/*metabolism

@article {pmid31534142,
year = {2019},
author = {Tarjan, DR and Flavahan, WA and Bernstein, BE},
title = {Epigenome editing strategies for the functional annotation of CTCF insulators.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {4258},
doi = {10.1038/s41467-019-12166-w},
pmid = {31534142},
issn = {2041-1723},
support = {DP1 CA216873/CA/NCI NIH HHS/United States ; },
mesh = {Brain Neoplasms/genetics/pathology ; CCCTC-Binding Factor/*genetics ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics/pathology ; Cell Line ; DNA (Cytosine-5-)-Methyltransferases/*genetics ; DNA Methylation ; Epigenesis, Genetic/genetics ; Gene Editing/*methods ; Genome, Human/genetics ; HEK293 Cells ; Humans ; Promoter Regions, Genetic/genetics ; Recombinant Fusion Proteins/*genetics ; Repressor Proteins/metabolism ; },
abstract = {The human genome is folded into regulatory units termed 'topologically-associated domains' (TADs). Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, partially-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing. Finally, we apply these strategies to simulate an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.},
}

Brain Neoplasms/genetics/pathology
CCCTC-Binding Factor/*genetics
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/genetics
Carcinogenesis/*genetics/pathology
Cell Line
DNA (Cytosine-5-)-Methyltransferases/*genetics
DNA Methylation
Epigenesis, Genetic/genetics
Gene Editing/*methods
Genome, Human/genetics
HEK293 Cells
Humans
Promoter Regions, Genetic/genetics
Recombinant Fusion Proteins/*genetics
Repressor Proteins/metabolism

@article {pmid31465870,
year = {2019},
author = {Kim, MS and Kim, KH},
title = {Effect of CRISPR/Cas9-mediated knockout of either Mx1 or ISG15 gene in EPC cells on resistance against VHSV infection.},
journal = {Fish & shellfish immunology},
volume = {93},
number = {},
pages = {1041-1046},
doi = {10.1016/j.fsi.2019.08.058},
pmid = {31465870},
issn = {1095-9947},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems/*immunology ; Cell Line, Tumor ; Cyprinidae/genetics/*immunology ; Disease Resistance/*genetics/immunology ; Fish Diseases/genetics/*immunology ; Fish Proteins/*genetics/*immunology ; *Gene Expression ; Gene Knockout Techniques/veterinary ; Hemorrhagic Septicemia, Viral/genetics/immunology ; Interferon Type I/genetics ; Novirhabdovirus/physiology ; Poly I-C/pharmacology ; Sequence Analysis, DNA/veterinary ; Sequence Analysis, Protein/veterinary ; },
abstract = {Although the type I interferon-mediated increase of Mx1 and ISG15 gene expression in Epithelioma papulosum cyprini (EPC) cells has been reported, the antiviral role of Mx1 and ISG15 in EPC cells has not been investigated. In this study, to know the anti-viral hemorrhagic septicemia virus (VHSV) role of Mx1 and ISG15 of EPC cells, either Mx1 or ISG15 gene was knocked-out using a CRISPR/Cas9 system, and the progression of cytopathic effects (CPE) and viral growth were analyzed. Mx1 gene and ISG15 gene knockout EPC cells were successfully produced via CRISPR/Cas9 coupled with a single-cell cloning. Through the sequence analysis, one clone showing two heterozygous indel patterns in Mx1 gene and a clone showing three heterozygous indel patterns in ISG15 gene were selected for further analyses. Mx1 knockout EPC cells did not show any differences in VHSV-mediated CPE progression, even when pre-treated with polyinosinic:polycytidylic acid (poly I:C), compared to control EPC cells. These results suggest that Mx1 in EPC cells may be unfunctional to cytoplasmic RNA viruses. In contrast to Mx1, ISG15 knockout cells showed clearly hampered anti-VHSV activity even when pre-treated with poly I:C, indicating that ISG15 plays an important role in type I interferon-mediated anti-viral activity in EPC cells, which allowed VHSV to replicate more efficiently in ISG15 knockout cells than Mx1 knockout and control cells.},
}

Amino Acid Sequence
Animals
Base Sequence
CRISPR-Cas Systems/*immunology
Cell Line, Tumor
Cyprinidae/genetics/*immunology
Disease Resistance/*genetics/immunology
Fish Diseases/genetics/*immunology
Fish Proteins/*genetics/*immunology
*Gene Expression
Gene Knockout Techniques/veterinary
Hemorrhagic Septicemia, Viral/genetics/immunology
Interferon Type I/genetics
Novirhabdovirus/physiology
Poly I-C/pharmacology
Sequence Analysis, DNA/veterinary
Sequence Analysis, Protein/veterinary