@article {pmid31232162,
year = {2019},
author = {Taylor, HN and Warner, EE and Armbrust, MJ and Crowley, VM and Olsen, KJ and Jackson, RN},
title = {Structural basis of Type IV CRISPR RNA biogenesis by a Cas6 endoribonuclease.},
journal = {RNA biology},
volume = {},
number = {},
pages = {},
doi = {10.1080/15476286.2019.1634965},
pmid = {31232162},
issn = {1555-8584},
abstract = {Prokaryotic CRISPR-Cas adaptive immune systems rely on small non-coding RNAs derived from CRISPR loci to recognize and destroy complementary nucleic acids. However, the mechanism of Type IV CRISPR RNA (crRNA) biogenesis is poorly understood. To dissect the mechanism of Type IV CRISPR RNA biogenesis, we determined the x-ray crystal structure of the putative Type IV CRISPR associated endoribonuclease Cas6 from Mahella australiensis (Ma Cas6-IV) and characterized its enzymatic activity with RNA cleavage assays. We show that Ma Cas6-IV specifically cleaves Type IV crRNA repeats at the 3' side of a predicted stem loop, with a metal-independent, single-turnover mechanism that relies on a histidine and a tyrosine located within the putative endonuclease active site. Structure and sequence alignments with Cas6 orthologs reveal that although Ma Cas6-IV shares little sequence homology with other Cas6 proteins, all share common structural features that bind distinct crRNA repeat sequences. This analysis of Type IV crRNA biogenesis provides a structural and biochemical framework for understanding the similarities and differences of crRNA biogenesis across multi-subunit Class 1 CRISPR immune systems.},
}

@article {pmid31231643,
year = {2019},
author = {Wasmer, M},
title = {Roads Forward for European GMO Policy-Uncertainties in Wake of ECJ Judgment Have to be Mitigated by Regulatory Reform.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {7},
number = {},
pages = {132},
doi = {10.3389/fbioe.2019.00132},
pmid = {31231643},
issn = {2296-4185},
abstract = {This article gives an overview of legal and procedural uncertainties regarding genome edited organisms and possible ways forward for European GMO policy. After a recent judgment by the European Court of Justice (ECJ judgment of 25 July 2018, C-528/16), organisms obtained by techniques of genome editing are GMOs and subject to the same obligations as transgenic organisms. Uncertainties emerge if genome edited organisms cannot be distinguished from organisms bred by conventional techniques, such as crossing or random mutagenesis. In this case, identical organisms can be subject to either GMO law or exempt from regulation because of the use of a technique that cannot be identified. Regulatory agencies might not be able to enforce GMO law for such cases in the long term. As other jurisdictions do not regulate such organisms as GMOs, accidental imports might occur and undermine European GMO regulation. In the near future, the EU Commission as well as European and national regulatory agencies will decide on how to apply the updated interpretation of the law. In order to mitigate current legal and procedural uncertainties, a first step forward lies in updating all guidance documents to specifically address genome editing specifically address genome editing, including a solution for providing a unique identifier. In part, the authorization procedure for GMO release can be tailored to different types of organisms by making use of existing flexibilities in GMO law. However, only an amendment to the regulations that govern the process of authorization for GMO release can substantially lower the burden for innovators. In a second step, any way forward has to aim at amending, supplementing or replacing the European GMO Directive (2001/18/EC). The policy options presented in this article presuppose political readiness for reform. This may not be realistic in the current political situation. However, if the problems of current GMO law are just ignored, European competitiveness and research in green biotechnology will suffer.},
}

@article {pmid31230714,
year = {2019},
author = {Rauch, S and He, E and Srienc, M and Zhou, H and Zhang, Z and Dickinson, BC},
title = {Programmable RNA-Guided RNA Effector Proteins Built from Human Parts.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2019.05.049},
pmid = {31230714},
issn = {1097-4172},
abstract = {Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS is not only smaller than naturally occurring CRISPR-Cas programmable RNA binding systems but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, and the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.},
}

@article {pmid31227825,
year = {2019},
author = {Tirronen, A and Hokkanen, K and Vuorio, T and Ylä-Herttuala, S},
title = {Recent advances in novel therapies for lipid disorders.},
journal = {Human molecular genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/hmg/ddz132},
pmid = {31227825},
issn = {1460-2083},
abstract = {The prevalence of lipid disorders is alarmingly increasing in the Western world. They are the result of either primary causes, such as unhealthy lifestyle choices or inherited risk factors, or secondary causes like other diseases or medication. Atypical changes in the synthesis, processing and catabolism of lipoprotein particles may lead to severe hypercholesterolemia, hypertriglyceridemia or elevated Lp(a). Although cholesterol-lowering drugs are the most prescribed medications, not all patients achieve guideline recommended cholesterol levels with the current treatment options, emphasising the need for new therapies. Also, some lipid disorders do not have any treatment options but rely only on stringent dietary restriction. Patients with untreated lipid disorders carry a severe risk of cardiovascular disease, diabetes, non-alcoholic fatty liver disease and pancreatitis among others. To achieve better treatment outcome, novel selective gene expression and epigenetic targeting therapies are constantly being developed. Therapeutic innovations employing targeted RNA technology utilise small interfering RNAs, antisense oligonucleotides, long non-coding RNAs and microRNAs to regulate target protein production whereas viral gene therapy provides functional therapeutic genes and CRISPR/Cas technology relies on gene editing and transcriptional regulation. In this brief review, we will discuss the latest advances in clinical trials for novel lipid-lowering therapies and potential new targets in pre-clinical phase.},
}

@article {pmid31160565,
year = {2019},
author = {Bandopadhayay, P and Piccioni, F and O'Rourke, R and Ho, P and Gonzalez, EM and Buchan, G and Qian, K and Gionet, G and Girard, E and Coxon, M and Rees, MG and Brenan, L and Dubois, F and Shapira, O and Greenwald, NF and Pages, M and Balboni Iniguez, A and Paolella, BR and Meng, A and Sinai, C and Roti, G and Dharia, NV and Creech, A and Tanenbaum, B and Khadka, P and Tracy, A and Tiv, HL and Hong, AL and Coy, S and Rashid, R and Lin, JR and Cowley, GS and Lam, FC and Goodale, A and Lee, Y and Schoolcraft, K and Vazquez, F and Hahn, WC and Tsherniak, A and Bradner, JE and Yaffe, MB and Milde, T and Pfister, SM and Qi, J and Schenone, M and Carr, SA and Ligon, KL and Kieran, MW and Santagata, S and Olson, JM and Gokhale, PC and Jaffe, JD and Root, DE and Stegmaier, K and Johannessen, CM and Beroukhim, R},
title = {Neuronal differentiation and cell-cycle programs mediate response to BET-bromodomain inhibition in MYC-driven medulloblastoma.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {2400},
doi = {10.1038/s41467-019-10307-9},
pmid = {31160565},
issn = {2041-1723},
mesh = {Animals ; Azepines/*pharmacology ; Basic Helix-Loop-Helix Transcription Factors/drug effects/metabolism ; CRISPR-Cas Systems ; Cell Cycle/*drug effects ; Cell Cycle Proteins/drug effects/metabolism ; Cell Line, Tumor ; Cell Lineage ; Cerebellar Neoplasms/*drug therapy/genetics ; Cyclin D2/drug effects/metabolism ; Cyclin-Dependent Kinase 4/antagonists & inhibitors ; Cyclin-Dependent Kinase 6/antagonists & inhibitors ; Drug Resistance, Neoplasm ; Gene Expression Profiling ; Humans ; Medulloblastoma/*drug therapy/genetics ; Mice ; Neural Stem Cells/drug effects/metabolism ; Neurogenesis/*drug effects ; Proteins/*antagonists & inhibitors ; Proto-Oncogene Proteins c-myc/genetics ; S Phase/drug effects ; Triazoles/*pharmacology ; },
abstract = {BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.},
}

Animals
Azepines/*pharmacology
Basic Helix-Loop-Helix Transcription Factors/drug effects/metabolism
CRISPR-Cas Systems
Cell Cycle/*drug effects
Cell Cycle Proteins/drug effects/metabolism
Cell Line, Tumor
Cell Lineage
Cerebellar Neoplasms/*drug therapy/genetics
Cyclin D2/drug effects/metabolism
Cyclin-Dependent Kinase 4/antagonists & inhibitors
Cyclin-Dependent Kinase 6/antagonists & inhibitors
Drug Resistance, Neoplasm
Gene Expression Profiling
Humans
Medulloblastoma/*drug therapy/genetics
Mice
Neural Stem Cells/drug effects/metabolism
Neurogenesis/*drug effects
Proteins/*antagonists & inhibitors
Proto-Oncogene Proteins c-myc/genetics
S Phase/drug effects
Triazoles/*pharmacology

@article {pmid31115652,
year = {2019},
author = {Lee, M and Kim, H},
title = {Therapeutic application of the CRISPR system: current issues and new prospects.},
journal = {Human genetics},
volume = {138},
number = {6},
pages = {563-590},
doi = {10.1007/s00439-019-02028-2},
pmid = {31115652},
issn = {1432-1203},
mesh = {*CRISPR-Cas Systems ; Epigenomics/methods ; Gene Editing/*methods ; Gene Rearrangement ; *Gene Transfer Techniques ; Genetic Therapy/methods ; Humans ; *Mutagenesis ; Reproducibility of Results ; },
abstract = {Since its discovery, the Clustered Regularly Interspaced Short Palindromic Repeat (the CRISPR) system has been increasingly applied to therapeutic genome editing. Employment of several viral and non-viral vectors has enabled efficient delivery of the CRISPR system to target cells or tissues. In addition, the CRISPR system is able to modulate the target gene's expression in various ways, such as mutagenesis, gene integration, epigenome regulation, chromosomal rearrangement, base editing and mRNA editing. However, there are still limitations hindering an ideal application of the system: inefficient delivery, dysregulation of the delivered gene, the immune response against the CRISPR system, the off-target effects or the unintended on-target mutations. In addition, there are recent discoveries that have not been yet applied to CRISPR-mediated therapeutic genome editing. Here, we review the overall principles related to the therapeutic application of the CRISPR system, along with new strategies for the further application and prospects to overcome the limitations.},
}

*CRISPR-Cas Systems
Epigenomics/methods
Gene Editing/*methods
Gene Rearrangement
*Gene Transfer Techniques
Genetic Therapy/methods
Humans
*Mutagenesis
Reproducibility of Results

@article {pmid30888429,
year = {2019},
author = {He, W and Chen, J and Gao, S},
title = {Mammalian haploid stem cells: establishment, engineering and applications.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {76},
number = {12},
pages = {2349-2367},
doi = {10.1007/s00018-019-03069-6},
pmid = {30888429},
issn = {1420-9071},
support = {31721003//NSFC/ ; 2016YFA0100400//Ministry of Science and Technology of the People's Republic of China/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cloning, Organism/*methods ; DNA Methylation ; Embryonic Stem Cells/*cytology/metabolism ; Female ; Gene Editing/methods ; *Haploidy ; Humans ; Male ; Spermatocytes/cytology/metabolism ; },
abstract = {Haploid embryonic stem cells (haESCs) contain only one set of genomes inherited from the sperm or egg and are termed AG- or PG-haESCs, respectively. Mammalian haESCs show genome-wide hypomethylation and dysregulated imprinting, whereas they can sustain genome integrity during derivation and long-term propagation. In addition, haESCs exhibit similar pluripotency to traditional diploid ESCs but are unique because they function as gametes and have been used to produce semi-cloned animals. More strikingly, unisexual reproduction has been achieved in mice by using haESCs. In combination with a gene editing or screening system, haESCs represent a powerful tool for studies of underlying gene functions and explorations of mechanisms of genetic and epigenetic regulation not only at the cellular level in vitro but also at the animal level in vivo. More importantly, genetically edited AG-haESC lines may further serve as an ideal candidate for the establishment of a sperm bank, which is a highly cost-effective approach, and a wide range of engineered semi-cloned mice have been produced. Here, we review the historical development, characteristics, advantages and disadvantages of haESCs. Additionally, we present an in-depth discussion of the recent advances in haESCs and their potential applications.},
}

Animals
CRISPR-Cas Systems
Cloning, Organism/*methods
DNA Methylation
Embryonic Stem Cells/*cytology/metabolism
Female
Gene Editing/methods
*Haploidy
Humans
Male
Spermatocytes/cytology/metabolism

@article {pmid30849393,
year = {2019},
author = {Bhate, A and Sun, T and Li, JB},
title = {ADAR1: A New Target for Immuno-oncology Therapy.},
journal = {Molecular cell},
volume = {73},
number = {5},
pages = {866-868},
doi = {10.1016/j.molcel.2019.02.021},
pmid = {30849393},
issn = {1097-4164},
mesh = {Adenosine Deaminase/genetics/immunology/*metabolism ; Animals ; CRISPR-Cas Systems ; Cell Survival ; Gene Editing ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Genetic Therapy/*methods ; Humans ; Immunity, Innate ; Immunotherapy/*methods ; Interferon-Induced Helicase, IFIH1/genetics/metabolism ; Neoplasms/enzymology/genetics/immunology/*therapy ; RNA Editing ; RNA, Double-Stranded/genetics/immunology/*metabolism ; RNA-Binding Proteins/genetics/immunology/*metabolism ; },
abstract = {Three recent studies by Ishizuka et al. (2019), Liu et al. (2019), and Gannon et al. (2018) show that deleting RNA editing enzyme ADAR1 could induce higher cell lethality and render tumor cells more vulnerable to immunotherapy, pinpointing ADAR1 as a new immuno-oncology target.},
}

Adenosine Deaminase/genetics/immunology/*metabolism
Animals
CRISPR-Cas Systems
Cell Survival
Gene Editing
Gene Expression Regulation, Enzymologic
Gene Expression Regulation, Neoplastic
Genetic Therapy/*methods
Humans
Immunity, Innate
Immunotherapy/*methods
Interferon-Induced Helicase, IFIH1/genetics/metabolism
Neoplasms/enzymology/genetics/immunology/*therapy
RNA Editing
RNA, Double-Stranded/genetics/immunology/*metabolism
RNA-Binding Proteins/genetics/immunology/*metabolism

@article {pmid30777520,
year = {2019},
author = {Argani, H},
title = {Genome Engineering for Stem Cell Transplantation.},
journal = {Experimental and clinical transplantation : official journal of the Middle East Society for Organ Transplantation},
volume = {17},
number = {Suppl 1},
pages = {31-37},
doi = {10.6002/ect.MESOT2018.L34},
pmid = {30777520},
issn = {2146-8427},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Diseases, Inborn/diagnosis/genetics/immunology/*therapy ; Genetic Predisposition to Disease ; *Genome, Human ; Humans ; Induced Pluripotent Stem Cells/immunology/*transplantation ; Phenotype ; Stem Cell Transplantation/adverse effects/*methods ; Treatment Outcome ; },
abstract = {To avoid the ethical issues of embryonic stem cells, genome engineering has focused on inducible pluripotent stem cells, which can develop into all 3 germ layers. The ability to detect methylation patterns in these cells allows research into pluripotency markers. The recently developed CRISPR system has allowed widespread application of genome engineering techniques. The CRISPR-Cas9 system, a potent system for genome editing, can be used for gene knockout or knock-in genome manipulations through substitution of a target genetic sequence with a desired donor sequence. Two types of genome engineering can be initiated: homologous or nonhomologous DNA repair by the Cas9 nuclease. Delivery of the CRISPR-Cas9 and target donor vectors in human pluripotent stem cells can be accomplished via viral and nonviral delivery methods. Nonviral delivery includes lipid-mediated transfection and electroporation. It has become the most common and efficient in vitro delivery method for human pluripotent stem cells. The CRISPR-Cas9 system can be combined with inducible pluripotent stem cells to generate single or multiple gene knockouts, correct mutations, or insert reporter transgenes. Knockouts can also be utilized to investigate epigenetic roles and targets, such as investigation of DNA methylation. CRISPR could be combined with human pluripotent stem cells to explore genetic determinants of lineage choice, differentiation, and stem cell fate, allowing investigators to study how various genes or noncoding elements contribute to specific processes and pathways. The CRISPR-Cas9 system can also be used to create null or nucleasedead Cas9, which has no enzymatic activity but has been utilized through fusion with other functional protein domains. In conclusion, RNA-guided genome targeting will have broad implications for synthetic biology, direct perturbation of gene networks, and targeted ex vivo and in vivo gene therapy.},
}

Animals
*CRISPR-Cas Systems
Gene Editing/*methods
Genetic Diseases, Inborn/diagnosis/genetics/immunology/*therapy
Genetic Predisposition to Disease
*Genome, Human
Humans
Induced Pluripotent Stem Cells/immunology/*transplantation
Phenotype
Stem Cell Transplantation/adverse effects/*methods
Treatment Outcome

@article {pmid30686591,
year = {2019},
author = {Mengwasser, KE and Adeyemi, RO and Leng, Y and Choi, MY and Clairmont, C and D'Andrea, AD and Elledge, SJ},
title = {Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets.},
journal = {Molecular cell},
volume = {73},
number = {5},
pages = {885-899.e6},
doi = {10.1016/j.molcel.2018.12.008},
pmid = {30686591},
issn = {1097-4164},
mesh = {Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; BRCA1 Protein/genetics/metabolism ; BRCA2 Protein/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Death ; Cell Line, Tumor ; DNA Damage ; DNA End-Joining Repair ; DNA-(Apurinic or Apyrimidinic Site) Lyase/*genetics/metabolism ; Flap Endonucleases/*genetics/metabolism ; Gene Expression Regulation, Neoplastic ; *Genes, Lethal ; Humans ; Neoplasms/drug therapy/enzymology/*genetics/pathology ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Proliferating Cell Nuclear Antigen/genetics/metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; *RNA Interference ; RNA, Small Interfering/genetics ; *Synthetic Lethal Mutations ; },
abstract = {BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.},
}

Ataxia Telangiectasia Mutated Proteins/genetics/metabolism
BRCA1 Protein/genetics/metabolism
BRCA2 Protein/*genetics/metabolism
*CRISPR-Cas Systems
Cell Death
Cell Line, Tumor
DNA Damage
DNA End-Joining Repair
DNA-(Apurinic or Apyrimidinic Site) Lyase/*genetics/metabolism
Flap Endonucleases/*genetics/metabolism
Gene Expression Regulation, Neoplastic
*Genes, Lethal
Humans
Neoplasms/drug therapy/enzymology/*genetics/pathology
Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
Proliferating Cell Nuclear Antigen/genetics/metabolism
Protein Binding
Protein Interaction Domains and Motifs
*RNA Interference
RNA, Small Interfering/genetics
*Synthetic Lethal Mutations

@article {pmid30639242,
year = {2019},
author = {Kovalski, JR and Bhaduri, A and Zehnder, AM and Neela, PH and Che, Y and Wozniak, GG and Khavari, PA},
title = {The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2.},
journal = {Molecular cell},
volume = {73},
number = {4},
pages = {830-844.e12},
doi = {10.1016/j.molcel.2018.12.001},
pmid = {30639242},
issn = {1097-4164},
support = {P30 CA124435/CA/NCI NIH HHS/United States ; R01 AR043799/AR/NIAMS NIH HHS/United States ; R01 CA142635/CA/NCI NIH HHS/United States ; S10 RR027425/RR/NCRR NIH HHS/United States ; },
mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems ; Caco-2 Cells ; Cell Cycle Checkpoints ; Cell Proliferation ; Cell Transformation, Neoplastic/genetics/*metabolism/pathology ; Female ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; Humans ; Mechanistic Target of Rapamycin Complex 2/genetics/*metabolism ; Mice, Hairless ; Mice, SCID ; Mice, Transgenic ; Mutation ; Neoplasms/genetics/*metabolism/pathology ; Protein Binding ; Protein Interaction Domains and Motifs ; *Proteome ; Proteomics/methods ; Tumor Burden ; ras Proteins/genetics/*metabolism ; },
abstract = {Proximity-dependent biotin labeling (BioID) may identify new targets for cancers driven by difficult-to-drug oncogenes such as Ras. Therefore, BioID was used with wild-type (WT) and oncogenic mutant (MT) H-, K-, and N-Ras, identifying known interactors, including Raf and PI3K, as well as a common set of 130 novel proteins proximal to all Ras isoforms. A CRISPR screen of these proteins for Ras dependence identified mTOR, which was also found proximal to MT Ras in human tumors. Oncogenic Ras directly bound two mTOR complex 2 (mTORC2) components, mTOR and MAPKAP1, to promote mTORC2 kinase activity at the plasma membrane. mTORC2 enabled the Ras pro-proliferative cell cycle transcriptional program, and perturbing the Ras-mTORC2 interaction impaired Ras-dependent neoplasia in vivo. Combining proximity-dependent proteomics with CRISPR screening identified a new set of functional Ras-associated proteins, defined mTORC2 as a new direct Ras effector, and offers a strategy for finding new proteins that cooperate with dominant oncogenes.},
}

Animals
Binding Sites
CRISPR-Cas Systems
Caco-2 Cells
Cell Cycle Checkpoints
Cell Proliferation
Cell Transformation, Neoplastic/genetics/*metabolism/pathology
Female
Gene Expression Regulation, Neoplastic
HEK293 Cells
Humans
Mechanistic Target of Rapamycin Complex 2/genetics/*metabolism
Mice, Hairless
Mice, SCID
Mice, Transgenic
Mutation
Neoplasms/genetics/*metabolism/pathology
Protein Binding
Protein Interaction Domains and Motifs
*Proteome
Proteomics/methods
Tumor Burden
ras Proteins/genetics/*metabolism

@article {pmid30581144,
year = {2019},
author = {Edraki, A and Mir, A and Ibraheim, R and Gainetdinov, I and Yoon, Y and Song, CQ and Cao, Y and Gallant, J and Xue, W and Rivera-Pérez, JA and Sontheimer, EJ},
title = {A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.},
journal = {Molecular cell},
volume = {73},
number = {4},
pages = {714-726.e4},
doi = {10.1016/j.molcel.2018.12.003},
pmid = {30581144},
issn = {1097-4164},
support = {UG3 HL147367/HL/NHLBI NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; R21 HD089566/HD/NICHD NIH HHS/United States ; R01 GM125797/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics/metabolism ; Dependovirus/genetics ; Embryo Transfer ; Female ; Gene Editing/*methods ; Genetic Vectors ; HEK293 Cells ; Humans ; K562 Cells ; Liver/*enzymology ; Mice, Inbred C57BL ; Neisseria meningitidis/*enzymology ; Nucleotide Motifs ; Proprotein Convertase 9/*genetics/metabolism ; RNA, Guide/genetics/metabolism ; Substrate Specificity ; Zygote/metabolism ; },
abstract = {CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.},
}

Animals
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA/*genetics/metabolism
Dependovirus/genetics
Embryo Transfer
Female
Gene Editing/*methods
Genetic Vectors
HEK293 Cells
Humans
K562 Cells
Liver/*enzymology
Mice, Inbred C57BL
Neisseria meningitidis/*enzymology
Nucleotide Motifs
Proprotein Convertase 9/*genetics/metabolism
RNA, Guide/genetics/metabolism
Substrate Specificity
Zygote/metabolism

@article {pmid30573836,
year = {2019},
author = {Strack, R},
title = {Precision genome editing.},
journal = {Nature methods},
volume = {16},
number = {1},
pages = {21},
doi = {10.1038/s41592-018-0286-6},
pmid = {30573836},
issn = {1548-7105},
mesh = {CRISPR-Cas Systems ; Fibroblasts/metabolism ; *Gene Editing ; Genome, Human ; Genotype ; Humans ; Machine Learning ; Mutation ; },
}

CRISPR-Cas Systems
Fibroblasts/metabolism
*Gene Editing
Genome, Human
Genotype
Humans
Machine Learning
Mutation

@article {pmid30559432,
year = {2019},
author = {Breinig, M and Schweitzer, AY and Herianto, AM and Revia, S and Schaefer, L and Wendler, L and Cobos Galvez, A and Tschaharganeh, DF},
title = {Multiplexed orthogonal genome editing and transcriptional activation by Cas12a.},
journal = {Nature methods},
volume = {16},
number = {1},
pages = {51-54},
doi = {10.1038/s41592-018-0262-1},
pmid = {30559432},
issn = {1548-7105},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; *Transcriptional Activation ; },
abstract = {CRISPR-Cas9-based combinatorial perturbation approaches for orthogonal knockout and gene activation have been impeded by complex vector designs and co-delivery of multiple constructs. Here, we demonstrate that catalytically active CRISPR-Cas12a fused to a transcriptional-activator domain enables flexible switching between genome editing and transcriptional activation by altering guide length. By leveraging Cas12a-mediated CRISPR-RNA array processing, we illustrate that Cas12a-VPR enables simplified multiplexed knockout and transcriptional activation in vitro and in vivo.},
}

Animals
*CRISPR-Cas Systems
Cell Line, Tumor
*Gene Editing
HEK293 Cells
Humans
Mice
*Transcriptional Activation

@article {pmid30554945,
year = {2019},
author = {Chakrabarti, AM and Henser-Brownhill, T and Monserrat, J and Poetsch, AR and Luscombe, NM and Scaffidi, P},
title = {Target-Specific Precision of CRISPR-Mediated Genome Editing.},
journal = {Molecular cell},
volume = {73},
number = {4},
pages = {699-713.e6},
doi = {10.1016/j.molcel.2018.11.031},
pmid = {30554945},
issn = {1097-4164},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Proliferation ; Chromatin/genetics/metabolism ; Chromatin Assembly and Disassembly ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics/metabolism ; *Gene Deletion ; Gene Editing/*methods ; HEK293 Cells ; Hep G2 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; *Mutagenesis, Insertional ; Nucleotide Motifs ; RNA, Guide/genetics/metabolism ; },
abstract = {The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms.},
}

CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
Cell Proliferation
Chromatin/genetics/metabolism
Chromatin Assembly and Disassembly
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA/*genetics/metabolism
*Gene Deletion
Gene Editing/*methods
HEK293 Cells
Hep G2 Cells
High-Throughput Nucleotide Sequencing
Humans
*Mutagenesis, Insertional
Nucleotide Motifs
RNA, Guide/genetics/metabolism

@article {pmid30551175,
year = {2018},
author = {Møller, HD and Lin, L and Xiang, X and Petersen, TS and Huang, J and Yang, L and Kjeldsen, E and Jensen, UB and Zhang, X and Liu, X and Xu, X and Wang, J and Yang, H and Church, GM and Bolund, L and Regenberg, B and Luo, Y},
title = {CRISPR-C: circularization of genes and chromosome by CRISPR in human cells.},
journal = {Nucleic acids research},
volume = {46},
number = {22},
pages = {e131},
doi = {10.1093/nar/gky767},
pmid = {30551175},
issn = {1362-4962},
mesh = {Base Sequence ; Biosensing Techniques ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; Chromosomes, Human, Pair 18/chemistry/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA End-Joining Repair ; DNA, Circular/*genetics/metabolism ; Fibroblasts ; Fluorescent Dyes/chemistry/metabolism ; Gene Editing/*methods ; Genes, Reporter ; Genetic Loci ; Genetic Vectors/chemistry/metabolism ; Genome, Human ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Humans ; Luminescent Proteins/genetics/metabolism ; RNA, Guide/genetics/metabolism ; },
abstract = {Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders. However, there is a lack of genetic engineering tools to recapitulate and study the biogenesis of eccDNAs. Here, we created a dual-fluorescence biosensor cassette, which upon the delivery of pairs of CRISPR/Cas9 guide RNAs, CRISPR-C, allows us to study the biogenesis of a specific fluorophore expressing eccDNA in human cells. We show that CRISPR-C can generate functional eccDNA, using the novel eccDNA biosensor system. We further reveal that CRISPR-C also can generate eccDNAs from intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts. EccDNAs mainly forms by end-joining mediated DNA-repair and we show that CRISPR-C is able to generate endogenous eccDNAs in sizes from a few hundred base pairs and ranging up to 207 kb. Even a 47.4 megabase-sized ring chromosome 18 can be created by CRISPR-C. Our study creates a new territory for CRISPR gene editing and highlights CRISPR-C as a useful tool for studying the cellular impact, persistence and function of eccDNAs.},
}

Base Sequence
Biosensing Techniques
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
Cell Line
Chromosomes, Human, Pair 18/chemistry/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA End-Joining Repair
DNA, Circular/*genetics/metabolism
Fibroblasts
Fluorescent Dyes/chemistry/metabolism
Gene Editing/*methods
Genes, Reporter
Genetic Loci
Genetic Vectors/chemistry/metabolism
Genome, Human
Green Fluorescent Proteins/genetics/metabolism
HEK293 Cells
Humans
Luminescent Proteins/genetics/metabolism
RNA, Guide/genetics/metabolism

@article {pmid30398641,
year = {2018},
author = {Chi, B and O'Connell, JD and Iocolano, AD and Coady, JA and Yu, Y and Gangopadhyay, J and Gygi, SP and Reed, R},
title = {The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex.},
journal = {Nucleic acids research},
volume = {46},
number = {22},
pages = {11939-11951},
doi = {10.1093/nar/gky1093},
pmid = {30398641},
issn = {1362-4962},
support = {R35 GM122524/GM/NIGMS NIH HHS/United States ; R01 GM043375/GM/NIGMS NIH HHS/United States ; },
mesh = {Amyotrophic Lateral Sclerosis/*genetics/metabolism/pathology ; CRISPR-Cas Systems ; Carrier Proteins/genetics/metabolism ; DNA Helicases/genetics/metabolism ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Humans ; Muscular Atrophy, Spinal/*genetics/metabolism/pathology ; Nuclear Matrix-Associated Proteins/deficiency/*genetics ; Nuclear Proteins/genetics/metabolism ; Positive Transcriptional Elongation Factor B/genetics/metabolism ; RNA Polymerase II/genetics/metabolism ; RNA, Transfer/genetics/metabolism ; RNA-Binding Protein EWS/deficiency/*genetics ; RNA-Binding Protein FUS/deficiency/*genetics ; RNA-Binding Proteins/*genetics ; Ribonucleoprotein, U1 Small Nuclear/genetics/metabolism ; Spliceosomes/chemistry/metabolism ; TATA-Binding Protein Associated Factors/deficiency/*genetics ; Transcription Factors, TFII/genetics/metabolism ; },
abstract = {Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.},
}

Amyotrophic Lateral Sclerosis/*genetics/metabolism/pathology
CRISPR-Cas Systems
Carrier Proteins/genetics/metabolism
DNA Helicases/genetics/metabolism
Gene Editing
Gene Expression Regulation
Gene Knockout Techniques
Humans
Muscular Atrophy, Spinal/*genetics/metabolism/pathology
Nuclear Matrix-Associated Proteins/deficiency/*genetics
Nuclear Proteins/genetics/metabolism
Positive Transcriptional Elongation Factor B/genetics/metabolism
RNA Polymerase II/genetics/metabolism
RNA, Transfer/genetics/metabolism
RNA-Binding Protein EWS/deficiency/*genetics
RNA-Binding Protein FUS/deficiency/*genetics
RNA-Binding Proteins/*genetics
Ribonucleoprotein, U1 Small Nuclear/genetics/metabolism
Spliceosomes/chemistry/metabolism
TATA-Binding Protein Associated Factors/deficiency/*genetics
Transcription Factors, TFII/genetics/metabolism

@article {pmid30335163,
year = {2018},
author = {Park, J and Lee, H and Han, N and Kwak, S and Lee, HT and Kim, JH and Kang, K and Youn, BH and Yang, JH and Jeong, HJ and Kang, JS and Kim, SY and Han, JW and Youn, HD and Cho, EJ},
title = {Long non-coding RNA ChRO1 facilitates ATRX/DAXX-dependent H3.3 deposition for transcription-associated heterochromatin reorganization.},
journal = {Nucleic acids research},
volume = {46},
number = {22},
pages = {11759-11775},
doi = {10.1093/nar/gky923},
pmid = {30335163},
issn = {1362-4962},
mesh = {Animals ; CRISPR-Cas Systems ; Carrier Proteins/*genetics/metabolism ; Cell Cycle Proteins/genetics/metabolism ; Cell Differentiation ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; Female ; Gene Editing ; Gene Expression Regulation, Developmental ; HEK293 Cells ; Heterochromatin/*chemistry/metabolism ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; Histones/*genetics/metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/*genetics/metabolism ; Male ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Mice ; Mice, Inbred C57BL ; Muscle Development/*genetics ; Muscle, Skeletal/cytology/growth & development/metabolism ; NIH 3T3 Cells ; Nuclear Proteins/*genetics/metabolism ; RNA, Long Noncoding/antagonists & inhibitors/*genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; Transcription, Genetic ; X-linked Nuclear Protein/*genetics/metabolism ; },
abstract = {Constitutive heterochromatin undergoes a dynamic clustering and spatial reorganization during myogenic differentiation. However the detailed mechanisms and its role in cell differentiation remain largely elusive. Here, we report the identification of a muscle-specific long non-coding RNA, ChRO1, involved in constitutive heterochromatin reorganization. ChRO1 is induced during terminal differentiation of myoblasts, and is specifically localized to the chromocenters in myotubes. ChRO1 is required for efficient cell differentiation, with global impacts on gene expression. It influences DNA methylation and chromatin compaction at peri/centromeric regions. Inhibition of ChRO1 leads to defects in the spatial fusion of chromocenters, and mislocalization of H4K20 trimethylation, Suv420H2, HP1, MeCP2 and cohesin. In particular, ChRO1 specifically associates with ATRX/DAXX/H3.3 complex at chromocenters to promote H3.3 incorporation and transcriptional induction of satellite repeats, which is essential for chromocenter clustering. Thus, our results unveil a mechanism involving a lncRNA that plays a role in large-scale heterochromatin reorganization and cell differentiation.},
}

Animals
CRISPR-Cas Systems
Carrier Proteins/*genetics/metabolism
Cell Cycle Proteins/genetics/metabolism
Cell Differentiation
Chromosomal Proteins, Non-Histone/genetics/metabolism
Female
Gene Editing
Gene Expression Regulation, Developmental
HEK293 Cells
Heterochromatin/*chemistry/metabolism
Histone-Lysine N-Methyltransferase/genetics/metabolism
Histones/*genetics/metabolism
Humans
Intracellular Signaling Peptides and Proteins/*genetics/metabolism
Male
Methyl-CpG-Binding Protein 2/genetics/metabolism
Mice
Mice, Inbred C57BL
Muscle Development/*genetics
Muscle, Skeletal/cytology/growth & development/metabolism
NIH 3T3 Cells
Nuclear Proteins/*genetics/metabolism
RNA, Long Noncoding/antagonists & inhibitors/*genetics/metabolism
RNA, Small Interfering/genetics/metabolism
Transcription, Genetic
X-linked Nuclear Protein/*genetics/metabolism

@article {pmid30006877,
year = {2018},
author = {Vetchinova, AS and Simonova, VV and Novosadova, EV and Manuilova, ES and Nenasheva, VV and Tarantul, VZ and Grivennikov, IA and Khaspekov, LG and Illarioshkin, SN},
title = {Cytogenetic Analysis of the Results of Genome Editing on the Cell Model of Parkinson's Disease.},
journal = {Bulletin of experimental biology and medicine},
volume = {165},
number = {3},
pages = {378-381},
doi = {10.1007/s10517-018-4174-y},
pmid = {30006877},
issn = {1573-8221},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cell Differentiation ; Fibroblasts/*metabolism/pathology ; Gene Editing/*methods ; Gene Expression ; Humans ; Induced Pluripotent Stem Cells/metabolism/pathology ; Karyotyping ; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/*genetics ; Mosaicism ; *Mutation ; Neurons/*metabolism/pathology ; Parkinson Disease/*genetics/metabolism/pathology ; Primary Cell Culture ; Tetraploidy ; },
abstract = {We performed a cytogenetic analysis of the results of CRISPR/Cas9-correction of G2019S mutation in LRRK2 gene associated with Parkinson's disease. Genome editing was performed on induced pluripotent stem cells derived from fibroblasts of a patient carrying this mutation. A mosaic variant of tetraploidy 92 XXYY/46,XY (24-43% cells from various clones) was found in neuronal precursors differentiated from the induced pluripotent stem cells after gene editing procedure. Solitary cases of translocations and chromosome breaks were observed. These data confirm the importance of the development of new approaches ensuring genome stability in CRISPR/Cas9-edited cultures.},
}

Base Sequence
CRISPR-Cas Systems
Cell Differentiation
Fibroblasts/*metabolism/pathology
Gene Editing/*methods
Gene Expression
Humans
Induced Pluripotent Stem Cells/metabolism/pathology
Karyotyping
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/*genetics
Mosaicism
*Mutation
Neurons/*metabolism/pathology
Parkinson Disease/*genetics/metabolism/pathology
Primary Cell Culture
Tetraploidy

@article {pmid29875502,
year = {2018},
author = {},
title = {Bacterial nanotechnology.},
journal = {Nature nanotechnology},
volume = {13},
number = {6},
pages = {435},
doi = {10.1038/s41565-018-0177-0},
pmid = {29875502},
issn = {1748-3395},
mesh = {Awards and Prizes ; Bacteria/*genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; Nanomedicine ; Norway ; RNA, Guide/genetics ; },
}

Awards and Prizes
Bacteria/*genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Humans
Nanomedicine
Norway
RNA, Guide/genetics

@article {pmid29869798,
year = {2018},
author = {Ma, S and Lv, J and Sun, J and Tang, P and Li, H and Zhou, H and Zhang, Z and Lin, Y and Rong, Z},
title = {iKA-CRISPR hESCs for inducible and multiplex orthogonal gene knockout and activation.},
journal = {FEBS letters},
volume = {592},
number = {13},
pages = {2238-2247},
doi = {10.1002/1873-3468.13127},
pmid = {29869798},
issn = {1873-3468},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Differentiation/genetics ; Cells, Cultured ; Gene Expression/genetics ; *Gene Knockout Techniques ; Human Embryonic Stem Cells/*metabolism/physiology ; Humans ; Mice ; Mice, Transgenic ; RNA, Guide/genetics/metabolism ; Transcriptional Activation/physiology ; },
abstract = {Human embryonic stem cells (hESCs) have a wide range of applications in early human embryonic development mimics, disease modeling, and cell therapy. To fulfill these applications, we established hESCs for inducible and multiplex orthogonal gene knockout and activation, which we named iKA-CRISPR hESCs. In cells, when complexed with a short guide RNA containing a 14-bp target sequence (14-bp gRNA) or a long 20-bp gRNA, the doxycycline-induced Cas9-p300 protein could activate gene transcription or cleave genomic DNA, respectively. We also demonstrate using iKA-CRISPR hESCs that knockout of OCT4 promoted differentiation, and developmentally relevant microRNAs and transcription factors could be efficiently activated. Thus, iKA-CRISPR hESCs provide a convenient platform to control gene expression networks and, therefore, facilitate the applications of hESCs in basic and translational biomedical research.},
}

Animals
CRISPR-Cas Systems/*genetics
Cell Differentiation/genetics
Cells, Cultured
Gene Expression/genetics
*Gene Knockout Techniques
Human Embryonic Stem Cells/*metabolism/physiology
Humans
Mice
Mice, Transgenic
RNA, Guide/genetics/metabolism
Transcriptional Activation/physiology

@article {pmid29852072,
year = {2018},
author = {Holtzman, L and Gersbach, CA},
title = {Editing the Epigenome: Reshaping the Genomic Landscape.},
journal = {Annual review of genomics and human genetics},
volume = {19},
number = {},
pages = {43-71},
doi = {10.1146/annurev-genom-083117-021632},
pmid = {29852072},
issn = {1545-293X},
support = {R41 GM119914/GM/NIGMS NIH HHS/United States ; U01 HG007900/HG/NHGRI NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; UM1 HG009428/HG/NHGRI NIH HHS/United States ; P30 AR066527/AR/NIAMS NIH HHS/United States ; R21 DA041878/DA/NIDA NIH HHS/United States ; R21 NS103007/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; DNA Methylation ; *Epigenomics ; Gene Editing ; Gene Expression Regulation ; Genetic Predisposition to Disease ; *Genome ; Histone Code ; Humans ; Protein Processing, Post-Translational ; },
abstract = {The eukaryotic epigenome has an instrumental role in determining and maintaining cell identity and function. Epigenetic components such as DNA methylation, histone tail modifications, chromatin accessibility, and DNA architecture are tightly correlated with central cellular processes, while their dysregulation manifests in aberrant gene expression and disease. The ability to specifically edit the epigenome holds the promise of enhancing understanding of how epigenetic modifications function and enabling manipulation of cell phenotype for research or therapeutic purposes. Genome engineering technologies use highly specific DNA-targeting tools to precisely deposit epigenetic changes in a locus-specific manner, creating diverse epigenome editing platforms. This review summarizes these technologies and insights from recent studies, describes the complex relationship between epigenetic components and gene regulation, and highlights caveats and promises of the emerging field of epigenome editing, including applications for translational purposes, such as epigenetic therapy and regenerative medicine.},
}

CRISPR-Cas Systems
DNA Methylation
*Epigenomics
Gene Editing
Gene Expression Regulation
Genetic Predisposition to Disease
*Genome
Histone Code
Humans
Protein Processing, Post-Translational

@article {pmid29570443,
year = {2018},
author = {Redel, BK and Beaton, BP and Spate, LD and Benne, JA and Murphy, SL and O'Gorman, CW and Spate, AM and Prather, RS and Wells, KD},
title = {Single step production of Cas9 mRNA for zygote injection.},
journal = {BioTechniques},
volume = {64},
number = {3},
pages = {118-124},
pmid = {29570443},
issn = {1940-9818},
support = {U42 OD011140/OD/NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cloning, Molecular/methods ; Microinjections ; RNA, Messenger/administration & dosage/*genetics ; Swine/genetics ; Zygote/*metabolism ; },
abstract = {Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.},
}

Animals
Animals, Genetically Modified/genetics
CRISPR-Associated Protein 9/*genetics
*CRISPR-Cas Systems
Cloning, Molecular/methods
Microinjections
RNA, Messenger/administration & dosage/*genetics
Swine/genetics
Zygote/*metabolism

@article {pmid31228550,
year = {2019},
author = {Gramelspacher, MJ and Hou, Z and Zhang, Y},
title = {Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems.},
journal = {Methods (San Diego, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymeth.2019.06.018},
pmid = {31228550},
issn = {1095-9130},
abstract = {The majority of bacteria and archaea rely on CRISPR-Cas systems for RNA-guided, adaptive immunity against mobile genetic elements. The Cas9 family of type II CRISPR-associated DNA endonucleases generates programmable double strand breaks in the CRISPR-complementary DNA targets flanked by the PAM motif. Nowadays, CRISPR-Cas9 provides a set of powerful tools for precise genome manipulation in eukaryotes and prokaryotes. Recently, a few Cas9 orthologs have been reported to possess intrinsic CRISPR-guided, sequence-specific ribonuclease activities. These discoveries fundamentally expanded the targeting capability of CRISPR-Cas9 systems, and promise to provide new CRISPR tools to manipulate specific cellular RNA transcripts. Here we present a detailed method for the biochemical characterization of Cas9's RNA-targeting potential.},
}

@article {pmid31226710,
year = {2019},
author = {Hampton, HG and Patterson, AG and Chang, JT and Taylor, C and Fineran, PC},
title = {GalK limits type I-F CRISPR-Cas expression in a CRP-dependent manner.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnz137},
pmid = {31226710},
issn = {1574-6968},
abstract = {CRISPR-Cas adaptive immune systems protect bacteria from phage predation, and other foreign genetic elements such as plasmids. Significant advances have been made regarding how CRISPR-Cas systems elicit immunity; however, comparatively little is known about their regulation. To study CRISPR-Cas regulation, we describe the construction of suicide lacZ-reporter plasmids with different antibiotic resistance cassettes. Through recombination into the host chromosome, single-copy expression can be achieved, thus preserving natural gene expression and maintaining a reporter expression output that reflects regulation within a normal genomic context. Previous work determined that the galactose metabolism gene GalM, decreased the expression of the cas operon in Pectobacterium atrosepticum. We used the new integrative reporters to investigate GalK, a gene that is located elsewhere in the genome and is responsible for the conversion of α-D-Galactose to Galactose-1-P during galactose metabolism. Deletion of galK led to elevated cas expression in a CRP-dependent manner but had no effect on CRISPR array expression. These results highlight that the metabolic status of the host cell is linked to the induction of CRISPR-Cas immunity.},
}

@article {pmid31225753,
year = {2019},
author = {Sontheimer, EJ},
title = {X-Tracting a New CRISPR-Cas Genome-Editing Platform from Metagenomic Data Sets.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {148-150},
doi = {10.1089/crispr.2019.29061.ejs},
pmid = {31225753},
issn = {2573-1602},
}

@article {pmid31223339,
year = {2019},
author = {Cairns, TC and Feurstein, C and Zheng, X and Zheng, P and Sun, J and Meyer, V},
title = {A quantitative image analysis pipeline for the characterization of filamentous fungal morphologies as a tool to uncover targets for morphology engineering: a case study using aplD in Aspergillus niger.},
journal = {Biotechnology for biofuels},
volume = {12},
number = {},
pages = {149},
doi = {10.1186/s13068-019-1473-0},
pmid = {31223339},
issn = {1754-6834},
abstract = {Background: Fungal fermentation is used to produce a diverse repertoire of enzymes, chemicals, and drugs for various industries. During submerged cultivation, filamentous fungi form a range of macromorphologies, including dispersed mycelia, clumped aggregates, or pellets, which have critical implications for rheological aspects during fermentation, gas/nutrient transfer, and, thus, product titres. An important component of strain engineering efforts is the ability to quantitatively assess fungal growth phenotypes, which will drive novel leads for morphologically optimized production strains.

Results: In this study, we developed an automated image analysis pipeline to quantify the morphology of pelleted and dispersed growth (MPD) which rapidly and reproducibly measures dispersed and pelleted macromorphologies from any submerged fungal culture. It (i) enables capture and analysis of several hundred images per user/day, (ii) is designed to quantitatively assess heterogeneous cultures consisting of dispersed and pelleted forms, (iii) gives a quantitative measurement of culture heterogeneity, (iv) automatically generates key Euclidian parameters for individual fungal structures including particle diameter, aspect ratio, area, and solidity, which are also assembled into a previously described dimensionless morphology number MN, (v) has an in-built quality control check which enables end-users to easily confirm the accuracy of the automated calls, and (vi) is easily adaptable to user-specified magnifications and macromorphological definitions. To concomitantly provide proof of principle for the utility of this image analysis pipeline, and provide new leads for morphologically optimized fungal strains, we generated a morphological mutant in the cell factory Aspergillus niger based on CRISPR-Cas technology. First, we interrogated a previously published co-expression networks for A. niger to identify a putative gamma-adaptin encoding gene (aplD) that was predicted to play a role in endosome cargo trafficking. Gene editing was used to generate a conditional aplD expression mutant under control of the titratable Tet-on system. Reduced aplD expression caused a hyperbranched growth phenotype and diverse defects in pellet formation with a putative increase in protein secretion. This possible protein hypersecretion phenotype could be correlated with increased dispersed mycelia, and both decreased pellet diameter and MN.

Conclusion: The MPD image analysis pipeline is a simple, rapid, and flexible approach to quantify diverse fungal morphologies. As an exemplar, we have demonstrated that the putative endosomal transport gene aplD plays a crucial role in A. niger filamentous growth and pellet formation during submerged culture. This suggests that endocytic components are underexplored targets for engineering fungal cell factories.},
}

@article {pmid30788791,
year = {2019},
author = {Wong, L and Holdridge, B and Engel, J and Xu, P},
title = {Genetic Tools for Streamlined and Accelerated Pathway Engineering in Yarrowia lipolytica.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1927},
number = {},
pages = {155-177},
doi = {10.1007/978-1-4939-9142-6_11},
pmid = {30788791},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cloning, Molecular ; DNA, Fungal ; *Gene Editing/methods ; Gene Expression ; Gene Library ; Genes, Reporter ; *Genetic Engineering ; *Metabolic Engineering/methods ; *Metabolic Networks and Pathways ; Plasmids/genetics ; Promoter Regions, Genetic ; RNA, Catalytic ; RNA, Guide ; Transformation, Genetic ; Yarrowia/*genetics/*metabolism ; },
abstract = {Yarrowia lipolytica is an industrial oleaginous yeast that has many attractive physiological and metabolic characteristics for various biotechnological applications. Although it has a long history of industrial applications, the number of genetic tools available to effectively and efficiently engineer Y. lipolytica still falls behind the vast number of tools available for common organisms such as Escherichia coli and Saccharomyces cerevisiae. In this protocol, we have developed a complete and versatile genetic toolkit tailored for facile genetic manipulation in Y. lipolytica. We created a versatile DNA assembly platform YaliBrick, which can streamline the cloning of large multigene pathways with reused genetic parts. We established a sensitive luciferase reporter assay to characterize a set of 12 native promoters. In addition, we used YaliBrick to generate different gene configurations in multigene constructs. The five-gene biosynthetic pathway of the anticancer, antimicrobial pigment violacein was rapidly assembled in 1 week to demonstrate the simplicity and effectiveness of integrating pathway-balancing strategies with our YaliBrick vectors. In the end, we incorporated CRISPR-Cas9 into our YaliBrick vectors and achieved indel mutation and frameshift gene deletion at the CAN1 (arginine permease) genomic loci of Yarrowia lipolytica. The reported protocol provides a standard procedure to streamline and accelerate metabolic pathway engineering in Yarrowia lipolytica.},
}

CRISPR-Cas Systems
Cloning, Molecular
DNA, Fungal
*Gene Editing/methods
Gene Expression
Gene Library
Genes, Reporter
*Genetic Engineering
*Metabolic Engineering/methods
*Metabolic Networks and Pathways
Plasmids/genetics
Promoter Regions, Genetic
RNA, Catalytic
RNA, Guide
Transformation, Genetic
Yarrowia/*genetics/*metabolism

@article {pmid30329048,
year = {2019},
author = {Tu, Z and Zhao, H and Li, B and Yan, S and Wang, L and Tang, Y and Li, Z and Bai, D and Li, C and Lin, Y and Li, Y and Liu, J and Xu, H and Guo, X and Jiang, YH and Zhang, YQ and Li, XJ},
title = {CRISPR/Cas9-mediated disruption of SHANK3 in monkey leads to drug-treatable autism-like symptoms.},
journal = {Human molecular genetics},
volume = {28},
number = {4},
pages = {561-571},
doi = {10.1093/hmg/ddy367},
pmid = {30329048},
issn = {1460-2083},
support = {R01 NS036232/NS/NINDS NIH HHS/United States ; R01 NS101701/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Autism Spectrum Disorder/diagnostic imaging/*drug therapy/genetics/pathology ; Behavior, Animal/drug effects ; Brain/diagnostic imaging/*drug effects/pathology ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Exons ; Fluoxetine/*administration & dosage ; Humans ; Interpersonal Relations ; Macaca fascicularis/genetics ; Mice ; Mutation ; Nerve Tissue Proteins/*genetics ; },
abstract = {Monogenic mutations in the SHANK3 gene, which encodes a postsynaptic scaffold protein, play a causative role in autism spectrum disorder (ASD). Although a number of mouse models with Shank3 mutations have been valuable for investigating the pathogenesis of ASD, species-dependent differences in behaviors and brain structures post considerable challenges to use small animals to model ASD and to translate experimental therapeutics to the clinic. We have used clustered regularly interspersed short palindromic repeat/CRISPR-associated nuclease 9 to generate a cynomolgus monkey model by disrupting SHANK3 at exons 6 and 12. Analysis of the live mutant monkey revealed the core behavioral abnormalities of ASD, including impaired social interaction and repetitive behaviors, and reduced brain network activities detected by positron-emission computed tomography (PET). Importantly, these abnormal behaviors and brain activities were alleviated by the antidepressant fluoxetine treatment. Our findings provide the first demonstration that the genetically modified non-human primate can be used for translational research of therapeutics for ASD.},
}

Animals
Autism Spectrum Disorder/diagnostic imaging/*drug therapy/genetics/pathology
Behavior, Animal/drug effects
Brain/diagnostic imaging/*drug effects/pathology
CRISPR-Cas Systems/genetics
Disease Models, Animal
Exons
Fluoxetine/*administration & dosage
Humans
Interpersonal Relations
Macaca fascicularis/genetics
Mice
Mutation
Nerve Tissue Proteins/*genetics

@article {pmid30307510,
year = {2019},
author = {Yang, N and Wu, N and Zhang, L and Zhao, Y and Liu, J and Liang, X and Ren, X and Li, W and Chen, W and Dong, S and Zhao, S and Lin, J and Xiang, H and Xue, H and Chen, L and Sun, H and Zhang, J and Shi, J and Zhang, S and Lu, D and Wu, X and Jin, L and Ding, J and Qiu, G and Wu, Z and Lupski, JR and Zhang, F},
title = {TBX6 compound inheritance leads to congenital vertebral malformations in humans and mice.},
journal = {Human molecular genetics},
volume = {28},
number = {4},
pages = {539-547},
doi = {10.1093/hmg/ddy358},
pmid = {30307510},
issn = {1460-2083},
support = {UM1 HG006542/HG/NHGRI NIH HHS/United States ; },
mesh = {Adolescent ; Alleles ; Animals ; CRISPR-Cas Systems/genetics ; Child ; Child, Preschool ; Congenital Abnormalities/diagnostic imaging/*genetics/physiopathology ; Disease Models, Animal ; Female ; Haploinsufficiency ; Humans ; Infant ; Male ; Mice ; Mutation ; Phenotype ; Scoliosis/diagnostic imaging/*genetics/physiopathology ; Spine/*abnormalities/diagnostic imaging/physiopathology ; T-Box Domain Proteins/*genetics ; },
abstract = {Congenital vertebral malformations (CVMs) are associated with human TBX6 compound inheritance that combines a rare null allele and a common hypomorphic allele at the TBX6 locus. Our previous in vitro evidence suggested that this compound inheritance resulted in a TBX6 gene dosage of less than haploinsufficiency (i.e. <50%) as a potential mechanism of TBX6-associated CVMs. To further investigate this pathogenetic model, we ascertained and collected 108 Chinese CVM cases and found that 10 (9.3%) of them carried TBX6 null mutations in combination with common hypomorphic variants at the second TBX6 allele. For in vivo functional verification and genetic analysis of TBX6 compound inheritance, we generated both null and hypomorphic mutations in mouse Tbx6 using the CRISPR-Cas9 method. These Tbx6 mutants are not identical to the patient variants at the DNA sequence level, but instead functionally mimic disease-associated TBX6 variants. Intriguingly, as anticipated by the compound inheritance model, a high penetrance of CVM phenotype was only observed in the mice with combined null and hypomorphic alleles of Tbx6. These findings are consistent with our experimental observations in humans and supported the dosage effect of TBX6 in CVM etiology. In conclusion, our findings in the newly collected human CVM subjects and Tbx6 mouse models consistently support the contention that TBX6 compound inheritance causes CVMs, potentially via a gene dosage-dependent mechanism. Furthermore, mouse Tbx6 mutants mimicking human CVM-associated variants will be useful models for further mechanistic investigations of CVM pathogenesis in the cases associated with TBX6.},
}

Adolescent
Alleles
Animals
CRISPR-Cas Systems/genetics
Child
Child, Preschool
Congenital Abnormalities/diagnostic imaging/*genetics/physiopathology
Disease Models, Animal
Female
Haploinsufficiency
Humans
Infant
Male
Mice
Mutation
Phenotype
Scoliosis/diagnostic imaging/*genetics/physiopathology
Spine/*abnormalities/diagnostic imaging/physiopathology
T-Box Domain Proteins/*genetics

@article {pmid28904339,
year = {2017},
author = {Liu, YJ and Liu, X and Chen, H and Zheng, P and Wang, W and Wang, L and Zhang, J and Tu, J},
title = {A Plastid-Localized Pentatricopeptide Repeat Protein is Required for Both Pollen Development and Plant Growth in Rice.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {11484},
pmid = {28904339},
issn = {2045-2322},
mesh = {CRISPR-Cas Systems ; Fatty Acids/biosynthesis ; Fluorescent Antibody Technique ; *Gene Expression Regulation, Plant ; Gene Targeting ; Lipid Metabolism ; Lipids/chemistry ; Mitochondrial Proteins/*genetics/metabolism ; Mutation ; Oryza/*physiology ; Phenotype ; Plant Development/*genetics ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Plastids/*metabolism ; Pollen/*metabolism ; Protein Biosynthesis ; Protein Transport ; RNA Interference ; RNA-Binding Proteins/*genetics/metabolism ; },
abstract = {Several mitochondrial-targeted pentatricopeptide repeat (PPR) proteins involved in pollen development have been reported to be fertility restorer (Rf) proteins. However, the roles of plastid-localized PPR proteins in plant male reproduction are poorly defined. Here, we described a plastid-localized PPR-SMR protein, OsPPR676, which is required for plant growth and pollen development in rice. In this study, OsPPR676 was confirmed to be an interacted protein with Osj10gBTF3, β-subunit of nascent polypeptide-associated complex (β-NAC), by bimolecular fluorescence complementation assays, indicating that both proteins are probably involved in the same regulatory pathway of pollen development. Compared with other chloroplast-rich tissues, OsPPR676 was only weakly expressed in anther, but in the Mei and YM stages of pollen development, its expression was relatively strong in the tapetum. Disruption of OsPPR676 resulted in growth retardation of plants and partial sterility of pollens. Phenotypic analysis of different osppr676 mutant lines implied that the SMR domain was not essential for the function of OsPPR676. We further demonstrated that OsPPR676 is essential for production of plastid atpB subunit, and then plays crucial roles in biosynthesis of fatty acids, carbohydrates, and other organic matters via affecting activity of ATP synthase.},
}

CRISPR-Cas Systems
Fatty Acids/biosynthesis
Fluorescent Antibody Technique
*Gene Expression Regulation, Plant
Gene Targeting
Lipid Metabolism
Lipids/chemistry
Mitochondrial Proteins/*genetics/metabolism
Mutation
Oryza/*physiology
Phenotype
Plant Development/*genetics
Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Plastids/*metabolism
Pollen/*metabolism
Protein Biosynthesis
Protein Transport
RNA Interference
RNA-Binding Proteins/*genetics/metabolism

@article {pmid28900124,
year = {2017},
author = {Yeh, YC and Kinoshita, M and Ng, TH and Chang, YH and Maekawa, S and Chiang, YA and Aoki, T and Wang, HC},
title = {Using CRISPR/Cas9-mediated gene editing to further explore growth and trade-off effects in myostatin-mutated F4 medaka (Oryzias latipes).},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {11435},
pmid = {28900124},
issn = {2045-2322},
mesh = {Animals ; Breeding ; *CRISPR-Cas Systems ; *Gene Editing ; Microinjections ; Mutation ; Myostatin/*genetics ; Oryzias/*genetics ; RNA, Guide ; RNA, Messenger/administration & dosage/genetics ; },
abstract = {Myostatin (MSTN) suppresses skeletal muscle development and growth in mammals, but its role in fish is less well understood. Here we used CRISPR/Cas9 to mutate the MSTN gene in medaka (Oryzias latipes) and evaluate subsequent growth performance. We produced mutant F0 fish that carried different frameshifts in the OlMSTN coding sequence and confirmed the heritability of the mutant genotypes to the F1 generation. Two F1 fish with the same heterozygous frame-shifted genomic mutations (a 22 bp insertion in one allele; a 32 bp insertion in the other) were then crossbred to produce subsequent generations (F2~F5). Body length and weight of the MSTN-/- F4 medaka were significantly higher than in the wild type fish, and muscle fiber density in the inner and outer compartments of the epaxial muscles was decreased, suggesting that MSTN null mutation induces muscle hypertrophy. From 3~4 weeks post hatching (wph), the expression of three major myogenic related factors (MRFs), MyoD, Myf5 and Myogenin, was also significantly upregulated. Some medaka had a spinal deformity, and we also observed a trade-off between growth and immunity in MSTN-/- F4 medaka. Reproduction was unimpaired in the fast-growth phenotypes.},
}

Animals
Breeding
*CRISPR-Cas Systems
*Gene Editing
Microinjections
Mutation
Myostatin/*genetics
Oryzias/*genetics
RNA, Guide
RNA, Messenger/administration & dosage/genetics

@article {pmid31220585,
year = {2019},
author = {Shen, J and Zhou, J and Xu, Y and Xiu, Z},
title = {Prophages contribute to remarkable genome plasticity of Klebsiella pneumoniae and may involve the chromosomal integration of ARGs in CG258.},
journal = {Genomics},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ygeno.2019.06.016},
pmid = {31220585},
issn = {1089-8646},
abstract = {Klebsiella pneumoniae is an important multidrug-resistant pathogen carrying prophages. Here we explore the contribution of prophages to bacterial evolution and fitness in silico. This study showed prophages contribute to remarkable genome plasticity of K. pneumoniae. The strains of CG258 possess several conserved prophages including the couple of P2-P4 prophages. CRISPR-Cas system has limited impact on the presence of prophages. The strong MLST-depended distribution of CRISPR-Cas and prophages and the high proportion of strains with self-targeting spacers may be the causes. Four core ARGs (blaSHV, fosA and oqxAB) were detected on almost all the chromosomes, but the acquired ARGs were only found in CG258 and CRISPR-positive strains. The factors influencing the chromosomal integration of ARGs in CG258 and CRISPR-positive strains may be different. In CG258, prophages may involve the chromosomal integration of ARGs. For CRISPR-positive strains, the immunity of CRISPR-Cas systems against invading ARG-bearing mobile genetic elements may accelerate the process.},
}

@article {pmid31220528,
year = {2019},
author = {Rahimi, S and Roushandeh, AM and Ebrahimi, A and Samdani, AA and Kuwahara, Y and Roudkenar, MH},
title = {CRISPR/Cas9-mediated knockout of Lcn2 effectively enhanced CDDP-induced apoptosis and reduced cell migration capacity of PC3 cells.},
journal = {Life sciences},
volume = {},
number = {},
pages = {116586},
doi = {10.1016/j.lfs.2019.116586},
pmid = {31220528},
issn = {1879-0631},
abstract = {AIMS: Lipocalin 2 (Lcn2/NGAL) belongs to lipocalin superfamily with diverse functions. The precise function of Lcn2, particularly in cancer development, remains to be elucidated yet. In an attempt to knockout of Lcn2 expression by CRISPR/Cas 9 technology in a highly aggressive and invasive prostate cancer cell line and to evaluate the combination therapy with cisplatin (CDDP), this study was conducted.

MAIN METHODS: Control CRISPR/Cas9 plasmid and homology-directed repair plasmid or validated human Lcn2 CRISPR/Cas9 KO plasmids were co-transfected into PC3 cells using fugene HD transfection reagent. The stable cells were selected in the presence of puromycin. Correspondingly, knock out of Lcn2 was evaluated by RT-PCR, ELISA, and immunocytochemistry. PC3-Scr (control) and Lcn2-KO (PC3 cells in which lcn2 has been knocked out) were treated with or without cisplatin (CDDP). Cell proliferative ability was measured by WST-1 and colony-formation assays. Apoptosis was evaluated by DAPI staining, in situ cell death detection (TUNEL) assay, and cell death detection ELISA plus methods. The migration capabilities were studied by wound healing/scratch and transwell assays.

KEY FINDINGS: Lcn2 knock out in a highly aggressive and invasive cancer cell like PC3 decreased cell proliferation and increased the sensitivity of CDDP. Conspicuously, loss of Lcn2 expression effectively enhanced CDDP-induced apoptosis in PC3 cells. Lcn2 knock out by CRISPR/Cas9 technology decreased the cell migration capacity of PC3 cells as well.

SIGNIFICANCE: Lcn2 not only is a valuable and useful biomarker for diagnosis and prognosis of prostate cancer but also and more importantly is a potential novel emerging therapeutic target.},
}

@article {pmid31219653,
year = {2019},
author = {Ahmadzadeh, V and Farajnia, S and Baghban, R and Rahbarnia, L and Zarredar, H},
title = {CRISPR-Cas system: Toward a more efficient technology for genome editing and beyond.},
journal = {Journal of cellular biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/jcb.29140},
pmid = {31219653},
issn = {1097-4644},
support = {58570//Drug Applied Research Center, Tabriz University of Medical Sciences/ ; },
abstract = {Genome engineering technology is of great interest for biomedical research that enables scientists to make specific manipulation in the DNA sequence. Early methods for introducing double-stranded DNA breaks relies on protein-based systems. These platforms have enabled fascinating advances, but all are costly and time-consuming to engineer, preventing these from gaining high-throughput applications. The CRISPR-Cas9 system, co-opted from bacteria, has generated considerable excitement in gene targeting. In this review, we describe gene targeting techniques with an emphasis on recent strategies to improve the specificities of CRISPR-Cas systems for nuclease and non-nuclease applications.},
}

@article {pmid31219587,
year = {2019},
author = {Grainy, J and Garrett, S and Graveley, BR and P Terns, M},
title = {CRISPR repeat sequences and relative spacing specify DNA integration by Pyrococcus furiosus Cas1 and Cas2.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkz548},
pmid = {31219587},
issn = {1362-4962},
abstract = {Acquiring foreign spacer DNA into the CRISPR locus is an essential primary step of the CRISPR-Cas pathway in prokaryotes for developing host immunity to mobile genetic elements. Here, we investigate spacer integration in vitro using proteins from Pyrococcus furiosus and demonstrate that Cas1 and Cas2 are sufficient to accurately integrate spacers into a minimal CRISPR locus. Using high-throughput sequencing, we identified high frequency spacer integration occurring at the same CRISPR repeat border sites utilized in vivo, as well as at several non-CRISPR plasmid sequences which share features with repeats. Analysis of non-CRISPR integration sites revealed that Cas1 and Cas2 are directed to catalyze full-site spacer integration at specific DNA stretches where guanines and/or cytosines are 30 base pairs apart and the intervening sequence harbors several positionally conserved bases. Moreover, assaying a series of CRISPR repeat mutations, followed by sequencing of the integration products, revealed that the specificity of integration is primarily directed by sequences at the leader-repeat junction as well as an adenine-rich sequence block in the mid-repeat. Together, our results indicate that P. furiosus Cas1 and Cas2 recognize multiple sequence features distributed over a 30 base pair DNA region for accurate spacer integration at the CRISPR repeat.},
}

@article {pmid31218106,
year = {2019},
author = {Ouyang, Q and Liu, Y and Tan, J and Li, J and Yang, D and Zeng, F and Huang, W and Kong, Y and Liu, Z and Zhou, H and Liu, Y},
title = {Loss of ZNF587B and SULF1 contributed to cisplatin resistance in ovarian cancer cell lines based on Genome-scale CRISPR/Cas9 screening.},
journal = {American journal of cancer research},
volume = {9},
number = {5},
pages = {988-998},
pmid = {31218106},
issn = {2156-6976},
abstract = {Ovarian cancer is one of the most lethal malignancies of the female reproductive system. Platinum-resistance is the major obstacle in the successful treatment of ovarian cancer. Previous studies largely failed to identify the key genes associated with platinum-resistance by using candidate genes testing, bioinformatic analysis and GWAS method. The aim of the study was to utilize the whole human Genome-scale CRISPR-Cas9 knockout (GeCKO) library to screen for novel genes involved in cisplatin resistance in ovarian cancer cell lines. The GeCKO library targeted 19052 genes with 122417 unique guide sequences. Six candidate genes had been screened out including one previously validated gene SULF1 and five novel genes ZNF587B, TADA1, SEMA4G, POTEC and USP17L20. After validated by CCK-8 and RT-PCR analysis, two genes (ZNF587B and SULF1) were discovered to be involved in cisplatin resistance. ZNF587B may serve as a new biomarker for predicting cisplatin resistance.},
}

@article {pmid30872531,
year = {2019},
author = {Chui, AJ and Okondo, MC and Rao, SD and Gai, K and Griswold, AR and Johnson, DC and Ball, DP and Taabazuing, CY and Orth, EL and Vittimberga, BA and Bachovchin, DA},
title = {N-terminal degradation activates the NLRP1B inflammasome.},
journal = {Science (New York, N.Y.)},
volume = {364},
number = {6435},
pages = {82-85},
doi = {10.1126/science.aau1208},
pmid = {30872531},
issn = {1095-9203},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 AI137168/AI/NIAID NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; T32 GM115327/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Antigens, Bacterial/*metabolism ; Apoptosis Regulatory Proteins/genetics/*metabolism ; Bacterial Toxins/*metabolism ; CRISPR-Cas Systems ; Caspase 1/metabolism ; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/antagonists & inhibitors ; Gene Knockout Techniques ; HEK293 Cells ; Host-Pathogen Interactions/immunology ; Humans ; Immunity, Innate ; Inflammasomes/*metabolism ; Mice ; Proteasome Endopeptidase Complex/metabolism ; *Proteolysis ; Pyroptosis/genetics/*physiology ; RAW 264.7 Cells ; Serine Proteinase Inhibitors/pharmacology ; THP-1 Cells ; Ubiquitin-Protein Ligases/genetics ; },
abstract = {Intracellular pathogens and danger signals trigger the formation of inflammasomes, which activate inflammatory caspases and induce pyroptosis. The anthrax lethal factor metalloprotease and small-molecule DPP8/9 inhibitors both activate the NLRP1B inflammasome, but the molecular mechanism of NLRP1B activation is unknown. In this study, we used genome-wide CRISPR-Cas9 knockout screens to identify genes required for NLRP1B-mediated pyroptosis. We discovered that lethal factor induces cell death via the N-end rule proteasomal degradation pathway. Lethal factor directly cleaves NLRP1B, inducing the N-end rule-mediated degradation of the NLRP1B N terminus and freeing the NLRP1B C terminus to activate caspase-1. DPP8/9 inhibitors also induce proteasomal degradation of the NLRP1B N terminus but not via the N-end rule pathway. Thus, N-terminal degradation is the common activation mechanism of this innate immune sensor.},
}

Animals
Antigens, Bacterial/*metabolism
Apoptosis Regulatory Proteins/genetics/*metabolism
Bacterial Toxins/*metabolism
CRISPR-Cas Systems
Caspase 1/metabolism
Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/antagonists & inhibitors
Gene Knockout Techniques
HEK293 Cells
Host-Pathogen Interactions/immunology
Humans
Immunity, Innate
Inflammasomes/*metabolism
Mice
Proteasome Endopeptidase Complex/metabolism
*Proteolysis
Pyroptosis/genetics/*physiology
RAW 264.7 Cells
Serine Proteinase Inhibitors/pharmacology
THP-1 Cells
Ubiquitin-Protein Ligases/genetics

@article {pmid29581186,
year = {2018},
author = {Bruurs, LJM and van der Net, MC and Zwakenberg, S and Rosendahl Huber, AKM and Post, A and Zwartkruis, FJ and Bos, JL},
title = {The Phosphatase PTPL1 Is Required for PTEN-Mediated Regulation of Apical Membrane Size.},
journal = {Molecular and cellular biology},
volume = {38},
number = {12},
pages = {},
pmid = {29581186},
issn = {1098-5549},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Membrane/*metabolism ; Cell Polarity/*physiology ; Epithelial Cells/physiology ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Male ; Mice ; Microvilli/genetics/*metabolism ; Neoplasms/pathology ; PTEN Phosphohydrolase/genetics/*metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 13/*genetics ; },
abstract = {PTEN is a tumor suppressor that is frequently lost in epithelial malignancies. A part of the tumor-suppressive properties of PTEN is attributed to its function in cell polarization and consequently its role in maintaining epithelial tissue integrity. However, surprisingly little is known about the function and regulation of PTEN during epithelial cell polarization. We used clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated gene disruption to delete PTEN in intestinal epithelial Ls174T:W4 cells, which upon differentiation form a microvillus-covered apical membrane (brush border) on a part of the cell cortex, independent of cell-cell junctions. We show that loss of PTEN results in the formation of a larger brush border that, in a fraction of the cells, even spans the entire plasma membrane, revealing that PTEN functions in the regulation of apical membrane size. Depletion of the phosphatase PTPL1 resulted in a similar defect. PTPL1 interacts with PTEN, and this interaction is necessary for apical membrane enrichment of PTEN. Importantly, phosphatase activity of PTPL1 is not required, indicating that PTPL1 functions as an anchor protein in this process. Our work thus demonstrates a novel function for PTEN during cell polarization in controlling apical membrane size and identifies PTPL1 as a critical apical membrane anchor for PTEN in this process.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Cell Membrane/*metabolism
Cell Polarity/*physiology
Epithelial Cells/physiology
Gene Knockout Techniques
HEK293 Cells
Humans
Male
Mice
Microvilli/genetics/*metabolism
Neoplasms/pathology
PTEN Phosphohydrolase/genetics/*metabolism
Protein Tyrosine Phosphatase, Non-Receptor Type 13/*genetics

@article {pmid29581185,
year = {2018},
author = {Nelson, ND and Dodson, LM and Escudero, L and Sukumar, AT and Williams, CL and Mihalek, I and Baldan, A and Baird, DM and Bertuch, AA},
title = {The C-Terminal Extension Unique to the Long Isoform of the Shelterin Component TIN2 Enhances Its Interaction with TRF2 in a Phosphorylation- and Dyskeratosis Congenita Cluster-Dependent Fashion.},
journal = {Molecular and cellular biology},
volume = {38},
number = {12},
pages = {},
pmid = {29581185},
issn = {1098-5549},
support = {F30 CA168144/CA/NCI NIH HHS/United States ; R01 HL131744/HL/NHLBI NIH HHS/United States ; T32 DK060445/DK/NIDDK NIH HHS/United States ; T32 GM008307/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Dyskeratosis Congenita/genetics ; Humans ; Mutation/genetics ; Phosphorylation ; Protein Isoforms/genetics/metabolism ; Telomere/metabolism ; Telomere-Binding Proteins/*genetics/*metabolism ; Telomeric Repeat Binding Protein 2/*genetics ; },
abstract = {TIN2 is central to the shelterin complex, linking the telomeric proteins TRF1 and TRF2 with TPP1/POT1. Mutations in TINF2, which encodes TIN2, that are found in dyskeratosis congenita (DC) result in very short telomeres and cluster in a region shared by the two TIN2 isoforms, TIN2S (short) and TIN2L (long). Here we show that TIN2L, but not TIN2S, is phosphorylated. TRF2 interacts more with TIN2L than TIN2S, and both the DC cluster and phosphorylation promote this enhanced interaction. The binding of TIN2L, but not TIN2S, is affected by TRF2-F120, which is also required for TRF2's interaction with end processing factors such as Apollo. Conversely, TRF1 interacts more with TIN2S than with TIN2L. A DC-associated mutation further reduces TIN2L-TRF1, but not TIN2S-TRF1, interaction. Cells overexpressing TIN2L or phosphomimetic TIN2L are permissive to telomere elongation, whereas cells overexpressing TIN2S or phosphodead TIN2L are not. Telomere lengths are unchanged in cell lines in which TIN2L expression has been eliminated by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated mutation. These results indicate that TIN2 isoforms are biochemically and functionally distinguishable and that shelterin composition could be fundamentally altered in patients with TINF2 mutations.},
}

CRISPR-Cas Systems/genetics
Cell Line
Dyskeratosis Congenita/genetics
Humans
Mutation/genetics
Phosphorylation
Protein Isoforms/genetics/metabolism
Telomere/metabolism
Telomere-Binding Proteins/*genetics/*metabolism
Telomeric Repeat Binding Protein 2/*genetics

@article {pmid30704043,
year = {2019},
author = {Kim, J and Koo, BK and Yoon, KJ},
title = {Modeling Host-Virus Interactions in Viral Infectious Diseases Using Stem-Cell-Derived Systems and CRISPR/Cas9 Technology.},
journal = {Viruses},
volume = {11},
number = {2},
pages = {},
doi = {10.3390/v11020124},
pmid = {30704043},
issn = {1999-4915},
mesh = {Adenoviridae/genetics ; Animals ; *CRISPR-Cas Systems ; Gene Editing ; Host Microbial Interactions/*genetics ; Humans ; Induced Pluripotent Stem Cells/*virology ; Mice ; Norovirus/genetics ; Organoids/*virology ; Virus Diseases/*genetics ; Viruses/genetics/*pathogenicity ; Zika Virus/genetics ; Zika Virus Infection ; },
abstract = {Pathologies induced by viral infections have undergone extensive study, with traditional model systems such as two-dimensional (2D) cell cultures and in vivo mouse models contributing greatly to our understanding of host-virus interactions. However, the technical limitations inherent in these systems have constrained efforts to more fully understand such interactions, leading to a search for alternative in vitro systems that accurately recreate in vivo physiology in order to advance the study of viral pathogenesis. Over the last decade, there have been significant technological advances that have allowed researchers to more accurately model the host environment when modeling viral pathogenesis in vitro, including induced pluripotent stem cells (iPSCs), adult stem-cell-derived organoid culture systems and CRISPR/Cas9-mediated genome editing. Such technological breakthroughs have ushered in a new era in the field of viral pathogenesis, where previously challenging questions have begun to be tackled. These include genome-wide analysis of host-virus crosstalk, identification of host factors critical for viral pathogenesis, and the study of viral pathogens that previously lacked a suitable platform, e.g., noroviruses, rotaviruses, enteroviruses, adenoviruses, and Zika virus. In this review, we will discuss recent advances in the study of viral pathogenesis and host-virus crosstalk arising from the use of iPSC, organoid, and CRISPR/Cas9 technologies.},
}

Adenoviridae/genetics
Animals
*CRISPR-Cas Systems
Gene Editing
Host Microbial Interactions/*genetics
Humans
Induced Pluripotent Stem Cells/*virology
Mice
Norovirus/genetics
Organoids/*virology
Virus Diseases/*genetics
Viruses/genetics/*pathogenicity
Zika Virus/genetics
Zika Virus Infection

@article {pmid30593630,
year = {2019},
author = {Qiao, L and Luo, GG},
title = {Functional Characterization of Apolipoproteins in the HCV Life Cycle.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1911},
number = {},
pages = {235-246},
doi = {10.1007/978-1-4939-8976-8_16},
pmid = {30593630},
issn = {1940-6029},
support = {P30 AI027767/AI/NIAID NIH HHS/United States ; },
mesh = {Antibodies, Neutralizing/metabolism ; Apolipoproteins/genetics/*metabolism ; Apolipoproteins E/genetics/metabolism ; Blotting, Western/methods ; CRISPR-Cas Systems ; Cell Line ; Hepacivirus/genetics/growth & development/*physiology ; Hepatitis C/*metabolism ; *Host-Pathogen Interactions ; Humans ; RNA Interference ; RNA, Small Interfering/genetics ; Real-Time Polymerase Chain Reaction/methods ; Transfection/methods ; Viral Envelope Proteins/genetics/*metabolism ; *Virus Assembly ; *Virus Attachment ; },
abstract = {Apolipoprotein E (apoE) plays dual functions in the HCV life cycle by promoting HCV infection and virion assembly and production. ApoE is a structural component on the HCV envelope. It mediates HCV cell attachment through specific interactions with the cell surface receptors such as syndecan-1 (SDC-1) and SDC-2 heparan sulfate proteoglycans (HSPGs). It also interacts with NS5A and E2, resulting in an enhancement of HCV morphogenesis. It can bind HCV extracellularly and promotes HCV infection. It is critical for HCV cell-to-cell transmission and may also play a role in HCV persistence by interfering with the action of HCV-neutralizing antibodies. Other apolipoproteins particularly apoB and apoC1 were also found on the HCV envelope, but their roles in the HCV life cycle remain unclear. In the last decade, a number of genomic, immunological, structural, and cell biology methodologies have been developed and used for determining the importance of apoE in the HCV life cycle. These methods and protocols will continue to be valuable to further understand the importance and the underlying molecular mechanism of various apolipoproteins in HCV infection and pathogenesis.},
}

Antibodies, Neutralizing/metabolism
Apolipoproteins/genetics/*metabolism
Apolipoproteins E/genetics/metabolism
Blotting, Western/methods
CRISPR-Cas Systems
Cell Line
Hepacivirus/genetics/growth & development/*physiology
Hepatitis C/*metabolism
*Host-Pathogen Interactions
Humans
RNA Interference
RNA, Small Interfering/genetics
Real-Time Polymerase Chain Reaction/methods
Transfection/methods
Viral Envelope Proteins/genetics/*metabolism
*Virus Assembly
*Virus Attachment

@article {pmid30593622,
year = {2019},
author = {Wu, X and Dao Thi, VL},
title = {Embryonic or Induced Pluripotent Stem Cell-Derived Hepatocellular Systems for HCV Culture.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1911},
number = {},
pages = {121-135},
doi = {10.1007/978-1-4939-8976-8_8},
pmid = {30593622},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Cell Culture Techniques/*methods ; *Cell Differentiation ; Cell Line ; Gene Editing/methods ; Hepacivirus/physiology ; Hepatitis C/genetics ; Hepatocytes/*cytology/metabolism ; Host-Pathogen Interactions ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; },
abstract = {Human-induced pluripotent stem cell-derived hepatocyte-like cells (iHeps) constitute a powerful tool for modeling hepatotropic pathogen infections in cell culture. Meanwhile, CRISPR-Cas9 technology enables precise editing of stem cell genomes to generate patient-specific disease models and thus development of personalized experimental systems. Here we present a detailed stepwise protocol for the differentiation of stem cells to hepatocyte-like cells for HCV studies in cell culture. We also outline the use of an inducible iCRISPR platform for the rapid and efficient modification of host factors of interest to better understand their function during HCV infection.},
}

CRISPR-Cas Systems
Cell Culture Techniques/*methods
*Cell Differentiation
Cell Line
Gene Editing/methods
Hepacivirus/physiology
Hepatitis C/genetics
Hepatocytes/*cytology/metabolism
Host-Pathogen Interactions
Humans
Induced Pluripotent Stem Cells/*cytology/metabolism

@article {pmid30414698,
year = {2019},
author = {Goodspeed, A and Jean, A and Costello, JC},
title = {A Whole-genome CRISPR Screen Identifies a Role of MSH2 in Cisplatin-mediated Cell Death in Muscle-invasive Bladder Cancer.},
journal = {European urology},
volume = {75},
number = {2},
pages = {242-250},
doi = {10.1016/j.eururo.2018.10.040},
pmid = {30414698},
issn = {1873-7560},
support = {P30 CA046934/CA/NCI NIH HHS/United States ; T32 GM007635/GM/NIGMS NIH HHS/United States ; },
mesh = {Antineoplastic Agents/*pharmacology ; Biomarkers, Tumor/*genetics ; *CRISPR-Cas Systems ; Cell Death/drug effects ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Databases, Genetic ; Drug Resistance, Neoplasm/genetics ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Humans ; MutS Homolog 2 Protein/*genetics ; Neoplasm Invasiveness ; Phenotype ; Urinary Bladder Neoplasms/*drug therapy/genetics/metabolism/pathology ; Whole Genome Sequencing/*methods ; },
abstract = {BACKGROUND: The response to first-line, platinum-based treatment of muscle-invasive bladder cancer has not improved in 3 decades.

OBJECTIVE: To identify genes that influence cisplatin resistance in bladder cancer.

We performed a whole-genome CRISPR screen in a bladder cancer cell line to identify genes that mediate resistance to cisplatin.

Targeted validation was performed in two bladder cancer cell lines. The top gene candidate was validated in a publicly available bladder cancer dataset.

RESULTS AND LIMITATIONS: From the CRISPR screen, we identified MSH2 as the most significantly enriched gene and mismatch repair as the most significantly enriched pathway that promoted resistance to cisplatin. Bladder cancer cells with knockdown of MSH2 showed a reduction in cisplatin-mediated apoptosis. MSH2 loss did not impact the sensitivity to other chemotherapies, including the cisplatin analog oxaliplatin. Bladder tumors with low MSH2 protein levels, quantified using reverse-phase protein array, showed poorer survival when treated with cisplatin- or carboplatin-based therapy; these results require future validation using immunohistochemistry. Additionally, results are retrospective from patients with primarily high-grade tumors; thus, validation in a controlled clinical trial is needed.

CONCLUSIONS: We generated in vitro evidence that bladder cancer cell lines depleted of MSH2 are more resistant to cisplatin. We additionally found an association between low MSH2 in bladder tumors and poorer patient survival when treated with platinum-based chemotherapy. If successfully validated prospectively, MSH2 protein level could assist in the selection of patients for chemotherapy.

PATIENT SUMMARY: We report the first evidence that MSH2 protein level may contribute to chemotherapy resistance observed in muscle-invasive bladder cancer. MSH2 has potential as a biomarker predictive of response to platinum-based therapy.},
}

Antineoplastic Agents/*pharmacology
Biomarkers, Tumor/*genetics
*CRISPR-Cas Systems
Cell Death/drug effects
Cell Line, Tumor
Cisplatin/*pharmacology
Databases, Genetic
Drug Resistance, Neoplasm/genetics
Genetic Predisposition to Disease
Genome-Wide Association Study
Humans
MutS Homolog 2 Protein/*genetics
Neoplasm Invasiveness
Phenotype
Urinary Bladder Neoplasms/*drug therapy/genetics/metabolism/pathology
Whole Genome Sequencing/*methods

@article {pmid31208775,
year = {2019},
author = {Kurtz, S and Petersen, B},
title = {Pre-determination of sex in pigs by application of CRISPR/Cas system for genome editing.},
journal = {Theriogenology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.theriogenology.2019.05.039},
pmid = {31208775},
issn = {1879-3231},
abstract = {In livestock industries, one sex is usually preferred because of the impact on the production (e.g. milk from cows, eggs from laying hens). Furthermore, in pig production, the male-specific boar taint is a big hurdle for consumer acceptance. Consequently, a shift in the ratio towards the desired sex would be a great benefit. The most widely applied method for pre-determination of the sex is fluorescence-activated sperm sorting, which relies on the different DNA content of the X- and Y-chromosomal sperm. However, the successful practical adaption of this method depends on its ease of use. At present, sperm sexing via fluorescence-activated cell sorting (FACS) has only reached commercial application in cattle. Nevertheless, sperm sexing technology still needs to be improved with respect to efficiency and reliability, to obtain high numbers of sexed sperm and less invasive sperm treatment to avoid damage. New genome editing technologies such as Zinc finger nucleases (ZFN), Transcription-activator like endonucleases (TALENs) and the CRISPR/Cas system have emerged and offer great potential to affect determination of the sex at the genome level. The sex-determining region on the Y chromosome (SRY) serves as a main genetic switch of male gender development. It was previously shown that a knockout of the SRY gene in mice and rabbits displayed suppressed testis development in the fetal gonadal ridges resulting in a female phenotype. These new technologies hold great opportunities to pre-determine sex in pigs. However, further investigations are needed to exploit their full potential for practical application.},
}

@article {pmid31169010,
year = {2019},
author = {, and Aicale, R and Tarantino, D and Maccauro, G and Peretti, GM and Maffulli, N},
title = {Genetics in orthopaedic practice.},
journal = {Journal of biological regulators and homeostatic agents},
volume = {33},
number = {2 Suppl. 1},
pages = {103-117},
pmid = {31169010},
issn = {0393-974X},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Profile ; Humans ; Musculoskeletal Diseases/*genetics ; Musculoskeletal System ; *Orthopedics ; Polymorphism, Single Nucleotide ; Tissue Engineering ; },
abstract = {DNA holds genetic information in the nucleus of eukaryotic cells; and has three different functions: replication, storage of hereditary information, and regulation of cell division. Most studies described the association of single nucleotide polymorphism (SNP) to common orthopaedics diseases and the susceptibility to develop musculoskeletal injuries. Several mutations are associated with osteoporosis, musculoskeletal ailments and other musculoskeletal deformity and conditions. Several strategies, including gene therapy and tissue engineering with mesenchymal stem cells (MSC), have been proposed to enhance healing of musculoskeletal tissues. Furthermore, a recent technique has revolutionized gene editing: clustered regulatory interspaced short palindromic repeat (CRISPR) technology is characterized by simplicity in target design, affordability, versatility, and high efficiency, but needs more studies to become the preferred platform for genome editing. Predictive genomics DNA profiling allows to understand which genetic advantage, if any, may be exploited, and why a given rehabilitation protocol can be more effective in some individual than others. In conclusion, a better understanding of the genetic influence on the function of the musculoskeletal system and healing of its ailments is needed to plan and develop patient specific management strategies.},
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Genetic Profile
Humans
Musculoskeletal Diseases/*genetics
Musculoskeletal System
*Orthopedics
Polymorphism, Single Nucleotide
Tissue Engineering

@article {pmid30737704,
year = {2019},
author = {Moravec, CE and Pelegri, FJ},
title = {An Accessible Protocol for the Generation of CRISPR-Cas9 Knockouts Using INDELs in Zebrafish.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1920},
number = {},
pages = {377-392},
doi = {10.1007/978-1-4939-9009-2_23},
pmid = {30737704},
issn = {1940-6029},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Computational Biology/methods ; Female ; *Gene Editing ; *Gene Knockout Techniques ; *Gene Targeting/methods ; *INDEL Mutation ; Male ; RNA, Guide/chemical synthesis/genetics ; Zebrafish/*genetics ; },
abstract = {The ability to create targeted mutations in specific genes, and therefore a loss-of-function condition, provides essential information about their endogenous functions during development and homeostasis. The discovery that CRISPR-Cas9 can target specific sequences according to base-pair complementarity and readily create knockouts in a desired gene has elevated the implementation of genetic analysis in numerous organisms. As CRISPR-Cas9 has become a powerful tool in a number of species, multiple methods for designing, creating, and screening editing efficiencies have been published, each of which has unique benefits. This chapter presents a cost-efficient, accessible protocol for creating knockout mutants in zebrafish using insertions/deletions (INDELS), from target site selection to mutant propagation, using basic laboratory supplies. The presented approach can be adapted to other systems, including any vertebrate species.},
}

Animals
Animals, Genetically Modified
*CRISPR-Cas Systems
Computational Biology/methods
Female
*Gene Editing
*Gene Knockout Techniques
*Gene Targeting/methods
*INDEL Mutation
Male
RNA, Guide/chemical synthesis/genetics
Zebrafish/*genetics

@article {pmid30633974,
year = {2019},
author = {Huang, H and Chai, C and Yang, S and Jiang, W and Gu, Y},
title = {Phage serine integrase-mediated genome engineering for efficient expression of chemical biosynthetic pathway in gas-fermenting Clostridium ljungdahlii.},
journal = {Metabolic engineering},
volume = {52},
number = {},
pages = {293-302},
doi = {10.1016/j.ymben.2019.01.005},
pmid = {30633974},
issn = {1096-7184},
mesh = {Attachment Sites, Microbiological/genetics ; Bacteriophages/*enzymology/*genetics ; Butyric Acid/metabolism ; CRISPR-Cas Systems ; Clostridium/*genetics/*metabolism ; DNA, Bacterial/genetics ; Fermentation ; Genome, Bacterial/genetics ; Integrases/*genetics/*metabolism ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; Plasmids/genetics ; Serine/metabolism ; },
abstract = {The real value of gas-fermenting clostridia, capable of using CO and CO2, resides in their potential of being developed into cell factories to produce various bulk chemicals and fuels. This process requires rapid chromosomal integration of heterologous chemical biosynthetic pathways, which is impeded by the absence of genetic tools competent for efficient genome engineering in these anaerobes. Here, we developed a phage serine integrase-mediated site-specific genome engineering technique in Clostridium ljungdahlii, one of the major acetogenic gas-fermenting microbes. Two heterologous phage attachment/integration (Att/Int) systems (from Clostridium difficile and Streptomyces) were introduced into C. ljungdahlii and proven to be highly active, achieving efficient chromosomal integration of a whole donor vector via single-crossover recombination. Based on this, we further realized markerless chromosomal integration of target DNA fragments through a "dual integrase cassette exchange" (DICE) strategy with the assistance of the CRISPR-Cas9 editing system. As a proof of concept, a butyric acid production pathway from Clostridium acetobutylicum was integrated into the C. ljungdahlii genome without the introduction of extra markers, enabling stable expression of the pathway genes. The resulting engineered strain produced 1.01 g/L of butyric acid within 3 days by fermenting synthesis gas (CO2/CO). More importantly, the engineered strain showed good genetic stability and maintained butyric acid production ability after continuous subculturing. The system developed in this study overcomes the deficiencies of currently available genetic tools in the chromosomal integration of large DNA fragments (rapid, markerless and stable) in C. ljungdahlii, and may be extended to other Clostridium species.},
}

Attachment Sites, Microbiological/genetics
Bacteriophages/*enzymology/*genetics
Butyric Acid/metabolism
CRISPR-Cas Systems
Clostridium/*genetics/*metabolism
DNA, Bacterial/genetics
Fermentation
Genome, Bacterial/genetics
Integrases/*genetics/*metabolism
Metabolic Engineering/*methods
Metabolic Networks and Pathways/genetics
Plasmids/genetics
Serine/metabolism

@article {pmid30513349,
year = {2019},
author = {Amann, T and Hansen, AH and Kol, S and Hansen, HG and Arnsdorf, J and Nallapareddy, S and Voldborg, B and Lee, GM and Andersen, MR and Kildegaard, HF},
title = {Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles.},
journal = {Metabolic engineering},
volume = {52},
number = {},
pages = {143-152},
doi = {10.1016/j.ymben.2018.11.014},
pmid = {30513349},
issn = {1096-7184},
mesh = {Animals ; Antigens, CD/biosynthesis/genetics ; CHO Cells/*metabolism ; CRISPR-Cas Systems ; Complement C1 Inhibitor Protein/*biosynthesis ; Cricetinae ; Cricetulus ; Glycosylation ; Humans ; Metabolic Engineering/*methods ; Recombinant Proteins/biosynthesis ; Sialyltransferases/biosynthesis/genetics ; alpha 1-Antitrypsin/*biosynthesis ; },
abstract = {Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.},
}

Animals
Antigens, CD/biosynthesis/genetics
CHO Cells/*metabolism
CRISPR-Cas Systems
Complement C1 Inhibitor Protein/*biosynthesis
Cricetinae
Cricetulus
Glycosylation
Humans
Metabolic Engineering/*methods
Recombinant Proteins/biosynthesis
Sialyltransferases/biosynthesis/genetics
alpha 1-Antitrypsin/*biosynthesis

@article {pmid30447330,
year = {2019},
author = {Kim, CL and Lee, GM},
title = {Improving recombinant bone morphogenetic protein-4 (BMP-4) production by autoregulatory feedback loop removal using BMP receptor-knockout CHO cell lines.},
journal = {Metabolic engineering},
volume = {52},
number = {},
pages = {57-67},
doi = {10.1016/j.ymben.2018.11.003},
pmid = {30447330},
issn = {1096-7184},
mesh = {Animals ; Antimetabolites/pharmacology ; Bone Morphogenetic Protein 4/*biosynthesis/*genetics ; Bone Morphogenetic Protein Receptors/antagonists & inhibitors/*genetics ; CHO Cells ; CRISPR-Cas Systems ; Cricetinae ; Cricetulus ; Feedback, Physiological ; Gene Expression Profiling ; Gene Knockout Techniques ; Homeostasis ; Methotrexate/pharmacology ; Pyrazoles/pharmacology ; Pyrimidines/pharmacology ; Recombinant Proteins ; },
abstract = {A Chinese hamster ovary (CHO) cell line producing recombinant human bone morphogenetic protein-4 (rhBMP-4) (CHO-BMP-4), which expresses essential components of BMP signal transduction, underwent autocrine BMP-4 signaling. RNA seq analysis on CHO host cells (DG44) treated with rhBMP-4 (20 µg/mL) suggested that rhBMP-4 induced signaling in CHO cells could be a critical factor in limiting rhBMP-4 production and should be removed to improve rhBMP-4 production in recombinant CHO (rCHO) cells. The inhibition of autocrine BMP signaling in CHO-BMP-4 cells by the addition of LDN-193189, a chemical inhibitor of BMP receptor type I, significantly increased the mRNA expression levels of rhBMP-4. To establish BMP signaling-free host cells, a BMP receptor, the BMPRIA or BMPRII gene in DG44 cells, was knocked out using CRISPR/Cas9 gene-editing technology. Using three different knockout (KO) host cell lines as well as a DG44 wild-type (wt) cell line, rCHO cell clones producing rhBMP-4 were generated by a stepwise selection with increasing methotrexate concentrations. KO-derived clones showed a significantly higher maximum rhBMP-4 concentration than wt-derived clones in both batch and fed-batch cultures. Unlike wt-derived clones, KO-derived cell clones were able to produce higher amounts of hBMP-4 transcripts and proteins in the stationary phase of growth and did not experience growth inhibition induced by rhBMP-4. The mean maximum rhBMP-4 concentration of KO host-derived clones was approximately 2.4-fold higher than that of wt-derived clones (P < 0.05). Taken together, the disruption of BMP signaling in CHO cells by knocking out the BMP receptor significantly improved rhBMP-4 production.},
}

Animals
Antimetabolites/pharmacology
Bone Morphogenetic Protein 4/*biosynthesis/*genetics
Bone Morphogenetic Protein Receptors/antagonists & inhibitors/*genetics
CHO Cells
CRISPR-Cas Systems
Cricetinae
Cricetulus
Feedback, Physiological
Gene Expression Profiling
Gene Knockout Techniques
Homeostasis
Methotrexate/pharmacology
Pyrazoles/pharmacology
Pyrimidines/pharmacology
Recombinant Proteins

@article {pmid30045945,
year = {2018},
author = {Stolte, B and Iniguez, AB and Dharia, NV and Robichaud, AL and Conway, AS and Morgan, AM and Alexe, G and Schauer, NJ and Liu, X and Bird, GH and Tsherniak, A and Vazquez, F and Buhrlage, SJ and Walensky, LD and Stegmaier, K},
title = {Genome-scale CRISPR-Cas9 screen identifies druggable dependencies in TP53 wild-type Ewing sarcoma.},
journal = {The Journal of experimental medicine},
volume = {215},
number = {8},
pages = {2137-2155},
pmid = {30045945},
issn = {1540-9538},
support = {T32 CA136432/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; R35 CA210030/CA/NCI NIH HHS/United States ; F30 CA221087/CA/NCI NIH HHS/United States ; T32 GM008313/GM/NIGMS NIH HHS/United States ; R35 CA197583/CA/NCI NIH HHS/United States ; R50 CA211399/CA/NCI NIH HHS/United States ; R01 CA211681/CA/NCI NIH HHS/United States ; },
mesh = {Aminopyridines/pharmacology ; Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Death/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival ; Dipeptides/pharmacology ; Drug Synergism ; Female ; *Genome, Human ; Humans ; Mice, Nude ; Mutation/genetics ; Neoplasm Proteins/metabolism ; Peptides, Cyclic/pharmacology ; Reproducibility of Results ; Sarcoma, Ewing/*genetics/pathology ; Thiophenes/pharmacology ; Transcription, Genetic/drug effects ; Tumor Suppressor Protein p53/*genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Ewing sarcoma is a pediatric cancer driven by EWS-ETS transcription factor fusion oncoproteins in an otherwise stable genomic background. The majority of tumors express wild-type TP53, and thus, therapies targeting the p53 pathway would benefit most patients. To discover targets specific for TP53 wild-type Ewing sarcoma, we used a genome-scale CRISPR-Cas9 screening approach and identified and validated MDM2, MDM4, USP7, and PPM1D as druggable dependencies. The stapled peptide inhibitor of MDM2 and MDM4, ATSP-7041, showed anti-tumor efficacy in vitro and in multiple mouse models. The USP7 inhibitor, P5091, and the Wip1/PPM1D inhibitor, GSK2830371, decreased the viability of Ewing sarcoma cells. The combination of ATSP-7041 with P5091, GSK2830371, and chemotherapeutic agents showed synergistic action on the p53 pathway. The effects of the inhibitors, including the specific USP7 inhibitor XL-188, were rescued by concurrent TP53 knockout, highlighting the essentiality of intact p53 for the observed cytotoxic activities.},
}

Aminopyridines/pharmacology
Animals
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Cell Death/drug effects
Cell Line, Tumor
Cell Proliferation/drug effects
Cell Survival
Dipeptides/pharmacology
Drug Synergism
Female
*Genome, Human
Humans
Mice, Nude
Mutation/genetics
Neoplasm Proteins/metabolism
Peptides, Cyclic/pharmacology
Reproducibility of Results
Sarcoma, Ewing/*genetics/pathology
Thiophenes/pharmacology
Transcription, Genetic/drug effects
Tumor Suppressor Protein p53/*genetics
Xenograft Model Antitumor Assays

@article {pmid30031067,
year = {2019},
author = {Nauroy, P and Guiraud, A and Chlasta, J and Malbouyres, M and Gillet, B and Hughes, S and Lambert, E and Ruggiero, F},
title = {Gene profile of zebrafish fin regeneration offers clues to kinetics, organization and biomechanics of basement membrane.},
journal = {Matrix biology : journal of the International Society for Matrix Biology},
volume = {75-76},
number = {},
pages = {82-101},
doi = {10.1016/j.matbio.2018.07.005},
pmid = {30031067},
issn = {1569-1802},
mesh = {Animal Fins/*growth & development/ultrastructure ; Animals ; Basement Membrane/*growth & development/ultrastructure ; CRISPR-Cas Systems ; Collagen/*genetics ; Extracellular Matrix/genetics/ultrastructure ; Extracellular Matrix Proteins/genetics ; Gene Expression Regulation, Developmental/genetics ; Kinetics ; Regeneration/*genetics ; Transcriptome/genetics ; Wound Healing/genetics ; Zebrafish/genetics/growth & development ; Zebrafish Proteins/*genetics ; },
abstract = {How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.},
}

Animal Fins/*growth & development/ultrastructure
Animals
Basement Membrane/*growth & development/ultrastructure
CRISPR-Cas Systems
Collagen/*genetics
Extracellular Matrix/genetics/ultrastructure
Extracellular Matrix Proteins/genetics
Gene Expression Regulation, Developmental/genetics
Kinetics
Regeneration/*genetics
Transcriptome/genetics
Wound Healing/genetics
Zebrafish/genetics/growth & development
Zebrafish Proteins/*genetics

@article {pmid29490262,
year = {2018},
author = {Finn, JD and Smith, AR and Patel, MC and Shaw, L and Youniss, MR and van Heteren, J and Dirstine, T and Ciullo, C and Lescarbeau, R and Seitzer, J and Shah, RR and Shah, A and Ling, D and Growe, J and Pink, M and Rohde, E and Wood, KM and Salomon, WE and Harrington, WF and Dombrowski, C and Strapps, WR and Chang, Y and Morrissey, DV},
title = {A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing.},
journal = {Cell reports},
volume = {22},
number = {9},
pages = {2227-2235},
doi = {10.1016/j.celrep.2018.02.014},
pmid = {29490262},
issn = {2211-1247},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Gene Transfer Techniques ; Lipids/*chemistry ; Liver/metabolism ; Mice ; Nanoparticles/*administration & dosage/*chemistry ; RNA, Guide/chemistry/genetics ; Rats ; },
abstract = {The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.},
}

Animals
Base Sequence
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
*Gene Editing
*Gene Transfer Techniques
Lipids/*chemistry
Liver/metabolism
Mice
Nanoparticles/*administration & dosage/*chemistry
RNA, Guide/chemistry/genetics
Rats

@article {pmid29440296,
year = {2018},
author = {Brunen, D and de Vries, RC and Lieftink, C and Beijersbergen, RL and Bernards, R},
title = {PIM Kinases Are a Potential Prognostic Biomarker and Therapeutic Target in Neuroblastoma.},
journal = {Molecular cancer therapeutics},
volume = {17},
number = {4},
pages = {849-857},
doi = {10.1158/1535-7163.MCT-17-0868},
pmid = {29440296},
issn = {1538-8514},
mesh = {Animals ; Apoptosis ; Biomarkers, Tumor ; Biphenyl Compounds/pharmacology ; CRISPR-Cas Systems ; Cell Proliferation ; Female ; Follow-Up Studies ; Humans ; Infant ; Male ; Mice ; Mice, Nude ; Neoplasm Recurrence, Local/drug therapy/metabolism/*pathology ; Neuroblastoma/drug therapy/metabolism/*pathology ; Neurofibromin 1/genetics/*metabolism ; Prognosis ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors/genetics/*metabolism ; Survival Rate ; Thiazolidines/pharmacology ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; },
abstract = {The majority of high-risk neuroblastoma patients are refractory to, or relapse on, current treatment regimens, resulting in 5-year survival rates of less than 50%. This emphasizes the urgent need to identify novel therapeutic targets. Here, we report that high PIM kinase expression is correlated with poor overall survival. Treatment of neuroblastoma cell lines with the pan-PIM inhibitors AZD1208 or PIM-447 suppressed proliferation through inhibition of mTOR signaling. In a panel of neuroblastoma cell lines, we observed a marked binary response to PIM inhibition, suggesting that specific genetic lesions control responses to PIM inhibition. Using a genome-wide CRISPR-Cas9 genetic screen, we identified NF1 loss as the major resistance mechanism to PIM kinase inhibitors. Treatment with AZD1208 impaired the growth of NF1 wild-type xenografts, while NF1 knockout cells were insensitive. Thus, our data indicate that PIM inhibition may be a novel targeted therapy in NF1 wild-type neuroblastoma. Mol Cancer Ther; 17(4); 849-57. ©2018 AACR.},
}

Animals
Apoptosis
Biomarkers, Tumor
Biphenyl Compounds/pharmacology
CRISPR-Cas Systems
Cell Proliferation
Female
Follow-Up Studies
Humans
Infant
Male
Mice
Mice, Nude
Neoplasm Recurrence, Local/drug therapy/metabolism/*pathology
Neuroblastoma/drug therapy/metabolism/*pathology
Neurofibromin 1/genetics/*metabolism
Prognosis
Protein Kinase Inhibitors/pharmacology
Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors/genetics/*metabolism
Survival Rate
Thiazolidines/pharmacology
Tumor Cells, Cultured
Xenograft Model Antitumor Assays

@article {pmid31205227,
year = {2019},
author = {Anderson, KL and Snyder, KM and Ito, D and Lins, DC and Mills, LJ and Weiskopf, K and Ring, NG and Ring, AM and Shimizu, Y and Mescher, MF and Weissman, IL and Modiano, JF},
title = {Evolutionarily conserved resistance to phagocytosis observed in melanoma cells is insensitive to upregulation of pro-phagocytic signals and to CD47 blockade.},
journal = {Melanoma research},
volume = {},
number = {},
pages = {},
doi = {10.1097/CMR.0000000000000629},
pmid = {31205227},
issn = {1473-5636},
abstract = {Therapeutic activation of macrophage phagocytosis has the ability to restrain tumour growth through phagocytic clearance of tumour cells and activation of the adaptive immune response. Our objective for this study was to evaluate the effects of modulating pro- and anti-phagocytic pathways in malignant melanoma. In order to identify evolutionarily conserved mechanisms of resistance that may be important for melanoma cell survival, we utilized a multi-species approach and examined the phagocytosis of human, mouse, and dog melanoma cells. We observed that melanoma cells from all three species displayed unexpected resistance to phagocytosis that could not be fully mitigated by blockade of the 'don't eat me' signal CD47 or by chemotherapeutic enhancement of known 'eat me' signals. Additionally, CD47 blockade failed to promote anti-melanoma immune responses or tumour regression in vivo. This melanoma resistance to phagocytosis was not mediated by soluble factors, and it was unaffected by siRNA-mediated knockdown of 47 prospective 'don't eat me' signals or by CRISPR-Cas-mediated CD47 knockout. Unexpectedly, CD47 knockout also did not enhance phagocytosis of lymphoma cells, but it eliminated the pro-phagocytic effect of CD47 blockade, suggesting that the pro-phagocytic effects of CD47 blockade are due in part to Fc receptor engagement. From this study, we conclude that melanoma cells possess an evolutionarily conserved resistance to macrophage phagocytosis. Further investigation will be needed to overcome the mechanisms that mediate melanoma cell resistance to innate immunity.This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.},
}

@article {pmid31203918,
year = {2019},
author = {Olivaes, J and Bonamino, MH and Markoski, MM},
title = {CRISPR/Cas 9 system for the treatment of dilated cardiomyopathy: A hypothesis related to function of a MAP kinase.},
journal = {Medical hypotheses},
volume = {128},
number = {},
pages = {91-93},
doi = {10.1016/j.mehy.2019.05.013},
pmid = {31203918},
issn = {1532-2777},
abstract = {Dilated cardiomyopathy (DCM) is a disease with high incidence and mortality rates. Its therapies have one primary goal, which is to minimize symptoms and it has only one effective approach to healing, the heart transplantation. As it is widely associated with genetic causes, the use of gene therapies, such as the CRISPR/Cas9 system, is a promising alternative to treat DCM. For this purpose, it is necessary to analyze possible target genes for this approach and what would be the implications of their use. Here, we hypothesized that cardiac troponin I type 3 interacting kinase (TNI3K), involved with superoxide production in DCM patients, besides other factors, could be a good target for the use of gene editing.},
}

@article {pmid31186565,
year = {2019},
author = {Cyranoski, D},
title = {Russian biologist plans more CRISPR-edited babies.},
journal = {Nature},
volume = {570},
number = {7760},
pages = {145-146},
doi = {10.1038/d41586-019-01770-x},
pmid = {31186565},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Homozygote ; Humans ; Receptors, CCR5 ; Russia ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Homozygote
Humans
Receptors, CCR5
Russia

@article {pmid30778165,
year = {2019},
author = {Ruf, S and Forner, J and Hasse, C and Kroop, X and Seeger, S and Schollbach, L and Schadach, A and Bock, R},
title = {High-efficiency generation of fertile transplastomic Arabidopsis plants.},
journal = {Nature plants},
volume = {5},
number = {3},
pages = {282-289},
doi = {10.1038/s41477-019-0359-2},
pmid = {30778165},
issn = {2055-0278},
mesh = {Arabidopsis/cytology/genetics/*physiology ; Bacterial Proteins/genetics ; Biolistics/methods ; *CRISPR-Cas Systems ; Cell Culture Techniques ; Chloroplasts/genetics ; Gene Editing ; Gene Knockout Techniques ; Genetic Vectors ; Luminescent Proteins/genetics ; Plant Roots/cytology/genetics ; *Plants, Genetically Modified ; Plastids/*genetics ; Transformation, Genetic ; },
abstract = {The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-generated knockout allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species.},
}

Arabidopsis/cytology/genetics/*physiology
Bacterial Proteins/genetics
Biolistics/methods
*CRISPR-Cas Systems
Cell Culture Techniques
Chloroplasts/genetics
Gene Editing
Gene Knockout Techniques
Genetic Vectors
Luminescent Proteins/genetics
Plant Roots/cytology/genetics
*Plants, Genetically Modified
Plastids/*genetics
Transformation, Genetic

@article {pmid30649572,
year = {2019},
author = {Danilo, B and Perrot, L and Mara, K and Botton, E and Nogué, F and Mazier, M},
title = {Efficient and transgene-free gene targeting using Agrobacterium-mediated delivery of the CRISPR/Cas9 system in tomato.},
journal = {Plant cell reports},
volume = {38},
number = {4},
pages = {459-462},
doi = {10.1007/s00299-019-02373-6},
pmid = {30649572},
issn = {1432-203X},
support = {ANR11-BTBR-0001-GENIUS//Agence Nationale de la Recherche/International ; },
mesh = {Agrobacterium/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Targeting/methods ; Lycopersicon esculentum/*genetics ; Transgenes/*genetics ; },
}

Agrobacterium/*genetics
CRISPR-Cas Systems/*genetics
Gene Targeting/methods
Lycopersicon esculentum/*genetics
Transgenes/*genetics

@article {pmid30610647,
year = {2019},
author = {Liang, Z and Chen, K and Gao, C},
title = {Biolistic Delivery of CRISPR/Cas9 with Ribonucleoprotein Complex in Wheat.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {327-335},
doi = {10.1007/978-1-4939-8991-1_24},
pmid = {30610647},
issn = {1940-6029},
mesh = {Biolistics/*methods ; CRISPR-Cas Systems/genetics/*physiology ; Gene Editing/methods ; Seedlings/genetics ; Triticum/*genetics ; },
abstract = {The great advances in exploiting the CRISPR/Cas9 system are paving the way for targeted genome engineering in plants. Genome editing by direct delivery of CRISPR/Cas9 ribonucleoprotein complexes (RNPs) into plant cells reduces off-target mutations and avoids the integration of foreign DNA fragments, thus providing an efficient and accurate method for precision crop breeding. Here we describe an RNP-based genome editing protocol for wheat. The protocol covers the in vitro transcription of sgRNA, purification of Cas9 protein, biolistic delivery of CRISPR/Cas9 RNPs, and tissue culture procedures for regenerating testable seedlings.},
}

Biolistics/*methods
CRISPR-Cas Systems/genetics/*physiology
Gene Editing/methods
Seedlings/genetics
Triticum/*genetics

@article {pmid30610646,
year = {2019},
author = {Mahas, A and Ali, Z and Tashkandi, M and Mahfouz, MM},
title = {Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {311-326},
doi = {10.1007/978-1-4939-8991-1_23},
pmid = {30610646},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genetic Engineering/methods ; Genome, Plant/*genetics ; Plants, Genetically Modified/genetics ; RNA Viruses/genetics ; },
abstract = {Targeted modification of plant genomes is a powerful strategy for investigating and engineering cellular systems, paving the way for the discovery and development of important, novel agricultural traits. Cas9, an RNA-guided DNA endonuclease from the type II adaptive immune CRISPR system of the prokaryote Streptococcus pyogenes, has gained widespread popularity as a genome-editing tool for use in a wide array of cells and organisms, including model and crop plants. Effective genome engineering requires the delivery of the Cas9 protein and guide RNAs into target cells. However, in planta genome modification faces many hurdles, including the difficulty in efficiently delivering genome engineering reagents to the desired tissues. We recently developed a Tobacco rattle virus (TRV)-mediated genome engineering system for Nicotiana benthamiana. Using this platform, genome engineering reagents can be delivered into all plant parts in a simple, efficient manner, facilitating the recovery of progeny plants with the desired genomic modifications, thus bypassing the need for transformation and tissue culture. This platform expands the utility of the CRISPR/Cas9 system for in planta, targeted genome modification. Here, we provide a detailed protocol explaining the methodologies used to develop and implement TRV-mediated genome engineering in N. benthamiana. The protocol described here can be extended to any other plant species susceptible to systemic infection by TRV. However, this approach is not limited to vectors derived from TRV, as other RNA viruses could be used to develop similar delivery platforms.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Genetic Engineering/methods
Genome, Plant/*genetics
Plants, Genetically Modified/genetics
RNA Viruses/genetics

@article {pmid30610645,
year = {2019},
author = {Shimatani, Z and Ariizumi, T and Fujikura, U and Kondo, A and Ezura, H and Nishida, K},
title = {Targeted Base Editing with CRISPR-Deaminase in Tomato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {297-307},
doi = {10.1007/978-1-4939-8991-1_22},
pmid = {30610645},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytidine Deaminase/genetics ; Gene Editing/*methods ; Lycopersicon esculentum/*genetics ; },
abstract = {The Target-AID system, consisting of a complex of cytidine deaminase and deficient CRISPR/Cas9, enables highly specific genomic nucleotide substitutions without the need for template DNA. The Cas9-fused cytidine deaminase is guided by sgRNAs and catalyzes the conversion of cytosine to uracil. The resulting U-G DNA mismatches trigger nucleotide substitutions (C to T or G to A) through DNA replication and repair pathways. Target-AID also retains the benefits of conventional CRISPR/Cas9 including robustness in various organisms, high targeting efficiency, and multiplex simultaneous gene editing. Our research group recently developed plant-optimized Target-AID system and demonstrated targeted base editing in tomato and rice. In this chapter, we introduce methods for Target-AID application in tomato.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Cytidine Deaminase/genetics
Gene Editing/*methods
Lycopersicon esculentum/*genetics

@article {pmid30610644,
year = {2019},
author = {Li, J and Meng, X and Li, J and Gao, C},
title = {Gene Replacement by Intron Targeting with CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {285-296},
doi = {10.1007/978-1-4939-8991-1_21},
pmid = {30610644},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; Gene Editing/*methods ; Introns/genetics ; Recombinational DNA Repair/*genetics ; },
abstract = {The CRISPR-Cas9 system has become the most widely adopted genome editing platform and is used in an expanding number of organisms, mainly by creating targeted knockouts through non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). It would also be highly desirable to be able to use homology-directed repair (HDR) to perform precise gene editing, for example, by replacing a small section of DNA to substitute one amino acid for another in a given gene product. However, this remains a serious challenge in plants. Here, we describe a recently developed intron-mediated site-specific gene replacement method acting through the NHEJ pathway in which Cas9 simultaneously introduces DSBs in adjacent introns and the donor template. This approach is of general use for replacing targeted gene fragments at specific genomic sites in plants.},
}

CRISPR-Cas Systems/*genetics
DNA Breaks, Double-Stranded
Gene Editing/*methods
Introns/genetics
Recombinational DNA Repair/*genetics

@article {pmid30610642,
year = {2019},
author = {Yin, X and Anand, A and Quick, P and Bandyopadhyay, A},
title = {Editing a Stomatal Developmental Gene in Rice with CRISPR/Cpf1.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {257-268},
doi = {10.1007/978-1-4939-8991-1_19},
pmid = {30610642},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Oryza/*genetics ; },
abstract = {CRISPR has arguably been the fastest growing genome editing tool so far. CRISPR/Cas9 (Cas9) has been proved to be efficient and precise in genome editing. However Cas9 has certain limitations. CRISPR/Cpf1 (Cpf1) has been discovered as an alternate approach that can overcome some of those limitations. Cpf1 allows targeting in AT-rich region, creating a staggered cleavage, and cutting at the distal end to the PAM (Protospacer Adjacent Motif) regions. We have successfully tested the efficiency of Cpf1 system in rice using OsEPFL9 which is a developmental gene known to regulate the stomatal density in leaf. Regulation of stomatal density and patterning is an important factor in regulating plant physiology, especially in improving the plant water use efficiency. We targeted the Exon1 of OsEPFL9 and the knockout lines were studied for several generations for establishment of stabilized editing, as well as transmission and segregation of edits through generations. The usage of Cpf1 as a genome editing tool to manipulate stomatal patterning may further help us gain more insight of the physiology of rice in stress conditions.},
}

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

@article {pmid30610641,
year = {2019},
author = {Zhang, Y and Zhang, Y and Qi, Y},
title = {Plant Gene Knockout and Knockdown by CRISPR-Cpf1 (Cas12a) Systems.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {245-256},
doi = {10.1007/978-1-4939-8991-1_18},
pmid = {30610641},
issn = {1940-6029},
mesh = {Arabidopsis/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Gene Knockout Techniques/*methods ; Oryza/genetics ; },
abstract = {CRISPR-Cpf1 (Cas12a) is a class II type V endonuclease, which has been used as a genome editing tool in different biological systems. Here we describe a fast, efficient, and user-friendly system for CRISPR-Cpf1 expression vector assembly. In this system, the Pol II promoter is used to drive the expression of both Cpf1 and its crRNA, with the crRNA flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozyme RNAs for precise crRNA processing. All the components of this system can be modified depending on plant species and experimental goals. Using this system, nearly 100% editing efficiency and 90% gene expression decrease were achieved in rice and Arabidopsis, respectively.},
}

Arabidopsis/genetics
CRISPR-Cas Systems/*genetics
Gene Editing
Gene Knockout Techniques/*methods
Oryza/genetics

@article {pmid30610640,
year = {2019},
author = {Jia, H and Zou, X and Orbovic, V and Wang, N},
title = {Genome Editing in Citrus Tree with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {235-241},
doi = {10.1007/978-1-4939-8991-1_17},
pmid = {30610640},
issn = {1940-6029},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems/*genetics ; Citrus/*genetics ; Gene Editing/*methods ; Plant Proteins/*genetics ; },
abstract = {CRISPR/Cas9 has been widely employed to edit genome in most of the organisms, including animal, plant, fungus, and microbe. Here we describe the modification of citrus gene CsLOB1 in transgenic citrus by Cas9/sgRNA, a two-component system derived from CRISPR-Cas9. Transgenic citrus plants can be created by Agrobacterium-mediated epicotyl transformation.},
}

Agrobacterium/genetics
CRISPR-Cas Systems/*genetics
Citrus/*genetics
Gene Editing/*methods
Plant Proteins/*genetics

@article {pmid30610639,
year = {2019},
author = {Liu, J and Gunapati, S and Mihelich, NT and Stec, AO and Michno, JM and Stupar, RM},
title = {Genome Editing in Soybean with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {217-234},
doi = {10.1007/978-1-4939-8991-1_16},
pmid = {30610639},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Electrophoresis, Polyacrylamide Gel ; Gene Editing/*methods ; Plant Roots/genetics ; Plants, Genetically Modified/*genetics ; Soybeans/*genetics ; Transformation, Genetic/genetics ; },
abstract = {CRISPR/Cas9 mediated genome editing technology has experienced rapid advances in recent years and has been applied to a wide variety of plant species, including soybean. Several platforms have been developed for designing and cloning of single CRISPR targets or multiple targets in a single destination vector. This chapter provides an updated working protocol for applying CRISPR/Cas9 technology to target a single gene or multiple genes simultaneously in soybean. We describe two platforms for cloning single CRISPR targets and multiplexing targets, respectively, and reagent delivery methodologies. The protocols address crucial limiting steps that can limit CRISPR editing in soybean hairy roots, composite plants, and tissue culture-based regenerated whole plants. To date, transgenic soybean plants with mutagenesis in up to three target genes have been obtained with this procedure.},
}

CRISPR-Cas Systems/*genetics
Electrophoresis, Polyacrylamide Gel
Gene Editing/*methods
Plant Roots/genetics
Plants, Genetically Modified/*genetics
Soybeans/*genetics
Transformation, Genetic/genetics

@article {pmid30610638,
year = {2019},
author = {Klimek-Chodacka, M and Oleszkiewicz, T and Baranski, R},
title = {Visual Assay for Gene Editing Using a CRISPR/Cas9 System in Carrot Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {203-215},
doi = {10.1007/978-1-4939-8991-1_15},
pmid = {30610638},
issn = {1940-6029},
mesh = {Anthocyanins/metabolism ; Bony Callus/metabolism ; CRISPR-Cas Systems/*genetics ; Daucus carota/*genetics ; Gene Editing/*methods ; RNA, Guide/genetics ; },
abstract = {The development of the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) system has advanced genome editing and has become widely adopted for this purpose in many species. Its efficient use requires the method adjustment and optimization. Here, we show the use of a model carrot callus system for demonstrating gene editing via CRISPR/Cas9 targeted mutagenesis. The system relies on the utilization of carrot tissue accumulating anthocyanin pigments responsible for a deep purple cell color and generation of knockout mutations in the flavanone-3-hydroxylase (F3H) gene in the anthocyanin biosynthesis pathway. F3H mutant cells targeted by Cas9/gRNA complexes are not able to synthesize anthocyanins and remain white, easily visually distinguished from purple wild-type cells. Mutations are either small indels or larger chromosomal deletions that can be identified by restriction fragment analysis and sequencing. This feasible system can also be applied for validating efficiency of CRISPR/Cas9 vectors.},
}

Anthocyanins/metabolism
Bony Callus/metabolism
CRISPR-Cas Systems/*genetics
Daucus carota/*genetics
Gene Editing/*methods
RNA, Guide/genetics

@article {pmid30610637,
year = {2019},
author = {Nadakuduti, SS and Starker, CG and Voytas, DF and Buell, CR and Douches, DS},
title = {Genome Editing in Potato with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {183-201},
doi = {10.1007/978-1-4939-8991-1_14},
pmid = {30610637},
issn = {1940-6029},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; RNA, Guide/genetics ; Solanum tuberosum/*genetics ; Tissue Culture Techniques ; Transformation, Genetic/genetics ; },
abstract = {Cultivated potato, Solanum tuberosum Group Tuberosum L. (2n = 4x = 48) is a heterozygous tetraploid crop that is clonally propagated, thereby resulting in identical genotypes. Due to the lack of sexual reproduction and its concomitant segregation of alleles, genetic engineering is an efficient way of introducing crop improvement traits in potato. In recent years, genome-editing via the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system for targeted genome modifications has emerged as the most powerful method due to the ease in designing and construction of gene-specific single guide RNA (sgRNA) vectors. These sgRNA vectors are easily reprogrammable to direct Streptococcus pyogenes Cas9 (SpCas9) to generate double stranded breaks (DSBs) in the target genomes that are then repaired by the cell via the error-prone non-homologous end-joining (NHEJ) pathway or by precise homologous recombination (HR) pathway. CRISPR/Cas9 technology has been successfully implemented in potato for targeted mutagenesis to generate knockout mutations (by means of NHEJ) as well as gene targeting to edit an endogenous gene (by HR). In this chapter, we describe procedures for designing sgRNAs, protocols to clone sgRNAs for CRISPR/Cas9 constructs to generate knockouts, design of donor repair templates and use geminivirus replicons (GVRs) to facilitate gene-editing by HR in potato. We also describe tissue culture procedures in potato for Agrobacterium-mediated transformation to generate gene-edited events along with their molecular characterization.},
}

Agrobacterium/genetics
CRISPR-Cas Systems/*genetics
Gene Editing/*methods
RNA, Guide/genetics
Solanum tuberosum/*genetics
Tissue Culture Techniques
Transformation, Genetic/genetics

@article {pmid30610636,
year = {2019},
author = {Reem, NT and Van Eck, J},
title = {Application of CRISPR/Cas9-Mediated Gene Editing in Tomato.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {171-182},
doi = {10.1007/978-1-4939-8991-1_13},
pmid = {30610636},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics/*physiology ; Gene Editing/*methods ; Genome, Plant/*genetics ; Lycopersicon esculentum/*genetics ; RNA, Guide/genetics ; },
abstract = {CRISPR-/Cas9-mediated gene editing has been demonstrated in a number of food crops including tomato. Tomato (Solanum lycopersicum) is both an important food crop and a model plant species that has been used extensively for studying gene function, especially as it relates to fruit biology. This duality in purpose combined with readily available resources (mutant populations, genome sequences, transformation methodology) makes tomato an ideal candidate for gene editing. The CRISPR/Cas9 system routinely used in our laboratory has been applied to various tomato genotypes and the wild species, Solanum pimpinellifolium. The vector system is based on Golden Gate cloning techniques. Cassettes that contain the neomycin phosphotransferase II (NPTII) selectable marker gene that confers resistance to kanamycin, a human codon-optimized Cas9 driven by the CaMV 35S promoter, and guide RNA (gRNA) under control of the Arabidopsis U6 polymerase promoter are assembled into a T-DNA vector. Generally, we design CRISPR/Cas9 constructs that contain two gRNAs per gene target. However, we have been successful with inclusion of up to eight gRNAs to simultaneously target multiple genes and regions. Introduction of CRISPR-/Cas9-designed constructs into tomato is accomplished by transformation methodology based on Agrobacterium tumefaciens infection of young cotyledon sections and selection on kanamycin-containing medium based on the presence of the NPTII gene. The approaches for the development of CRISPR/Cas9 constructs and genotypic analyses (PCR-based amplicon sequencing and T7 endonuclease) are detailed in this chapter.},
}

CRISPR-Cas Systems/genetics/*physiology
Gene Editing/*methods
Genome, Plant/*genetics
Lycopersicon esculentum/*genetics
RNA, Guide/genetics

@article {pmid30610635,
year = {2019},
author = {Lawrenson, T and Hundleby, P and Harwood, W},
title = {Creating Targeted Gene Knockouts in Brassica oleracea Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {155-170},
doi = {10.1007/978-1-4939-8991-1_12},
pmid = {30610635},
issn = {1940-6029},
support = {BB/N019466/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P013511/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Brassica/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing ; Mutagenesis ; Plants, Genetically Modified/*genetics ; },
abstract = {While public and political views on genetic modification (inserting "foreign" genes to elicit new traits) have resulted in limited exploitation of the technology in some parts of the world, the new era of genome editing (to edit existing genes to gain new traits/genetic variation) has the potential to change the biotech landscape. Genome editing offers a faster and simpler approach to gene knockout in both single and multiple genetic locations, within a single or small number of generations, in a way that has not been possible through alternative breeding methods. Here we describe an Agrobacterium-mediated delivery approach to deliver Cas9 and dual sgRNAs into 4-day-old cotyledonary petioles of Brassica oleracea. Mutations are detected in approximately 10% of primary transgenic plants and go on in subsequent T1 and T2 generations to segregate away from the T-DNA. This enables the recovery of non-transgenic, genome-edited plants carrying a variety of mutations at the target locus.},
}

Brassica/*genetics
CRISPR-Cas Systems/*genetics
Gene Editing
Mutagenesis
Plants, Genetically Modified/*genetics

@article {pmid30610634,
year = {2019},
author = {Yu, H and Zhao, Y},
title = {Fluorescence Marker-Assisted Isolation of Cas9-Free and CRISPR-Edited Arabidopsis Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {147-154},
doi = {10.1007/978-1-4939-8991-1_11},
pmid = {30610634},
issn = {1940-6029},
support = {R01 GM114660/GM/NIGMS NIH HHS/United States ; },
mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Luminescent Proteins/genetics ; Plants, Genetically Modified/genetics ; RNA Editing/genetics ; RNA, Guide/*genetics ; },
abstract = {CRISPR/Cas9 gene editing technology has successfully introduced modifications at target DNA sequences in many plant species including Arabidopsis. After the target gene is edited, the CRISPR/Cas9 construct needs to be removed to ensure genetic stability and to gain any regulatory approval for commercial applications. However, removal of the transgenes by genetic segregation, backcross, and genotyping is very laborious and time-consuming. The methods we report here allow fast and effective isolation of transgene-free T2 Arabidopsis plants with the desired modifications at the target genes. We express a fluorescence protein mCherry under the control of a seed-specific promoter At2S3 and placed the cassette into the CRISPR/Cas9 vector. Therefore, we can use mCherry as a proxy for the presence of Cas9, and we are able to visually isolate the Cas9-free Arabidopsis plants with heritable mutations at the T2 generation. We targeted two sites in the ABP1 gene to demonstrate the effectiveness of our approach.},
}

Arabidopsis/*genetics
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/methods
Luminescent Proteins/genetics
Plants, Genetically Modified/genetics
RNA Editing/genetics
RNA, Guide/*genetics

@article {pmid30610632,
year = {2019},
author = {Zhou, J and Zhong, Z and Chen, H and Li, Q and Zheng, X and Qi, Y and Zhang, Y},
title = {Knocking Out MicroRNA Genes in Rice with CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {109-119},
doi = {10.1007/978-1-4939-8991-1_9},
pmid = {30610632},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Genome, Plant/*genetics ; MicroRNAs/*genetics ; Oryza/*genetics ; },
abstract = {BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs that play important roles in plant development and stress responses. Loss-of-function analysis of miRNA genes has been traditionally challenging due to lack of appropriate knockout tools. In this chapter, we describe a method of using CRISPR-Cas9 for knocking out microRNA genes in rice by Agrobacterium-mediated transformation. We also demonstrate single-strand conformation polymorphism (SSCP) as an effective genotyping method for screening CRISPR-Cas9-induced mutations.},
}

CRISPR-Cas Systems/genetics/physiology
Genome, Plant/*genetics
MicroRNAs/*genetics
Oryza/*genetics

@article {pmid30610631,
year = {2019},
author = {Gu, W and Zhang, D and Qi, Y and Yuan, Z},
title = {Generating Photoperiod-Sensitive Genic Male Sterile Rice Lines with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {97-107},
doi = {10.1007/978-1-4939-8991-1_8},
pmid = {30610631},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Oryza/*genetics/*physiology ; *Photoperiod ; Plant Breeding ; Plant Infertility/genetics/*physiology ; },
abstract = {Obtaining photoperiod-sensitive genic male sterility (PGMS) lines is one of the most important steps in two-line hybrid rice breeding. Traditionally, such lines were screened and developed with a classic rice breeding system under both long-day and short-day conditions. The isolation and backcross process used for this could easily last for more than 3 years with a very low success rate. Here, we describe a straightforward method for generating csa-based PGMS lines by using the CRISPR-Cas9 technology in rice.},
}

CRISPR-Cas Systems/*genetics
Oryza/*genetics/*physiology
*Photoperiod
Plant Breeding
Plant Infertility/genetics/*physiology

@article {pmid30610630,
year = {2019},
author = {Malzahn, A and Zhang, Y and Qi, Y},
title = {CRISPR-Act2.0: An Improved Multiplexed System for Plant Transcriptional Activation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {83-93},
doi = {10.1007/978-1-4939-8991-1_7},
pmid = {30610630},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods ; Gene Expression Regulation, Plant/genetics ; Plant Immunity/genetics/physiology ; Transcriptional Activation/genetics/physiology ; },
abstract = {CRISPR systems have greatly promoted research in genome editing and transcriptional regulation. CRISPR-based transcriptional repression and activation systems will be valuable for applications in engineering plant immunity, boosting metabolic production, and enhancing our knowledge of gene regulatory networks. Multiplexing of CRISPR allows multiple genes to be targeted without significant additional effort. Here, we describe our CRISPR-Act2.0 system which is an improved multiplexing transcriptional activation system in plants.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/*methods
Gene Expression Regulation, Plant/genetics
Plant Immunity/genetics/physiology
Transcriptional Activation/genetics/physiology

@article {pmid30610629,
year = {2019},
author = {Tang, X and Zhong, Z and Ren, Q and Liu, B and Zhang, Y},
title = {A Single Transcript CRISPR-Cas9 System for Multiplex Genome Editing in Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {75-82},
doi = {10.1007/978-1-4939-8991-1_6},
pmid = {30610629},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Plants/*genetics ; Promoter Regions, Genetic/genetics ; RNA Polymerase II/genetics ; RNA, Guide/genetics ; },
abstract = {The CRISPR-Cas9 system has been widely adopted in genome editing. By changing the 20 bp guide sequence, it can easily edit any sequence adjacent to a protospacer adjacent motif (PAM) in a genome. Multiplex genome editing could be accomplished with simultaneous expression of multiple single-guide RNAs (sgRNA). Given that sgRNAs are expressed by Pol III promoters, multiplex genome editing is conventionally done by assembly of multiple complete sgRNA expression cassettes together, which can be a challenge in vector construction. Here, we described a multiplex genome editing system based on a single transcript unit CRISPR-Cas9 (STU CRISPR-Cas9) expression system driven by a single Pol II promoter. It represents a novel approach for multiplex genome editing.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Genome, Plant/*genetics
Plants/*genetics
Promoter Regions, Genetic/genetics
RNA Polymerase II/genetics
RNA, Guide/genetics

@article {pmid30610628,
year = {2019},
author = {Xie, K and Yang, Y},
title = {A Multiplexed CRISPR/Cas9 Editing System Based on the Endogenous tRNA Processing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {63-73},
doi = {10.1007/978-1-4939-8991-1_5},
pmid = {30610628},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; RNA, Guide/genetics ; RNA, Transfer/*genetics ; },
abstract = {The CRISPR-Cas9 system has become a powerful and popular tool for genome editing due to its efficiency and simplicity. Multiplex genome editing is an important feature of the CRISPR-Cas9 system and requires simultaneous expression of multiple guide RNAs (gRNAs). Here we describe a general method to efficiently produce many gRNAs from a single gene transcript based on the endogenous tRNA-processing system. A step-by-step protocol is provided for the design and construction of the polycistronic tRNA-gRNA (PTG) gene. The PTG method has been demonstrated to be highly efficient for multiplex genome editing in various plant, animal, and microbial species.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/*methods
RNA, Guide/genetics
RNA, Transfer/*genetics

@article {pmid30610627,
year = {2019},
author = {Li, R and Char, SN and Yang, B},
title = {Creating Large Chromosomal Deletions in Rice Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {47-61},
doi = {10.1007/978-1-4939-8991-1_4},
pmid = {30610627},
issn = {1940-6029},
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Chromosome Deletion ; Gene Editing/methods ; Genome, Plant/*genetics ; Oryza/*genetics ; Plants, Genetically Modified/*genetics ; RNA, Guide/genetics ; },
abstract = {Engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) is an efficient and the most popularly used tool for genome engineering in eukaryotic organisms including plants, especially in crop plants. This system has been effectively used to introduce mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. CRISPR/Cas9 hence presents great value in basic and applied research for improving the performance of crop plants in various aspects such as increasing grain yields, improving nutritional content, and better combating biotic and abiotic stresses. Besides above applications, CRISPR/Cas9 system has been shown to be very effective in creating large chromosomal deletions in plants, which is useful for genetic analysis of chromosomal fragments, functional study of gene clusters in biological processes, and so on. Here, we present a protocol of creating large chromosomal deletions in rice using CRISPR/Cas9 system, including detailed information about single-guide RNA design, vector construction, plant transformation, and large deletion screening processes in rice.},
}

Agrobacterium/genetics
CRISPR-Cas Systems
Chromosome Deletion
Gene Editing/methods
Genome, Plant/*genetics
Oryza/*genetics
Plants, Genetically Modified/*genetics
RNA, Guide/genetics

@article {pmid30610626,
year = {2019},
author = {Xie, X and Ma, X and Liu, YG},
title = {Decoding Sanger Sequencing Chromatograms from CRISPR-Induced Mutations.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {33-43},
doi = {10.1007/978-1-4939-8991-1_3},
pmid = {30610626},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Mutation/genetics ; Plants, Genetically Modified/*genetics ; },
abstract = {In many diploid organisms, the majority mutations induced by clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing are non- chimeric, including biallelic, homozygous, and heterozygous mutations. Direct Sanger sequencing of the PCR amplicons containing non-homozygous mutations superimposes sequencing chromatograms, displaying overlapping peaks beginning from the mutation sites. In this chapter we describe the degenerate sequence decoding (DSD) strategy and its automatic web-based tool, DSDecodeM, for decoding the Sanger sequencing chromatograms from different types of targeted mutations. DSDecodeM, as a convenient and versatile tool, can considerably facilitate the genotyping work of CRISPR-induced mutants.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/*methods
Genome, Plant/*genetics
Mutation/genetics
Plants, Genetically Modified/*genetics

@article {pmid30610625,
year = {2019},
author = {Wang, C and Wang, K},
title = {Rapid Screening of CRISPR/Cas9-Induced Mutants Using the ACT-PCR Method.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1917},
number = {},
pages = {27-32},
doi = {10.1007/978-1-4939-8991-1_2},
pmid = {30610625},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Polymerase Chain Reaction/*methods ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in targeted mutagenesis. However, a significant amount of time and cost is required to screen for the CRISPR/Cas9-induced mutants from a typically large number of initial samples. Here, we describe a cost-effective and sensitive screening technique based on conventional polymerase chain reaction (PCR), termed "annealing at critical temperature PCR" (ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants.},
}

CRISPR-Cas Systems/genetics/physiology
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/methods
Polymerase Chain Reaction/*methods

@article {pmid30559070,
year = {2019},
author = {Recchia, A},
title = {AAV-CRISPR Persistence in the Eye of the Beholder.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {1},
pages = {12-14},
doi = {10.1016/j.ymthe.2018.12.007},
pmid = {30559070},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Campylobacter jejuni/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dependovirus/*genetics ; Gene Editing ; Mice ; Retina ; },
}

Animals
CRISPR-Cas Systems
Campylobacter jejuni/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Dependovirus/*genetics
Gene Editing
Mice
Retina

@article {pmid30540740,
year = {2018},
author = {Kallimasioti-Pazi, EM and Thelakkad Chathoth, K and Taylor, GC and Meynert, A and Ballinger, T and Kelder, MJE and Lalevée, S and Sanli, I and Feil, R and Wood, AJ},
title = {Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair.},
journal = {PLoS biology},
volume = {16},
number = {12},
pages = {e2005595},
doi = {10.1371/journal.pbio.2005595},
pmid = {30540740},
issn = {1545-7885},
support = {102560/Z/13/Z//Wellcome Trust/United Kingdom ; //Medical Research Council/United Kingdom ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics/physiology ; DNA Breaks, Double-Stranded ; DNA Repair/genetics/*physiology ; Endonucleases/metabolism ; Gene Editing/methods ; Genome ; Heterochromatin/*genetics/*physiology ; Mice ; Mice, Inbred C57BL ; Mouse Embryonic Stem Cells/physiology ; Mutagenesis, Insertional ; Mutagens ; Mutation/genetics ; Recombinational DNA Repair/physiology ; Sequence Deletion ; },
abstract = {Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.},
}

Animals
Base Sequence
CRISPR-Cas Systems/genetics/physiology
DNA Breaks, Double-Stranded
DNA Repair/genetics/*physiology
Endonucleases/metabolism
Gene Editing/methods
Genome
Heterochromatin/*genetics/*physiology
Mice
Mice, Inbred C57BL
Mouse Embryonic Stem Cells/physiology
Mutagenesis, Insertional
Mutagens
Mutation/genetics
Recombinational DNA Repair/physiology
Sequence Deletion

@article {pmid30481169,
year = {2018},
author = {Tian, S and Muneeruddin, K and Choi, MY and Tao, L and Bhuiyan, RH and Ohmi, Y and Furukawa, K and Furukawa, K and Boland, S and Shaffer, SA and Adam, RM and Dong, M},
title = {Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation.},
journal = {PLoS biology},
volume = {16},
number = {11},
pages = {e2006951},
pmid = {30481169},
issn = {1545-7885},
support = {R01 AI132387/AI/NIAID NIH HHS/United States ; R01 NS080833/NS/NINDS NIH HHS/United States ; R01 AI139087/AI/NIAID NIH HHS/United States ; R21 NS106159/NS/NINDS NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; P30 HD018655/HD/NICHD NIH HHS/United States ; },
mesh = {Bacterial Toxins/metabolism ; CRISPR-Cas Systems ; Endosomes/metabolism ; Genome-Wide Association Study/methods ; Glycolipids/metabolism ; Glycosphingolipids ; Glycosylation ; Golgi Apparatus/metabolism/physiology ; HEK293 Cells ; HeLa Cells ; Humans ; Loss of Function Mutation/genetics ; Membrane Proteins/metabolism ; Membrane Transport Proteins/metabolism/physiology ; Oncogene Proteins/metabolism ; Protein Transport ; Ricin/*genetics/metabolism ; Shiga Toxins/*genetics/metabolism ; Trihexosylceramides/metabolism/physiology ; },
abstract = {Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific "activator" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.},
}

Bacterial Toxins/metabolism
CRISPR-Cas Systems
Endosomes/metabolism
Genome-Wide Association Study/methods
Glycolipids/metabolism
Glycosphingolipids
Glycosylation
Golgi Apparatus/metabolism/physiology
HEK293 Cells
HeLa Cells
Humans
Loss of Function Mutation/genetics
Membrane Proteins/metabolism
Membrane Transport Proteins/metabolism/physiology
Oncogene Proteins/metabolism
Protein Transport
Ricin/*genetics/metabolism
Shiga Toxins/*genetics/metabolism
Trihexosylceramides/metabolism/physiology

@article {pmid30420783,
year = {2019},
author = {Cheng, AZ and Yockteng-Melgar, J and Jarvis, MC and Malik-Soni, N and Borozan, I and Carpenter, MA and McCann, JL and Ebrahimi, D and Shaban, NM and Marcon, E and Greenblatt, J and Brown, WL and Frappier, L and Harris, RS},
title = {Epstein-Barr virus BORF2 inhibits cellular APOBEC3B to preserve viral genome integrity.},
journal = {Nature microbiology},
volume = {4},
number = {1},
pages = {78-88},
pmid = {30420783},
issn = {2058-5276},
support = {F30 CA200432/CA/NCI NIH HHS/United States ; R21 CA206309/CA/NCI NIH HHS/United States ; T32 CA009138/CA/NCI NIH HHS/United States ; //Howard Hughes Medical Institute/United States ; T32 GM008244/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Catalytic Domain/genetics ; Cell Line ; Cytidine Deaminase/*antagonists & inhibitors ; Genome, Viral/genetics ; HEK293 Cells ; Herpesvirus 4, Human/growth & development/*metabolism ; Herpesvirus 8, Human/*metabolism ; Humans ; Minor Histocompatibility Antigens ; RNA Interference ; RNA, Small Interfering/genetics ; Ribonucleotide Reductases/genetics/*metabolism ; Viral Proteins/genetics/*metabolism ; },
abstract = {The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC) family of single-stranded DNA (ssDNA) cytosine deaminases provides innate immunity against virus and transposon replication1-4. A well-studied mechanism is APOBEC3G restriction of human immunodeficiency virus type 1, which is counteracted by a virus-encoded degradation mechanism1-4. Accordingly, most work has focused on retroviruses with obligate ssDNA replication intermediates and it is unclear whether large double-stranded DNA (dsDNA) viruses may be similarly susceptible to restriction. Here, we show that the large dsDNA herpesvirus Epstein-Barr virus (EBV), which is the causative agent of infectious mononucleosis and multiple cancers5, utilizes a two-pronged approach to counteract restriction by APOBEC3B. Proteomics studies and immunoprecipitation experiments showed that the ribonucleotide reductase large subunit of EBV, BORF26,7, binds APOBEC3B. Mutagenesis mapped the interaction to the APOBEC3B catalytic domain, and biochemical studies demonstrated that BORF2 stoichiometrically inhibits APOBEC3B DNA cytosine deaminase activity. BORF2 also caused a dramatic relocalization of nuclear APOBEC3B to perinuclear bodies. On lytic reactivation, BORF2-null viruses were susceptible to APOBEC3B-mediated deamination as evidenced by lower viral titres, lower infectivity and hypermutation. The Kaposi's sarcoma-associated herpesvirus homologue, ORF61, also bound APOBEC3B and mediated relocalization. These data support a model where the genomic integrity of human γ-herpesviruses is maintained by active neutralization of the antiviral enzyme APOBEC3B.},
}

CRISPR-Cas Systems
Catalytic Domain/genetics
Cell Line
Cytidine Deaminase/*antagonists & inhibitors
Genome, Viral/genetics
HEK293 Cells
Herpesvirus 4, Human/growth & development/*metabolism
Herpesvirus 8, Human/*metabolism
Humans
Minor Histocompatibility Antigens
RNA Interference
RNA, Small Interfering/genetics
Ribonucleotide Reductases/genetics/*metabolism
Viral Proteins/genetics/*metabolism

@article {pmid30361258,
year = {2018},
author = {McDiarmid, TA and Au, V and Loewen, AD and Liang, J and Mizumoto, K and Moerman, DG and Rankin, CH},
title = {CRISPR-Cas9 human gene replacement and phenomic characterization in Caenorhabditis elegans to understand the functional conservation of human genes and decipher variants of uncertain significance.},
journal = {Disease models & mechanisms},
volume = {11},
number = {12},
pages = {},
doi = {10.1242/dmm.036517},
pmid = {30361258},
issn = {1754-8411},
support = {MOP 130287//CIHR/Canada ; },
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/chemistry/genetics ; Chemotaxis/drug effects ; Choice Behavior/drug effects ; *Conserved Sequence ; Gene Deletion ; Gene Dosage ; Gene Editing ; *Gene Targeting ; Genes, Helminth ; *Genetic Variation ; Humans ; Neurons/drug effects/metabolism ; PTEN Phosphohydrolase/chemistry/genetics ; Phenotype ; Sodium Chloride/pharmacology ; },
abstract = {Our ability to sequence genomes has vastly surpassed our ability to interpret the genetic variation we discover. This presents a major challenge in the clinical setting, where the recent application of whole-exome and whole-genome sequencing has uncovered thousands of genetic variants of uncertain significance. Here, we present a strategy for targeted human gene replacement and phenomic characterization, based on CRISPR-Cas9 genome engineering in the genetic model organism Caenorhabditis elegans, that will facilitate assessment of the functional conservation of human genes and structure-function analysis of disease-associated variants with unprecedented precision. We validate our strategy by demonstrating that direct single-copy replacement of the C. elegans ortholog (daf-18) with the critical human disease-associated gene phosphatase and tensin homolog (PTEN) is sufficient to rescue multiple phenotypic abnormalities caused by complete deletion of daf-18, including complex chemosensory and mechanosensory impairments. In addition, we used our strategy to generate animals harboring a single copy of the known pathogenic lipid phosphatase inactive PTEN variant (PTEN-G129E), and showed that our automated in vivo phenotypic assays could accurately and efficiently classify this missense variant as loss of function. The integrated nature of the human transgenes allows for analysis of both homozygous and heterozygous variants and greatly facilitates high-throughput precision medicine drug screens. By combining genome engineering with rapid and automated phenotypic characterization, our strategy streamlines the identification of novel conserved gene functions in complex sensory and learning phenotypes that can be used as in vivo functional assays to decipher variants of uncertain significance.},
}

Amino Acid Sequence
Animals
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Caenorhabditis elegans/*genetics
Caenorhabditis elegans Proteins/chemistry/genetics
Chemotaxis/drug effects
Choice Behavior/drug effects
*Conserved Sequence
Gene Deletion
Gene Dosage
Gene Editing
*Gene Targeting
Genes, Helminth
*Genetic Variation
Humans
Neurons/drug effects/metabolism
PTEN Phosphohydrolase/chemistry/genetics
Phenotype
Sodium Chloride/pharmacology

@article {pmid30287957,
year = {2018},
author = {Lemmon, ZH and Reem, NT and Dalrymple, J and Soyk, S and Swartwood, KE and Rodriguez-Leal, D and Van Eck, J and Lippman, ZB},
title = {Rapid improvement of domestication traits in an orphan crop by genome editing.},
journal = {Nature plants},
volume = {4},
number = {10},
pages = {766-770},
doi = {10.1038/s41477-018-0259-x},
pmid = {30287957},
issn = {2055-0278},
mesh = {Arabidopsis/genetics ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Crop Production/*methods ; *Domestication ; Gene Editing/*methods ; Lycopersicon esculentum/genetics ; Physalis/*genetics/growth & development ; Plants, Genetically Modified ; },
abstract = {Genome editing holds great promise for increasing crop productivity, and there is particular interest in advancing breeding in orphan crops, which are often burdened by undesirable characteristics resembling wild relatives. We developed genomic resources and efficient transformation in the orphan Solanaceae crop 'groundcherry' (Physalis pruinosa) and used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) to mutate orthologues of tomato domestication and improvement genes that control plant architecture, flower production and fruit size, thereby improving these major productivity traits. Thus, translating knowledge from model crops enables rapid creation of targeted allelic diversity and novel breeding germplasm in distantly related orphan crops.},
}

Arabidopsis/genetics
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Crop Production/*methods
*Domestication
Gene Editing/*methods
Lycopersicon esculentum/genetics
Physalis/*genetics/growth & development
Plants, Genetically Modified

@article {pmid30171624,
year = {2019},
author = {Wu, W and Yang, Y and Lei, H},
title = {Progress in the application of CRISPR: From gene to base editing.},
journal = {Medicinal research reviews},
volume = {39},
number = {2},
pages = {665-683},
doi = {10.1002/med.21537},
pmid = {30171624},
issn = {1098-1128},
support = {R01 EY012509/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Endonucleases/metabolism ; *Gene Editing ; Genome ; HEK293 Cells ; Humans ; Mice ; *Mutation ; Point Mutation ; },
abstract = {The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated endonucleases (Cas) has been utilized for genome editing with great accuracy and high efficiency in generating gene knockout, knockin, and point mutations in eukaryotic genomes. However, traditional CRISPR/Cas9 technology introduces double-stranded DNA breaks (DSBs) at a target locus as the first step to make gene corrections, which easily results in undesired mutations. Thus, it is necessary to develop new methods for correcting the unwanted mutations. In this review, we summarize the recent developments and a new approach to genome and base editing by using CRISPR/Cas9. This methodology renders a conversion of one target base into another, for example, C to T (or G to A), and A to G (or T to C) without producing DSBs, requiring a donor DNA template, or generating excessive insertions and deletions. Furthermore, CRISPR/Cas9-derived base editing also improves efficiency in repairing point mutations in the genome.},
}

Animals
*CRISPR-Cas Systems
DNA Breaks, Double-Stranded
Endonucleases/metabolism
*Gene Editing
Genome
HEK293 Cells
Humans
Mice
*Mutation
Point Mutation

@article {pmid30078389,
year = {2019},
author = {Lee, WB and Choi, WY and Lee, DH and Shim, H and Kim-Ha, J and Kim, YJ},
title = {OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages.},
journal = {BMB reports},
volume = {52},
number = {2},
pages = {133-138},
pmid = {30078389},
issn = {1976-670X},
mesh = {2',5'-Oligoadenylate Synthetase/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Chemokines/*biosynthesis/genetics ; Endoribonucleases/genetics/metabolism ; Gene Expression Regulation ; Gene Knockout Techniques ; Humans ; Interferons/*biosynthesis/genetics ; Lipopolysaccharides/pharmacology ; Macrophages/metabolism/*physiology ; Poly I-C/biosynthesis/genetics ; RNA, Double-Stranded/genetics/metabolism ; RNA, Viral/genetics/metabolism ; Transfection ; Virus Replication/genetics ; },
abstract = {Upon viral infection, the 2', 5'-oligoadenylate synthetase (OAS)-ribonuclease L (RNaseL) system works to cleave viral RNA, thereby blocking viral replication. However, it is unclear whether OAS proteins have a role in regulating gene expression. Here, we show that OAS1 and OAS3 act as negative regulators of the expression of chemokines and interferonresponsive genes in human macrophages. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology was used to engineer human myeloid cell lines in which the OAS1 or OAS3 gene was deleted. Neither OAS1 nor OAS3 was exclusively responsible for the degradation of rRNA in macrophages stimulated with poly(I:C), a synthetic surrogate for viral double-stranded (ds)RNA. An mRNA sequencing analysis revealed that genes related to type I interferon signaling and chemokine activity were increased in OAS1-/- and OAS3-/- macrophages treated with intracellular poly(I:C). Indeed, retinoic-acid-inducible gene (RIG)-I- and interferon-induced helicase C domain-containing protein (IFIH1 or MDA5)-mediated induction of chemokines and interferon-stimulated genes was regulated by OAS3, but Toll-like receptor 3 (TLR3)- and TLR4-mediated induction of those genes was modulated by OAS1 in macrophages. However, stimulation of these cells with type I interferons had no effect on OAS1- or OAS3-mediated chemokine secretion. These data suggest that OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages. [BMB Reports 2019; 52(2): 133-138].},
}

2',5'-Oligoadenylate Synthetase/*genetics/metabolism
CRISPR-Cas Systems
Cell Line
Chemokines/*biosynthesis/genetics
Endoribonucleases/genetics/metabolism
Gene Expression Regulation
Gene Knockout Techniques
Humans
Interferons/*biosynthesis/genetics
Lipopolysaccharides/pharmacology
Macrophages/metabolism/*physiology
Poly I-C/biosynthesis/genetics
RNA, Double-Stranded/genetics/metabolism
RNA, Viral/genetics/metabolism
Transfection
Virus Replication/genetics

@article {pmid29786424,
year = {2018},
author = {Mariscal, AM and Kakizawa, S and Hsu, JY and Tanaka, K and González-González, L and Broto, A and Querol, E and Lluch-Senar, M and Piñero-Lambea, C and Sun, L and Weyman, PD and Wise, KS and Merryman, C and Tse, G and Moore, AJ and Hutchison, CA and Smith, HO and Tomita, M and Venter, JC and Glass, JI and Piñol, J and Suzuki, Y},
title = {Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells.},
journal = {ACS synthetic biology},
volume = {7},
number = {6},
pages = {1538-1552},
doi = {10.1021/acssynbio.8b00028},
pmid = {29786424},
issn = {2161-5063},
mesh = {Aminoglycosides/pharmacology ; Bacterial Proteins/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Expression Regulation, Bacterial ; Gene Regulatory Networks ; Genetic Engineering/*methods ; Luminescent Proteins/genetics ; Methyltransferases/genetics ; Microorganisms, Genetically-Modified ; Mycoplasma/drug effects/*genetics ; Riboswitch/genetics ; Tetracycline/pharmacology ; },
abstract = {Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.},
}

Aminoglycosides/pharmacology
Bacterial Proteins/genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Expression Regulation, Bacterial
Gene Regulatory Networks
Genetic Engineering/*methods
Luminescent Proteins/genetics
Methyltransferases/genetics
Microorganisms, Genetically-Modified
Mycoplasma/drug effects/*genetics
Riboswitch/genetics
Tetracycline/pharmacology

@article {pmid29659822,
year = {2018},
author = {Zhang, C and Quan, R and Wang, J},
title = {Development and application of CRISPR/Cas9 technologies in genomic editing.},
journal = {Human molecular genetics},
volume = {27},
number = {R2},
pages = {R79-R88},
doi = {10.1093/hmg/ddy120},
pmid = {29659822},
issn = {1460-2083},
mesh = {Animals ; CRISPR-Cas Systems/*genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods/*trends ; Genetic Therapy ; Genomics/methods/trends ; Humans ; },
abstract = {Genomic editing to correct disease-causing mutations is a promising approach for the treatment of human diseases. As a simple and programmable nuclease-based genomic editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has substantially improved the ability to make precise changes in the human genome. Rapid development of CRISPR-based technologies in recent years has expanded its application scope and promoted CRISPR-based therapies in preclinical trails. Here, we review the application of the CRISPR system over the last 2 years; including its development and application in base editing, transcription modulation and epigenetic editing, genomic-scale screening, and cell and embryo therapy. Finally, the prospects and challenges related to application of CRISPR/Cas9 technologies are discussed.},
}

Animals
CRISPR-Cas Systems/*genetics/physiology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/*methods/*trends
Genetic Therapy
Genomics/methods/trends
Humans

@article {pmid29625203,
year = {2018},
author = {Hyams, G and Abadi, S and Lahav, S and Avni, A and Halperin, E and Shani, E and Mayrose, I},
title = {CRISPys: Optimal sgRNA Design for Editing Multiple Members of a Gene Family Using the CRISPR System.},
journal = {Journal of molecular biology},
volume = {430},
number = {15},
pages = {2184-2195},
doi = {10.1016/j.jmb.2018.03.019},
pmid = {29625203},
issn = {1089-8638},
mesh = {Algorithms ; Base Sequence ; *CRISPR-Cas Systems ; Computer Simulation ; Gene Editing/*methods ; Gene Knockout Techniques ; Genes, Plant/genetics ; Genome, Plant/genetics ; Genomics/*methods ; Lycopersicon esculentum/genetics ; Models, Genetic ; RNA, Guide/*genetics/metabolism ; },
abstract = {The development of the CRISPR-Cas9 system in recent years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simple and effective task. The system is directed to a genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with it, subsequently leading to site-specific modifications. However, many gene families in eukaryotic genomes exhibit partially overlapping functions, and thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR-Cas9 system, which may lead to the modification of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. We introduce CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the efficiency to edit the induced targets is maximized. We suggest several approaches for designing the optimal individual sgRNA and an approach to compute the optimal set of sgRNAs for cases when the experimental platform allows for more than one. The latter may optionally account for the homologous relationships among gene-family members. We further show that CRISPys outperforms simpler alignment-based techniques by in silico examination over all gene families in the Solanum lycopersicum genome.},
}

Algorithms
Base Sequence
*CRISPR-Cas Systems
Computer Simulation
Gene Editing/*methods
Gene Knockout Techniques
Genes, Plant/genetics
Genome, Plant/genetics
Genomics/*methods
Lycopersicon esculentum/genetics
Models, Genetic
RNA, Guide/*genetics/metabolism

@article {pmid29100008,
year = {2017},
author = {Ruzo, A and Brivanlou, AH},
title = {At Last: Gene Editing in Human Embryos to Understand Human Development.},
journal = {Cell stem cell},
volume = {21},
number = {5},
pages = {564-565},
doi = {10.1016/j.stem.2017.10.008},
pmid = {29100008},
issn = {1875-9777},
mesh = {Animals ; Blastocyst ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryo, Mammalian ; *Gene Editing ; Humans ; },
abstract = {Our understanding of early human development is typically based on inference from animal models, which may not fully recapitulate human embryonic features. As proof of concept, Fogarty et al. (2017) used CRISPR/Cas9 to genetically ablate the OCT4 gene in human preimplantation embryos and found key differences from its function in model systems.},
}

Animals
Blastocyst
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Embryo, Mammalian
*Gene Editing
Humans

@article {pmid28985519,
year = {2017},
author = {Shin, J and Corn, JE},
title = {E Pluribus Unum ("Out of Many, One"): CRISPR Modeling of Myeloid Expansion.},
journal = {Cell stem cell},
volume = {21},
number = {4},
pages = {415-416},
doi = {10.1016/j.stem.2017.09.013},
pmid = {28985519},
issn = {1875-9777},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Hematopoiesis ; Hematopoietic Stem Cells ; Humans ; Neoplasms/*genetics ; },
abstract = {In this issue of Cell Stem Cell, Tothova et al. (2017) demonstrate a promising way to model the complex genetics of clonal hematopoiesis and myeloid disorders using CRISPR-Cas9 genome editing in human hematopoietic stem and progenitor cells. Their approach opens the door to genotype-specific pharmacologic testing.},
}

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Hematopoiesis
Hematopoietic Stem Cells
Humans
Neoplasms/*genetics

@article {pmid28886363,
year = {2017},
author = {Li, XJ and Tu, Z and Yang, W and Li, S},
title = {CRISPR: Established Editor of Human Embryos?.},
journal = {Cell stem cell},
volume = {21},
number = {3},
pages = {295-296},
pmid = {28886363},
issn = {1875-9777},
support = {R01 NS036232/NS/NINDS NIH HHS/United States ; R01 NS095181/NS/NINDS NIH HHS/United States ; R01 NS095279/NS/NINDS NIH HHS/United States ; R01 NS101701/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; Mosaicism ; Mutation ; },
abstract = {Off-target effects and mosaicism are major concerns for applying CRISPR-Cas9 to correct genetic mutations. A recent article in Nature by Ma et al. (2017) uses an elegant CRISPR-Cas9 approach that repairs a genetic mutation in human embryos with negligible mosaicism and no off-target effects, bringing this editing tool closer to clinical application.},
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
Humans
Mosaicism
Mutation

@article {pmid31202125,
year = {2019},
author = {Yang, J and Wang, X},
title = {Role of long non-coding RNAs in lymphoma: A systematic review and clinical perspectives.},
journal = {Critical reviews in oncology/hematology},
volume = {141},
number = {},
pages = {13-22},
doi = {10.1016/j.critrevonc.2019.05.007},
pmid = {31202125},
issn = {1879-0461},
abstract = {Long non-coding RNAs (lncRNAs), are over 200 nucleotides in length, and they rarely act as templates for protein synthesis. Mounting studies have shown that lncRNAs play a crucial regulatory role in various processes that sustain life, such as epigenetic regulation, cell cycle control, splicing, and post-transcriptional regulation. LncRNAs were aberrantly expressed in most hematological malignancies including lymphoma, participating in tumor suppression or promoting oncogenesis and modulating key genes in different pathways. The specific expression patterns of lncRNAs in lymphoma make them good candidates to be used as diagnostic biomarkers or as therapeutic targets. LncRNAs can be targeted by multiple approaches including nucleic acid therapeutics, CRISPR/Cas genome editing techniques, small molecule inhibitors, and gene therapy. Efforts are made to develop therapeutic strategies aimed at targeting lncRNAs, but there are still some avenues to be covered before they can be applied to the clinical treatment of lymphoma.},
}