@article {pmid30656391,
year = {2019},
author = {Plavec, TV and Berlec, A},
title = {Engineering of lactic acid bacteria for delivery of therapeutic proteins and peptides.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00253-019-09628-y},
pmid = {30656391},
issn = {1432-0614},
support = {J4-9327//Javna Agencija za Raziskovalno Dejavnost RS/ ; },
abstract = {Lactic acid bacteria (LAB) have a long-term history of use in food industry and are becoming attractive for use in therapy on account of their safety, intrinsic beneficial health effects, and considerable biotechnological potential. The established systems for engineering are combined with novel approaches, such as CRISPR-Cas, to enable the use of LAB as vectors for delivery of various therapeutic molecules. The latter are either secreted or surface displayed and can be used for the treatment or prevention of numerous conditions: inflammatory bowel diseases, infections, autoimmune diseases, and even cancer. This review presents some recent data on engineering of LAB, with the emphasis on the most commonly used genera Lactococcus and Lactobacillus. Their use for the delivery of therapeutic proteins is discussed, while a special focus is given to the delivery of therapeutic peptides. Therapeutically relevant improvements of engineered LAB, such as containment systems, ability to visualize bacteria, or target specific host cells are also addressed. Future engineering of LAB for therapy will adopt the capabilities of synthetic biology, with first examples already emerging.},
}

@article {pmid30655519,
year = {2019},
author = {Díez-Villaseñor, C and Rodriguez-Valera, F},
title = {CRISPR analysis suggests that small circular single-stranded DNA smacoviruses infect Archaea instead of humans.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {294},
doi = {10.1038/s41467-018-08167-w},
pmid = {30655519},
issn = {2041-1723},
abstract = {Smacoviridae is a family of small (~2.5 Kb) CRESS-DNA (Circular Rep Encoding Single-Stranded (ss) DNA) viruses. These viruses have been found in faeces, were thought to infect eukaryotes and are suspected to cause gastrointestinal disease in humans. CRISPR-Cas systems are adaptive immune systems in prokaryotes, wherein snippets of genomes from invaders are stored as spacers that are interspersed between a repeated CRISPR sequence. Here we report several spacer sequences in the faecal archaeon Candidatus Methanomassiliicoccus intestinalis matching smacoviruses, implicating the archaeon as a firm candidate for a host. This finding may be relevant to understanding the potential origin of smacovirus-associated human diseases. Our results support that CRESS-DNA viruses can infect non-eukaryotes, which would mean that smacoviruses are the viruses with the smallest genomes to infect prokaryotes known to date. A probable target strand bias suggests that, in addition to double-stranded DNA, the CRISPR-Cas system can target ssDNA.},
}

@article {pmid30654914,
year = {2019},
author = {Li, Y and Li, S and Wang, J and Liu, G},
title = {CRISPR/Cas Systems towards Next-Generation Biosensing.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2018.12.005},
pmid = {30654914},
issn = {1879-3096},
abstract = {Beyond its remarkable genome editing ability, the CRISPR/Cas9 effector has also been utilized in biosensing applications. The recent discovery of the collateral RNA cleavage activity of the Cas13a effector has sparked even greater interest in developing novel biosensing technologies for nucleic acid detection and promised significant advances in CRISPR diagnostics. Now, along with the discovery of Cas12 collateral cleavage activities on single-stranded DNA (ssDNA), several CRISPR/Cas systems have been established for detecting various targets, including bacteria, viruses, cancer mutations, and others. Based on key Cas effectors, we provide a detailed classification of CRISPR/Cas biosensing systems and propose their future utility. As the field continues to mature, CRISPR/Cas systems have the potential to become promising candidates for next-generation diagnostic biosensing platforms.},
}

@article {pmid30274819,
year = {2018},
author = {Song, Y and Zhang, Y and Chen, M and Deng, J and Sui, T and Lai, L and Li, Z},
title = {Functional validation of the albinism-associated tyrosinase T373K SNP by CRISPR/Cas9-mediated homology-directed repair (HDR) in rabbits.},
journal = {EBioMedicine},
volume = {36},
number = {},
pages = {517-525},
pmid = {30274819},
issn = {2352-3964},
mesh = {Albinism, Oculocutaneous/*genetics/metabolism ; *Amino Acid Substitution ; Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Disease Models, Animal ; Enzyme Activation ; Female ; Gene Editing ; Genetic Association Studies ; Genetic Loci ; Genetic Predisposition to Disease ; Melanins ; Monophenol Monooxygenase/*genetics/metabolism ; Mutation ; *Polymorphism, Single Nucleotide ; Rabbits ; *Recombinational DNA Repair ; },
abstract = {BACKGROUND: Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by reduced melanin that are caused by mutations in the gene encoding tyrosinase (TYR), which is the rate-limiting enzyme in the production of the pigment melanin. Many studies or meta-analyses have suggested an association between the TYR T373K SNP and OCA1, but there is limited biochemical and genetic evidence to support this association.

METHODS: We overexpressed TYR-WT and TYR-T373K mutants on HK293T cells and tested the changes of melanin production and tyrosinase activity. Then we generated TYR-K373T knock-in (KI) rabbits by microinjection of ssDNA and synthesized RNAs targeting C1118A using CRISPR/Cas9-HDR to observe the formation of melanin.

FINDINGS: We demonstrated that the T373K mutation in TYR can reduce tyrosinase activity, leading to an absence of melanin synthesis at the cell-level. The gene-edited TYR-K373T rabbits exhibited rescued melanin production in hair follicles and irises, as inferred from the evident decrease in pigmentation in TYR-T373K rabbits, thus providing functional validation of the albinism-associated T373K SNP at the animal level.

INTERPRETATION: Our study provides the first animal-level functional validation of the albinism-associated TYR K373T SNP in rabbits, and these results will facilitate gene therapy of OCA1 in pre-clinical settings in the future. FUND: The National Key Research and Development Program of China Stem Cell and Translational Research, the Strategic Priority Research Program of the Chinese Academy of Sciences, the Guangdong Province Science and Technology Plan Project, and the Program for JLU Science and Technology Innovative Research Team.},
}

Albinism, Oculocutaneous/*genetics/metabolism
*Amino Acid Substitution
Animals
*CRISPR-Cas Systems
Cells, Cultured
Disease Models, Animal
Enzyme Activation
Female
Gene Editing
Genetic Association Studies
Genetic Loci
Genetic Predisposition to Disease
Melanins
Monophenol Monooxygenase/*genetics/metabolism
Mutation
*Polymorphism, Single Nucleotide
Rabbits
*Recombinational DNA Repair

@article {pmid30097473,
year = {2018},
author = {Després, PC and Dubé, AK and Nielly-Thibault, L and Yachie, N and Landry, CR},
title = {Double Selection Enhances the Efficiency of Target-AID and Cas9-Based Genome Editing in Yeast.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {10},
pages = {3163-3171},
pmid = {30097473},
issn = {2160-1836},
support = {299432//CIHR/Canada ; 324265//CIHR/Canada ; 387697//CIHR/Canada ; 364920//CIHR/Canada ; 384483//CIHR/Canada ; },
mesh = {Algorithms ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Order ; *Gene Targeting ; Genetic Loci ; Genetic Vectors/genetics ; *Genome, Fungal ; *Genomics/methods ; Models, Genetic ; *Selection, Genetic ; Yeasts/*genetics ; },
abstract = {CRISPR-Cas9 loss of function (LOF) and base editing screens are powerful tools in genetics and genomics. Yeast is one of the main models in these fields, but has only recently started to adopt this new toolkit for high throughput experiments. We developed a double selection strategy based on co-selection that increases LOF mutation rates using the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection experiments, and show that the improvement in Target-AID efficiency generalizes across loci. Using modeling, we show that this improvement in efficiency provides the required increased in detection power to measure the fitness effects of thousands of mutations in typical yeast pooled screens. We show that double selection can also improve Cas9 mediated LOF rates, but that this multiplex genome editing causes programmable chromosomal translocations at high frequency. This suggests that multiplex LOF editing should be performed with caution and that base-editors could be preferable tools for some screens in yeast. Base editing using double selection is simple and straightforward and provides an alternative to homology directed repair based high throughput variant strain construction methods.},
}

Algorithms
*CRISPR-Cas Systems
*Gene Editing
Gene Order
*Gene Targeting
Genetic Loci
Genetic Vectors/genetics
*Genome, Fungal
*Genomics/methods
Models, Genetic
*Selection, Genetic
Yeasts/*genetics

@article {pmid30654190,
year = {2018},
author = {Moses, C and Nugent, F and Waryah, CB and Garcia-Bloj, B and Harvey, AR and Blancafort, P},
title = {Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System.},
journal = {Molecular therapy. Nucleic acids},
volume = {14},
number = {},
pages = {287-300},
doi = {10.1016/j.omtn.2018.12.003},
pmid = {30654190},
issn = {2162-2531},
abstract = {PTEN expression is lost in many cancers, and even small changes in PTEN activity affect susceptibility and prognosis in a range of highly aggressive malignancies, such as melanoma and triple-negative breast cancer (TNBC). Loss of PTEN expression occurs via multiple mechanisms, including mutation, transcriptional repression and epigenetic silencing. Transcriptional repression of PTEN contributes to resistance to inhibitors used in the clinic, such as B-Raf inhibitors in BRAF mutant melanoma. We aimed to activate PTEN expression using the CRISPR system, specifically dead (d) Cas9 fused to the transactivator VP64-p65-Rta (VPR). dCas9-VPR was directed to the PTEN proximal promoter by single-guide RNAs (sgRNAs), in cancer cells that exhibited low levels of PTEN expression. The dCas9-VPR system increased PTEN expression in melanoma and TNBC cell lines, without transcriptional regulation at predicted off-target sgRNA binding sites. PTEN activation significantly repressed downstream oncogenic pathways, including AKT, mTOR, and MAPK signaling. BRAF V600E mutant melanoma cells transduced with dCas9-VPR displayed reduced migration, as well as diminished colony formation in the presence of B-Raf inhibitors, PI3K/mTOR inhibitors, and with combined PI3K/mTOR and B-Raf inhibition. CRISPR-mediated targeted activation of PTEN may provide an alternative therapeutic approach for highly aggressive cancers that are refractory to current treatments.},
}

@article {pmid30652832,
year = {2018},
author = {Węgleński, P},
title = {[Gene editing].},
journal = {Postepy biochemii},
volume = {64},
number = {1},
pages = {9-12},
pmid = {30652832},
issn = {0032-5422},
abstract = {Development of the gene engineering techniques has raised worries that they will be used for construction of organism endangering humans and environment. In 1975 at the Asilomar conference, geneticists from many countries decided that genetic engineering brings more benefits than threats. In last years a new CRISPR-Cas technique emerged . It allows to make the precise changes in genomes, e.g. to inactivate particular genes or to replace mutated genes by their wild-type alleles. Inactivation in mice of genes corresponding to those whose mutations cause the genetic diseases in man allows to get model organisms for studying the etiology of given disease and for working out the methods of its curing. This technique can be applied for repairing genes whose mutations result in metabolic diseases and cancer. Some voices were raised that the technique can be potentially used for the "improvement" of man, what would create many ethical and social problems. Geneticists, ethicists and lawyers gathered in 2015 at the Washington conference, discussed these problems and proposed rules for their solving.},
}

@article {pmid30652289,
year = {2019},
author = {Sander, JD},
title = {Gene Editing in Sorghum Through Agrobacterium.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1931},
number = {},
pages = {155-168},
doi = {10.1007/978-1-4939-9039-9_11},
pmid = {30652289},
issn = {1940-6029},
abstract = {The application of CRISPR/Cas to introduce targeted genomic edits is powering research and discovery across the genetic frontier. Applying CRISPR/Cas in sorghum can facilitate the study of gene function and unlock our understanding of this robust crop that serves as a staple for some of the most food insecure regions on the planet. When paired with recent advances in sorghum tissue culture and Agrobacteria technology, CRISPR/Cas can be used to introduce desirable changes and natural genetic variations directly into agriculturally relevant sorghum lines facilitating product development. This chapter describes CRISPR/Cas gene editing and provides high-level strategies and expectations for applying this technology using Agrobacterium in sorghum.},
}

@article {pmid30648020,
year = {2019},
author = {García-Zea, JA and de la Herrán, R and Robles Rodríguez, F and Navajas-Pérez, R and Ruiz Rejón, C},
title = {Detection and variability analyses of CRISPR-like loci in the H. pylori genome.},
journal = {PeerJ},
volume = {7},
number = {},
pages = {e6221},
doi = {10.7717/peerj.6221},
pmid = {30648020},
issn = {2167-8359},
abstract = {Helicobacter pylori is a human pathogenic bacterium with a high genomic plasticity. Although the functional CRISPR-Cas system has not been found in its genome, CRISPR-like loci have been recently identified. In this work, 53 genomes from different geographical areas are analyzed for the search and analysis of variability of this type of structure. We confirm the presence of a locus that was previously described in the VlpC gene in al lgenomes, and we characterize new CRISPR-like loci in other genomic locations. By studying the variability and gene location of these loci, the evolution and the possible roles of these sequences are discussed. Additionally, the usefulness of this type of sequences as a phylogenetic marker has been demonstrated, associating the different strains by geographical area.},
}

@article {pmid30647128,
year = {2019},
author = {Alimov, I and Menon, S and Cochran, N and Maher, R and Wang, Q and Alford, J and Concannon, JB and Yang, Z and Harrington, E and Llamas, L and Lindeman, A and Hoffman, G and Schuhmann, T and Russ, C and Reece-Hoyes, J and Canham, SM and Cai, X},
title = {Bile acid analogues are activators of Pyrin inflammasome.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA118.005103},
pmid = {30647128},
issn = {1083-351X},
abstract = {Bile acids are critical metabolites in the gastrointestinal tract which contribute to maintaining intestinal immune homeostasis through crosstalk with the gut microbiota. The conversion of bile acids by the gut microbiome is now recognized as a factor affecting both host metabolism and immune responses, but its physiological roles remain unclear. We conducted a screen for microbiome metabolites that would function as inflammasome activators and herein report the identification of 12-oxo-lithocholic acid, (BAA485), a potential microbiome-derived bile acid metabolite. We demonstrate the more potent analog 11-oxo-12S-hydroxy lithocholic acid methyl ester (BAA473) can induce secretion of interleukin-18 (IL18) through activation of the inflammasome in both myeloid and intestinal epithelial cells. Using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen with compound induced pyroptosis in THP-1 cells, we identified inflammasome activation by BAA473 is Pyrin-dependent (MEFV). To our knowledge, the bile acid analogues BAA485 and BAA473 are the first small molecule activators of the Pyrin inflammasome. We surmise that Pyrin inflammasome activation through microbiota-modified bile acid metabolites such as BAA473 and BAA485 plays a role in gut microbiota regulated intestinal immune response. The discovery of these two bioactive compounds may help further unveil the importance of Pyrin in gut homeostasis and autoimmune diseases.},
}

@article {pmid30518324,
year = {2018},
author = {Nishihara, M and Higuchi, A and Watanabe, A and Tasaki, K},
title = {Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri.},
journal = {BMC plant biology},
volume = {18},
number = {1},
pages = {331},
pmid = {30518324},
issn = {1471-2229},
support = {15H04453//Japanese Society for the Promotion of Science/ ; 16K18654//Japanese Society for the Promotion of Science/ ; },
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Color ; Flowers/anatomy & histology/*genetics ; Gene Editing/*methods ; Genes, Plant/genetics ; Lamiales/anatomy & histology/*genetics ; Plants, Genetically Modified ; Sequence Analysis, DNA ; },
abstract = {BACKGROUND: CRISPR/Cas9 technology is one of the most powerful and useful tools for genome editing in various living organisms. In higher plants, the system has been widely exploited not only for basic research, such as gene functional analysis, but also for applied research such as crop breeding. Although the CRISPR/Cas9 system has been used to induce mutations in genes involved in various plant developmental processes, few studies have been performed to modify the color of ornamental flowers. We therefore attempted to use this system to modify flower color in the model plant torenia (Torenia fournieri L.).

RESULTS: We attempted to induce mutations in the torenia flavanone 3-hydroxylase (F3H) gene, which encodes a key enzyme involved in flavonoid biosynthesis. Application of the CRISPR/Cas9 system successfully generated pale blue (almost white) flowers at a high frequency (ca. 80% of regenerated lines) in transgenic torenia T0 plants. Sequence analysis of PCR amplicons by Sanger and next-generation sequencing revealed the occurrence of mutations such as base substitutions and insertions/deletions in the F3H target sequence, thus indicating that the obtained phenotype was induced by the targeted mutagenesis of the endogenous F3H gene.

CONCLUSIONS: These results clearly demonstrate that flower color modification by genome editing with the CRISPR/Cas9 system is easily and efficiently achievable. Our findings further indicate that this system may be useful for future research on flower pigmentation and/or functional analyses of additional genes in torenia.},
}

CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Color
Flowers/anatomy & histology/*genetics
Gene Editing/*methods
Genes, Plant/genetics
Lamiales/anatomy & histology/*genetics
Plants, Genetically Modified
Sequence Analysis, DNA

@article {pmid30291237,
year = {2018},
author = {Xu, C and Lu, Z and Luo, Y and Liu, Y and Cao, Z and Shen, S and Li, H and Liu, J and Chen, K and Chen, Z and Yang, X and Gu, Z and Wang, J},
title = {Targeting of NLRP3 inflammasome with gene editing for the amelioration of inflammatory diseases.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {4092},
pmid = {30291237},
issn = {2041-1723},
mesh = {Amino Acid Sequence ; Animals ; Apoptosis Regulatory Proteins/metabolism ; CRISPR-Cas Systems ; Calcium-Binding Proteins/metabolism ; Caspase Activation and Recruitment Domain ; Female ; *Gene Editing ; *Genetic Therapy ; Inflammasomes/*genetics ; Inflammation/*therapy ; Male ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/*genetics/metabolism ; Peritonitis/immunology ; Protein Conformation ; },
abstract = {The NLRP3 inflammasome is a well-studied target for the treatment of multiple inflammatory diseases, but how to promote the current therapeutics remains a large challenge. CRISPR/Cas9, as a gene editing tool, allows for direct ablation of NLRP3 at the genomic level. In this study, we screen an optimized cationic lipid-assisted nanoparticle (CLAN) to deliver Cas9 mRNA (mCas9) and guide RNA (gRNA) into macrophages. By using CLAN encapsulating mCas9 and gRNA-targeting NLRP3 (gNLRP3) (CLANmCas9/gNLRP3), we disrupt NLRP3 of macrophages, inhibiting the activation of the NLRP3 inflammasome in response to diverse stimuli. After intravenous injection, CLANmCas9/gNLRP3 mitigates acute inflammation of LPS-induced septic shock and monosodium urate crystal (MSU)-induced peritonitis. In addition, CLANmCas9/gNLRP3 treatment improves insulin sensitivity and reduces adipose inflammation of high-fat-diet (HFD)-induced type 2 diabetes (T2D). Thus, our study provides a promising strategy for treating NLRP3-dependent inflammatory diseases and provides a carrier for delivering CRISPR/Cas9 into macrophages.},
}

Amino Acid Sequence
Animals
Apoptosis Regulatory Proteins/metabolism
CRISPR-Cas Systems
Calcium-Binding Proteins/metabolism
Caspase Activation and Recruitment Domain
Female
*Gene Editing
*Genetic Therapy
Inflammasomes/*genetics
Inflammation/*therapy
Male
Mice, Inbred C57BL
NLR Family, Pyrin Domain-Containing 3 Protein/*genetics/metabolism
Peritonitis/immunology
Protein Conformation

@article {pmid30260951,
year = {2018},
author = {Hara, T and Maejima, I and Akuzawa, T and Hirai, R and Kobayashi, H and Tsukamoto, S and Tsunoda, M and Ono, A and Yamakoshi, S and Oikawa, S and Sato, K},
title = {Rer1-mediated quality control system is required for neural stem cell maintenance during cerebral cortex development.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007647},
pmid = {30260951},
issn = {1553-7404},
mesh = {Amyloid Precursor Protein Secretases/*metabolism ; Animals ; Behavior, Animal ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cerebral Cortex/*growth & development/metabolism ; Chromosome Deletion ; Chromosome Disorders/genetics ; Chromosomes, Human, Pair 1/genetics ; Disease Models, Animal ; Female ; *Gene Expression Regulation, Developmental ; Humans ; Lysosomes/metabolism ; Male ; Membrane Glycoproteins/genetics/*metabolism ; Mice ; Mice, Knockout ; Neural Stem Cells ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Receptors, Notch/metabolism ; },
abstract = {Rer1 is a retrieval receptor for endoplasmic reticulum (ER) retention of various ER membrane proteins and unassembled or immature components of membrane protein complexes. However, its physiological functions during mammalian development remain unclear. This study aimed to investigate the role of Rer1-mediated quality control system in mammalian development. We show that Rer1 is required for the sufficient cell surface expression and activity of γ-secretase complex, which modulates Notch signaling during mouse cerebral cortex development. When Rer1 was depleted in the mouse cerebral cortex, the number of neural stem cells decreased significantly, and malformation of the cerebral cortex was observed. Rer1 loss reduced γ-secretase activity and downregulated Notch signaling in the developing cerebral cortex. In Rer1-deficient cells, a subpopulation of γ-secretase complexes and components was transported to and degraded in lysosomes, thereby significantly reducing the amount of γ-secretase complex on the cell surface. These results suggest that Rer1 maintains Notch signaling by maintaining sufficient expression of the γ-secretase complex on the cell surface and regulating neural stem cell maintenance during cerebral cortex development.},
}

Amyloid Precursor Protein Secretases/*metabolism
Animals
Behavior, Animal
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Cerebral Cortex/*growth & development/metabolism
Chromosome Deletion
Chromosome Disorders/genetics
Chromosomes, Human, Pair 1/genetics
Disease Models, Animal
Female
*Gene Expression Regulation, Developmental
Humans
Lysosomes/metabolism
Male
Membrane Glycoproteins/genetics/*metabolism
Mice
Mice, Knockout
Neural Stem Cells
Receptors, Cytoplasmic and Nuclear/genetics/*metabolism
Receptors, Notch/metabolism

@article {pmid30248107,
year = {2018},
author = {Wu, S and Zhang, M and Yang, X and Peng, F and Zhang, J and Tan, J and Yang, Y and Wang, L and Hu, Y and Peng, Q and Li, J and Liu, Y and Guan, Y and Chen, C and Hamer, MA and Nijsten, T and Zeng, C and Adhikari, K and Gallo, C and Poletti, G and Schuler-Faccini, L and Bortolini, MC and Canizales-Quinteros, S and Rothhammer, F and Bedoya, G and González-José, R and Li, H and Krutmann, J and Liu, F and Kayser, M and Ruiz-Linares, A and Tang, K and Xu, S and Zhang, L and Jin, L and Wang, S},
title = {Genome-wide association studies and CRISPR/Cas9-mediated gene editing identify regulatory variants influencing eyebrow thickness in humans.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007640},
pmid = {30248107},
issn = {1553-7404},
support = {BB/I021213/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Cas Systems/genetics ; Chromosomes, Human/genetics ; Eyebrows/*growth & development ; Forkhead Transcription Factors/genetics ; Gene Editing ; Genetic Loci/*genetics ; Genome-Wide Association Study ; Genotype ; Humans ; *Phenotype ; Polymorphism, Single Nucleotide ; SOXB1 Transcription Factors/genetics ; Selection, Genetic ; },
abstract = {Hair plays an important role in primates and is clearly subject to adaptive selection. While humans have lost most facial hair, eyebrows are a notable exception. Eyebrow thickness is heritable and widely believed to be subject to sexual selection. Nevertheless, few genomic studies have explored its genetic basis. Here, we performed a genome-wide scan for eyebrow thickness in 2961 Han Chinese. We identified two new loci of genome-wide significance, at 3q26.33 near SOX2 (rs1345417: P = 6.51×10(-10)) and at 5q13.2 near FOXD1 (rs12651896: P = 1.73×10(-8)). We further replicated our findings in the Uyghurs, a population from China characterized by East Asian-European admixture (N = 721), the CANDELA cohort from five Latin American countries (N = 2301), and the Rotterdam Study cohort of Dutch Europeans (N = 4411). A meta-analysis combining the full GWAS results from the three cohorts of full or partial Asian descent (Han Chinese, Uyghur and Latin Americans, N = 5983) highlighted a third signal of genome-wide significance at 2q12.3 (rs1866188: P = 5.81×10(-11)) near EDAR. We performed fine-mapping and prioritized four variants for further experimental verification. CRISPR/Cas9-mediated gene editing provided evidence that rs1345417 and rs12651896 affect the transcriptional activity of the nearby SOX2 and FOXD1 genes, which are both involved in hair development. Finally, suitable statistical analyses revealed that none of the associated variants showed clear signals of selection in any of the populations tested. Contrary to popular speculation, we found no evidence that eyebrow thickness is subject to strong selective pressure.},
}

CRISPR-Cas Systems/genetics
Chromosomes, Human/genetics
Eyebrows/*growth & development
Forkhead Transcription Factors/genetics
Gene Editing
Genetic Loci/*genetics
Genome-Wide Association Study
Genotype
Humans
*Phenotype
Polymorphism, Single Nucleotide
SOXB1 Transcription Factors/genetics
Selection, Genetic

@article {pmid30242271,
year = {2018},
author = {Guo, Y and Jardin, BD and Zhou, P and Sethi, I and Akerberg, BN and Toepfer, CN and Ai, Y and Li, Y and Ma, Q and Guatimosim, S and Hu, Y and Varuzhanyan, G and VanDusen, NJ and Zhang, D and Chan, DC and Yuan, GC and Seidman, CE and Seidman, JG and Pu, WT},
title = {Hierarchical and stage-specific regulation of murine cardiomyocyte maturation by serum response factor.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3837},
pmid = {30242271},
issn = {2041-1723},
support = {U01 HL131003/HL/NHLBI NIH HHS/United States ; 2UM1 HL098166//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/International ; 17IRG33410894//American Heart Association (American Heart Association, Inc.)/International ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems ; Chromatin/metabolism ; Female ; Gene Expression Regulation ; Male ; Mice ; Mice, Knockout ; Mutagenesis ; Myocytes, Cardiac/*physiology ; Serum Response Factor/*metabolism ; Transcriptome ; },
abstract = {After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal's lifespan. How this maturation process is regulated and coordinated is poorly understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly.},
}

Animals
Animals, Newborn
CRISPR-Cas Systems
Chromatin/metabolism
Female
Gene Expression Regulation
Male
Mice
Mice, Knockout
Mutagenesis
Myocytes, Cardiac/*physiology
Serum Response Factor/*metabolism
Transcriptome

@article {pmid30222779,
year = {2018},
author = {Shi, CH and Rubel, C and Soss, SE and Sanchez-Hodge, R and Zhang, S and Madrigal, SC and Ravi, S and McDonough, H and Page, RC and Chazin, WJ and Patterson, C and Mao, CY and Willis, MS and Luo, HY and Li, YS and Stevens, DA and Tang, MB and Du, P and Wang, YH and Hu, ZW and Xu, YM and Schisler, JC},
title = {Disrupted structure and aberrant function of CHIP mediates the loss of motor and cognitive function in preclinical models of SCAR16.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007664},
pmid = {30222779},
issn = {1553-7404},
support = {R37 HL065619/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Behavior, Animal ; CRISPR-Cas Systems/genetics ; *Cognition ; Disease Models, Animal ; Female ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Models, Molecular ; Motor Activity/*genetics ; Mutagenesis, Site-Directed ; Phenotype ; Point Mutation ; Protein Domains/*genetics ; Protein Multimerization/genetics ; Rats ; Rats, Sprague-Dawley ; Spinocerebellar Ataxias/congenital/*genetics ; Ubiquitin-Protein Ligases/*genetics/metabolism ; },
abstract = {CHIP (carboxyl terminus of heat shock 70-interacting protein) has long been recognized as an active member of the cellular protein quality control system given the ability of CHIP to function as both a co-chaperone and ubiquitin ligase. We discovered a genetic disease, now known as spinocerebellar autosomal recessive 16 (SCAR16), resulting from a coding mutation that caused a loss of CHIP ubiquitin ligase function. The initial mutation describing SCAR16 was a missense mutation in the ubiquitin ligase domain of CHIP (p.T246M). Using multiple biophysical and cellular approaches, we demonstrated that T246M mutation results in structural disorganization and misfolding of the CHIP U-box domain, promoting oligomerization, and increased proteasome-dependent turnover. CHIP-T246M has no ligase activity, but maintains interactions with chaperones and chaperone-related functions. To establish preclinical models of SCAR16, we engineered T246M at the endogenous locus in both mice and rats. Animals homozygous for T246M had both cognitive and motor cerebellar dysfunction distinct from those observed in the CHIP null animal model, as well as deficits in learning and memory, reflective of the cognitive deficits reported in SCAR16 patients. We conclude that the T246M mutation is not equivalent to the total loss of CHIP, supporting the concept that disease-causing CHIP mutations have different biophysical and functional repercussions on CHIP function that may directly correlate to the spectrum of clinical phenotypes observed in SCAR16 patients. Our findings both further expand our basic understanding of CHIP biology and provide meaningful mechanistic insight underlying the molecular drivers of SCAR16 disease pathology, which may be used to inform the development of novel therapeutics for this devastating disease.},
}

Animals
Behavior, Animal
CRISPR-Cas Systems/genetics
*Cognition
Disease Models, Animal
Female
Humans
Male
Mice
Mice, Inbred C57BL
Models, Molecular
Motor Activity/*genetics
Mutagenesis, Site-Directed
Phenotype
Point Mutation
Protein Domains/*genetics
Protein Multimerization/genetics
Rats
Rats, Sprague-Dawley
Spinocerebellar Ataxias/congenital/*genetics
Ubiquitin-Protein Ligases/*genetics/metabolism

@article {pmid30010246,
year = {2018},
author = {Martin Gonzalez, J and Baudet, A and Abelechian, S and Bonderup, K and d'Altri, T and Porse, B and Brakebusch, C and Juliusson, G and Cammenga, J},
title = {A new genetic tool to improve immune-compromised mouse models: Derivation and CRISPR/Cas9-mediated targeting of NRG embryonic stem cell lines.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {56},
number = {9},
pages = {e23238},
doi = {10.1002/dvg.23238},
pmid = {30010246},
issn = {1526-968X},
support = {//Cancerfonden/International ; //Barncancerfonden/International ; //Linköping University/International ; //NovoNordisk Foundation (The NovoNordisk Foundation Section for Stem Cell Biology in Human Disease)/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Cell Line ; *Embryonic Stem Cells ; Fertilization in Vitro ; GATA2 Transcription Factor/genetics ; Gene Targeting ; Immunocompromised Host/genetics ; Mice ; Mice, Inbred NOD/*genetics/immunology ; Models, Biological ; },
abstract = {Development of human hematopoietic stem cells and differentiation of embryonic stem (ES) cells/induced pluripotent stem (iPS) cells to hematopoietic stem cells are poorly understood. NOD (Non-obese diabetic)-derived mouse strains, such as NSG (NOD-Scid-il2Rg) or NRG (NOD-Rag1-il2Rg), are the best available models for studying the function of fetal and adult human hematopoietic cells as well as ES/iPS cell-derived hematopoietic stem cells. Unfortunately, engraftment of human hematopoietic stem cells is very variable in these models. Introduction of additional permissive mutations into these complex genetic backgrounds of the NRG/NSG mice by natural breeding is a very demanding task in terms of time and resources. Specifically, since the genetic elements defining the NSG/NRG phenotypes have not yet been fully characterized, intense backcrossing is required to ensure transmission of the full phenotype. Here we describe the derivation of embryonic stem cell (ESC) lines from NRG pre-implantation embryos generated by in vitro fertilization followed by the CRISPR/CAS9 targeting of the Gata-2 locus. After injection into morula stage embryos, cells from three tested lines gave rise to chimeric adult mice showing high contribution of the ESCs (70%-100%), assessed by coat color. Moreover, these lines have been successfully targeted using Cas9/CRISPR technology, and the mutant cells have been shown to remain germ line competent. Therefore, these new NRG ESC lines combined with genome editing nucleases bring a powerful genetic tool that facilitates the generation of new NOD-based mouse models with the aim to improve the existing xenograft models.},
}

Animals
*CRISPR-Cas Systems
*Cell Line
*Embryonic Stem Cells
Fertilization in Vitro
GATA2 Transcription Factor/genetics
Gene Targeting
Immunocompromised Host/genetics
Mice
Mice, Inbred NOD/*genetics/immunology
Models, Biological

@article {pmid29968715,
year = {2018},
author = {Matsuda, Y and Bai, T and Phippen, CBW and Nødvig, CS and Kjærbølling, I and Vesth, TC and Andersen, MR and Mortensen, UH and Gotfredsen, CH and Abe, I and Larsen, TO},
title = {Novofumigatonin biosynthesis involves a non-heme iron-dependent endoperoxide isomerase for orthoester formation.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2587},
pmid = {29968715},
issn = {2041-1723},
support = {VKR023427//Villum Fonden (Villum Foundation)/International ; JP15H01836//Japan Society for the Promotion of Science (JSPS)/International ; JP16H06443//Japan Society for the Promotion of Science (JSPS)/International ; },
mesh = {Aspergillus/genetics/*metabolism ; Biosynthetic Pathways ; CRISPR-Cas Systems ; Catalysis ; Fungal Proteins/genetics/*metabolism ; Gene Deletion ; Iron/metabolism ; Ketoglutaric Acids/metabolism ; Peroxides/metabolism ; Prostaglandin-E Synthases/genetics/*metabolism ; Terpenes/*metabolism ; },
abstract = {Novofumigatonin (1), isolated from the fungus Aspergillus novofumigatus, is a heavily oxygenated meroterpenoid containing a unique orthoester moiety. Despite the wide distribution of orthoesters in nature and their biological importance, little is known about the biogenesis of orthoesters. Here we show the elucidation of the biosynthetic pathway of 1 and the identification of key enzymes for the orthoester formation by a series of CRISPR-Cas9-based gene-deletion experiments and in vivo and in vitro reconstitutions of the biosynthesis. The novofumigatonin pathway involves endoperoxy compounds as key precursors for the orthoester synthesis, in which the Fe(II)/α-ketoglutarate-dependent enzyme NvfI performs the endoperoxidation. NvfE, the enzyme catalyzing the orthoester synthesis, is an Fe(II)-dependent, but cosubstrate-free, endoperoxide isomerase, despite the fact that NvfE shares sequence homology with the known Fe(II)/α-ketoglutarate-dependent dioxygenases. NvfE thus belongs to a class of enzymes that gained an isomerase activity by losing the α-ketoglutarate-binding ability.},
}

Aspergillus/genetics/*metabolism
Biosynthetic Pathways
CRISPR-Cas Systems
Catalysis
Fungal Proteins/genetics/*metabolism
Gene Deletion
Iron/metabolism
Ketoglutaric Acids/metabolism
Peroxides/metabolism
Prostaglandin-E Synthases/genetics/*metabolism
Terpenes/*metabolism

@article {pmid29779931,
year = {2018},
author = {Sedlyarov, V and Eichner, R and Girardi, E and Essletzbichler, P and Goldmann, U and Nunes-Hasler, P and Srndic, I and Moskovskich, A and Heinz, LX and Kartnig, F and Bigenzahn, JW and Rebsamen, M and Kovarik, P and Demaurex, N and Superti-Furga, G},
title = {The Bicarbonate Transporter SLC4A7 Plays a Key Role in Macrophage Phagosome Acidification.},
journal = {Cell host & microbe},
volume = {23},
number = {6},
pages = {766-774.e5},
pmid = {29779931},
issn = {1934-6069},
support = {//European Research Council/International ; },
mesh = {Bicarbonates/*metabolism ; CRISPR-Cas Systems ; Cation Transport Proteins/metabolism ; Cytoplasm/metabolism ; Gene Knockout Techniques ; Homeostasis ; Humans ; Hydrogen-Ion Concentration ; Macrophages/*metabolism ; Phagocytosis ; Phagosomes/*metabolism ; Sodium-Bicarbonate Symporters/genetics/*physiology ; THP-1 Cells ; Transcriptome ; U937 Cells ; },
abstract = {Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.},
}

Bicarbonates/*metabolism
CRISPR-Cas Systems
Cation Transport Proteins/metabolism
Cytoplasm/metabolism
Gene Knockout Techniques
Homeostasis
Humans
Hydrogen-Ion Concentration
Macrophages/*metabolism
Phagocytosis
Phagosomes/*metabolism
Sodium-Bicarbonate Symporters/genetics/*physiology
THP-1 Cells
Transcriptome
U937 Cells

@article {pmid29685891,
year = {2018},
author = {Wu, J and Wang, P and Li, L and You, C and Wang, Y},
title = {Cytotoxic and mutagenic properties of minor-groove O2-alkylthymidine lesions in human cells.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {22},
pages = {8638-8644},
pmid = {29685891},
issn = {1083-351X},
support = {R01 ES025121/ES/NIEHS NIH HHS/United States ; },
mesh = {Alkylation ; CRISPR-Cas Systems ; DNA/*chemistry ; *DNA Damage ; *DNA Replication ; DNA-Directed DNA Polymerase/chemistry/genetics/*metabolism ; Gene Editing ; HEK293 Cells ; Humans ; Mutagenesis ; Thymidine/*chemistry ; },
abstract = {Endogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, are recognized by the DNA replication machinery in human cells. We found that the replication bypass efficiencies of these lesions decrease with increasing length of the alkyl chain, and that these lesions induce substantial frequencies of T→A and T→G mutations. Replication experiments using isogenic cells deficient in specific translesion synthesis (TLS) DNA polymerases revealed that the absence of polymerase η or polymerase ζ, but not polymerase κ or polymerase ι, significantly decreased both the bypass efficiencies and the mutation frequencies for those O2-alkyldT lesions carrying a straight-chain alkyl group. Moreover, the mutagenic properties of the O2-alkyldT lesions were influenced by the length and topology of the alkyl chain and by TLS polymerases. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of O2-alkyldT lesions, and illustrate the roles of TLS polymerases in replicative bypass of these lesions in human cells.},
}

Alkylation
CRISPR-Cas Systems
DNA/*chemistry
*DNA Damage
*DNA Replication
DNA-Directed DNA Polymerase/chemistry/genetics/*metabolism
Gene Editing
HEK293 Cells
Humans
Mutagenesis
Thymidine/*chemistry

@article {pmid29674317,
year = {2018},
author = {Takahashi, C and Miyatake, K and Kusakabe, M and Nishida, E},
title = {The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {22},
pages = {8342-8361},
pmid = {29674317},
issn = {1083-351X},
mesh = {Animals ; Breast Neoplasms/enzymology/*pathology ; CRISPR-Cas Systems ; Cell Adhesion ; Cell Membrane ; Cells, Cultured ; Embryo, Nonmammalian/cytology/*enzymology ; Epithelial Cells/*chemistry/enzymology/pathology ; Female ; Hep G2 Cells ; Humans ; Mitogen-Activated Protein Kinase 6/antagonists & inhibitors/genetics/*metabolism ; Tight Junctions ; Transcription Factor AP-2/antagonists & inhibitors/genetics/*metabolism ; Xenopus laevis/embryology/*physiology ; },
abstract = {Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A.},
}

Animals
Breast Neoplasms/enzymology/*pathology
CRISPR-Cas Systems
Cell Adhesion
Cell Membrane
Cells, Cultured
Embryo, Nonmammalian/cytology/*enzymology
Epithelial Cells/*chemistry/enzymology/pathology
Female
Hep G2 Cells
Humans
Mitogen-Activated Protein Kinase 6/antagonists & inhibitors/genetics/*metabolism
Tight Junctions
Transcription Factor AP-2/antagonists & inhibitors/genetics/*metabolism
Xenopus laevis/embryology/*physiology

@article {pmid29358088,
year = {2018},
author = {Marrone, L and Poser, I and Casci, I and Japtok, J and Reinhardt, P and Janosch, A and Andree, C and Lee, HO and Moebius, C and Koerner, E and Reinhardt, L and Cicardi, ME and Hackmann, K and Klink, B and Poletti, A and Alberti, S and Bickle, M and Hermann, A and Pandey, UB and Hyman, AA and Sterneckert, JL},
title = {Isogenic FUS-eGFP iPSC Reporter Lines Enable Quantification of FUS Stress Granule Pathology that Is Rescued by Drugs Inducing Autophagy.},
journal = {Stem cell reports},
volume = {10},
number = {2},
pages = {375-389},
pmid = {29358088},
issn = {2213-6711},
support = {R01 NS081303/NS/NINDS NIH HHS/United States ; R21 NS094921/NS/NINDS NIH HHS/United States ; R21 NS098379/NS/NINDS NIH HHS/United States ; R21 NS100055/NS/NINDS NIH HHS/United States ; },
mesh = {Amyotrophic Lateral Sclerosis/*genetics/metabolism/pathology ; Animals ; Antidepressive Agents/pharmacology ; Antipyretics/pharmacology ; Autophagy/genetics ; CRISPR-Cas Systems ; Drosophila ; Drosophila Proteins/*genetics ; Drug Evaluation, Preclinical ; Green Fluorescent Proteins/genetics ; Heterogeneous-Nuclear Ribonucleoprotein Group F-H/*genetics ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Motor Neurons/metabolism/pathology ; Mutation ; Phosphatidylinositol 3-Kinases/genetics ; Proto-Oncogene Proteins c-akt/genetics ; RNA-Binding Protein FUS/*genetics ; Signal Transduction/drug effects ; TOR Serine-Threonine Kinases/genetics ; },
abstract = {Perturbations in stress granule (SG) dynamics may be at the core of amyotrophic lateral sclerosis (ALS). Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments.},
}

Amyotrophic Lateral Sclerosis/*genetics/metabolism/pathology
Animals
Antidepressive Agents/pharmacology
Antipyretics/pharmacology
Autophagy/genetics
CRISPR-Cas Systems
Drosophila
Drosophila Proteins/*genetics
Drug Evaluation, Preclinical
Green Fluorescent Proteins/genetics
Heterogeneous-Nuclear Ribonucleoprotein Group F-H/*genetics
Humans
Induced Pluripotent Stem Cells/*metabolism
Motor Neurons/metabolism/pathology
Mutation
Phosphatidylinositol 3-Kinases/genetics
Proto-Oncogene Proteins c-akt/genetics
RNA-Binding Protein FUS/*genetics
Signal Transduction/drug effects
TOR Serine-Threonine Kinases/genetics

@article {pmid29307579,
year = {2018},
author = {Steyer, B and Bu, Q and Cory, E and Jiang, K and Duong, S and Sinha, D and Steltzer, S and Gamm, D and Chang, Q and Saha, K},
title = {Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection.},
journal = {Stem cell reports},
volume = {10},
number = {2},
pages = {642-654},
pmid = {29307579},
issn = {2213-6711},
support = {T32 GM008692/GM/NIGMS NIH HHS/United States ; T32 HG002760/HG/NHGRI NIH HHS/United States ; F30 EY027699/EY/NEI NIH HHS/United States ; R03 HD086523/HD/NICHD NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; U54 HD090256/HD/NICHD NIH HHS/United States ; R01 EY024588/EY/NEI NIH HHS/United States ; },
mesh = {Acetyltransferases/chemistry/*genetics ; CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; DNA, Single-Stranded/genetics ; Gene Editing/*methods ; Gene Expression Regulation/genetics ; Genetic Vectors/genetics ; Genome, Human/genetics ; Humans ; *Induced Pluripotent Stem Cells ; },
abstract = {Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.},
}

Acetyltransferases/chemistry/*genetics
CRISPR-Cas Systems/genetics
Cell Differentiation/*genetics
DNA, Single-Stranded/genetics
Gene Editing/*methods
Gene Expression Regulation/genetics
Genetic Vectors/genetics
Genome, Human/genetics
Humans
*Induced Pluripotent Stem Cells

@article {pmid28694465,
year = {2017},
author = {Zhang, W and Wang, G and Wang, Y and Jin, Y and Zhao, L and Xiong, Q and Zhang, L and Mou, L and Li, R and Yang, H and Dai, Y},
title = {Generation of complement protein C3 deficient pigs by CRISPR/Cas9-mediated gene targeting.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {5009},
pmid = {28694465},
issn = {2045-2322},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Complement C3/*deficiency ; Fibroblasts/cytology/metabolism ; Gene Knockout Techniques/*methods ; Genetic Engineering ; Male ; Nuclear Transfer Techniques/*veterinary ; Swine ; },
abstract = {Complement protein C3 is the pivotal component of the complement system. Previous studies have demonstrated that C3 has implications in various human diseases and exerts profound functions under certain conditions. However, the delineation of pathological and physiological roles of C3 has been hampered by the insufficiency of suitable animal models. In the present study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the C3 gene in porcine fetal fibroblasts. Our results indicated that CRISPR/Cas9 targeting efficiency was as high as 84.7%, and the biallelic mutation efficiency reached at 45.7%. The biallelic modified colonies were used as donor for somatic cell nuclear transfer (SCNT) technology to generate C3 targeted piglets. A total of 19 C3 knockout (KO) piglets were produced and their plasma C3 protein was undetectable by western blot analysis and ELISA. The hemolytic complement activity and complement-dependent cytotoxicity assay further confirmed that C3 was disrupted in these piglets. These C3 KO pigs could be utilized as a valuable large animal model for the elucidation of the roles of C3.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems
Complement C3/*deficiency
Fibroblasts/cytology/metabolism
Gene Knockout Techniques/*methods
Genetic Engineering
Male
Nuclear Transfer Techniques/*veterinary
Swine

@article {pmid30643127,
year = {2019},
author = {Nakamura, M and Srinivasan, P and Chavez, M and Carter, MA and Dominguez, AA and La Russa, M and Lau, MB and Abbott, TR and Xu, X and Zhao, D and Gao, Y and Kipniss, NH and Smolke, CD and Bondy-Denomy, J and Qi, LS},
title = {Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {194},
doi = {10.1038/s41467-018-08158-x},
pmid = {30643127},
issn = {2041-1723},
abstract = {Repurposed CRISPR-Cas molecules provide a useful tool set for broad applications of genomic editing and regulation of gene expression in prokaryotes and eukaryotes. Recent discovery of phage-derived proteins, anti-CRISPRs, which serve to abrogate natural CRISPR anti-phage activity, potentially expands the ability to build synthetic CRISPR-mediated circuits. Here, we characterize a panel of anti-CRISPR molecules for expanded applications to counteract CRISPR-mediated gene activation and repression of reporter and endogenous genes in various cell types. We demonstrate that cells pre-engineered with anti-CRISPR molecules become resistant to gene editing, thus providing a means to generate "write-protected" cells that prevent future gene editing. We further show that anti-CRISPRs can be used to control CRISPR-based gene regulation circuits, including implementation of a pulse generator circuit in mammalian cells. Our work suggests that anti-CRISPR proteins should serve as widely applicable tools for synthetic systems regulating the behavior of eukaryotic cells.},
}

@article {pmid30199527,
year = {2018},
author = {Gay, S and Bugeon, J and Bouchareb, A and Henry, L and Delahaye, C and Legeai, F and Montfort, J and Le Cam, A and Siegel, A and Bobe, J and Thermes, V},
title = {MiR-202 controls female fecundity by regulating medaka oogenesis.},
journal = {PLoS genetics},
volume = {14},
number = {9},
pages = {e1007593},
pmid = {30199527},
issn = {1553-7404},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Female ; Fertility/*genetics ; Gene Editing ; Gene Expression Profiling ; *Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Granulosa Cells ; Male ; MicroRNAs/*physiology ; Oocytes/growth & development/metabolism ; Oogenesis/*genetics ; Oryzias/*physiology ; Ovary/cytology/growth & development/metabolism ; },
abstract = {Female gamete production relies on coordinated molecular and cellular processes that occur in the ovary throughout oogenesis. In fish, as in other vertebrates, these processes have been extensively studied both in terms of endocrine/paracrine regulation and protein expression and activity. The role of small non-coding RNAs in the regulation of animal reproduction remains however largely unknown and poorly investigated, despite a growing interest for the importance of miRNAs in a wide variety of biological processes. Here, we analyzed the role of miR-202, a miRNA predominantly expressed in male and female gonads in several vertebrate species. We studied its expression in the medaka ovary and generated a mutant line (using CRISPR/Cas9 genome editing) to determine its importance for reproductive success with special interest for egg production. Our results show that miR-202-5p is the most abundant mature form of the miRNA and that it is expressed in granulosa cells and in the unfertilized egg. The knock out (KO) of mir-202 gene resulted in a strong phenotype both in terms of number and quality of eggs produced. Mutant females exhibited either no egg production or produced a dramatically reduced number of eggs that could not be fertilized, ultimately leading to no reproductive success. We quantified the size distribution of the oocytes in the ovary of KO females and performed a large-scale transcriptomic analysis approach to identified dysregulated molecular pathways. Together, cellular and molecular analyses indicate that the lack of miR-202 impairs the early steps of oogenesis/folliculogenesis and decreases the number of large (i.e. vitellogenic) follicles, ultimately leading to dramatically reduced female fecundity. This study sheds new light on the regulatory mechanisms that control the early steps of follicular development, including possible targets of miR-202-5p, and provides the first in vivo functional evidence that a gonad-predominant microRNA may have a major role in female reproduction.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems
Female
Fertility/*genetics
Gene Editing
Gene Expression Profiling
*Gene Expression Regulation, Developmental
Gene Knockout Techniques
Granulosa Cells
Male
MicroRNAs/*physiology
Oocytes/growth & development/metabolism
Oogenesis/*genetics
Oryzias/*physiology
Ovary/cytology/growth & development/metabolism

@article {pmid30110323,
year = {2018},
author = {Ravi, P and Trivedi, D and Hasan, G},
title = {FMRFa receptor stimulated Ca2+ signals alter the activity of flight modulating central dopaminergic neurons in Drosophila melanogaster.},
journal = {PLoS genetics},
volume = {14},
number = {8},
pages = {e1007459},
pmid = {30110323},
issn = {1553-7404},
support = {P40 OD018537/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Calcium Signaling ; Dopaminergic Neurons/*physiology ; Drosophila Proteins/genetics/*physiology ; Drosophila melanogaster/genetics/*physiology ; Flight, Animal/*physiology ; Gene Expression Regulation ; Male ; Receptors, Invertebrate Peptide/genetics/*physiology ; },
abstract = {Neuropeptide signaling influences animal behavior by modulating neuronal activity and thus altering circuit dynamics. Insect flight is a key innate behavior that very likely requires robust neuromodulation. Cellular and molecular components that help modulate flight behavior are therefore of interest and require investigation. In a genetic RNAi screen for G-protein coupled receptors that regulate flight bout durations, we earlier identified several receptors, including the receptor for the neuropeptide FMRFa (FMRFaR). To further investigate modulation of insect flight by FMRFa we generated CRISPR-Cas9 mutants in the gene encoding the Drosophila FMRFaR. The mutants exhibit significant flight deficits with a focus in dopaminergic cells. Expression of a receptor specific RNAi in adult central dopaminergic neurons resulted in progressive loss of sustained flight. Further, genetic and cellular assays demonstrated that FMRFaR stimulates intracellular calcium signaling through the IP3R and helps maintain neuronal excitability in a subset of dopaminergic neurons for positive modulation of flight bout durations.},
}

Animals
CRISPR-Cas Systems
*Calcium Signaling
Dopaminergic Neurons/*physiology
Drosophila Proteins/genetics/*physiology
Drosophila melanogaster/genetics/*physiology
Flight, Animal/*physiology
Gene Expression Regulation
Male
Receptors, Invertebrate Peptide/genetics/*physiology

@article {pmid30076291,
year = {2018},
author = {Yang, D and Zheng, X and Chen, S and Wang, Z and Xu, W and Tan, J and Hu, T and Hou, M and Wang, W and Gu, Z and Wang, Q and Zhang, R and Zhang, Y and Liu, Q},
title = {Sensing of cytosolic LPS through caspy2 pyrin domain mediates noncanonical inflammasome activation in zebrafish.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3052},
pmid = {30076291},
issn = {2041-1723},
mesh = {Animals ; Bacterial Infections/*immunology ; CRISPR-Cas Systems ; Caspases/*drug effects/genetics/immunology/*metabolism ; Cytosol/*metabolism ; Disease Models, Animal ; Fibroblasts/immunology ; Gastrointestinal Tract/pathology ; Gene Knockdown Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Immunity, Innate ; Inflammasomes/*metabolism ; Inflammation ; Lipopolysaccharides/*toxicity ; NLR Family, Pyrin Domain-Containing 3 Protein ; Pyrin Domain/*physiology ; Pyroptosis/immunology ; Sepsis/microbiology ; Zebrafish/*immunology/microbiology ; Zebrafish Proteins/*drug effects/genetics/*metabolism ; },
abstract = {The noncanonical inflammasome is critical for cytosolic sensing of Gram-negative pathogens. Here, we show that bacterial infection induces caspy2 activation in zebrafish fibroblasts, which mediates pyroptosis via a caspase-5-like activity. Zebrafish caspy2 binds directly to lipopolysaccharide via the N-terminal pyrin death domain, resulting in caspy2 oligomerization, which is critical for pyroptosis. Furthermore, we show that caspy2 is highly expressed in the zebrafish gut and is activated during infection. Knockdown of caspy2 expression impairs the ability of zebrafish to restrict bacterial invasion in vivo, and protects larvae from lethal sepsis. Collectively, our results identify a crucial event in the evolution of pattern recognition into the death domain superfamily-mediated intracellular lipopolysaccharide-sensing pathway in innate immunity.},
}

Animals
Bacterial Infections/*immunology
CRISPR-Cas Systems
Caspases/*drug effects/genetics/immunology/*metabolism
Cytosol/*metabolism
Disease Models, Animal
Fibroblasts/immunology
Gastrointestinal Tract/pathology
Gene Knockdown Techniques
HEK293 Cells
HeLa Cells
Humans
Immunity, Innate
Inflammasomes/*metabolism
Inflammation
Lipopolysaccharides/*toxicity
NLR Family, Pyrin Domain-Containing 3 Protein
Pyrin Domain/*physiology
Pyroptosis/immunology
Sepsis/microbiology
Zebrafish/*immunology/microbiology
Zebrafish Proteins/*drug effects/genetics/*metabolism

@article {pmid29751067,
year = {2018},
author = {Shi, X and Kitano, A and Jiang, Y and Luu, V and Hoegenauer, KA and Nakada, D},
title = {Clonal expansion and myeloid leukemia progression modeled by multiplex gene editing of murine hematopoietic progenitor cells.},
journal = {Experimental hematology},
volume = {64},
number = {},
pages = {33-44.e5},
pmid = {29751067},
issn = {1873-2399},
support = {R01 DK107413/DK/NIDDK NIH HHS/United States ; R01 CA193235/CA/NCI NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; P30 AI036211/AI/NIAID NIH HHS/United States ; S10 RR024574/RR/NCRR NIH HHS/United States ; },
mesh = {Animals ; Bone Marrow Transplantation ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Clone Cells/*pathology ; Disease Models, Animal ; Disease Progression ; Female ; Gene Editing/*methods ; Genes, Neoplasm ; Hematopoietic Stem Cells/*pathology ; Humans ; INDEL Mutation ; Leukemia, Myeloid, Acute/genetics/*pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mutation ; Neoplastic Stem Cells/*pathology ; Preleukemia/genetics/pathology ; Ribonucleoproteins/genetics ; Specific Pathogen-Free Organisms ; },
abstract = {Recent advances in next-generation sequencing have identified novel mutations and revealed complex genetic architectures in human hematological malignancies. Moving forward, new methods to quickly generate animal models that recapitulate the complex genetics of human hematological disorders are needed to transform the genetic information to new therapies. Here, we used a ribonucleoprotein-based CRISPR/Cas9 system to model human clonal hematopoiesis of indeterminate potential and acute myeloid leukemia (AML). We edited multiple genes recurrently mutated in hematological disorders, including those encoding epigenetic regulators, transcriptional regulators, and signaling components in murine hematopoietic stem/progenitor cells. Tracking the clonal dynamics by sequencing the indels induced by CRISPR/Cas9 revealed clonal expansion in some recipient mice that progressed to AML initiated by leukemia-initiating cells. Our results establish that the CRISPR/Cas9-mediated multiplex mutagenesis can be used to engineer a variety of murine models of hematological malignancies with complex genetic architectures seen in human disease.},
}

Animals
Bone Marrow Transplantation
CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Clone Cells/*pathology
Disease Models, Animal
Disease Progression
Female
Gene Editing/*methods
Genes, Neoplasm
Hematopoietic Stem Cells/*pathology
Humans
INDEL Mutation
Leukemia, Myeloid, Acute/genetics/*pathology
Male
Mice
Mice, Inbred C57BL
Mutation
Neoplastic Stem Cells/*pathology
Preleukemia/genetics/pathology
Ribonucleoproteins/genetics
Specific Pathogen-Free Organisms

@article {pmid29468553,
year = {2018},
author = {Naert, T and Vleminckx, K},
title = {CRISPR/Cas9-Mediated Knockout of Rb1 in Xenopus tropicalis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1726},
number = {},
pages = {177-193},
doi = {10.1007/978-1-4939-7565-5_16},
pmid = {29468553},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Knockout Techniques/*methods ; *Genome ; Retinoblastoma Protein/*antagonists & inhibitors/genetics ; Xenopus/classification/*genetics ; },
abstract = {At this time, no molecular targeted therapies exist for treatment of retinoblastoma. This can be, in part, attributed to the lack of animal models that allow for both rapid identification of novel therapeutic targets and hypothesis driven drug testing. Within this scope, we have recently reported the first genuine genetic nonmammalian retinoblastoma cancer model within the aquatic model organism Xenopus tropicalis (Naert et al., Sci Rep 6: 35263, 2016). Here we describe the methods to generate rb1 mosaic mutant Xenopus tropicalis by employing the CRISPR/Cas9 technology. In depth, we discuss short guide RNA (sgRNA) design parameters, generation, quality control, quantification, and delivery followed by several methods for assessing genome editing efficiencies. As such the reader should be capable, by minor changes to the methods described here, to (co-) target rb1 or any one or multiple gene(s) within the Xenopus tropicalis genome by multiplex CRISPR/Cas9 methodology.},
}

Animals
*CRISPR-Cas Systems
Gene Knockout Techniques/*methods
*Genome
Retinoblastoma Protein/*antagonists & inhibitors/genetics
Xenopus/classification/*genetics

@article {pmid29177429,
year = {2017},
author = {Rahim, K and Huo, L and Li, C and Zhang, P and Basit, A and Xiang, B and Ting, B and Hao, X and Zhu, X},
title = {Identification of a basidiomycete-specific Vilse-like GTPase activating proteins (GAPs) and its roles in the production of virulence factors in Cryptococcus neoformans.},
journal = {FEMS yeast research},
volume = {17},
number = {8},
pages = {},
doi = {10.1093/femsyr/fox089},
pmid = {29177429},
issn = {1567-1364},
mesh = {Biomarkers ; CRISPR-Cas Systems ; Cell Wall/metabolism ; Cryptococcus neoformans/classification/*physiology ; GTPase-Activating Proteins/*genetics/*metabolism ; Gene Editing ; Gene Expression Regulation, Fungal ; Humans ; Melanins/metabolism ; Mutation ; Osmotic Pressure ; Phylogeny ; Signal Transduction ; Virulence Factors ; },
abstract = {Cryptococcus neoformans is a basidiomycetous pathogenic yeast that causes fatal infections in both immunocompetent and immunocompromised patients. Regulation on the production of its virulence factors is not fully understood. Here we reported the characterization of a gene, named CVH1(CNA06260), encoding a Drosophila Vilse-like RhoGAP homolog, which is hallmarked by three conserved functional domains: WW, MyTH4 and RhoGAP. Phylogenetic analysis suggests that CVH1 is highly conserved from protists to mammals and interestingly in basidiomycetes, but absent in plants or Ascomycota and other lower fungi. This phylogenetic distribution indicates an evolutionary link among these groups of organisms. Functional analyses demonstrated that CVH1 was involved in stress tolerance and virulence factor production. By disrupting CVH1, we created a second mutant cvh1Δ with the CRISPR-Cas9 editing tool. The mutant strain exhibited hypersensitivity to osmotic stress by 2 M sorbitol and NaCl, suggesting defects in the HOG signaling pathway and an interaction of Cvh1 with the HOG pathway. Hypersensitivity of cvh1Δ to 1% Congo red and 0.01% SDS suggests that the cell wall integrity was impaired in the mutant. And cvh1Δ hardly produced the pigment melanin and capsule. Our study for the first time demonstrates that the fungal Vilse-like RhoGAP CVH1 is an important regulator of multiple biological processes in C. neoformans, and provides novel insights into the regulatory circuit of stress resistance/cell wall integrity, and laccase and capsule synthesis in C. neoformans.},
}

Biomarkers
CRISPR-Cas Systems
Cell Wall/metabolism
Cryptococcus neoformans/classification/*physiology
GTPase-Activating Proteins/*genetics/*metabolism
Gene Editing
Gene Expression Regulation, Fungal
Humans
Melanins/metabolism
Mutation
Osmotic Pressure
Phylogeny
Signal Transduction
Virulence Factors

@article {pmid29145596,
year = {2017},
author = {Mans, R and Hassing, EJ and Wijsman, M and Giezekamp, A and Pronk, JT and Daran, JM and van Maris, AJA},
title = {A CRISPR/Cas9-based exploration into the elusive mechanism for lactate export in Saccharomyces cerevisiae.},
journal = {FEMS yeast research},
volume = {17},
number = {8},
pages = {},
doi = {10.1093/femsyr/fox085},
pmid = {29145596},
issn = {1567-1364},
mesh = {Bioreactors ; *CRISPR-Cas Systems ; Fermentation ; Gene Deletion ; Gene Editing ; Gene Knockout Techniques ; Glucose/metabolism ; Lactic Acid/*metabolism ; *Metabolic Engineering ; Metabolic Networks and Pathways ; Saccharomyces cerevisiae/*genetics/*metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S. cerevisiae strain was constructed with deletions in 25 genes encoding transport proteins, including the complete aqua(glycero)porin family and all known carboxylic acid transporters. The 25-deletion strain was then transformed with an expression cassette for Lactobacillus casei lactate dehydrogenase (LcLDH). In anaerobic, glucose-grown batch cultures this strain exhibited a lower specific growth rate (0.15 vs. 0.25 h-1) and biomass-specific lactate production rate (0.7 vs. 2.4 mmol g biomass-1 h-1) than an LcLDH-expressing reference strain. However, a comparison of the two strains in anaerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) showed identical lactate production rates. These results indicate that, although deletion of the 25 transporter genes affected the maximum specific growth rate, it did not impact lactate export rates when analysed at a fixed specific growth rate. The 25-deletion strain provides a first step towards a 'minimal transportome' yeast platform, which can be applied for functional analysis of specific (heterologous) transport proteins as well as for evaluation of metabolic engineering strategies.},
}

Bioreactors
*CRISPR-Cas Systems
Fermentation
Gene Deletion
Gene Editing
Gene Knockout Techniques
Glucose/metabolism
Lactic Acid/*metabolism
*Metabolic Engineering
Metabolic Networks and Pathways
Saccharomyces cerevisiae/*genetics/*metabolism
Saccharomyces cerevisiae Proteins/genetics/metabolism

@article {pmid29099923,
year = {2017},
author = {Borodina, I and Zhao, ZK},
title = {Editorial: Yeast cell factories for production of fuels and chemicals.},
journal = {FEMS yeast research},
volume = {17},
number = {8},
pages = {},
doi = {10.1093/femsyr/fox082},
pmid = {29099923},
issn = {1567-1364},
mesh = {*Biofuels ; CRISPR-Cas Systems ; Gene Editing ; Genome, Fungal ; Genomics/methods ; *Metabolic Engineering ; Saccharomyces cerevisiae/*physiology ; },
}

*Biofuels
CRISPR-Cas Systems
Gene Editing
Genome, Fungal
Genomics/methods
*Metabolic Engineering
Saccharomyces cerevisiae/*physiology

@article {pmid30639735,
year = {2019},
author = {Gürtler, S and Wolke, C and Otto, O and Heise, N and Scholz, F and Laporte, A and Elsner, M and Jörns, A and Weinert, S and Döring, M and Jansing, S and Gardemann, A and Lendeckel, U and Schild, L},
title = {Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions, and cellular proliferation.},
journal = {Biochimica et biophysica acta. Molecular and cell biology of lipids},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.bbalip.2019.01.006},
pmid = {30639735},
issn = {1879-2618},
abstract = {The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function and, more recently, with cellular proliferation. Tafazzin, an acyltransferase with key functions in CL remodeling determining actual CL composition, affects mitochondrial oxidative phosphorylation. Here, we show that the CRISPR-Cas9 mediated knock-out of tafazzin (Taz) is associated with substantial alterations of various mitochondrial and cellular characteristics in C6 glioma cells. The knock-out of tafazzin substantially changed the profile of fatty acids incorporated in CL and the distribution of molecular CL species. Taz knock-out was further associated with decreased capacity of oxidative phosphorylation that mainly originates from impaired complex I associated energy metabolism in C6 glioma cells. The lack of tafazzin switched energy metabolism from oxidative phosphorylation to glycolysis indicated by lower respiration rates, membrane potential and higher levels of mitochondria-derived reactive oxygen species but keeping the cellular ATP content unchanged. The impact of tafazzin on mitochondria was also indicated by altered morphology and arrangement in tafazzin deficient C6 glioma cells. In the cells we observed tafazzin-dependent changes in the distribution of cellular fatty acids as an indication of altered lipid metabolism as well as in stability/morphology. Most impressive is the dramatic reduction in cell proliferation in tafazzin deficient C6 glioma cells that is not mediated by reactive oxygen species. Our data clearly indicate that defects in CL phospholipid remodeling trigger a cascade of events including modifications in CL linked to subsequent alterations in mitochondrial and cellular functions.},
}

@article {pmid30639408,
year = {2019},
author = {Takeshita, D and Sato, M and Inanaga, H and Numata, T},
title = {Crystal Structures of Csm2 and Csm3 in the Type III-A CRISPR-Cas Effector Complex.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jmb.2019.01.009},
pmid = {30639408},
issn = {1089-8638},
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (Cas) genes encode CRISPR RNAs (crRNA) and Cas proteins, respectively, which play important roles in the adaptive immunity system (CRISPR-Cas system) in prokaryotes. The crRNA and Cas proteins form ribonucleoprotein effector complexes to capture and degrade invading genetic materials with base complementarity to the crRNA guide sequences. The Csm complex, a type III-A effector complex, comprises five Cas proteins (Csm1-Csm5) and a crRNA, which co-transcriptionally degrades invading DNA and RNA. Here we report the crystal structures of the Staphylococcus epidermidis Csm2 (SeCsm2) and Thermoplasma volcanium Csm3 (TvCsm3) at 2.4 and 2.7 Å resolutions, respectively. SeCsm2 adopts a monomeric globular fold by itself, in striking contrast to the previously reported Thermotoga maritima Csm2, which adopted an extended conformation and formed a dimeric structure. We propose that the globular monomeric form is the bona fide structure of Csm2. TvCsm3 forms a filamentous structure in the crystals. The molecular arrangement of TvCsm3 is similar to that of the stacked Cmr4 proteins in the Cmr complex, suggesting the functionally relevant architecture of the present Csm3 structure. We constructed model structures of the Csm complex, which revealed that Csm3 binds the crRNA and periodically deforms the crRNA-target duplex by a similar mechanism to that of Cmr4 in the Cmr complex. The model and mutational analysis suggest that the conserved lysine residue of Csm2 is important for target RNA binding, and Csm2 stabilizes the active structure of the Csm complex to facilitate the reaction.},
}

@article {pmid30639240,
year = {2018},
author = {Swarts, DC and Jinek, M},
title = {Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2018.11.021},
pmid = {30639240},
issn = {1097-4164},
abstract = {CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.},
}

@article {pmid30637337,
year = {2018},
author = {Bayer, K and Jahn, MT and Slaby, BM and Moitinho-Silva, L and Hentschel, U},
title = {Marine Sponges as Chloroflexi Hot Spots: Genomic Insights and High-Resolution Visualization of an Abundant and Diverse Symbiotic Clade.},
journal = {mSystems},
volume = {3},
number = {6},
pages = {},
doi = {10.1128/mSystems.00150-18},
pmid = {30637337},
issn = {2379-5077},
abstract = {Members of the widespread bacterial phylum Chloroflexi can dominate high-microbial-abundance (HMA) sponge microbiomes. In the Sponge Microbiome Project, Chloroflexi sequences amounted to 20 to 30% of the total microbiome of certain HMA sponge genera with the classes/clades SAR202, Caldilineae, and Anaerolineae being the most prominent. We performed metagenomic and single-cell genomic analyses to elucidate the functional gene repertoire of Chloroflexi symbionts of Aplysina aerophoba. Eighteen draft genomes were reconstructed and placed into phylogenetic context of which six were investigated in detail. Common genomic features of Chloroflexi sponge symbionts were related to central energy and carbon converting pathways, amino acid and fatty acid metabolism, and respiration. Clade-specific metabolic features included a massively expanded genomic repertoire for carbohydrate degradation in Anaerolineae and Caldilineae genomes, but only amino acid utilization by SAR202. While Anaerolineae and Caldilineae import cofactors and vitamins, SAR202 genomes harbor genes encoding components involved in cofactor biosynthesis. A number of features relevant to symbiosis were further identified, including CRISPR-Cas systems, eukaryote-like repeat proteins, and secondary metabolite gene clusters. Chloroflexi symbionts were visualized in the sponge extracellular matrix at ultrastructural resolution by the fluorescence in situ hybridization-correlative light and electron microscopy (FISH-CLEM) method. Carbohydrate degradation potential was reported previously for "Candidatus Poribacteria" and SAUL, typical symbionts of HMA sponges, and we propose here that HMA sponge symbionts collectively engage in degradation of dissolved organic matter, both labile and recalcitrant. Thus, sponge microbes may not only provide nutrients to the sponge host, but they may also contribute to dissolved organic matter (DOM) recycling and primary productivity in reef ecosystems via a pathway termed the sponge loop. IMPORTANCEChloroflexi represent a widespread, yet enigmatic bacterial phylum with few cultivated members. We used metagenomic and single-cell genomic approaches to characterize the functional gene repertoire of Chloroflexi symbionts in marine sponges. The results of this study suggest clade-specific metabolic specialization and that Chloroflexi symbionts have the genomic potential for dissolved organic matter (DOM) degradation from seawater. Considering the abundance and dominance of sponges in many benthic environments, we predict that the role of sponge symbionts in biogeochemical cycles is larger than previously thought.},
}

@article {pmid30545858,
year = {2018},
author = {Dzau, VJ and McNutt, M and Bai, C},
title = {Wake-up call from Hong Kong.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6420},
pages = {1215},
doi = {10.1126/science.aaw3127},
pmid = {30545858},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*ethics/*standards ; *Genome, Human ; Hong Kong ; Humans ; Stakeholder Participation ; },
}

*CRISPR-Cas Systems
Gene Editing/*ethics/*standards
*Genome, Human
Hong Kong
Humans
Stakeholder Participation

@article {pmid30511631,
year = {2018},
author = {Farooq, R and Hussain, K and Nazir, S and Javed, MR and Masood, N},
title = {CRISPR/Cas9; A robust technology for producing genetically engineered plants.},
journal = {Cellular and molecular biology (Noisy-le-Grand, France)},
volume = {64},
number = {14},
pages = {31-38},
pmid = {30511631},
issn = {1165-158X},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Genetic Engineering/*methods ; Mutation/genetics ; Plants, Genetically Modified/*genetics ; Transgenes ; },
abstract = {CRISPR/Cas9 is a technology evolved from modified type II immune system of bacteria and archaea. Exploitation of this bacterial immune system in all eukaryotes including plants may lead to site-specific targeted genome engineering. Genome engineering is objectively utilized to express/silence a trait harbouring gene in the plant genome. In this review, different genetic engineering techniques including classical breeding, RNAi and genetic transformation and synthetic sequence-specific nucleases (zinc finger nucleases; ZFNs and transcription activator-like effector nuclease; TALENs) techniques have been described and compared with advanced genome editing technique CRISPR/Cas9, on the basis of their merits and drawbacks. This revolutionary genome engineering technology has edge over all other approaches because of its simplicity, stability, specificity of the target and multiple genes can be engineered at a time. CRISPR/Cas9 requires only Cas9 endonuclease and single guide RNA, which are directly delivered into plant cells via either vector-mediated stable transformation or transient delivery of ribonucleoproteins (RNPs) and generate double-strand breaks (DSBs) at target site. These DSBs are further repaired by cell endogenous repairing pathways via HDR or NHEJ. The major advantage of CRISPR/Cas9 system is that engineered plants are considered Non-GM; can be achieved using in vitro expressed RNPs transient delivery. Different variants of Cas9 genes cloned in different plasmid vectors can be used to achieve different objectives of genome editing including double-stranded DNA break, single-stranded break, activate/repress the gene expression. Fusion of Cas9 with fluorescent protein can lead to visualize the expression of the CRISPR/Cas9 system. The applications of this technology in plant genome editing to improve different plant traits are comprehensively described.},
}

CRISPR-Cas Systems/*genetics
Gene Editing
Genetic Engineering/*methods
Mutation/genetics
Plants, Genetically Modified/*genetics
Transgenes

@article {pmid30417500,
year = {2019},
author = {Wahiduzzaman, M and Karnan, S and Ota, A and Hanamura, I and Murakami, H and Inoko, A and Rahman, ML and Hyodo, T and Konishi, H and Tsuzuki, S and Hosokawa, Y},
title = {Establishment and characterization of CRISPR/Cas9-mediated NF2-/- human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma.},
journal = {Cancer science},
volume = {110},
number = {1},
pages = {180-193},
doi = {10.1111/cas.13871},
pmid = {30417500},
issn = {1349-7006},
support = {//Hirose International Scholarship Foundation/ ; },
mesh = {Adolescent ; Adult ; Aged ; Aged, 80 and over ; Base Sequence ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Child, Preschool ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms/*genetics/metabolism/pathology ; Male ; Mesothelioma/*genetics/metabolism/pathology ; Middle Aged ; Neurofibromin 2/*genetics/metabolism ; Pleural Neoplasms/*genetics/metabolism/pathology ; Receptor, Fibroblast Growth Factor, Type 2/*genetics ; Sequence Homology, Nucleic Acid ; Young Adult ; },
abstract = {Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2-knockout human mesothelial cell line, MeT-5A (NF2-KO). In NF2-KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2-WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2-WT and NF2-KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c-Jun, and retinoblastoma (Rb) in NF2-KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2-KO clone. In addition, the disruption of FGFR2 in the NF2-KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c-Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2-negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2-positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.},
}

Adolescent
Adult
Aged
Aged, 80 and over
Base Sequence
*CRISPR-Cas Systems
Cell Line, Tumor
Cell Movement/genetics
Cell Proliferation/genetics
Child, Preschool
Female
Gene Expression Profiling
Gene Expression Regulation, Neoplastic
Humans
Lung Neoplasms/*genetics/metabolism/pathology
Male
Mesothelioma/*genetics/metabolism/pathology
Middle Aged
Neurofibromin 2/*genetics/metabolism
Pleural Neoplasms/*genetics/metabolism/pathology
Receptor, Fibroblast Growth Factor, Type 2/*genetics
Sequence Homology, Nucleic Acid
Young Adult

@article {pmid30326628,
year = {2018},
author = {Chen, G and Xiong, L and Wang, Y and He, L and Huang, R and Liao, L and Zhu, Z and Wang, Y},
title = {ITGB1b-Deficient Rare Minnows Delay Grass Carp Reovirus (GCRV) Entry and Attenuate GCRV-Triggered Apoptosis.},
journal = {International journal of molecular sciences},
volume = {19},
number = {10},
pages = {},
pmid = {30326628},
issn = {1422-0067},
support = {31721005 and 31702332//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Apoptosis ; CRISPR-Cas Systems ; Carps/*genetics/*virology ; Clathrin/metabolism ; Endocytosis ; Fish Diseases/*genetics/*virology ; Gene Knockout Techniques ; Integrin beta1/*genetics ; Phenotype ; Phylogeny ; Reoviridae/classification/*physiology ; *Virus Internalization ; },
abstract = {Integrin β-1 (ITGB1) is a transmembrane protein belonging to the integrin family and it plays an important role in viral entry. In this study, the itgb1b gene of the rare minnow, Gobiocypris rarus, was cloned and analyzed. To investigate the possible role of itgb1b on grass carp reovirus (GCRV) infection, we generated an ITGB1b-deficient rare minnow (ITGB1b-/-) using the CRISPR/Cas9 system. Following stimulation with GCRV, the survival time of the -ITGB1b-/- rare minnows was extended in comparison to the wild-type minnows. Moreover, the relative copy number of GCRV and the level of clathrin-mediated endocytosis-associated and apoptosis-related gene expression in the ITGB1b-/- rare minnows was significantly lower than that of the wild-type minnows. These results suggested that the absence of itgb1b reduced viral entry efficiency and the expression of apoptosis-related genes. Moreover, the data suggested that itgb1b played an important role in mediating the entry of viruses into the cells via clathrin. Therefore, these findings provide novel insight into the function of itgb1b in the process of GCRV infection.},
}

Animals
*Apoptosis
CRISPR-Cas Systems
Carps/*genetics/*virology
Clathrin/metabolism
Endocytosis
Fish Diseases/*genetics/*virology
Gene Knockout Techniques
Integrin beta1/*genetics
Phenotype
Phylogeny
Reoviridae/classification/*physiology
*Virus Internalization

@article {pmid30254261,
year = {2018},
author = {Serif, M and Dubois, G and Finoux, AL and Teste, MA and Jallet, D and Daboussi, F},
title = {One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3924},
pmid = {30254261},
issn = {2041-1723},
mesh = {Adenine Phosphoribosyltransferase/genetics/metabolism ; Algal Proteins/genetics/metabolism ; Amino Acid Sequence ; Base Sequence ; CRISPR-Cas Systems ; Diatoms/*genetics/metabolism ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Microalgae/genetics/metabolism ; Multienzyme Complexes/genetics/metabolism ; Orotate Phosphoribosyltransferase/genetics/metabolism ; Orotidine-5'-Phosphate Decarboxylase/genetics/metabolism ; Reproducibility of Results ; Ribonucleoproteins/genetics/metabolism ; Sequence Homology, Amino Acid ; Transfection/*methods ; },
abstract = {Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.},
}

Adenine Phosphoribosyltransferase/genetics/metabolism
Algal Proteins/genetics/metabolism
Amino Acid Sequence
Base Sequence
CRISPR-Cas Systems
Diatoms/*genetics/metabolism
Gene Editing/*methods
Gene Knockout Techniques/*methods
Microalgae/genetics/metabolism
Multienzyme Complexes/genetics/metabolism
Orotate Phosphoribosyltransferase/genetics/metabolism
Orotidine-5'-Phosphate Decarboxylase/genetics/metabolism
Reproducibility of Results
Ribonucleoproteins/genetics/metabolism
Sequence Homology, Amino Acid
Transfection/*methods

@article {pmid30110327,
year = {2018},
author = {Xiao, D and Wang, H and Hao, L and Guo, X and Ma, X and Qian, Y and Chen, H and Ma, J and Zhang, J and Sheng, W and Shou, W and Huang, G and Ma, D},
title = {The roles of SMYD4 in epigenetic regulation of cardiac development in zebrafish.},
journal = {PLoS genetics},
volume = {14},
number = {8},
pages = {e1007578},
pmid = {30110327},
issn = {1553-7404},
support = {P01 HL134599/HL/NHLBI NIH HHS/United States ; },
mesh = {Adolescent ; Animals ; CRISPR-Cas Systems ; Child ; Child, Preschool ; Cohort Studies ; Disease Models, Animal ; Embryonic Development/drug effects ; *Epigenesis, Genetic ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Heart/drug effects/embryology ; Heart Defects, Congenital/genetics ; Histone Deacetylase 1/genetics/physiology ; Histone-Lysine N-Methyltransferase/genetics/*physiology ; Humans ; Infant ; Male ; Mutation, Missense ; Protein Conformation ; Sequence Analysis, RNA ; Transcriptome ; Whole Exome Sequencing ; Zebrafish/embryology/*genetics ; Zebrafish Proteins/genetics/*physiology ; },
abstract = {SMYD4 belongs to a family of lysine methyltransferases. We analyzed the role of smyd4 in zebrafish development by generating a smyd4 mutant zebrafish line (smyd4L544Efs*1) using the CRISPR/Cas9 technology. The maternal and zygotic smyd4L544Efs*1 mutants demonstrated severe cardiac malformations, including defects in left-right patterning and looping and hypoplastic ventricles, suggesting that smyd4 was critical for heart development. Importantly, we identified two rare SMYD4 genetic variants in a 208-patient cohort with congenital heart defects. Both biochemical and functional analyses indicated that SMYD4(G345D) was pathogenic. Our data suggested that smyd4 functions as a histone methyltransferase and, by interacting with HDAC1, also serves as a potential modulator for histone acetylation. Transcriptome and bioinformatics analyses of smyd4L544Efs*1 and wild-type developing hearts suggested that smyd4 is a key epigenetic regulator involved in regulating endoplasmic reticulum-mediated protein processing and several important metabolic pathways in developing zebrafish hearts.},
}

Adolescent
Animals
CRISPR-Cas Systems
Child
Child, Preschool
Cohort Studies
Disease Models, Animal
Embryonic Development/drug effects
*Epigenesis, Genetic
Female
Gene Expression Profiling
Gene Expression Regulation, Developmental
Heart/drug effects/embryology
Heart Defects, Congenital/genetics
Histone Deacetylase 1/genetics/physiology
Histone-Lysine N-Methyltransferase/genetics/*physiology
Humans
Infant
Male
Mutation, Missense
Protein Conformation
Sequence Analysis, RNA
Transcriptome
Whole Exome Sequencing
Zebrafish/embryology/*genetics
Zebrafish Proteins/genetics/*physiology

@article {pmid30080860,
year = {2018},
author = {Torbey, P and Thierion, E and Collombet, S and de Cian, A and Desmarquet-Trin-Dinh, C and Dura, M and Concordet, JP and Charnay, P and Gilardi-Hebenstreit, P},
title = {Cooperation, cis-interactions, versatility and evolutionary plasticity of multiple cis-acting elements underlie krox20 hindbrain regulation.},
journal = {PLoS genetics},
volume = {14},
number = {8},
pages = {e1007581},
pmid = {30080860},
issn = {1553-7404},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems ; Chromatin/metabolism ; Early Growth Response Protein 2/*genetics/physiology ; Enhancer Elements, Genetic ; Evolution, Molecular ; *Gene Expression Regulation, Developmental ; Genetic Loci ; Morphogenesis/genetics ; Rhombencephalon/*metabolism ; Transcriptional Activation ; Zebrafish/embryology/*genetics ; },
abstract = {Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes.},
}

Amino Acid Sequence
Animals
CRISPR-Cas Systems
Chromatin/metabolism
Early Growth Response Protein 2/*genetics/physiology
Enhancer Elements, Genetic
Evolution, Molecular
*Gene Expression Regulation, Developmental
Genetic Loci
Morphogenesis/genetics
Rhombencephalon/*metabolism
Transcriptional Activation
Zebrafish/embryology/*genetics

@article {pmid29991786,
year = {2018},
author = {Callaway, E},
title = {Controversial CRISPR 'gene drives' tested in mammals for the first time.},
journal = {Nature},
volume = {559},
number = {7713},
pages = {164},
doi = {10.1038/d41586-018-05665-1},
pmid = {29991786},
issn = {1476-4687},
mesh = {Animals ; Animals, Wild/genetics ; *CRISPR-Cas Systems ; Culicidae/genetics ; Female ; Gene Drive Technology/*methods ; *Gene Editing ; Introduced Species ; Malaria/prevention & control/transmission ; Male ; Mice ; Pest Control, Biological/*methods ; Sex Characteristics ; Uncertainty ; },
}

Animals
Animals, Wild/genetics
*CRISPR-Cas Systems
Culicidae/genetics
Female
Gene Drive Technology/*methods
*Gene Editing
Introduced Species
Malaria/prevention & control/transmission
Male
Mice
Pest Control, Biological/*methods
Sex Characteristics
Uncertainty

@article {pmid29985941,
year = {2018},
author = {Iyer, V and Boroviak, K and Thomas, M and Doe, B and Riva, L and Ryder, E and Adams, DJ},
title = {No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice.},
journal = {PLoS genetics},
volume = {14},
number = {7},
pages = {e1007503},
pmid = {29985941},
issn = {1553-7404},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Biological Variation, Population/genetics ; CRISPR-Cas Systems/*genetics ; DNA Mutational Analysis/methods ; Female ; Gene Editing/*methods ; Genome/genetics ; Hair Color/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Monophenol Monooxygenase/*genetics ; Mutagenesis/*genetics ; Pedigree ; RNA, Guide/genetics ; Research Design ; Whole Genome Sequencing/*methods ; },
abstract = {CRISPR-Cas9 technologies have transformed genome-editing of experimental organisms and have immense therapeutic potential. Despite significant advances in our understanding of the CRISPR-Cas9 system, concerns remain over the potential for off-target effects. Recent studies have addressed these concerns using whole-genome sequencing (WGS) of gene-edited embryos or animals to search for de novo mutations (DNMs), which may represent candidate changes introduced by poor editing fidelity. Critically, these studies used strain-matched, but not pedigree-matched controls and thus were unable to reliably distinguish generational or colony-related differences from true DNMs. Here we used a trio design and whole genome sequenced 8 parents and 19 embryos, where 10 of the embryos were mutagenised with well-characterised gRNAs targeting the coat colour Tyrosinase (Tyr) locus. Detailed analyses of these whole genome data allowed us to conclude that if CRISPR mutagenesis were causing SNV or indel off-target mutations in treated embryos, then the number of these mutations is not statistically distinguishable from the background rate of DNMs occurring due to other processes.},
}

Animals
Biological Variation, Population/genetics
CRISPR-Cas Systems/*genetics
DNA Mutational Analysis/methods
Female
Gene Editing/*methods
Genome/genetics
Hair Color/genetics
Male
Mice
Mice, Inbred C57BL
Mice, Transgenic
Monophenol Monooxygenase/*genetics
Mutagenesis/*genetics
Pedigree
RNA, Guide/genetics
Research Design
Whole Genome Sequencing/*methods

@article {pmid29973717,
year = {2018},
author = {Zimmermann, M and Murina, O and Reijns, MAM and Agathanggelou, A and Challis, R and Tarnauskaitė, Ž and Muir, M and Fluteau, A and Aregger, M and McEwan, A and Yuan, W and Clarke, M and Lambros, MB and Paneesha, S and Moss, P and Chandrashekhar, M and Angers, S and Moffat, J and Brunton, VG and Hart, T and de Bono, J and Stankovic, T and Jackson, AP and Durocher, D},
title = {CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.},
journal = {Nature},
volume = {559},
number = {7713},
pages = {285-289},
pmid = {29973717},
issn = {1476-4687},
support = {MC_PC_U127580972//Medical Research Council/United Kingdom ; },
mesh = {Animals ; BRCA1 Protein/deficiency/genetics ; *CRISPR-Cas Systems ; Cell Line ; *DNA Damage/drug effects ; DNA Repair/genetics ; DNA Replication ; DNA Topoisomerases, Type I/metabolism ; Female ; *Gene Editing ; Genes, BRCA1 ; Genome/genetics ; HeLa Cells ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy/enzymology/genetics/pathology ; Male ; Mice ; Neoplasms/drug therapy/enzymology/*genetics/*pathology ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Poly (ADP-Ribose) Polymerase-1/deficiency/genetics/*metabolism ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Prostatic Neoplasms/drug therapy/enzymology/pathology ; Ribonuclease H/deficiency/genetics/metabolism ; Ribonucleotides/*genetics ; Synthetic Lethal Mutations ; Xenograft Model Antitumor Assays ; },
abstract = {The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1-4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.},
}

Animals
BRCA1 Protein/deficiency/genetics
*CRISPR-Cas Systems
Cell Line
*DNA Damage/drug effects
DNA Repair/genetics
DNA Replication
DNA Topoisomerases, Type I/metabolism
Female
*Gene Editing
Genes, BRCA1
Genome/genetics
HeLa Cells
Humans
Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy/enzymology/genetics/pathology
Male
Mice
Neoplasms/drug therapy/enzymology/*genetics/*pathology
Phthalazines/pharmacology
Piperazines/pharmacology
Poly (ADP-Ribose) Polymerase-1/deficiency/genetics/*metabolism
Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
Prostatic Neoplasms/drug therapy/enzymology/pathology
Ribonuclease H/deficiency/genetics/metabolism
Ribonucleotides/*genetics
Synthetic Lethal Mutations
Xenograft Model Antitumor Assays

@article {pmid29874289,
year = {2018},
author = {Xie, S and Cooley, A and Armendariz, D and Zhou, P and Hon, GC},
title = {Frequent sgRNA-barcode recombination in single-cell perturbation assays.},
journal = {PloS one},
volume = {13},
number = {6},
pages = {e0198635},
pmid = {29874289},
issn = {1932-6203},
support = {DP2 GM128203/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; K562 Cells ; Lentivirus/genetics ; RNA, Guide/*genetics ; Sequence Analysis, RNA ; Single-Cell Analysis/*methods ; Transfection/*methods ; },
abstract = {Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.},
}

CRISPR-Cas Systems/genetics
Genetic Vectors/genetics
HEK293 Cells
Humans
K562 Cells
Lentivirus/genetics
RNA, Guide/*genetics
Sequence Analysis, RNA
Single-Cell Analysis/*methods
Transfection/*methods

@article {pmid29684399,
year = {2018},
author = {Qian, P and Wang, X and Yang, Z and Li, Z and Gao, H and Su, XZ and Cui, H and Yuan, J},
title = {A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.},
journal = {Molecular and biochemical parasitology},
volume = {222},
number = {},
pages = {21-28},
doi = {10.1016/j.molbiopara.2018.04.003},
pmid = {29684399},
issn = {1872-9428},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems ; Female ; Gene Deletion ; Gene Editing/*methods ; Gene Knock-In Techniques ; Life Cycle Stages ; Malaria/parasitology/*veterinary ; Male ; Mice ; Mice, Inbred ICR ; Plasmids/genetics/metabolism ; Plasmodium yoelii/*genetics/growth & development/metabolism ; Rodent Diseases/*parasitology ; },
abstract = {The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii.},
}

Animals
CRISPR-Associated Protein 9/genetics/*metabolism
CRISPR-Cas Systems
Female
Gene Deletion
Gene Editing/*methods
Gene Knock-In Techniques
Life Cycle Stages
Malaria/parasitology/*veterinary
Male
Mice
Mice, Inbred ICR
Plasmids/genetics/metabolism
Plasmodium yoelii/*genetics/growth & development/metabolism
Rodent Diseases/*parasitology

@article {pmid28944418,
year = {2018},
author = {Nitika, and Truman, AW},
title = {Endogenous epitope tagging of heat shock protein 70 isoform Hsc70 using CRISPR/Cas9.},
journal = {Cell stress & chaperones},
volume = {23},
number = {3},
pages = {347-355},
pmid = {28944418},
issn = {1466-1268},
support = {R15 CA208773/CA/NCI NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; *Epitope Mapping ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; HSC70 Heat-Shock Proteins/genetics/*metabolism ; Humans ; Promoter Regions, Genetic/genetics ; Protein Binding ; Protein Isoforms/metabolism ; RNA, Guide/metabolism ; Reproducibility of Results ; },
abstract = {Heat shock protein 70 (Hsp70) is an evolutionarily well-conserved molecular chaperone involved in several cellular processes such as folding of proteins, modulating protein-protein interactions, and transport of proteins across the membrane. Binding partners of Hsp70 (known as "clients") are identified on an individual basis as researchers discover their particular protein of interest binds to Hsp70. A full complement of Hsp70 interactors under multiple stress conditions remains to be determined. A promising approach to characterizing the Hsp70 "interactome" is the use of protein epitope tagging and then affinity purification followed by mass spectrometry (AP-MS/MS). AP-MS analysis is a widely used method to decipher protein-protein interaction networks and identifying protein functions. Conventionally, the proteins are overexpressed ectopically which interferes with protein complex stoichiometry, skewing AP-MS/MS data. In an attempt to solve this issue, we used CRISPR/Cas9-mediated gene editing to integrate a tandem-affinity (TAP) epitope tag into the genomic locus of HSC70. This system offers several benefits over existing expression systems including native expression, no requirement for selection, and homogeneity between cells. This cell line, freely available to chaperone researchers, will aid in small and large-scale protein interaction studies as well as the study of biochemical activities and structure-function relationships of the Hsc70 protein.},
}

Base Sequence
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
DNA Breaks, Double-Stranded
*Epitope Mapping
Green Fluorescent Proteins/metabolism
HEK293 Cells
HSC70 Heat-Shock Proteins/genetics/*metabolism
Humans
Promoter Regions, Genetic/genetics
Protein Binding
Protein Isoforms/metabolism
RNA, Guide/metabolism
Reproducibility of Results

@article {pmid28117345,
year = {2017},
author = {Jiang, C and Mei, M and Li, B and Zhu, X and Zu, W and Tian, Y and Wang, Q and Guo, Y and Dong, Y and Tan, X},
title = {A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo.},
journal = {Cell research},
volume = {27},
number = {3},
pages = {440-443},
pmid = {28117345},
issn = {1748-7838},
support = {R35 GM119679/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA, Viral/*metabolism ; Gene Targeting ; HEK293 Cells ; Hepatitis B virus/*genetics ; Humans ; Lipids/chemistry ; Liver/metabolism ; Mice ; Mice, Inbred C57BL ; NIH 3T3 Cells ; Nanoparticles/chemistry ; Proprotein Convertase 9/*metabolism ; },
}

Animals
Base Sequence
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
DNA, Viral/*metabolism
Gene Targeting
HEK293 Cells
Hepatitis B virus/*genetics
Humans
Lipids/chemistry
Liver/metabolism
Mice
Mice, Inbred C57BL
NIH 3T3 Cells
Nanoparticles/chemistry
Proprotein Convertase 9/*metabolism

@article {pmid28084328,
year = {2017},
author = {Zhou, Y and Wang, P and Tian, F and Gao, G and Huang, L and Wei, W and Xie, XS},
title = {Painting a specific chromosome with CRISPR/Cas9 for live-cell imaging.},
journal = {Cell research},
volume = {27},
number = {2},
pages = {298-301},
pmid = {28084328},
issn = {1748-7838},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Survival ; *Chromosome Painting ; Chromosomes, Human, Pair 9/*genetics ; HeLa Cells ; Humans ; *Imaging, Three-Dimensional ; RNA, Guide/metabolism ; },
}

CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Cell Survival
*Chromosome Painting
Chromosomes, Human, Pair 9/*genetics
HeLa Cells
Humans
*Imaging, Three-Dimensional
RNA, Guide/metabolism

@article {pmid27910851,
year = {2017},
author = {Liu, X and Zhang, Y and Cheng, C and Cheng, AW and Zhang, X and Li, N and Xia, C and Wei, X and Liu, X and Wang, H},
title = {CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells.},
journal = {Cell research},
volume = {27},
number = {1},
pages = {154-157},
pmid = {27910851},
issn = {1748-7838},
mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Exons/genetics ; *Gene Editing ; Humans ; *Immunotherapy, Adoptive ; Mice ; RNA, Guide/metabolism ; T-Lymphocytes/*metabolism ; },
}

Animals
Base Sequence
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
Exons/genetics
*Gene Editing
Humans
*Immunotherapy, Adoptive
Mice
RNA, Guide/metabolism
T-Lymphocytes/*metabolism

@article {pmid30635671,
year = {2019},
author = {Schuster, JA and Vogel, RF and Ehrmann, MA},
title = {Characterization and distribution of CRISPR-Cas systems in Lactobacillus sakei.},
journal = {Archives of microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00203-019-01619-x},
pmid = {30635671},
issn = {1432-072X},
support = {18552 N//AiF/ ; },
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR)-Cas (CRISPR-associated) structures, known as prokaryotes 'immune system', have been successfully applied for genetic engineering and genotyping purposes for a variety of microorganisms. Here we investigated 50 Lactobacillus (L.) sakei genomes and found 13 of them as CRISPR-Cas positive. The majority of positive genomes contain type II-A system, which appears to be widespread across food born lactic acid bacteria. However, a type II-C system with low similarity in Cas protein sequence to related II-C structures is rarely present in the genomes. We depicted a correlation between prophages integrated in the genomes and the presence/absence of CRISPR-Cas systems and identified the novel protospacer adjunction motifs (PAMs) (a/g)AAA for the II-A and (g/a)(c/t)AC for the II-C system including the corresponding tracrRNAs, creating the basis for the development of new Cas-mediated genome editing tools. Moreover, we performed a PCR screening for 81 selected L. sakei isolates and identified 25 (31%) isolates as CRISPR-Cas positive with hypervariable spacer content. Comparative sequence analysis of 33 repeat-spacer arrays resulted in 18 CRISPR genotypes, revealing insights into evolutionary relationships between different strains and illustrating possible applications for the research and development of starter cultures, e.g., the usage for strain differentiation in assertiveness experiments or the development of bacteriophage-resistant strains.},
}

@article {pmid30633905,
year = {2019},
author = {Oakes, BL and Fellmann, C and Rishi, H and Taylor, KL and Ren, SM and Nadler, DC and Yokoo, R and Arkin, AP and Doudna, JA and Savage, DF},
title = {CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification.},
journal = {Cell},
volume = {176},
number = {1-2},
pages = {254-267.e16},
doi = {10.1016/j.cell.2018.11.052},
pmid = {30633905},
issn = {1097-4172},
abstract = {The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.},
}

@article {pmid30486613,
year = {2018},
author = {Lee, SH and Kim, S and Hur, JK},
title = {CRISPR and Target-Specific DNA Endonucleases for Efficient DNA Knock-in in Eukaryotic Genomes.},
journal = {Molecules and cells},
volume = {41},
number = {11},
pages = {943-952},
pmid = {30486613},
issn = {0219-1032},
mesh = {Animals ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/*genetics ; DNA Repair ; Deoxyribonuclease I/*metabolism ; Eukaryota/genetics ; Gene Editing ; Gene Knock-In Techniques/*methods ; Genetic Engineering ; Genetic Therapy ; Genome/*genetics ; Humans ; },
abstract = {The discovery and mechanistic understanding of target-specific genome engineering technologies has led to extremely effective and specific genome editing in higher organisms. Target-specific genetic modification technology is expected to have a leading position in future gene therapy development, and has a ripple effect on various basic and applied studies. However, several problems remain and hinder efficient and specific editing of target genomic loci. The issues are particularly critical in precise targeted insertion of external DNA sequences into genomes. Here, we discuss some recent efforts to overcome such problems and present a perspective of future genome editing technologies.},
}

Animals
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA/*genetics
DNA Repair
Deoxyribonuclease I/*metabolism
Eukaryota/genetics
Gene Editing
Gene Knock-In Techniques/*methods
Genetic Engineering
Genetic Therapy
Genome/*genetics
Humans

@article {pmid30232196,
year = {2018},
author = {Nachmanson, D and Lian, S and Schmidt, EK and Hipp, MJ and Baker, KT and Zhang, Y and Tretiakova, M and Loubet-Senear, K and Kohrn, BF and Salk, JJ and Kennedy, SR and Risques, RA},
title = {Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).},
journal = {Genome research},
volume = {28},
number = {10},
pages = {1589-1599},
pmid = {30232196},
issn = {1549-5469},
support = {R01 CA160674/CA/NCI NIH HHS/United States ; R01 CA181308/CA/NCI NIH HHS/United States ; R44 CA221426/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; DNA Fragmentation ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Ovarian Neoplasms/*genetics ; Sequence Analysis, DNA/*methods ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.},
}

*CRISPR-Cas Systems
DNA/genetics
DNA Fragmentation
Female
High-Throughput Nucleotide Sequencing
Humans
Ovarian Neoplasms/*genetics
Sequence Analysis, DNA/*methods
Tumor Suppressor Protein p53/*genetics

@article {pmid30208656,
year = {2018},
author = {Sun, Q and Lin, L and Liu, D and Wu, D and Fang, Y and Wu, J and Wang, Y},
title = {CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.},
journal = {International journal of molecular sciences},
volume = {19},
number = {9},
pages = {},
pmid = {30208656},
issn = {1422-0067},
support = {2016YFD0102000//National Key Research and Development Program of China/ ; 2015CB150201//National Key Basic Research Program of China/ ; 31330057//National Natural Science Foundation of China/ ; 31601330//National Natural Science Foundation of China/ ; 2015M581867//China Postdoctoral Science Foundation/ ; 2016T90514//China Postdoctoral Science Foundation/ ; },
mesh = {Ascomycota/physiology ; Brassica napus/*genetics ; *CRISPR-Cas Systems ; Disease Resistance ; *Gene Editing ; Gene Expression Regulation, Plant ; Genome, Plant ; Mutagenesis ; Mutation ; Plant Diseases/*genetics/microbiology ; Plant Proteins/*genetics ; Plants, Genetically Modified/*genetics ; Transcription Factors/*genetics ; },
abstract = {Targeted genome editing is a desirable means of basic science and crop improvement. The clustered, regularly interspaced, palindromic repeat (CRISPR)/Cas9 (CRISPR-associated 9) system is currently the simplest and most commonly used system in targeted genomic editing in plants. Single and multiplex genome editing in plants can be achieved under this system. In Arabidopsis, AtWRKY11 and AtWRKY70 genes were involved in JA- and SA-induced resistance to pathogens, in rapeseed (Brassica napus L.), BnWRKY11 and BnWRKY70 genes were found to be differently expressed after inoculated with the pathogenic fungus, Sclerotinia sclerotiorum (Lib.) de Bary. In this study, two Cas9/sgRNA constructs targeting two copies of BnWRKY11 and four copies of BnWRKY70 were designed to generate BnWRKY11 and BnWRKY70 mutants respectively. As a result, twenty-two BnWRKY11 and eight BnWRKY70 independent transformants (T₀) were obtained, with the mutation ratios of 54.5% (12/22) and 50% (4/8) in BnWRKY11 and BnWRKY70 transformants respectively. Eight and two plants with two copies of mutated BnWRKY11 and BnWRKY70 were obtained respectively. In T₁ generation of each plant examined, new mutations on target genes were detected with high efficiency. The vast majority of BnWRKY70 mutants showed editing in three copies of BnWRKY70 in examined T₁ plants. BnWRKY70 mutants exhibited enhanced resistance to Sclerotinia, while BnWRKY11 mutants showed no significant difference in Sclerotinia resistance when compared to non-transgenic plants. In addition, plants that overexpressed BnWRKY70 showed increased sensitivity when compared to non-transgenic plants. Altogether, our results demonstrated that BnWRKY70 may function as a regulating factor to negatively control the Sclerotinia resistance and CRISPR/Cas9 system could be used to generate germplasm in B. napus with high resistance against Sclerotinia.},
}

Ascomycota/physiology
Brassica napus/*genetics
*CRISPR-Cas Systems
Disease Resistance
*Gene Editing
Gene Expression Regulation, Plant
Genome, Plant
Mutagenesis
Mutation
Plant Diseases/*genetics/microbiology
Plant Proteins/*genetics
Plants, Genetically Modified/*genetics
Transcription Factors/*genetics

@article {pmid30175907,
year = {2018},
author = {Alper, HS and Beisel, CL},
title = {Advances in CRISPR Technologies for Microbial Strain Engineering.},
journal = {Biotechnology journal},
volume = {13},
number = {9},
pages = {e1800460},
doi = {10.1002/biot.201800460},
pmid = {30175907},
issn = {1860-7314},
mesh = {Bacteria/*genetics ; CRISPR-Cas Systems ; Fungi/*genetics ; Gene Editing/methods ; Genetic Engineering/*methods ; },
}

Bacteria/*genetics
CRISPR-Cas Systems
Fungi/*genetics
Gene Editing/methods
Genetic Engineering/*methods

@article {pmid30111655,
year = {2018},
author = {Liu, K and Yu, W and Tang, M and Tang, J and Liu, X and Liu, Q and Li, Y and He, L and Zhang, L and Evans, SM and Tian, X and Lui, KO and Zhou, B},
title = {A dual genetic tracing system identifies diverse and dynamic origins of cardiac valve mesenchyme.},
journal = {Development (Cambridge, England)},
volume = {145},
number = {18},
pages = {},
doi = {10.1242/dev.167775},
pmid = {30111655},
issn = {1477-9129},
mesh = {Animals ; Antigens, CD/metabolism ; CRISPR-Cas Systems/genetics ; Cadherins/metabolism ; DNA Nucleotidyltransferases/*metabolism ; Endothelial Cells/cytology ; Gene Knock-In Techniques ; Genetic Engineering/*methods ; Heart Valves/*embryology ; Mesoderm/*embryology ; Mice ; Mice, Inbred C57BL ; },
abstract = {In vivo genomic engineering is instrumental for studying developmental biology and regenerative medicine. Development of novel systems with more site-specific recombinases (SSRs) that complement with the commonly used Cre-loxP would be valuable for more precise lineage tracing and genome editing. Here, we introduce a new SSR system via Nigri-nox. By generating tissue-specific Nigri knock-in and its responding nox reporter mice, we show that the Nigri-nox system works efficiently in vivo by targeting specific tissues. As a new orthogonal system to Cre-loxP, Nigri-nox provides an additional control of genetic manipulation. We also demonstrate how the two orthogonal systems Nigri-nox and Cre-loxP could be used simultaneously to map the cell fate of two distinct developmental origins of cardiac valve mesenchyme in the mouse heart, providing dynamics of cellular contribution from different origins for cardiac valve mesenchyme during development. This work provides a proof-of-principle application of the Nigri-nox system for in vivo mouse genomic engineering. Coupled with other SSR systems, Nigri-nox would be valuable for more precise delineation of origins and cell fates during development, diseases and regeneration.},
}

Animals
Antigens, CD/metabolism
CRISPR-Cas Systems/genetics
Cadherins/metabolism
DNA Nucleotidyltransferases/*metabolism
Endothelial Cells/cytology
Gene Knock-In Techniques
Genetic Engineering/*methods
Heart Valves/*embryology
Mesoderm/*embryology
Mice
Mice, Inbred C57BL

@article {pmid29969727,
year = {2018},
author = {Ryu, N and Kim, MA and Park, D and Lee, B and Kim, YR and Kim, KH and Baek, JI and Kim, WJ and Lee, KY and Kim, UK},
title = {Effective PEI-mediated delivery of CRISPR-Cas9 complex for targeted gene therapy.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {14},
number = {7},
pages = {2095-2102},
doi = {10.1016/j.nano.2018.06.009},
pmid = {29969727},
issn = {1549-9642},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Proliferation ; *Drug Delivery Systems ; *Genetic Therapy ; Mice ; Neuroblastoma/genetics/*therapy ; *Plasmids ; Polyethyleneimine/*chemistry ; Sulfate Transporters/*antagonists & inhibitors/genetics ; Tumor Cells, Cultured ; },
abstract = {The-state-of-art CRISPR/Cas9 is one of the most powerful among the approaches being developed to rescue fundamental causes of gene-based inheritable diseases. Several strategies for delivering such genome editing materials have been developed, but the safety, efficacy over time, cost of production, and gene size limitations are still under debate and must be addressed to further improve applications. In this study, we evaluated branched forms of the polyethylenimine (PEI) - branched PEI 25 kDa (BPEI-25K) - and found that it could efficiently deliver CRISPR/Cas9 plasmids. Plasmid DNA expressing both guide RNA and Cas9 to target the Slc26a4 locus was successfully delivered into Neuro2a cells and meditated genome editing within the targeted locus. Our results demonstrated that BPEI-25K is a promising non-viral vector to deliver the CRISPR/Cas9 system in vitro to mediate targeted gene therapy, and these findings contribute to an understanding of CRISPR/Cas9 delivery that may enable development of successful in vivo techniques.},
}

Animals
*CRISPR-Cas Systems
Cell Proliferation
*Drug Delivery Systems
*Genetic Therapy
Mice
Neuroblastoma/genetics/*therapy
*Plasmids
Polyethyleneimine/*chemistry
Sulfate Transporters/*antagonists & inhibitors/genetics
Tumor Cells, Cultured

@article {pmid29950674,
year = {2018},
author = {Lu, IL and Chen, C and Tung, CY and Chen, HH and Pan, JP and Chang, CH and Cheng, JS and Chen, YA and Wang, CH and Huang, CW and Kang, YN and Chang, HY and Li, LL and Chang, KP and Shih, YH and Lin, CH and Kwan, SY and Tsai, JW},
title = {Identification of genes associated with cortical malformation using a transposon-mediated somatic mutagenesis screen in mice.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {2498},
pmid = {29950674},
issn = {2041-1723},
mesh = {Adolescent ; Adult ; Animals ; Biomarkers/analysis ; CRISPR-Cas Systems ; Cerebral Cortex/*abnormalities/cytology/diagnostic imaging/pathology ; Child ; Child, Preschool ; DNA Transposable Elements/genetics ; Disease Models, Animal ; Epilepsy/diagnosis/*genetics/pathology ; Female ; Functional Neuroimaging ; Gene Knockdown Techniques ; Genetic Testing/*methods ; Humans ; Male ; Malformations of Cortical Development, Group I/diagnosis/*genetics/pathology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mutagenesis/genetics ; Mutation ; Neurons/*pathology ; RNA Interference ; RNA, Small Interfering/metabolism ; Young Adult ; },
abstract = {Mutations in genes involved in the production, migration, or differentiation of cortical neurons often lead to malformations of cortical development (MCDs). However, many genetic mutations involved in MCD pathogenesis remain unidentified. Here we developed a genetic screening paradigm based on transposon-mediated somatic mutagenesis by in utero electroporation and the inability of mutant neuronal precursors to migrate to the cortex and identified 33 candidate MCD genes. Consistent with the screen, several genes have already been implicated in neural development and disorders. Functional disruption of the candidate genes by RNAi or CRISPR/Cas9 causes altered neuronal distributions that resemble human cortical dysplasia. To verify potential clinical relevance of these candidate genes, we analyzed somatic mutations in brain tissue from patients with focal cortical dysplasia and found that mutations are enriched in these candidate genes. These results demonstrate that this approach is able to identify potential mouse genes involved in cortical development and MCD pathogenesis.},
}

Adolescent
Adult
Animals
Biomarkers/analysis
CRISPR-Cas Systems
Cerebral Cortex/*abnormalities/cytology/diagnostic imaging/pathology
Child
Child, Preschool
DNA Transposable Elements/genetics
Disease Models, Animal
Epilepsy/diagnosis/*genetics/pathology
Female
Functional Neuroimaging
Gene Knockdown Techniques
Genetic Testing/*methods
Humans
Male
Malformations of Cortical Development, Group I/diagnosis/*genetics/pathology
Mice
Mice, Inbred C57BL
Mice, Inbred ICR
Mutagenesis/genetics
Mutation
Neurons/*pathology
RNA Interference
RNA, Small Interfering/metabolism
Young Adult

@article {pmid29917323,
year = {2018},
author = {Cao, M and Gao, M and Ploessl, D and Song, C and Shao, Z},
title = {CRISPR-Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis.},
journal = {Biotechnology journal},
volume = {13},
number = {9},
pages = {e1700598},
doi = {10.1002/biot.201700598},
pmid = {29917323},
issn = {1860-7314},
support = {4782024//Iowa Energy Center/ ; EEC-0813570//National Science Foundation/ ; EPS-1101284//National Science Foundation/ ; MCB 1716837//National Science Foundation/ ; //Regents Innovation Fund/ ; },
mesh = {CRISPR-Cas Systems ; DNA End-Joining Repair ; Fungal Proteins/*genetics ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Gene Knockout Techniques ; Saccharomycetales/*genetics ; },
abstract = {Scheffersomyces stipitis, renowned for its native xylose-utilizing capacity, has recently demonstrated its potential in producing health-promoting shikimate pathway derivatives. However, its broader application is hampered by the low transformation efficiency and the lack of genetic engineering tools to enable sophisticated genomic manipulations. S. stipitis employs the predominant non-homologous end joining (NHEJ) mechanism for repairing DNA double-strand breaks (DSB), which is less desired due to its incompetence in achieving precise genome editing. Using CRISPR technology, here a ku70Δku80Δ deficient strain in which homologous recombination (HR)-based genome editing appeared dominant for the first time in S. stipitis is constructed. To build all essential tools for efficiently manipulating this highly promising nonconventional microbial host, the gene knockdown tool is also established, and repression efficiency is improved by incorporating a transcriptional repressor Mxi1 into the CRISPR-dCas9 platform. All these results are obtained with the improved transformation efficiency, which is 191-fold higher than that obtained with the traditional parameters used in yeast transformation. This work paves the way for advancing a new microbial chassis and provides a guideline for developing efficient CRISPR tools in other nonconventional yeasts.},
}

CRISPR-Cas Systems
DNA End-Joining Repair
Fungal Proteins/*genetics
Gene Editing/*methods
Gene Expression Regulation, Bacterial
Gene Knockout Techniques
Saccharomycetales/*genetics

@article {pmid29864154,
year = {2018},
author = {Stratigopoulos, G and De Rosa, MC and LeDuc, CA and Leibel, RL and Doege, CA},
title = {DMSO increases efficiency of genome editing at two non-coding loci.},
journal = {PloS one},
volume = {13},
number = {6},
pages = {e0198637},
pmid = {29864154},
issn = {1932-6203},
support = {P30 DK026687/DK/NIDDK NIH HHS/United States ; R01 DK052431/DK/NIDDK NIH HHS/United States ; },
mesh = {Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics ; CRISPR-Cas Systems/*genetics ; Cell Line ; Dimethyl Sulfoxide/*chemistry ; Gene Editing/*methods ; Genetic Loci/*genetics ; Human Embryonic Stem Cells ; Humans ; Introns/*genetics ; Polymorphism, Single Nucleotide/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) has become the tool of choice for genome editing. Despite the fact that it has evolved as a highly efficient means to edit/replace coding sequence, CRISPR/Cas9 efficiency for "clean" editing of non-coding DNA remains low. We set out to introduce a single base-pair substitution in two intronic SNPs at the FTO locus without altering nearby non-coding sequence. Substitution efficiency increased up to 10-fold by treatment of human embryonic stem cells (ESC) with non-toxic levels of DMSO (1%) before CRISPR/Cas9 delivery. Treatment with DMSO did not result in CRISPR/Cas9 off-target effects or compromise the chromosomal stability of the ESC. Twenty-four hour treatment of human ESC with DMSO before CRISPR/Cas9 delivery may prove a simple means to increase editing efficiency of non-coding DNA without incorporation of undesirable mutations.},
}

Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics
CRISPR-Cas Systems/*genetics
Cell Line
Dimethyl Sulfoxide/*chemistry
Gene Editing/*methods
Genetic Loci/*genetics
Human Embryonic Stem Cells
Humans
Introns/*genetics
Polymorphism, Single Nucleotide/genetics

@article {pmid29862648,
year = {2018},
author = {Zhao, Y and Li, L and Zheng, G and Jiang, W and Deng, Z and Wang, Z and Lu, Y},
title = {CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces.},
journal = {Biotechnology journal},
volume = {13},
number = {9},
pages = {e1800121},
doi = {10.1002/biot.201800121},
pmid = {29862648},
issn = {1860-7314},
support = {ZSYS-016//The Derivative Bank of Chinese Biological Resources, CAS/ ; 31570072//The National Natural Science Foundation of China/ ; 31770088//The National Natural Science Foundation of China/ ; 16490712100//The Science and Technology Commission of Shanghai Municipality/ ; },
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems ; *Down-Regulation ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Metabolic Engineering ; Promoter Regions, Genetic ; Streptomyces coelicolor/genetics/*growth & development ; },
abstract = {Streptomycetes are Gram-positive bacteria with the capacity to produce copious bioactive secondary metabolites, which are the main source of medically and industrially relevant drugs. However, genetic manipulation of Streptomyces strains is much more difficult than other model microorganisms like Escherichia coli and Saccharomyces cerevisiae. Recently, CRISPR/Cas9 or dCas9-mediated genetic manipulation tools have been developed and facilitated Streptomyces genome editing. However, till now, CRISPR/dCas9-based interference system (CRISPRi) is only designed to repress single gene expression. Herein, the authors developed a novel CRISPRi system for multiplex gene repression in the model strain Streptomyces coelicolor. In this system, the integrative plasmid pSET152 is used as the backbone for the expression of the dCas9/sgRNA complex and both dCas9 and sgRNAs are designed to be under the control of constitutive promoters. Using the integrative CRISPRi system, the authors achieved efficient repression of multiple genes simultaneously; the mRNA levels of four targets are reduced to 2-32% of the control. Furthermore, it is successfully employed for functional gene screening, and an orphan response regulator (RR) (encoded by SCO2013) containing an RNA-binding ANTAR domain is identified being involved in bacterial growth. Collectively, this integrative CRISPRi system is very effective for multiplex gene repression in S. coelicolor, which could be extended to other Streptomyces strains for functional gene screening as well as for metabolic engineering.},
}

Bacterial Proteins/genetics
CRISPR-Cas Systems
*Down-Regulation
Gene Editing/*methods
Gene Expression Regulation, Bacterial
Metabolic Engineering
Promoter Regions, Genetic
Streptomyces coelicolor/genetics/*growth & development

@article {pmid29809313,
year = {2018},
author = {Gao, D and Smith, S and Spagnuolo, M and Rodriguez, G and Blenner, M},
title = {Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.},
journal = {Biotechnology journal},
volume = {13},
number = {9},
pages = {e1700590},
doi = {10.1002/biot.201700590},
pmid = {29809313},
issn = {1860-7314},
support = {NNX15AU46G//National Aeronautics and Space Administration/ ; 1403099//Division of Chemical, Bioengineering, Environmental, and Transport Systems/ ; 1706134//Division of Chemical, Bioengineering, Environmental, and Transport Systems/ ; },
mesh = {CRISPR-Cas Systems ; DNA End-Joining Repair ; Fungal Proteins/*genetics ; Gene Editing/*methods ; Yarrowia/*genetics ; },
abstract = {CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica.},
}

CRISPR-Cas Systems
DNA End-Joining Repair
Fungal Proteins/*genetics
Gene Editing/*methods
Yarrowia/*genetics

@article {pmid29480475,
year = {2018},
author = {Grenier, A and Sujobert, P and Olivier, S and Guermouche, H and Mondésir, J and Kosmider, O and Viollet, B and Tamburini, J},
title = {Knockdown of Human AMPK Using the CRISPR/Cas9 Genome-Editing System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1732},
number = {},
pages = {171-194},
doi = {10.1007/978-1-4939-7598-3_11},
pmid = {29480475},
issn = {1940-6029},
mesh = {AMP-Activated Protein Kinases/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Knockdown Techniques/*methods ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Protein Subunits/genetics ; RNA, Guide/genetics ; Signal Transduction/genetics ; Transfection/methods ; },
abstract = {AMP-activated protein kinase (AMPK) is a critical energy sensor, regulating signaling networks involved in pathology including metabolic diseases and cancer. This increasingly recognized role of AMPK has prompted tremendous research efforts to develop new pharmacological AMPK activators. To precisely study the role of AMPK, and the specificity and activity of AMPK activators in cellular models, genetic AMPK inactivating tools are required. We report here methods for genetic inactivation of AMPK α1/α2 catalytic subunits in human cell lines by the CRISPR/Cas9 technology, a recent breakthrough technique for genome editing.},
}

AMP-Activated Protein Kinases/*genetics
CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Gene Knockdown Techniques/*methods
Genetic Vectors/genetics
HEK293 Cells
Humans
Lentivirus/genetics
Protein Subunits/genetics
RNA, Guide/genetics
Signal Transduction/genetics
Transfection/methods

@article {pmid29330802,
year = {2018},
author = {Xin, Y and Duan, C},
title = {Microinjection of Antisense Morpholinos, CRISPR/Cas9 RNP, and RNA/DNA into Zebrafish Embryos.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1742},
number = {},
pages = {205-211},
doi = {10.1007/978-1-4939-7665-2_18},
pmid = {29330802},
issn = {1940-6029},
mesh = {Animals ; CRISPR-Cas Systems ; Embryonic Development ; Female ; Gene Expression Regulation, Developmental ; Genetic Engineering/*instrumentation ; Microinjections/*methods ; Models, Animal ; Morpholinos/administration & dosage ; Oligonucleotides, Antisense/administration & dosage ; RNA, Guide/administration & dosage ; Ribonucleoproteins/administration & dosage ; Thionucleotides ; Zebrafish/*embryology/genetics/growth & development ; Zygote/growth & development ; },
abstract = {In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.},
}

Animals
CRISPR-Cas Systems
Embryonic Development
Female
Gene Expression Regulation, Developmental
Genetic Engineering/*instrumentation
Microinjections/*methods
Models, Animal
Morpholinos/administration & dosage
Oligonucleotides, Antisense/administration & dosage
RNA, Guide/administration & dosage
Ribonucleoproteins/administration & dosage
Thionucleotides
Zebrafish/*embryology/genetics/growth & development
Zygote/growth & development

@article {pmid29329593,
year = {2018},
author = {Sun, S and Xiao, J and Huo, J and Geng, Z and Ma, K and Sun, X and Fu, X},
title = {Targeting ectodysplasin promotor by CRISPR/dCas9-effector effectively induces the reprogramming of human bone marrow-derived mesenchymal stem cells into sweat gland-like cells.},
journal = {Stem cell research & therapy},
volume = {9},
number = {1},
pages = {8},
pmid = {29329593},
issn = {1757-6512},
mesh = {Animals ; Bone Marrow Cells/cytology ; Burns/*therapy ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line ; Cell- and Tissue-Based Therapy/*methods ; Cellular Reprogramming Techniques/*methods ; Cyclin D1/metabolism ; Disease Models, Animal ; Doxycycline/pharmacology ; Ectodysplasins/biosynthesis/*genetics ; Gene Editing ; HEK293 Cells ; Hedgehog Proteins/metabolism ; Humans ; Male ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stem Cells/*cytology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Promoter Regions, Genetic/genetics ; RNA, Guide/genetics ; Sweat Glands/*cytology ; },
abstract = {BACKGROUND: Patients with a deep burn injury are characterized by losing the function of perspiration and being unable to regenerate the sweat glands. Because of their easy accession, multipotency, and lower immunogenicity, bone marrow-derived mesenchymal stem cells (BM-MSCs) represent as an ideal biological source for cell therapy. The aim of this study was to identify whether targeting the promotor of ectodysplasin (EDA) by CRISPR/dCas9-effector (dCas9-E) could induce the BM-MSCs to differentiate into sweat gland-like cells (SGCs).

METHODS: Activation of EDA transcription in BM-MSCs was attained by transfection of naive BM-MSCs with the lenti-CRISPR/dCas9-effector and single-guide RNAs (sgRNAs). The impact of dCas9-E BM-MSCs on the formation of SGCs and repair of burn injury was identified and evaluated both in vitro and in a mouse model.

RESULTS: After transfection with sgRNA-guided dCas9-E, the BM-MSCs acquired significantly higher transcription and expression of EDA by doxycycline (Dox) induction. Intriguingly, the specific markers (CEA, CK7, CK14, and CK19) of sweat glands were also positive in the transfected BM-MSCs, suggesting that EDA plays a critical role in promoting BM-MSC differentiation into sweat glands. Furthermore, when the dCas9-E BM-MSCs with Dox induction were implanted into a wound in a laboratory animal model, iodine-starch perspiration tests revealed that the treated paws were positive for perspiration, while the paws treated with saline showed a negative manifestation. For the regulatory mechanism, the expression of downstream genes of NF-κB (Shh and cyclin D1) was also enhanced accordingly.

CONCLUSIONS: These results suggest that EDA is a pivotal factor for sweat gland regeneration from BM-MSCs and may also offer a new approach for destroyed sweat glands and extensive deep burns.},
}

Animals
Bone Marrow Cells/cytology
Burns/*therapy
CRISPR-Cas Systems
Cell Differentiation/genetics
Cell Line
Cell- and Tissue-Based Therapy/*methods
Cellular Reprogramming Techniques/*methods
Cyclin D1/metabolism
Disease Models, Animal
Doxycycline/pharmacology
Ectodysplasins/biosynthesis/*genetics
Gene Editing
HEK293 Cells
Hedgehog Proteins/metabolism
Humans
Male
*Mesenchymal Stem Cell Transplantation
Mesenchymal Stem Cells/*cytology
Mice
Mice, Inbred BALB C
Mice, Nude
Promoter Regions, Genetic/genetics
RNA, Guide/genetics
Sweat Glands/*cytology

@article {pmid30628160,
year = {2019},
author = {Hai, L and Szwarc, MM and Lanza, DG and Heaney, JD and Lydon, JP},
title = {Using CRISPR/Cas9 engineering to generate a mouse with a conditional knockout allele for the promyelocytic leukemia zinc finger transcription factor.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {},
number = {},
pages = {e23281},
doi = {10.1002/dvg.23281},
pmid = {30628160},
issn = {1526-968X},
abstract = {The promyelocytic leukemia zinc finger (PLZF) transcription factor mediates a wide-range of biological processes. Accordingly, perturbation of PLZF function results in a myriad of physiologic defects, the most conspicuous of which is abnormal skeletal patterning. Although whole body knockout of Plzf in the mouse (PlzfKO) has significantly expanded our understanding of Plzf function in vivo, a conditional knockout mouse model that enables tissue or cell-type specific ablation of Plzf has not been developed. Therefore, we used CRISPR/Cas 9 gene editing to generate a mouse model in which exon 2 of the murine Plzf gene is specifically flanked (or floxed) by LoxP sites (Plzff/f). Crossing our Plzff/f mouse with a global cre-driver mouse to generate the Plzfd/d bigenic mouse, we demonstrate that exon 2 of the Plzf gene is ablated in the Plzfd/d bigenic. Similar to the previously reported PlzfKO mouse, the Plzfd/d mouse exhibits a severe defect in skeletal patterning of the hindlimb, indicating that the Plzff/f mouse functions as designed. Therefore, studies in this short technical report demonstrate that the Plzff/f mouse will be useful to investigators who wish to explore the role of the Plzf transcription factor in a specific tissue or cell-type. This article is protected by copyright. All rights reserved.},
}

@article {pmid30298816,
year = {2018},
author = {Libby, AR and Joy, DA and So, PL and Mandegar, MA and Muncie, JM and Mendoza-Camacho, FN and Weaver, VM and Conklin, BR and McDevitt, TC},
title = {Spatiotemporal mosaic self-patterning of pluripotent stem cells using CRISPR interference.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30298816},
issn = {2050-084X},
support = {P01 HL089707/HL/NHLBI NIH HHS/United States ; Center Core Grant, NIH P30 AI027763/NH/NIH HHS/United States ; LAI-C1408015//California Institute for Regenerative Medicine/International ; P01HL089707/NH/NIH HHS/United States ; U01 HL100406/HL/NHLBI NIH HHS/United States ; NSF-CBET 0939511//Emergent Behaviors of Integrated Cellular Systems/International ; S10 RR028962/RR/NCRR NIH HHS/United States ; R01 HL130533/HL/NHLBI NIH HHS/United States ; Instrumentation Grant, NIH S10 RR028962/NH/NIH HHS/United States ; F31HL140907/HL/NHLBI NIH HHS/United States ; P30 AI027763/AI/NIAID NIH HHS/United States ; U01HL100406/NH/NIH HHS/United States ; R01HL130533/NH/NIH HHS/United States ; F31 HL140907/HL/NHLBI NIH HHS/United States ; },
mesh = {Antigens, CD/*genetics ; CRISPR-Cas Systems/genetics ; Cadherins/*genetics ; Cell Communication/genetics ; Cell Differentiation/*genetics ; Cell Lineage/genetics ; Gene Knockdown Techniques ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism ; Morphogenesis/genetics ; rho-Associated Kinases/*genetics ; },
abstract = {Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis.},
}

Antigens, CD/*genetics
CRISPR-Cas Systems/genetics
Cadherins/*genetics
Cell Communication/genetics
Cell Differentiation/*genetics
Cell Lineage/genetics
Gene Knockdown Techniques
Humans
Induced Pluripotent Stem Cells/*cytology/metabolism
Morphogenesis/genetics
rho-Associated Kinases/*genetics

@article {pmid29855762,
year = {2018},
author = {Montoliu, L and Whitelaw, CBA},
title = {Unexpected mutations were expected and unrelated to CRISPR-Cas9 activity.},
journal = {Transgenic research},
volume = {27},
number = {4},
pages = {315-319},
pmid = {29855762},
issn = {1573-9368},
support = {//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Mice ; Mutagenesis ; Mutation/*genetics ; *Polymorphism, Genetic ; },
abstract = {The scientific journal Nature Methods have just retracted a publication that reported numerous unexpected mutations after a CRISPR-Cas9 experiment based on collecting whole genome sequencing information from one control and two experimental genome edited mice. In the intervening 10 months since publication the data presented have been strongly contested and criticized by the scientific and biotech communities, through publications, open science channels and social networks. The criticism focused on the animal used as control, which was derived from the same mouse strain as the experimental individuals but from an unrelated sub-colony, hence control and experimental mice were genetically divergent. The most plausible explanation for the vast majority of the reported unexpected mutations were the expected underlying genetic polymorphisms that normally accumulate in two different colonies of the same mouse strain which occur as a result of spontaneous mutations and genetic drift. Therefore, the reported mutations were most likely not related to CRISPR-Cas9 activity.},
}

Animals
CRISPR-Cas Systems/*genetics
Mice
Mutagenesis
Mutation/*genetics
*Polymorphism, Genetic

@article {pmid29797189,
year = {2018},
author = {D'Ambrosio, C and Stigliani, AL and Giorio, G},
title = {CRISPR/Cas9 editing of carotenoid genes in tomato.},
journal = {Transgenic research},
volume = {27},
number = {4},
pages = {367-378},
pmid = {29797189},
issn = {1573-9368},
mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; Carotenoids/biosynthesis/*genetics ; Genome, Plant ; Lycopersicon esculentum/*genetics/growth & development ; Mutation ; Phenotype ; Plants, Genetically Modified/*genetics/growth & development ; },
abstract = {CRISPR/Cas9 technology is rapidly spreading as genome editing system in crop breeding. The efficacy of CRISPR/Cas9 in tomato was tested on Psy1 and CrtR-b2, two key genes of carotenoid biosynthesis. Carotenoids are plant secondary metabolites that must be present in the diet of higher animals because they exert irreplaceable functions in important physiological processes. Psy1 and CrtR-b2 were chosen because their impairment is easily detectable as a change of fruit or flower color. Two CRISPR/Cas9 constructs were designed to target neighboring sequences on the first exon of each gene. Thirty-four out of forty-nine (69%) transformed plants showed the expected loss-of-function phenotypes due to the editing of both alleles of a locus. However, by including the seven plants edited only at one of the two homologs and showing a normal phenotype, the editing rate reaches the 84%. Although none chimeric phenotype was observed, the cloning of target region amplified fragments revealed that in the 40% of analyzed DNA samples were present more than two alleles. As concerning the type of mutation, it was possible to identify 34 new different alleles across the four transformation experiments. The sequence characterization of the CRISPR/Cas9-induced mutations showed that the most frequent repair errors were the insertion and the deletion of one base. The results of this study prove that the CRISPRCas9 system can be an efficient and quick method for the generation of useful mutations in tomato to be implemented in breeding programs.},
}

Alleles
CRISPR-Cas Systems/*genetics
Carotenoids/biosynthesis/*genetics
Genome, Plant
Lycopersicon esculentum/*genetics/growth & development
Mutation
Phenotype
Plants, Genetically Modified/*genetics/growth & development

@article {pmid29427756,
year = {2018},
author = {Zhang, Z and Ursin, R and Mahapatra, S and Gallicano, GI},
title = {CRISPR/CAS9 ablation of individual miRNAs from a miRNA family reveals their individual efficacies for regulating cardiac differentiation.},
journal = {Mechanisms of development},
volume = {150},
number = {},
pages = {10-20},
doi = {10.1016/j.mod.2018.02.002},
pmid = {29427756},
issn = {1872-6356},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/*genetics ; DNA-Binding Proteins/genetics ; Embryonic Stem Cells/cytology/metabolism ; Gene Expression Regulation, Developmental/genetics ; Gene Knockout Techniques ; Heart/*growth & development ; Humans ; Mice ; MicroRNAs/*genetics ; Myocytes, Cardiac/cytology ; Organogenesis/*genetics ; Transcription Factors/genetics ; },
abstract = {Although it is well understood that genetic mutations, chromosomal abnormalities, and epigenetic miscues can cause congenital birth defects, many defects are still labeled idiopathic, meaning their origin is not yet understood. microRNAs are quickly entering the causal fray of developmental defects. miRNAs use a 7-8 base-pair seed sequence to target a corresponding sequence on one or multiple mRNAs resulting in rapid down-regulation of translation. miRNAs can also control protein 'amounts' in cells. As a result if miRNAs are over or under expressed during development protein homeostasis can be compromised resulting in defects in the development of organ systems. Here, we show that during differentiation of embryonic stem cells, individual miRNAs that reside in the miRNA17 family (composed of 14 miRNAs) do not share the same function even though they have the same seed sequence. The advent of CRISPR/CAS9 technology has not only yielded a true observation of individual miRNA function, it has also reconnected advanced molecular biology approaches to classical cell biology approaches such as gene rescue. We show that miRNA106a and to a lesser extent miR17 and 93 target the cardiac suppressor gene Fog2, which specifically suppress Gata-4 and Coup-TF2. However, when each miRNA is knocked out, we find that their targeting efficacies for Fog2 differ resulting in varying degrees of cardiac differentiation.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Differentiation/*genetics
DNA-Binding Proteins/genetics
Embryonic Stem Cells/cytology/metabolism
Gene Expression Regulation, Developmental/genetics
Gene Knockout Techniques
Heart/*growth & development
Humans
Mice
MicroRNAs/*genetics
Myocytes, Cardiac/cytology
Organogenesis/*genetics
Transcription Factors/genetics

@article {pmid28676649,
year = {2017},
author = {Heinze, SD and Kohlbrenner, T and Ippolito, D and Meccariello, A and Burger, A and Mosimann, C and Saccone, G and Bopp, D},
title = {CRISPR-Cas9 targeted disruption of the yellow ortholog in the housefly identifies the brown body locus.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {4582},
pmid = {28676649},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Gene Editing ; Houseflies/*genetics ; Insect Proteins/genetics/metabolism ; Mice ; *Quantitative Trait Loci ; *Quantitative Trait, Heritable ; },
abstract = {The classic brown body (bwb) mutation in the housefly Musca domestica impairs normal melanization of the adult cuticle. In Drosophila melanogaster, a reminiscent pigmentation defect results from mutations in the yellow gene encoding dopachrome conversion enzyme (DCE). Here, we demonstrate that the bwb locus structurally and functionally represents the yellow ortholog of Musca domestica, MdY. In bwb Musca strains, we identified two mutant MdY alleles that contain lesions predicted to result in premature truncation of the MdY open reading frame. We targeted wildtype MdY by CRISPR-Cas9 RNPs and generated new mutant alleles that fail to complement existing MdY alleles, genetically confirming that MdY is the bwb locus. We further found evidence for Cas9-mediated interchromosomal recombination between wildtype and mutant bwb alleles. Our work resolves the molecular identity of the classic bwb mutation in Musca domestica and establishes the feasibility of Cas9-mediated genome editing in the Musca model.},
}

Animals
*CRISPR-Cas Systems
Female
*Gene Editing
Houseflies/*genetics
Insect Proteins/genetics/metabolism
Mice
*Quantitative Trait Loci
*Quantitative Trait, Heritable

@article {pmid28667332,
year = {2017},
author = {Van der Hoek, KH and Eyre, NS and Shue, B and Khantisitthiporn, O and Glab-Ampi, K and Carr, JM and Gartner, MJ and Jolly, LA and Thomas, PQ and Adikusuma, F and Jankovic-Karasoulos, T and Roberts, CT and Helbig, KJ and Beard, MR},
title = {Viperin is an important host restriction factor in control of Zika virus infection.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {4475},
pmid = {28667332},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Disease Models, Animal ; Female ; Gene Editing ; *Host-Pathogen Interactions/genetics/immunology ; Humans ; Immunity, Innate ; Macrophages/immunology/metabolism/virology ; Mice ; Mice, Knockout ; Monocytes/immunology/metabolism/virology ; Neural Stem Cells/metabolism/virology ; Placenta/metabolism/virology ; Pregnancy ; Proteins/genetics/*metabolism ; Virus Replication ; Zika Virus/*physiology ; Zika Virus Infection/genetics/immunology/*metabolism/*virology ; },
abstract = {Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-β and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin-/- mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.},
}

Animals
CRISPR-Cas Systems
Cell Line
Disease Models, Animal
Female
Gene Editing
*Host-Pathogen Interactions/genetics/immunology
Humans
Immunity, Innate
Macrophages/immunology/metabolism/virology
Mice
Mice, Knockout
Monocytes/immunology/metabolism/virology
Neural Stem Cells/metabolism/virology
Placenta/metabolism/virology
Pregnancy
Proteins/genetics/*metabolism
Virus Replication
Zika Virus/*physiology
Zika Virus Infection/genetics/immunology/*metabolism/*virology

@article {pmid30622340,
year = {2019},
author = {Domenici, G and Aurrekoetxea-Rodríguez, I and Simões, BM and Rábano, M and Lee, SY and Millán, JS and Comaills, V and Oliemuller, E and López-Ruiz, JA and Zabalza, I and Howard, BA and Kypta, RM and Vivanco, MD},
title = {A Sox2-Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells.},
journal = {Oncogene},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41388-018-0656-7},
pmid = {30622340},
issn = {1476-5594},
support = {PI14/01328//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; SAF2017-84934-R//Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness)/ ; SAF2017-90604-REDT//Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness)/ ; SAF2017-84092-R//Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness)/ ; },
abstract = {Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.},
}

@article {pmid30621855,
year = {2019},
author = {Homanics, GE},
title = {Gene-edited CRISPy Critters for alcohol research.},
journal = {Alcohol (Fayetteville, N.Y.)},
volume = {74},
number = {},
pages = {11-19},
doi = {10.1016/j.alcohol.2018.03.001},
pmid = {30621855},
issn = {1873-6823},
abstract = {Genetically engineered animals are powerful tools that have provided invaluable insights into mechanisms of alcohol action and alcohol-use disorder. Traditionally, production of gene-targeted animals was a tremendously expensive, time consuming, and technically demanding undertaking. However, the recent advent of facile methods for editing the genome at very high efficiency is revolutionizing how these animals are made. While pioneering approaches to create gene-edited animals first used zinc finger nucleases and subsequently used transcription activator-like effector nucleases, these approaches have been largely supplanted in an extremely short period of time with the recent discovery and precocious maturation of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. CRISPR uses a short RNA sequence to guide a non-specific CRISPR-associated nuclease (Cas) to a precise, single location in the genome. Because the CRISPR/Cas system can be cheaply, rapidly, and easily reprogrammed to target nearly any genomic locus of interest simply by recoding the sequence of the guide RNA, this gene-editing system has been rapidly adopted by numerous labs around the world. With CRISPR/Cas, it is now possible to perform gene editing directly in early embryos from every species of animals that is of interest to the alcohol field. Techniques have been developed that enable the rapid production of animals in which a gene has been inactivated (knockout) or modified to harbor specific nucleotide changes (knockins). This system has also been used to insert specific DNA sequences such as reporter or recombinase genes into specific loci of interest. Genetically engineered animals created with the CRISPR/Cas system (CRISPy Critters) are being produced at an astounding pace. Animal production is no longer a significant bottleneck to new discoveries. CRISPy animal studies are just beginning to appear in the alcohol literature, but their use is expected to explode in the near future. CRISPy mice, rats, and other model organisms are sure to facilitate advances in our understanding of alcohol-use disorder.},
}

@article {pmid30394035,
year = {2018},
author = {Chen, F and Yang, P and Zhu, J},
title = {[Construction and application of PGRN and Rev-erbβ double genes knockout HEK293 cell lines].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {34},
number = {10},
pages = {1679-1692},
doi = {10.13345/j.cjb.180033},
pmid = {30394035},
issn = {1000-3061},
mesh = {Blotting, Western ; *CRISPR-Cas Systems ; ErbB Receptors/genetics ; Gene Expression ; Gene Expression Regulation ; *Gene Knockout Techniques ; Gene Products, rev/*genetics ; HEK293 Cells ; Humans ; Lentivirus ; Progranulins/*genetics ; Promoter Regions, Genetic ; RNA, Messenger ; },
abstract = {In order to study the molecular mechanism and physiological significance of the interaction between PGRN and Rev-erbβ, the PGRN gene in HEK293 (Rev-erbβ-/-) marked as C3-6 cell lines was knocked out by CRISPR/Cas9 system to generate the Rev-erbβ and PGRN double genes knockout HEK293 cell lines. First, four sgRNAs were designed for PGRN gene, and PGRN sgRNA2 and sgRNA3 with the higher activity were used to construct the Lentiviral vector, pLenti/CMV-Loxp-Cas9-sgRNA2-U6-sgRNA3-U6-Loxp-EF1α-Puro. Then, the lentivirus vector carrying Cas9 and double PGRN sgRNA were used to infect HEK293 C3-6 cells. Through drug screening, cloning and sequencing, we obtained the monoclonal HEK293 (Rev-erbβ-/-; PGRN-/-) marked as C3-6/23 cell lines. Using qRT-PCR and Western blotting, we detected PGRN mRNA and protein expression in C3-6/23 cell lines. Finally, genetic complementation was used to study the effect of PGRN-mediated Rev-erbβ on the regulation of the target gene promoter transcriptional activity in the C3-6/23 cell lines. In HEK293 C3-6/23 cell lines, the two DNA chains of PGRN gene were both deletion mutagenesis, and the expression mRNA and protein of PGRN did not reach the detection level. At the same time, the interaction between PGRN and Rev-erbβ enhanced the regulation of Rev-erbβ on the transcription of target gene promoter in the cell lines. Using CRISPR/Cas9 system, we successfully constructed the double knockout HEK293 (Rev-erbβ-/-; PGRN-/-) monoclonal cell lines. The study found that PGRN could affect Rev-erbβ on the regulation of target gene promoter transcription in the C3-6/23 cell lines; however, the mechanism of PGRN involvement in mediating Rev-erbβ in transcriptional regulation remains to be further studied.},
}

Blotting, Western
*CRISPR-Cas Systems
ErbB Receptors/genetics
Gene Expression
Gene Expression Regulation
*Gene Knockout Techniques
Gene Products, rev/*genetics
HEK293 Cells
Humans
Lentivirus
Progranulins/*genetics
Promoter Regions, Genetic
RNA, Messenger

@article {pmid30166439,
year = {2018},
author = {Amoasii, L and Hildyard, JCW and Li, H and Sanchez-Ortiz, E and Mireault, A and Caballero, D and Harron, R and Stathopoulou, TR and Massey, C and Shelton, JM and Bassel-Duby, R and Piercy, RJ and Olson, EN},
title = {Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy.},
journal = {Science (New York, N.Y.)},
volume = {362},
number = {6410},
pages = {86-91},
pmid = {30166439},
issn = {1095-9203},
support = {//Wellcome Trust/United Kingdom ; R01 AR067294/AR/NIAMS NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Adenoviridae ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Dogs ; Dystrophin/*genetics/metabolism ; Female ; Gene Editing/*methods ; Gene Transfer Techniques ; Male ; Muscular Dystrophy, Duchenne/*therapy ; },
abstract = {Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational "hotspot" in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.},
}

Adenoviridae
Animals
CRISPR-Cas Systems
Disease Models, Animal
Dogs
Dystrophin/*genetics/metabolism
Female
Gene Editing/*methods
Gene Transfer Techniques
Male
Muscular Dystrophy, Duchenne/*therapy

@article {pmid30102814,
year = {2019},
author = {Ivanov Kavkova, E and Blöchl, C and Tenhaken, R},
title = {The Myo-inositol pathway does not contribute to ascorbic acid synthesis.},
journal = {Plant biology (Stuttgart, Germany)},
volume = {21 Suppl 1},
number = {},
pages = {95-102},
doi = {10.1111/plb.12898},
pmid = {30102814},
issn = {1438-8677},
support = {P25339//Austrian Science Fund/ ; },
mesh = {Arabidopsis/metabolism ; Arabidopsis Proteins/genetics/metabolism ; Ascorbic Acid/*biosynthesis ; Base Sequence ; Biosynthetic Pathways ; CRISPR-Cas Systems/genetics ; DNA, Bacterial/genetics ; Glucuronic Acid/metabolism ; Inositol/*metabolism ; Metabolome ; Mutagenesis, Insertional/genetics ; Mutation/genetics ; Plant Leaves/metabolism ; Tritium/metabolism ; },
abstract = {Ascorbic acid (AsA) biosynthesis in plants predominantly occurs via a pathway with d-mannose and l-galactose as intermediates. One alternative pathway for AsA synthesis, which is similar to the biosynthesis route in mammals, is controversially discussed for plants. Here, myo-inositol is cleaved to glucuronic acid and then converted via l-gulonate to AsA. In contrast to animals, plants have an effective recycling pathway for glucuronic acid, being a competitor for the metabolic rate. Recycling involves a phosphorylation at C1 by the enzyme glucuronokinase. Two previously described T-DNA insertion lines in the gene coding for glucuronokinase1 show wild type-like expression levels of the mRNA in our experiments and do not accumulate glucuronic acid in labelling experiments disproving that these lines are true knockouts. As suitable T-DNA insertion lines were not available, we generated frameshift mutations in the major expressed isoform glucuronokinase1 (At3g01640) to potentially redirect metabolites to AsA. However, radiotracer experiments with 3 H-myo-inositol revealed that the mutants in glucuronokinase1 accumulate only glucuronic acid and incorporate less metabolite into cell wall polymers. AsA was not labelled, suggesting that Arabidopsis cannot efficiently use glucuronic acid for AsA biosynthesis. All four mutants in glucuronokinase as well as the wild type have the same level of AsA in leaves.},
}

Arabidopsis/metabolism
Arabidopsis Proteins/genetics/metabolism
Ascorbic Acid/*biosynthesis
Base Sequence
Biosynthetic Pathways
CRISPR-Cas Systems/genetics
DNA, Bacterial/genetics
Glucuronic Acid/metabolism
Inositol/*metabolism
Metabolome
Mutagenesis, Insertional/genetics
Mutation/genetics
Plant Leaves/metabolism
Tritium/metabolism

@article {pmid29167381,
year = {2018},
author = {Reber, S and Mechtersheimer, J and Nasif, S and Benitez, JA and Colombo, M and Domanski, M and Jutzi, D and Hedlund, E and Ruepp, MD},
title = {CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells.},
journal = {Molecular biology of the cell},
volume = {29},
number = {2},
pages = {75-83},
pmid = {29167381},
issn = {1939-4586},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques/*methods ; HEK293 Cells ; HeLa Cells ; Humans ; },
abstract = {CRISPR/Cas9-based genome editing offers the possibility to knock out almost any gene of interest in an affordable and simple manner. The most common strategy is the introduction of a frameshift into the open reading frame (ORF) of the target gene which truncates the coding sequence (CDS) and targets the corresponding transcript for degradation by nonsense-mediated mRNA decay (NMD). However, we show that transcripts containing premature termination codons (PTCs) are not always degraded efficiently and can generate C-terminally truncated proteins which might have residual or dominant negative functions. Therefore, we recommend an alternative approach for knocking out genes, which combines CRISPR/Cas9 with gene traps (CRISPR-Trap) and is applicable to ∼50% of all spliced human protein-coding genes and a large subset of lncRNAs. CRISPR-Trap completely prevents the expression of the ORF and avoids expression of C-terminal truncated proteins. We demonstrate the feasibility of CRISPR-Trap by utilizing it to knock out several genes in different human cell lines. Finally, we also show that this approach can be used to efficiently generate gene replacements allowing for modulation of protein levels for otherwise lethal knockouts (KOs). Thus, CRISPR-Trap offers several advantages over conventional KO approaches and allows for generation of clean CRISPR/Cas9-based KOs.},
}

*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Knockout Techniques/*methods
HEK293 Cells
HeLa Cells
Humans

@article {pmid30619231,
year = {2018},
author = {Berges, M and Michel, AM and Lassek, C and Nuss, AM and Beckstette, M and Dersch, P and Riedel, K and Sievers, S and Becher, D and Otto, A and Maaß, S and Rohde, M and Eckweiler, D and Borrero-de Acuña, JM and Jahn, M and Neumann-Schaal, M and Jahn, D},
title = {Iron Regulation in Clostridioides difficile.},
journal = {Frontiers in microbiology},
volume = {9},
number = {},
pages = {3183},
doi = {10.3389/fmicb.2018.03183},
pmid = {30619231},
issn = {1664-302X},
abstract = {The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (15 μM) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron and siderophore transporter genes were induced by Fur during low iron (0.2 μM) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some intermediates (phenylpyruvate, 2-oxo-isocaproate, 3-hydroxy-butyryl-CoA, crotonyl-CoA) accumulated, while end products like isocaproate and butyrate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB) of the F0F1-type was induced while the formation of a ATP-consuming, proton-pumping V-type ATPase (atpDBAFCEKI) was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. The fur mutant showed an increased sensitivity to vancomycin and polymyxin B. An intensive remodeling of the cell wall was observed, Polyamine biosynthesis (spe) was induced leading to an accumulation of spermine, spermidine, and putrescine. The fur mutant lost most of its flagella and motility. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, a significant replacement of iron requiring metabolic pathways and the restructuring of the cell surface for protection during the complex adaptation phase and was only partly directly regulated by Fur.},
}

@article {pmid30400152,
year = {2018},
author = {Koslová, A and Kučerová, D and Reinišová, M and Geryk, J and Trefil, P and Hejnar, J},
title = {Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells.},
journal = {Viruses},
volume = {10},
number = {11},
pages = {},
pmid = {30400152},
issn = {1999-4915},
mesh = {Animals ; Avian Leukosis/*genetics/*virology ; Avian Leukosis Virus/*physiology ; Base Sequence ; *CRISPR-Cas Systems ; Cell Line ; Chickens ; Disease Resistance/*genetics ; *Gene Editing ; Genes, Viral ; Genetic Techniques ; Genetic Vectors/genetics ; RNA, Guide ; Receptors, Virus/*genetics/metabolism ; },
abstract = {Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.},
}

Animals
Avian Leukosis/*genetics/*virology
Avian Leukosis Virus/*physiology
Base Sequence
*CRISPR-Cas Systems
Cell Line
Chickens
Disease Resistance/*genetics
*Gene Editing
Genes, Viral
Genetic Techniques
Genetic Vectors/genetics
RNA, Guide
Receptors, Virus/*genetics/metabolism

@article {pmid29496999,
year = {2018},
author = {Yokoyama, CC and Baldridge, MT and Leung, DW and Zhao, G and Desai, C and Liu, TC and Diaz-Ochoa, VE and Huynh, JP and Kimmey, JM and Sennott, EL and Hole, CR and Idol, RA and Park, S and Storek, KM and Wang, C and Hwang, S and Viehmann Milam, A and Chen, E and Kerrinnes, T and Starnbach, MN and Handley, SA and Mysorekar, IU and Allen, PM and Monack, DM and Dinauer, MC and Doering, TL and Tsolis, RM and Dworkin, JE and Stallings, CL and Amarasinghe, GK and Micchelli, CA and Virgin, HW},
title = {LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {16},
pages = {6022-6038},
pmid = {29496999},
issn = {1083-351X},
support = {R01 AI102882/AI/NIAID NIH HHS/United States ; R01 AI109799/AI/NIAID NIH HHS/United States ; U19 AI113187/AI/NIAID NIH HHS/United States ; T32 AI007163/AI/NIAID NIH HHS/United States ; T32 GM007067/GM/NIGMS NIH HHS/United States ; T32 AI007172/AI/NIAID NIH HHS/United States ; F32 AI108089/AI/NIAID NIH HHS/United States ; R01 DK100644/DK/NIDDK NIH HHS/United States ; R01 AI095396/AI/NIAID NIH HHS/United States ; U19 AI109725/AI/NIAID NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; P30 AR048335/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Autoantigens/analysis ; Bacterial Infections/genetics/immunology ; CRISPR-Cas Systems ; Female ; *Gene Deletion ; Immunity, Innate ; Inflammation/genetics/immunology ; Male ; Membrane Proteins/analysis ; Mice ; Mycoses/genetics/immunology ; Phylogeny ; Virus Diseases/genetics/immunology ; },
abstract = {Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.},
}

Animals
Autoantigens/analysis
Bacterial Infections/genetics/immunology
CRISPR-Cas Systems
Female
*Gene Deletion
Immunity, Innate
Inflammation/genetics/immunology
Male
Membrane Proteins/analysis
Mice
Mycoses/genetics/immunology
Phylogeny
Virus Diseases/genetics/immunology

@article {pmid29378572,
year = {2018},
author = {Peng, R and Wang, Y and Feng, WW and Yue, XJ and Chen, JH and Hu, XZ and Li, ZF and Sheng, DH and Zhang, YM and Li, YZ},
title = {CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus.},
journal = {Microbial cell factories},
volume = {17},
number = {1},
pages = {15},
pmid = {29378572},
issn = {1475-2859},
support = {31471183//National Natural Science Foundation of China/ ; 31670076//National Natural Science Foundation of China/ ; 31370123//National Natural Science Foundation of China/ ; ZR2016QZ002//Key Program of Shandong Natural Science Foundation/ ; },
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Epothilones/*biosynthesis/genetics ; *Multigene Family ; Myxococcus xanthus/*genetics/metabolism ; Promoter Regions, Genetic ; RNA, Guide/genetics ; Recombinant Proteins/*genetics ; Secondary Metabolism ; *Transcription, Genetic ; Transcriptional Activation ; },
abstract = {BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites.

RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects.

CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.},
}

CRISPR-Associated Protein 9/genetics/metabolism
CRISPR-Cas Systems/*genetics
Epothilones/*biosynthesis/genetics
*Multigene Family
Myxococcus xanthus/*genetics/metabolism
Promoter Regions, Genetic
RNA, Guide/genetics
Recombinant Proteins/*genetics
Secondary Metabolism
*Transcription, Genetic
Transcriptional Activation

@article {pmid29316926,
year = {2018},
author = {Park, J and Shin, H and Lee, SM and Um, Y and Woo, HM},
title = {RNA-guided single/double gene repressions in Corynebacterium glutamicum using an efficient CRISPR interference and its application to industrial strain.},
journal = {Microbial cell factories},
volume = {17},
number = {1},
pages = {4},
pmid = {29316926},
issn = {1475-2859},
support = {2017M1A8A1072034//National Research Foundation of Korea/ ; 2017R1A2B2002566//National Research Foundation of Korea/ ; },
mesh = {CRISPR-Cas Systems ; Citrate (si)-Synthase/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Corynebacterium glutamicum/*genetics ; Gene Expression Regulation, Bacterial ; Gene Silencing/*physiology ; Genetic Vectors ; Metabolic Engineering/methods ; Plasmids ; RNA, Guide/*genetics ; Synthetic Biology/*methods ; },
abstract = {BACKGROUND: The construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering. Recently, RNA-guided CRISPR technologies have been developed for metabolic engineering of industrially-relevant host.

RESULTS: To demonstrate the application of the CRISPR interference (CRISPRi), we developed two-plasmid CRISPRi vectors and applied the CRISPRi in Corynebacterium glutamicum to repress single target genes and double target genes simultaneously. Four-different single genes (the pyc, gltA, idsA, and glgC genes) repressions were successfully performed using the CRISPRi vectors, resulting significant mRNA reductions of the targets compared to a control. Subsequently, the phenotypes for the target gene-repressed strains were analyzed, showing the expected cell growth behaviors with different carbon sources. In addition, double gene repression (the idsA and glgC genes in a different order) by the CRISPRi resulted in an independent gene repression to each target gene simultaneously. To demonstrate an industrial application of the CRISPRi, citrate synthase (CS)-targeting DM1919 (L-lysine producer) strains with a sgRNA-gltA-r showed reduced CS activity, resulting in the improvement of L-lysine yield by 1.39-fold than the parental DM1919 (a lysine producer).

CONCLUSIONS: Single or double gene repression were successfully performed using the CRISPRi vectors and sequence specific sgRNAs. The CRISPRi can be applied for multiplex metabolic engineering to enhanced lysine production and it will promote the further rapid development of microbial cell factories of C. glutamicum.},
}

CRISPR-Cas Systems
Citrate (si)-Synthase/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Corynebacterium glutamicum/*genetics
Gene Expression Regulation, Bacterial
Gene Silencing/*physiology
Genetic Vectors
Metabolic Engineering/methods
Plasmids
RNA, Guide/*genetics
Synthetic Biology/*methods

@article {pmid30615645,
year = {2019},
author = {Price, MA and Cruz, R and Baxter, S and Escalettes, F and Rosser, SJ},
title = {CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis.},
journal = {PloS one},
volume = {14},
number = {1},
pages = {e0210121},
doi = {10.1371/journal.pone.0210121},
pmid = {30615645},
issn = {1932-6203},
abstract = {CRISPR-Cas systems have become widely used across all fields of biology as a genome engineering tool. With its recent demonstration in the Gram positive industrial workhorse Bacillus subtilis, this tool has become an attractive option for rapid, markerless strain engineering of industrial production hosts. Previously described strategies for CRISPR-Cas9 genome editing in B. subtilis have involved chromosomal integrations of Cas9 and single guide RNA expression cassettes, or construction of large plasmids for simultaneous transformation of both single guide RNA and donor DNA. Here we use a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity.},
}

@article {pmid30251625,
year = {2018},
author = {Emmer, BT and Hesketh, GG and Kotnik, E and Tang, VT and Lascuna, PJ and Xiang, J and Gingras, AC and Chen, XW and Ginsburg, D},
title = {The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30251625},
issn = {2050-084X},
support = {R35 HL135793/HL/NHLBI NIH HHS/United States ; T32 HL007853/HL/NHLBI NIH HHS/United States ; R35-HL135793T/HL/NHLBI NIH HHS/United States ; T32-HL007853/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Biotinylation ; CRISPR-Cas Systems/genetics ; Endoplasmic Reticulum/metabolism ; Gene Deletion ; HEK293 Cells ; Humans ; Membrane Proteins/*metabolism ; Mutagenesis/genetics ; Proprotein Convertase 9/*metabolism ; Protein Binding ; Recombinant Fusion Proteins/metabolism ; Secretory Pathway ; },
abstract = {PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 secretion in HEK293T cells. The functional contribution of SURF4 to PCSK9 secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extracellular secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient cellular secretion of PCSK9.},
}

Biotinylation
CRISPR-Cas Systems/genetics
Endoplasmic Reticulum/metabolism
Gene Deletion
HEK293 Cells
Humans
Membrane Proteins/*metabolism
Mutagenesis/genetics
Proprotein Convertase 9/*metabolism
Protein Binding
Recombinant Fusion Proteins/metabolism
Secretory Pathway

@article {pmid30202008,
year = {2018},
author = {Palin, K and Pitkänen, E and Turunen, M and Sahu, B and Pihlajamaa, P and Kivioja, T and Kaasinen, E and Välimäki, N and Hänninen, UA and Cajuso, T and Aavikko, M and Tuupanen, S and Kilpivaara, O and van den Berg, L and Kondelin, J and Tanskanen, T and Katainen, R and Grau, M and Rauanheimo, H and Plaketti, RM and Taira, A and Sulo, P and Hartonen, T and Dave, K and Schmierer, B and Botla, S and Sokolova, M and Vähärautio, A and Gladysz, K and Ongen, H and Dermitzakis, E and Bramsen, JB and Ørntoft, TF and Andersen, CL and Ristimäki, A and Lepistö, A and Renkonen-Sinisalo, L and Mecklin, JP and Taipale, J and Aaltonen, LA},
title = {Contribution of allelic imbalance to colorectal cancer.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3664},
pmid = {30202008},
issn = {2041-1723},
mesh = {*Allelic Imbalance ; CRISPR-Cas Systems ; Chromosome Aberrations ; Chromosomes, Human, Pair 8 ; Colorectal Neoplasms/*genetics/pathology ; DNA Copy Number Variations ; Denmark ; Gene Expression Profiling ; *Genetic Predisposition to Disease ; Genomics ; Genotype ; Humans ; Loss of Heterozygosity ; Microsatellite Repeats ; Phenotype ; Point Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; RNA, Small Interfering/genetics ; Transcription Factors/genetics ; Tumor Suppressor Protein p53/genetics ; Whole Genome Sequencing ; },
abstract = {Point mutations in cancer have been extensively studied but chromosomal gains and losses have been more challenging to interpret due to their unspecific nature. Here we examine high-resolution allelic imbalance (AI) landscape in 1699 colorectal cancers, 256 of which have been whole-genome sequenced (WGSed). The imbalances pinpoint 38 genes as plausible AI targets based on previous knowledge. Unbiased CRISPR-Cas9 knockout and activation screens identified in total 79 genes within AI peaks regulating cell growth. Genetic and functional data implicate loss of TP53 as a sufficient driver of AI. The WGS highlights an influence of copy number aberrations on the rate of detected somatic point mutations. Importantly, the data reveal several associations between AI target genes, suggesting a role for a network of lineage-determining transcription factors in colorectal tumorigenesis. Overall, the results unravel the contribution of AI in colorectal cancer and provide a plausible explanation why so few genes are commonly affected by point mutations in cancers.},
}

*Allelic Imbalance
CRISPR-Cas Systems
Chromosome Aberrations
Chromosomes, Human, Pair 8
Colorectal Neoplasms/*genetics/pathology
DNA Copy Number Variations
Denmark
Gene Expression Profiling
*Genetic Predisposition to Disease
Genomics
Genotype
Humans
Loss of Heterozygosity
Microsatellite Repeats
Phenotype
Point Mutation
Proto-Oncogene Proteins p21(ras)/genetics
RNA, Small Interfering/genetics
Transcription Factors/genetics
Tumor Suppressor Protein p53/genetics
Whole Genome Sequencing

@article {pmid30178747,
year = {2018},
author = {Li, R and Bernau, K and Sandbo, N and Gu, J and Preissl, S and Sun, X},
title = {Pdgfra marks a cellular lineage with distinct contributions to myofibroblasts in lung maturation and injury response.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30178747},
issn = {2050-084X},
support = {R01 HL097134/HL/NHLBI NIH HHS/United States ; R01 HL122406/HL/NHLBI NIH HHS/United States ; R01 HL142215/HL/NHLBI NIH HHS/United States ; RO1 HL142215,RO1 HL097134,RO1 HL122406/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Bleomycin ; CRISPR-Cas Systems/genetics ; *Cell Lineage ; Cell Proliferation ; Disease Models, Animal ; Gene Deletion ; Gene Expression Profiling ; Gene Knock-In Techniques ; Green Fluorescent Proteins/metabolism ; Homologous Recombination/genetics ; Hyperoxia/pathology ; Lung/*growth & development/metabolism/*pathology ; Mice, Transgenic ; Myofibroblasts/*metabolism/*pathology ; Pulmonary Fibrosis/pathology ; Receptor, Platelet-Derived Growth Factor alpha/*metabolism ; Single-Cell Analysis ; },
abstract = {Pdgfra-expressing (Pdgfra+) cells have been implicated as progenitors in many mesenchymal tissues. To determine lineage potential, we generated PdgfrartTA knockin mice using CRISPR/Cas9. During lung maturation, counter to a prior study reporting that Pdgfra+ cells give rise equally to myofibroblasts and lipofibroblasts, lineage tracing using PdgfrartTA;tetO-cre mice indicated that ~95% of the lineaged cells are myofibroblasts. Genetic ablation of Pdgfra+ cells using PdgfrartTA-driven diphtheria toxin (DTA) led to alveolar simplification, demonstrating that these cells are essential for building the gas exchange surface area. In the adult bleomycin model of lung fibrosis, lineaged cells increased to contribute to pathological myofibroblasts. In contrast, in a neonatal hyperoxia model of bronchopulmonary dysplasia (BPD), lineaged cells decreased and do not substantially contribute to pathological myofibroblasts. Our findings revealed complexity in the behavior of the Pdgfra-lineaged cells as exemplified by their distinct contributions to myofibroblasts in normal maturation, BPD and adult fibrosis.},
}

Animals
Bleomycin
CRISPR-Cas Systems/genetics
*Cell Lineage
Cell Proliferation
Disease Models, Animal
Gene Deletion
Gene Expression Profiling
Gene Knock-In Techniques
Green Fluorescent Proteins/metabolism
Homologous Recombination/genetics
Hyperoxia/pathology
Lung/*growth & development/metabolism/*pathology
Mice, Transgenic
Myofibroblasts/*metabolism/*pathology
Pulmonary Fibrosis/pathology
Receptor, Platelet-Derived Growth Factor alpha/*metabolism
Single-Cell Analysis

@article {pmid30170115,
year = {2018},
author = {Pankowicz, FP and Barzi, M and Kim, KH and Legras, X and Martins, CS and Wooton-Kee, CR and Lagor, WR and Marini, JC and Elsea, SH and Bissig-Choisat, B and Moore, DD and Bissig, KD},
title = {Rapid Disruption of Genes Specifically in Livers of Mice Using Multiplex CRISPR/Cas9 Editing.},
journal = {Gastroenterology},
volume = {155},
number = {6},
pages = {1967-1970.e6},
doi = {10.1053/j.gastro.2018.08.037},
pmid = {30170115},
issn = {1528-0012},
support = {R01 DK115461/DK/NIDDK NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; R01 HL134510/HL/NHLBI NIH HHS/United States ; R56 DK115461/DK/NIDDK NIH HHS/United States ; T32 HL092332/HL/NHLBI NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; },
mesh = {ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics ; Animals ; Argininosuccinate Lyase/*genetics ; CRISPR-Associated Protein 9/*administration & dosage ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Gene Editing/*methods ; Hepatocytes/enzymology/physiology ; Liver/*enzymology ; Mice ; Mice, Knockout ; Oxidoreductases/genetics ; Phenotype ; Plasmids/genetics ; },
abstract = {BACKGROUND & AIMS: Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts.

METHODS: In this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product.

RESULTS: We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse.

CONCLUSIONS: This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.},
}

ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics
Animals
Argininosuccinate Lyase/*genetics
CRISPR-Associated Protein 9/*administration & dosage
CRISPR-Cas Systems/*genetics
Disease Models, Animal
Gene Editing/*methods
Hepatocytes/enzymology/physiology
Liver/*enzymology
Mice
Mice, Knockout
Oxidoreductases/genetics
Phenotype
Plasmids/genetics

@article {pmid30082838,
year = {2018},
author = {Lee, JK and Jeong, E and Lee, J and Jung, M and Shin, E and Kim, YH and Lee, K and Jung, I and Kim, D and Kim, S and Kim, JS},
title = {Directed evolution of CRISPR-Cas9 to increase its specificity.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3048},
pmid = {30082838},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; DNA/genetics ; *Directed Molecular Evolution ; Escherichia coli/genetics ; Gene Editing ; HEK293 Cells ; Humans ; Plasmids/metabolism ; RNA, Guide/genetics ; Recombinant Proteins/chemistry ; Ribonucleoproteins/chemistry ; Substrate Specificity ; },
abstract = {The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.},
}

*CRISPR-Cas Systems
DNA/genetics
*Directed Molecular Evolution
Escherichia coli/genetics
Gene Editing
HEK293 Cells
Humans
Plasmids/metabolism
RNA, Guide/genetics
Recombinant Proteins/chemistry
Ribonucleoproteins/chemistry
Substrate Specificity

@article {pmid30082728,
year = {2018},
author = {Elster, D and Tollot, M and Schlegelmilch, K and Ori, A and Rosenwald, A and Sahai, E and von Eyss, B},
title = {TRPS1 shapes YAP/TEAD-dependent transcription in breast cancer cells.},
journal = {Nature communications},
volume = {9},
number = {1},
pages = {3115},
pmid = {30082728},
issn = {2041-1723},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Acetylation ; Animals ; Binding Sites ; Breast Neoplasms/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chromatin/metabolism ; DNA-Binding Proteins/*genetics/*metabolism ; Enhancer Elements, Genetic ; Epigenesis, Genetic ; Female ; *Gene Expression Regulation, Neoplastic ; Genomics ; HEK293 Cells ; Humans ; MCF-7 Cells ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Promoter Regions, Genetic ; RNA, Small Interfering/metabolism ; Tissue Array Analysis ; Transcription Factors/*genetics/*metabolism ; Transcriptional Activation ; },
abstract = {Yes-associated protein (YAP), the downstream transducer of the Hippo pathway, is a key regulator of organ size, differentiation and tumorigenesis. To uncover Hippo-independent YAP regulators, we performed a genome-wide CRISPR screen that identifies the transcriptional repressor protein Trichorhinophalangeal Syndrome 1 (TRPS1) as a potent repressor of YAP-dependent transactivation. We show that TRPS1 globally regulates YAP-dependent transcription by binding to a large set of joint genomic sites, mainly enhancers. TRPS1 represses YAP-dependent function by recruiting a spectrum of corepressor complexes to joint sites. Loss of TRPS1 leads to activation of enhancers due to increased H3K27 acetylation and an altered promoter-enhancer interaction landscape. TRPS1 is commonly amplified in breast cancer, which suggests that restrained YAP activity favours tumour growth. High TRPS1 activity is associated with decreased YAP activity and leads to decreased frequency of tumour-infiltrating immune cells. Our study uncovers TRPS1 as an epigenetic regulator of YAP activity in breast cancer.},
}

Acetylation
Animals
Binding Sites
Breast Neoplasms/*genetics/metabolism
CRISPR-Cas Systems
Cell Line, Tumor
Chromatin/metabolism
DNA-Binding Proteins/*genetics/*metabolism
Enhancer Elements, Genetic
Epigenesis, Genetic
Female
*Gene Expression Regulation, Neoplastic
Genomics
HEK293 Cells
Humans
MCF-7 Cells
Mice
Mice, Inbred BALB C
Neoplasm Transplantation
Promoter Regions, Genetic
RNA, Small Interfering/metabolism
Tissue Array Analysis
Transcription Factors/*genetics/*metabolism
Transcriptional Activation

@article {pmid29786095,
year = {2018},
author = {Guo, X and Chavez, A and Tung, A and Chan, Y and Kaas, C and Yin, Y and Cecchi, R and Garnier, SL and Kelsic, ED and Schubert, M and DiCarlo, JE and Collins, JJ and Church, GM},
title = {High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast.},
journal = {Nature biotechnology},
volume = {36},
number = {6},
pages = {540-546},
pmid = {29786095},
issn = {1546-1696},
support = {P50 HG005550/HG/NHGRI NIH HHS/United States ; RM1 HG008525/HG/NHGRI NIH HHS/United States ; T32 CA009216/CA/NCI NIH HHS/United States ; },
mesh = {Base Sequence ; Biotechnology ; CRISPR-Cas Systems ; Conserved Sequence ; DNA, Fungal/*genetics ; *Gene Library ; Genetic Variation ; High-Throughput Nucleotide Sequencing ; Open Reading Frames ; Point Mutation ; RecQ Helicases/genetics ; Saccharomyces cerevisiae/enzymology/*genetics/growth & development ; Saccharomyces cerevisiae Proteins/genetics ; Sequence Deletion ; },
abstract = {Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80-100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.},
}

Base Sequence
Biotechnology
CRISPR-Cas Systems
Conserved Sequence
DNA, Fungal/*genetics
*Gene Library
Genetic Variation
High-Throughput Nucleotide Sequencing
Open Reading Frames
Point Mutation
RecQ Helicases/genetics
Saccharomyces cerevisiae/enzymology/*genetics/growth & development
Saccharomyces cerevisiae Proteins/genetics
Sequence Deletion

@article {pmid29734295,
year = {2018},
author = {Bao, Z and HamediRad, M and Xue, P and Xiao, H and Tasan, I and Chao, R and Liang, J and Zhao, H},
title = {Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision.},
journal = {Nature biotechnology},
volume = {36},
number = {6},
pages = {505-508},
pmid = {29734295},
issn = {1546-1696},
mesh = {Amino Acid Sequence ; Base Sequence ; Biotechnology ; CRISPR-Cas Systems ; DNA, Fungal/genetics ; Directed Molecular Evolution ; Gene Editing/methods ; Genetic Engineering/*methods ; Genome, Fungal ; Models, Molecular ; Mutagenesis ; Recombinational DNA Repair ; Saccharomyces cerevisiae/enzymology/*genetics ; Saccharomyces cerevisiae Proteins/chemistry/genetics/metabolism ; Ubiquitin-Protein Ligases/chemistry/genetics/metabolism ; },
abstract = {We developed a CRISPR-Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. We validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply our method to improve tolerance to growth inhibitors.},
}

Amino Acid Sequence
Base Sequence
Biotechnology
CRISPR-Cas Systems
DNA, Fungal/genetics
Directed Molecular Evolution
Gene Editing/methods
Genetic Engineering/*methods
Genome, Fungal
Models, Molecular
Mutagenesis
Recombinational DNA Repair
Saccharomyces cerevisiae/enzymology/*genetics
Saccharomyces cerevisiae Proteins/chemistry/genetics/metabolism
Ubiquitin-Protein Ligases/chemistry/genetics/metabolism

@article {pmid29734294,
year = {2018},
author = {Roy, KR and Smith, JD and Vonesch, SC and Lin, G and Tu, CS and Lederer, AR and Chu, A and Suresh, S and Nguyen, M and Horecka, J and Tripathi, A and Burnett, WT and Morgan, MA and Schulz, J and Orsley, KM and Wei, W and Aiyar, RS and Davis, RW and Bankaitis, VA and Haber, JE and Salit, ML and St Onge, RP and Steinmetz, LM},
title = {Multiplexed precision genome editing with trackable genomic barcodes in yeast.},
journal = {Nature biotechnology},
volume = {36},
number = {6},
pages = {512-520},
pmid = {29734294},
issn = {1546-1696},
support = {P01 HG000205/HG/NHGRI NIH HHS/United States ; 294542//European Research Council/International ; R01 GM044530/GM/NIGMS NIH HHS/United States ; U01 GM110706/GM/NIGMS NIH HHS/United States ; R01 GM121932/GM/NIGMS NIH HHS/United States ; R01 GM061766/GM/NIGMS NIH HHS/United States ; R37 GM020056/GM/NIGMS NIH HHS/United States ; },
mesh = {Amino Acid Substitution ; Biotechnology ; CRISPR-Cas Systems ; DNA Barcoding, Taxonomic/*methods ; DNA, Fungal/genetics ; Gene Editing/*methods ; Genome, Fungal ; Homologous Recombination ; Phospholipid Transfer Proteins/genetics ; Plasmids/genetics ; RNA, Fungal/genetics ; Saccharomyces cerevisiae/*genetics ; Saccharomyces cerevisiae Proteins/genetics ; },
abstract = {Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR-Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.},
}

Amino Acid Substitution
Biotechnology
CRISPR-Cas Systems
DNA Barcoding, Taxonomic/*methods
DNA, Fungal/genetics
Gene Editing/*methods
Genome, Fungal
Homologous Recombination
Phospholipid Transfer Proteins/genetics
Plasmids/genetics
RNA, Fungal/genetics
Saccharomyces cerevisiae/*genetics
Saccharomyces cerevisiae Proteins/genetics