@article {pmid31121300,
year = {2019},
author = {Wandera, KG and Collins, SP and Wimmer, F and Marshall, R and Noireaux, V and Beisel, CL},
title = {An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system.},
journal = {Methods (San Diego, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymeth.2019.05.014},
pmid = {31121300},
issn = {1095-9130},
abstract = {The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.},
}

@article {pmid31119685,
year = {2019},
author = {Bae, T and Hur, JW and Kim, D and Hur, JK},
title = {Recent trends in CRISPR-Cas system: genome, epigenome, and transcriptome editing and CRISPR delivery systems.},
journal = {Genes & genomics},
volume = {},
number = {},
pages = {},
doi = {10.1007/s13258-019-00830-w},
pmid = {31119685},
issn = {2092-9293},
support = {NRF-2018-M3A9H3021707//National Research Foundation of Korea/ ; NRF-2018-M3A9H3021707//National Research Foundation of Korea/ ; NRF-2018-R1A6A1A03025124//National Research Foundation of Korea/ ; NRF-2018-R1D1A1B07043383//National Research Foundation of Korea/ ; NRF-2017R1D1A1B03035094//National Research Foundation of Korea/ ; NRF-2017R1E1A1A01074529//National Research Foundation of Korea/ ; },
abstract = {BACKGROUND: The CRISPR-Cas systems have emerged as a robust genome editing tool useful in various fields of research. With the discovery and development of the orthologous CRISPR-Cas systems, their genome editing efficiency have improved.

OBJECTIVE: In this review, we aim to present the recent developments and applications of the CRISPR-Cas systems.

METHODS: First, we introduce how the advancements of CRISPR technology enabled genome editing to single base precision. Then, we discuss the CRISPR based methods for targeted transcriptional regulation, epigenome editing, and RNA editing. Finally, we review the CRISPR delivery systems highlighting recent attempts to integrate nanotechnology to develop novel CRISPR delivery modalities.

CONCLUSION: Here, we review the recent trends in CRISPR-based biotechnologies, encompassing genome editing, epigenome regulation and direct RNA targeting and provide an overview of methods employed for CRISPR delivery with an emphasis on the most recent nanotechnology-based delivery strategies. We anticipate that the development of CRISPR based technology will continue to explore novel methods.},
}

@article {pmid31119529,
year = {2019},
author = {Mitsui, R and Yamada, R and Ogino, H},
title = {Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution.},
journal = {Applied biochemistry and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s12010-019-03040-y},
pmid = {31119529},
issn = {1559-0291},
support = {18K14069//The Japan Society for the Promotion of Science/ ; },
abstract = {In bioprocesses, a microorganism with high tolerance to various stresses would be advantageous for efficient bio-based chemical production. Yeast Saccharomyces cerevisiae has long been used in the food industry because of its safety and convenience, and genetically engineered S. cerevisiae strains have been constructed and used for the production of various bio-based chemicals. In this study, we developed a novel genome shuffling method for S. cerevisiae using CRISPR-Cas. By using this, the thermotolerant mutant strain T8-292, which can grow well at 39 °C, was successfully created. The strain also showed higher cell viability in low pH and high ethanol concentration. In addition, the differences in genome structure between mutant and parent strains were suggested by random amplified polymorphic DNA PCR method. Our genome shuffling method could be a promising strategy for improvement of various stress tolerance in S. cerevisiae.},
}

@article {pmid31119128,
year = {2019},
author = {Eckerstorfer, MF and Dolezel, M and Heissenberger, A and Miklau, M and Reichenbecher, W and Steinbrecher, RA and Waßmann, F},
title = {Corrigendum: An EU Perspective on Biosafety Considerations for Plants Developed by Genome Editing and Other New Genetic Modification Techniques (nGMs).},
journal = {Frontiers in bioengineering and biotechnology},
volume = {7},
number = {},
pages = {90},
doi = {10.3389/fbioe.2019.00090},
pmid = {31119128},
issn = {2296-4185},
abstract = {[This corrects the article DOI: 10.3389/fbioe.2019.00031.].},
}

@article {pmid30559100,
year = {2018},
author = {Xin, GW and Hu, XX and Wang, KJ and Wang, XC},
title = {[Cas9 protein variant VQR recognizes NGAC protospacer adjacent motif in rice].},
journal = {Yi chuan = Hereditas},
volume = {40},
number = {12},
pages = {1112-1119},
doi = {10.16288/j.yczz.18-126},
pmid = {30559100},
issn = {0253-9772},
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Oryza/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is the third-generation genome editing tools that was developed and widely used in recent years. However, Streptococcus pyogenes Cas9 (SpCas9) in this system could only recognize NGG PAM (protospacer adjacent motif), which largely restricts the range of genome editing. The VQR (D1135V/R1335Q/T1337R) variant of SpCas9 could recognize NGAA, NGAG and NGAT PAMs in rice. However, whether VQR variant could recognize NGAC PAM remains unclear. In this study, three low editing efficiency sites of the VQR variant, NAL1-Q1, NAL1-Q2 and LPA1-Q, were selected for genome editing using the improved CRISPR/VQR system. The improved CRISPR/VQR system effectively edited these target sites, and the gene editing efficiency was 9.75%, 43.90% and 29.26% respectively. To ensure the recognition of NGAC PAM by the improved CRISPR/VQR system, two NGAC PAM containing sites (NAL-C and GL1-C) in the NARROW LEAF 1 (NAL1) for leaf length and GLOSSY1 (GL1) genes for wax biosynthesis were selected for genome editing in rice in this study, and 57 transgenic plants were obtained. The PCR amplification and sequencing results showed that 27 plants (47.36%) had mutation in the NAL1-C site, 44 plants (77.19%) had mutation in the GL1 gene, and 26 plants (45.61%) had mutation in the NAL-C and GL1-C sites. Further analysis revealed that there were four types of mutations caused by the CRISPR/VQR system, respectively for the hybrid mutation, biallelic mutation, chimeric mutation and homozygous mutations. Among them, heterozygous mutation and biallelic mutation were dominant changes. These results indicated that the improved CRISPR/VQR system could efficiently edit the NGAC PAM sites of the rice and produce abundant mutant types. This study provides a theoretical basis for NGAC PAM editing in rice and other related plants.},
}

CRISPR-Associated Protein 9/*genetics
*CRISPR-Cas Systems
*Gene Editing
Oryza/*genetics

@article {pmid30559097,
year = {2018},
author = {Liu, CX and Geng, LZ and Xu, JP},
title = {[Detection methods of genome editing in plants].},
journal = {Yi chuan = Hereditas},
volume = {40},
number = {12},
pages = {1075-1091},
doi = {10.16288/j.yczz.18-079},
pmid = {30559097},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genetic Engineering ; Genome, Plant ; Plants/*genetics ; },
abstract = {The life science has entered a new chapter with the revolutionary implementation of the CRISPR/Cas9 genome editing technology in various living organisms. With the unique flexibility, feasibility and extendibility, the CRISPR/Cas9 technology greatly accelerates genetic engineering research, as well as plant molecular breeding. However, it has become a challenge to screen for and identify genome-edited plants at early stages in a rapid and high-throughput fashion, due to the massive number of plants produced from transformation process. In this review, we summarize the molecular methods developed in recent years to identify genome-edited plants. We compare their advantages and disadvantages, and the scope of application. In addition, we provide insights of the development trend of detection methods for plant genome editing. This review will serve as a reference for future genome editing research in plants.},
}

*CRISPR-Cas Systems
*Gene Editing
*Genetic Engineering
Genome, Plant
Plants/*genetics

@article {pmid30507925,
year = {2018},
author = {Evans, BA and Pickerill, ES and Vyas, VK and Bernstein, DA},
title = {CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {141},
pages = {},
doi = {10.3791/58764},
pmid = {30507925},
issn = {1940-087X},
support = {R15 AI130950/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Candida albicans/*genetics ; Gene Editing/*methods ; Humans ; Plasmids ; RNA, Guide/genetics ; },
abstract = {This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.},
}

CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
Candida albicans/*genetics
Gene Editing/*methods
Humans
Plasmids
RNA, Guide/genetics

@article {pmid30507915,
year = {2018},
author = {Bialek, JK and Walther, T and Hauber, J and Lange, UC},
title = {CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {141},
pages = {},
doi = {10.3791/58572},
pmid = {30507915},
issn = {1940-087X},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; *Genome ; HIV Infections/*genetics ; HIV-1/*genetics ; Humans ; Jurkat Cells ; RNA, Guide/genetics ; *Virus Integration ; },
abstract = {Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites. In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described. A targeting vector with homology arms is constructed and transfected into Jurkat T cells. The reporter sequence is targeted to the selected genomic site by homologous recombination facilitated by a Cas9-mediated double-strand break at the target site. Single-cell clones are generated and screened for targeting events by flow cytometry and PCR. Selected clones are then expanded, and correct targeting is verified by PCR, sequencing, and Southern blotting. Potential off-target events of CRISPR-Cas9-mediated genome engineering are analyzed. By using this protocol, novel cell culture systems that model HIV infection at clinically relevant integration sites can be generated. Although the generation of single-cell clones and verification of correct reporter sequence integration is time-consuming, the resulting clonal lines are powerful tools to functionally analyze proviral integration site choice.},
}

CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
*Gene Editing
*Genome
HIV Infections/*genetics
HIV-1/*genetics
Humans
Jurkat Cells
RNA, Guide/genetics
*Virus Integration

@article {pmid29959122,
year = {2018},
author = {Li, HQ and Chen, C and Chen, RR and Song, XW and Li, JN and Zhu, YM and Ding, XD},
title = {[Preliminary analysis of the role of GmSnRK1.1 and GmSnRK1.2 in the ABA and alkaline stress response of the soybean using the CRISPR/Cas9-based gene double-knockout system].},
journal = {Yi chuan = Hereditas},
volume = {40},
number = {6},
pages = {496-507},
doi = {10.16288/j.yczz.17-424},
pmid = {29959122},
issn = {0253-9772},
mesh = {Agrobacterium ; Alkalies ; *CRISPR-Cas Systems ; Gene Knockdown Techniques ; Hydrogen-Ion Concentration ; Plant Proteins/*genetics ; Plant Roots ; Plants, Genetically Modified/genetics/physiology ; Protein-Serine-Threonine Kinases/*genetics ; Soybeans/*genetics/physiology ; Stress, Physiological ; },
abstract = {Sucrose non-fermenting related protein kinases (SnRKs) are a ubiquitous Ser/Thr protein kinase in the plant kingdom. These kinases play important roles in plant growth, development, metabolism and resistance to environmental stresses. The soybean (Glycine max L.) genome has four SnRK1 genes, of which GmSnRK1.1 and GmSnRK1.2 are predominant and participate in multiple stress response pathways. To dissect the mechanism of the role of GmSnRK1.1 and GmSnRK1.2 proteins in response to ABA and alkaline stresses, we constructed a dual-gRNA CRISPR vector to specifically knock out GmSnRK1.1 and GmSnRK1.2. The resultant constructs were transformed into soybean cotyledon nodes to induce hairy roots by agrobacteria (Agrobacterium rhizogenes). The soybean hairy roots obtained were genotyped, and the results showed that GmSnRK1.1 and GmSnRK1.2 were efficiently doubly knocked out in 48.6% hairy roots. We also generated control hairy roots that over-expressed GmSnRK1. The materials were treated with 25 μmol/L ABA for 15 days and the results showed that the growths of wild-type and GmSnRK1 over-expressed roots were significantly inhibited than GmSnRK1.1 GmSnRK1.2 double-knockout roots, as the controls displayed less root lengths and fresh weights. However, after treating with 50 mmol/L NaHCO3 for 15 days, we found that the growths of GmSnRK1.1 GmSnRK1.2 double-knockout roots were significantly inhibited than the wild-type and GmSnRK1 over-expressed control roots, as the knockout groups contained less root lengths and fresh weights. These results implied that the GmSnRK1.1 GmSnRK1.2 double knockout mitigated hairy root sensitivity to ABA and resistance to alkaline stress. Taken together, we established the CRISPR/Cas9 system to perform gene double knockout in the soybean and by using this technique, we determined the roles of GmSnRK1.1 and GmSnRK1.2 in response of abiotic stresses.},
}

Agrobacterium
Alkalies
*CRISPR-Cas Systems
Gene Knockdown Techniques
Hydrogen-Ion Concentration
Plant Proteins/*genetics
Plant Roots
Plants, Genetically Modified/genetics/physiology
Protein-Serine-Threonine Kinases/*genetics
Soybeans/*genetics/physiology
Stress, Physiological

@article {pmid29959120,
year = {2018},
author = {Tang, JB and Cao, HW and Xu, R and Zhang, DD and Huang, J},
title = {[Mutant generation of the testis genes and phenotype analyses in Drosophila].},
journal = {Yi chuan = Hereditas},
volume = {40},
number = {6},
pages = {478-487},
doi = {10.16288/j.yczz.18-055},
pmid = {29959120},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila/*genetics/growth & development ; *Genes, Insect ; Loss of Function Mutation ; Male ; Phenotype ; Testis/*growth & development ; },
abstract = {Multiple genes work together to maintain the normal functions of the reproductive system. However, for many of these genes, little is known about their specific functions and mechanisms. In the present study, eight Drosophila genes, including CG4161, CG11475, CG2921, CG10541, CG7276, CG3800, CG8117 and CG16779, were selected for detailed studies based on their testis expression, undefined functions, and having highly homologous and conserved genes in humans (Homo sapiens) and mouse (Mus musculus). We analyzed their expression levels in different tissues, and determined their probably functions in male reproduction. The results showed that the first five genes were mainly expressed in testis, while other three showed ubiquitous expression in all tissues examined. Using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) and homology-directed repair (HDR) strategies, we have systematically generated the mutants of these genes and studied their functions in male reproduction. Loss of function of CG7276 and CG3800 resulted in partial sterility and reduction of the offspring number, while other genes showed no significant impact on fertility. CG7276 -/- and CG3800 -/- mutants were partial sterile and showed various types of abnormities, including seminal vesicle atrophy, spermatogonial stem cell reduction and cellular distribution disorders. Results of DNA and F-actin staining also indicated that CG7276 and CG3800 could play important roles in spermiogenesis. The establishments of these mutants have provided means to unravel the functions and mechanisms of these genes in animal models.},
}

Animals
*CRISPR-Cas Systems
Drosophila/*genetics/growth & development
*Genes, Insect
Loss of Function Mutation
Male
Phenotype
Testis/*growth & development

@article {pmid31114911,
year = {2019},
author = {Béguin, P and Chekli, Y and Sezonov, G and Forterre, P and Krupovic, M},
title = {Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkz447},
pmid = {31114911},
issn = {1362-4962},
abstract = {Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1-Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3' end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR-Cas systems.},
}

@article {pmid30923122,
year = {2019},
author = {Kim, H and Bojar, D and Fussenegger, M},
title = {A CRISPR/Cas9-based central processing unit to program complex logic computation in human cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {15},
pages = {7214-7219},
doi = {10.1073/pnas.1821740116},
pmid = {30923122},
issn = {1091-6490},
mesh = {*CRISPR-Cas Systems ; *Computers, Molecular ; *Gene Expression Regulation ; HEK293 Cells ; Humans ; },
abstract = {Controlling gene expression with sophisticated logic gates has been and remains one of the central aims of synthetic biology. However, conventional implementations of biocomputers use central processing units (CPUs) assembled from multiple protein-based gene switches, limiting the programming flexibility and complexity that can be achieved within single cells. Here, we introduce a CRISPR/Cas9-based core processor that enables different sets of user-defined guide RNA inputs to program a single transcriptional regulator (dCas9-KRAB) to perform a wide range of bitwise computations, from simple Boolean logic gates to arithmetic operations such as the half adder. Furthermore, we built a dual-core CPU combining two orthogonal core processors in a single cell. In principle, human cells integrating multiple orthogonal CRISPR/Cas9-based core processors could offer enormous computational capacity.},
}

*CRISPR-Cas Systems
*Computers, Molecular
*Gene Expression Regulation
HEK293 Cells
Humans

@article {pmid30663071,
year = {2019},
author = {Jang, YJ and Kim, SA and Seo, SO and Li, L and Han, NS},
title = {Plasmid curing resulted in improved heterologous gene expression in Leuconostoc citreum EFEL2700.},
journal = {Letters in applied microbiology},
volume = {68},
number = {5},
pages = {430-436},
doi = {10.1111/lam.13118},
pmid = {30663071},
issn = {1472-765X},
support = {//Korean Ministry of Science/ ; //ICT/ ; 2013M3A6A8073553//Future Planning/ ; 918006-04-1SB010//Ministry of Agriculture, Food and Rural Affairs (MAFRA)/ ; },
mesh = {CRISPR-Cas Systems/genetics ; Gene Expression/genetics ; Gene Expression Regulation, Bacterial/*genetics ; Leuconostoc/*genetics/*metabolism ; Metabolic Engineering ; Plasmids/*genetics ; beta-Glucosidase/metabolism ; },
abstract = {Leuconostoc citreum EFEL2700 isolated from kimchi was used as a host strain for genetic and metabolic engineering in our previous studies, but the cells of EFEL2700 contained a cryptic plasmid (P-cells). Thus, we created plasmid-free cells (F-cells) using the CRISPR/Cas9 system. In this study, we compared the microbial characteristics of P- and F-cells in terms of growth rate, biochemical properties, transformation efficiency, plasmid copy number and protein expression level. When the growth rate was measured in MRS medium at 30°C, no significant difference (P > 0·01) was observed. Biochemical properties, tested using an API 50CHL kit, showed no differences. Transformation efficiency of F-cells, measured using pCB4270, was higher (1·3 × 104 CFU per μg DNA) than that of P-cells (5·0 × 103 CFU per μg DNA). Copy number after transformation of pCBBgl was 4-fold higher for F-cells than for P-cells. When β-glucosidase activity was assayed in the above experiment, F-cells showed 3·4-fold higher values than P-cells. In conclusion, this study demonstrates that plasmid curing in L. citreum EFEL2700 improves its characteristics as a gene expression host. SIGNIFICANCE AND IMPACT OF THE STUDY: Leuconostoc citreum EFEL2700 (P-cell) isolated from kimchi is a useful food-grade host for expressing heterologous genes. The presence of a cryptic plasmid is thought to limit efficient gene expression. In this study, we compared the microbial and genetic changes after plasmid curing in this strain. The plasmid-free strain showed improved levels of transformation efficiency, copy number and heterologous gene expression without alterations in phenotypes such as the growth rates and biochemical properties. The resulting strain of L. citreum EFEL2701 (F-cell) can be used as an efficient host for genetic engineering.},
}

CRISPR-Cas Systems/genetics
Gene Expression/genetics
Gene Expression Regulation, Bacterial/*genetics
Leuconostoc/*genetics/*metabolism
Metabolic Engineering
Plasmids/*genetics
beta-Glucosidase/metabolism

@article {pmid30538335,
year = {2019},
author = {Hatzi, K and Geng, H and Doane, AS and Meydan, C and LaRiviere, R and Cardenas, M and Duy, C and Shen, H and Vidal, MNC and Baslan, T and Mohammad, HP and Kruger, RG and Shaknovich, R and Haberman, AM and Inghirami, G and Lowe, SW and Melnick, AM},
title = {Histone demethylase LSD1 is required for germinal center formation and BCL6-driven lymphomagenesis.},
journal = {Nature immunology},
volume = {20},
number = {1},
pages = {86-96},
pmid = {30538335},
issn = {1529-2916},
support = {K99 CA212276/CA/NCI NIH HHS/United States ; R01 CA187109/CA/NCI NIH HHS/United States ; R35 CA220499/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; B-Lymphocytes/*physiology ; CRISPR-Cas Systems ; Carcinogenesis ; DNA, Intergenic/genetics ; Germinal Center/immunology/*pathology ; Histone Demethylases/genetics/*metabolism ; Hyperplasia ; Immunological Synapses/genetics ; Introns/genetics ; Lymphoma/genetics/*metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Proto-Oncogene Proteins c-bcl-6/genetics/*metabolism ; },
abstract = {Germinal center (GC) B cells feature repression of many gene enhancers to establish their characteristic transcriptome. Here we show that conditional deletion of Lsd1 in GCs significantly impaired GC formation, associated with failure to repress immune synapse genes linked to GC exit, which are also direct targets of the transcriptional repressor BCL6. We found that BCL6 directly binds LSD1 and recruits it primarily to intergenic and intronic enhancers. Conditional deletion of Lsd1 suppressed GC hyperplasia caused by constitutive expression of BCL6 and significantly delayed BCL6-driven lymphomagenesis. Administration of catalytic inhibitors of LSD1 had little effect on GC formation or GC-derived lymphoma cells. Using a CRISPR-Cas9 domain screen, we found instead that the LSD1 Tower domain was critical for dependence on LSD1 in GC-derived B cells. These results indicate an essential role for LSD1 in the humoral immune response, where it modulates enhancer function by forming repression complexes with BCL6.},
}

Animals
B-Lymphocytes/*physiology
CRISPR-Cas Systems
Carcinogenesis
DNA, Intergenic/genetics
Germinal Center/immunology/*pathology
Histone Demethylases/genetics/*metabolism
Hyperplasia
Immunological Synapses/genetics
Introns/genetics
Lymphoma/genetics/*metabolism
Mice
Mice, Inbred C57BL
Mice, Knockout
Proto-Oncogene Proteins c-bcl-6/genetics/*metabolism

@article {pmid30111373,
year = {2018},
author = {Yang, M and Yu, X and Li, X and Luo, B and Yang, W and Lin, Y and Li, D and Gan, Z and Xu, J and He, T},
title = {TNFAIP3 is required for FGFR1 activation-promoted proliferation and tumorigenesis of premalignant DCIS.COM human mammary epithelial cells.},
journal = {Breast cancer research : BCR},
volume = {20},
number = {1},
pages = {97},
pmid = {30111373},
issn = {1465-542X},
support = {CA112403/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breast/cytology/pathology ; Breast Neoplasms/genetics/*pathology ; CRISPR-Cas Systems ; Carcinoma, Intraductal, Noninfiltrating/genetics/*pathology ; Cell Line, Tumor ; Cell Movement/genetics ; Cell Proliferation/genetics ; Cell Transformation, Neoplastic/genetics/*pathology ; Epithelial Cells ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; MAP Kinase Signaling System ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness/pathology ; Receptor, Fibroblast Growth Factor, Type 1/*metabolism ; Spheroids, Cellular ; Tumor Necrosis Factor alpha-Induced Protein 3/genetics/*metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {BACKGROUND: Although ductal carcinoma in situ (DCIS) is a non-invasive breast cancer, many DCIS lesions may progress to invasive cancer and the genes and pathways responsible for its progression are largely unknown. FGFR1 plays an important role in cell proliferation, differentiation and carcinogenesis. The purpose of this study is to examine the roles of FGFR1 signaling in gene expression, cell proliferation, tumor growth and progression in a non-invasive DCIS model.

METHODS: DCIS.COM cells were transfected with an empty vector to generate DCIS-Ctrl cells. DCIS-iFGFR1 cells were transfected with an AP20187-inducible iFGFR1 vector to generate DCIS-iFGFR1 cells. iFGFR1 consists of the v-Src myristoylation membrane-targeting sequence, FGFR1 cytoplasmic domain and the AP20187-inducible FKBP12 dimerization domain, which simulates FGFR1 signaling. The CRISPR/Cas9 system was employed to knockout ERK1, ERK2 or TNFAIP3 in DCIS-iFGFR1 cells. Established cell lines were treated with/without AP20187 and with/without FGFR1, MEK, or ERK1/2 inhibitor. The effects of these treatments were determined by Western blot, RNA-Seq, real-time RT-PCR, cell proliferation, mammosphere growth, xenograft tumor growth, and tumor histopathological assays.

RESULTS: Activation of iFGFR1 signaling in DCIS-iFGFR1 cells enhanced ERK1/2 activities, induced partial epithelial-to-mesenchymal transition (EMT) and increased cell proliferation. Activation of iFGFR1 signaling promoted DCIS growth and progression to invasive cancer derived from DCIS-iFGFR1 cells in mice. Activation of iFGFR1 signaling also altered expression levels of 946 genes involved in cell proliferation, migration, cancer pathways, and other molecular and cellular functions. TNFAIP3, a ubiquitin-editing enzyme, is upregulated by iFGFR1 signaling in a FGFR1 kinase activity and in an ERK2-dependent manner. Importantly, TNFAIP3 knockout not only inhibited the AP20187-induced proliferation and tumor growth of DCIS-iFGFR1 cells, but also further reduced baseline proliferation and tumor growth of DCIS-iFGFR1 cells without AP20187 treatment.

CONCLUSIONS: Activation of iFGFR1 promotes ERK1/2 activity, EMT, cell proliferation, tumor growth, DCIS progression to invasive cancer, and altered the gene expression profile of DCIS-iFGFR1 cells. Activation of iFGFR1 upregulated TNFAIP3 in an ERK2-dependent manner and TNFAIP3 is required for iFGFR1 activation-promoted DCIS.COM cell proliferation, mammosphere growth, tumor growth and progression. These results suggest that TNFAIP3 may be a potential target for inhibiting DCIS growth and progression promoted by FGFR1 signaling.},
}

Animals
Breast/cytology/pathology
Breast Neoplasms/genetics/*pathology
CRISPR-Cas Systems
Carcinoma, Intraductal, Noninfiltrating/genetics/*pathology
Cell Line, Tumor
Cell Movement/genetics
Cell Proliferation/genetics
Cell Transformation, Neoplastic/genetics/*pathology
Epithelial Cells
Female
Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Humans
MAP Kinase Signaling System
Mice, Inbred BALB C
Mice, Nude
Neoplasm Invasiveness/pathology
Receptor, Fibroblast Growth Factor, Type 1/*metabolism
Spheroids, Cellular
Tumor Necrosis Factor alpha-Induced Protein 3/genetics/*metabolism
Xenograft Model Antitumor Assays

@article {pmid29483174,
year = {2018},
author = {Chadwick, AC and Evitt, NH and Lv, W and Musunuru, K},
title = {Reduced Blood Lipid Levels With In Vivo CRISPR-Cas9 Base Editing of ANGPTL3.},
journal = {Circulation},
volume = {137},
number = {9},
pages = {975-977},
pmid = {29483174},
issn = {1524-4539},
support = {R01 HL118744/HL/NHLBI NIH HHS/United States ; R01 HL126875/HL/NHLBI NIH HHS/United States ; T32 GM007170/GM/NIGMS NIH HHS/United States ; T32 HL007843/HL/NHLBI NIH HHS/United States ; },
mesh = {Angiopoietin-like Proteins/*genetics ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Female ; *Gene Editing ; Hyperlipidemias/blood/*genetics/*therapy ; Lipids/*blood ; Loss of Function Mutation ; Male ; Mice ; Mice, Inbred C57BL ; Proprotein Convertase 9/genetics ; },
}

Angiopoietin-like Proteins/*genetics
Animals
CRISPR-Cas Systems
Disease Models, Animal
Female
*Gene Editing
Hyperlipidemias/blood/*genetics/*therapy
Lipids/*blood
Loss of Function Mutation
Male
Mice
Mice, Inbred C57BL
Proprotein Convertase 9/genetics

@article {pmid29292383,
year = {2018},
author = {Zhang, H and Li, Y and Wang, HB and Zhang, A and Chen, ML and Fang, ZX and Dong, XD and Li, SB and Du, Y and Xiong, D and He, JY and Li, MZ and Liu, YM and Zhou, AJ and Zhong, Q and Zeng, YX and Kieff, E and Zhang, Z and Gewurz, BE and Zhao, B and Zeng, MS},
title = {Ephrin receptor A2 is an epithelial cell receptor for Epstein-Barr virus entry.},
journal = {Nature microbiology},
volume = {3},
number = {2},
pages = {1-8},
doi = {10.1038/s41564-017-0080-8},
pmid = {29292383},
issn = {2058-5276},
mesh = {Animals ; Benzoates/antagonists & inhibitors ; CHO Cells ; CRISPR-Cas Systems ; Cricetulus ; Ephrin-A2/*metabolism ; Epithelial Cells/*virology ; Gene Knockdown Techniques ; HEK293 Cells ; Herpesvirus 4, Human/*pathogenicity ; Humans ; Membrane Glycoproteins ; Molecular Chaperones ; RNA Interference ; Receptor, EphA2/drug effects/genetics/*metabolism ; Viral Proteins ; *Virus Internalization ; },
abstract = {Epstein-Barr virus (EBV) is causally associated with nasopharyngeal carcinoma, 10% of gastric carcinoma and various B cell lymphomas 1 . EBV infects both B cells and epithelial cells 2 . Recently, we reported that epidermal growth factor and Neuropilin 1 markedly enhanced EBV entry into nasopharyngeal epithelial cells 3 . However, knowledge of how EBV infects epithelial cells remains incomplete. To understand the mechanisms through which EBV infects epithelial cells, we integrated microarray and RNA interference screen analyses and found that Ephrin receptor A2 (EphA2) is important for EBV entry into the epithelial cells. EphA2 short interfering RNA knockdown or CRISPR-Cas9 knockout markedly reduced EBV epithelial cell infection, which was mostly restored by EphA2 complementary DNA rescue. EphA2 overexpression increased epithelial cell EBV infection. Soluble EphA2 protein, antibodies against EphA2, soluble EphA2 ligand EphrinA1, or the EphA2 inhibitor 2,5-dimethylpyrrolyl benzoic acid efficiently blocked EBV epithelial cell infection. Mechanistically, EphA2 interacted with EBV entry proteins gH/gL and gB to facilitate EBV internalization and fusion. The EphA2 Ephrin-binding domain and fibronectin type III repeats domain were essential for EphA2-mediated EBV infection, while the intracellular domain was dispensable. This is distinct from Kaposi's sarcoma-associated herpesvirus infection through EphA2 4 . Taken together, our results identify EphA2 as a critical player for EBV epithelial cell entry.},
}

Animals
Benzoates/antagonists & inhibitors
CHO Cells
CRISPR-Cas Systems
Cricetulus
Ephrin-A2/*metabolism
Epithelial Cells/*virology
Gene Knockdown Techniques
HEK293 Cells
Herpesvirus 4, Human/*pathogenicity
Humans
Membrane Glycoproteins
Molecular Chaperones
RNA Interference
Receptor, EphA2/drug effects/genetics/*metabolism
Viral Proteins
*Virus Internalization

@article {pmid29287762,
year = {2018},
author = {Fenini, G and Grossi, S and Gehrke, S and Beer, HD and Satoh, TK and Contassot, E and French, LE},
title = {The p38 Mitogen-Activated Protein Kinase Critically Regulates Human Keratinocyte Inflammasome Activation.},
journal = {The Journal of investigative dermatology},
volume = {138},
number = {6},
pages = {1380-1390},
doi = {10.1016/j.jid.2017.10.037},
pmid = {29287762},
issn = {1523-1747},
mesh = {CARD Signaling Adaptor Proteins/immunology/metabolism ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Enzyme Activation ; Humans ; Inflammasomes/*immunology/metabolism ; Interleukin-1beta/immunology/metabolism ; JNK Mitogen-Activated Protein Kinases ; Keratinocytes/*immunology/metabolism ; Mitogen-Activated Protein Kinase 13/antagonists & inhibitors/genetics/*metabolism ; Mitogen-Activated Protein Kinase 14/antagonists & inhibitors/genetics/*metabolism ; Mitogen-Activated Protein Kinase 8/antagonists & inhibitors/genetics/metabolism ; Myeloid Cells/immunology/metabolism ; Phosphorylation ; Primary Cell Culture ; Protein Multimerization/immunology ; RNA Interference ; RNA, Small Interfering/metabolism ; Signal Transduction/*immunology ; },
abstract = {Inflammasomes are key intracellular signaling platforms involved in innate immune responses to micro-organisms and danger signals. Extracellular signal-regulated kinase, Jun N-terminal kinase, and p38 mitogen-activated protein kinase family members are activated by numerous environmental stresses. Recently, it has been reported that Jun N-terminal kinase is involved in inflammasome activation in myeloid immune cells. To date, the role of mitogen-activated protein kinase in inflammasome activity in keratinocytes has not been investigated. Here, we show that, in primary human keratinocytes, p38 mitogen-activated protein kinase is required for inflammasome activation and IL-1β secretion. Using selective small molecule inhibitors, small interfering RNA gene silencing, and CRISPR/Cas9-based deletion, we demonstrate the above and identify p38α and p38δ as critical regulators of ASC oligomerization, inflammasome activation, and IL-1β secretion in keratinocytes. Furthermore, our data suggest that the nature of the mitogen-activated protein kinase regulating inflammasome activity exhibits a certain cell specificity, with p38 playing a predominant role in keratinocytes and Jun N-terminal kinase 1 in cells of myeloid origin.},
}

CARD Signaling Adaptor Proteins/immunology/metabolism
CRISPR-Cas Systems/genetics
Cells, Cultured
Enzyme Activation
Humans
Inflammasomes/*immunology/metabolism
Interleukin-1beta/immunology/metabolism
JNK Mitogen-Activated Protein Kinases
Keratinocytes/*immunology/metabolism
Mitogen-Activated Protein Kinase 13/antagonists & inhibitors/genetics/*metabolism
Mitogen-Activated Protein Kinase 14/antagonists & inhibitors/genetics/*metabolism
Mitogen-Activated Protein Kinase 8/antagonists & inhibitors/genetics/metabolism
Myeloid Cells/immunology/metabolism
Phosphorylation
Primary Cell Culture
Protein Multimerization/immunology
RNA Interference
RNA, Small Interfering/metabolism
Signal Transduction/*immunology

@article {pmid29216043,
year = {2017},
author = {Chakradhar, S},
title = {The Yearbook.},
journal = {Nature medicine},
volume = {23},
number = {12},
pages = {1386},
doi = {10.1038/nm1217-1386},
pmid = {29216043},
issn = {1546-170X},
mesh = {Bioethics ; CRISPR-Cas Systems ; Gene Editing/methods ; Humans ; *Patents as Topic ; *Publishing ; },
}

Bioethics
CRISPR-Cas Systems
Gene Editing/methods
Humans
*Patents as Topic
*Publishing

@article {pmid31110048,
year = {2019},
author = {Johnson, K and Learn, BA and Estrella, MA and Bailey, S},
title = {-Target sequence requirements of a type III-B CRISPR-Cas immune system.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA119.008728},
pmid = {31110048},
issn = {1083-351X},
abstract = {CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids. Type III CRISPR systems are unique, as their targeting mechanism requires target transcription. Upon transcript binding, DNA cleavage by type III effector complexes is activated. Type III systems must differentiate between invader and native transcripts in order to prevent autoimmunity. Transcript origin is dictated by the sequence that flanks the 3' end of the RNA target site (called the PFS). However, how the PFS is recognized may vary among different type III systems. Here, using purified proteins and in vitro assays, we define how the type III-B effector from the hyperthermophilic bacterium Thermotoga maritima discriminates between native and invader transcripts. We show that native transcripts are recognized by base pairing at positions -2 to -5 of the PFS and by a guanine at position -1, which is not recognized by base pairing. We also show that mismatches with the RNA target are highly tolerated in this system, except for those nucleotides adjacent to the PFS. These findings define the target requirement for the type III-B system from T. maritima and provide a framework for understanding the target requirements of type III systems as a whole.},
}

@article {pmid30894482,
year = {2019},
author = {Quinney, KB and Frankel, EB and Shankar, R and Kasberg, W and Luong, P and Audhya, A},
title = {Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {14},
pages = {6858-6867},
doi = {10.1073/pnas.1817898116},
pmid = {30894482},
issn = {1091-6490},
support = {P30 CA014520/CA/NCI NIH HHS/United States ; R01 GM088151/GM/NIGMS NIH HHS/United States ; T32 GM007215/GM/NIGMS NIH HHS/United States ; T32 GM008688/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Transformed ; Endosomal Sorting Complexes Required for Transport/*genetics/metabolism ; Gene Editing ; Humans ; Intercellular Signaling Peptides and Proteins/genetics/*metabolism ; Multivesicular Bodies/genetics/*metabolism ; Protein Transport/physiology ; },
abstract = {The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.},
}

CRISPR-Cas Systems
Cell Line, Transformed
Endosomal Sorting Complexes Required for Transport/*genetics/metabolism
Gene Editing
Humans
Intercellular Signaling Peptides and Proteins/genetics/*metabolism
Multivesicular Bodies/genetics/*metabolism
Protein Transport/physiology

@article {pmid30858603,
year = {2019},
author = {Park, H and Oh, J and Shim, G and Cho, B and Chang, Y and Kim, S and Baek, S and Kim, H and Shin, J and Choi, H and Yoo, J and Kim, J and Jun, W and Lee, M and Lengner, CJ and Oh, YK and Kim, J},
title = {In vivo neuronal gene editing via CRISPR-Cas9 amphiphilic nanocomplexes alleviates deficits in mouse models of Alzheimer's disease.},
journal = {Nature neuroscience},
volume = {22},
number = {4},
pages = {524-528},
doi = {10.1038/s41593-019-0352-0},
pmid = {30858603},
issn = {1546-1726},
mesh = {Alzheimer Disease/*genetics/*metabolism/therapy ; Amyloid Precursor Protein Secretases/*genetics/metabolism ; Amyloid beta-Peptides/*metabolism ; Animals ; Aspartic Acid Endopeptidases/*genetics/metabolism ; *CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Therapy/methods ; Hippocampus/metabolism ; Male ; Mice, Transgenic ; Nanoparticles/administration & dosage ; Neurons/*metabolism ; },
abstract = {In vivo gene editing in post-mitotic neurons of the adult brain may be a useful strategy for treating neurological diseases. Here, we develop CRISPR-Cas9 nanocomplexes and show they were effective in the adult mouse brain, with minimal off-target effects. Using this system to target Bace1 suppressed amyloid beta (Aβ)-associated pathologies and cognitive deficits in two mouse models of Alzheimer's disease. These results broaden the potential application of CRISPR-Cas9 systems to neurodegenerative diseases.},
}

Alzheimer Disease/*genetics/*metabolism/therapy
Amyloid Precursor Protein Secretases/*genetics/metabolism
Amyloid beta-Peptides/*metabolism
Animals
Aspartic Acid Endopeptidases/*genetics/metabolism
*CRISPR-Cas Systems
Disease Models, Animal
Gene Editing/*methods
Genetic Therapy/methods
Hippocampus/metabolism
Male
Mice, Transgenic
Nanoparticles/administration & dosage
Neurons/*metabolism

@article {pmid29867128,
year = {2018},
author = {Kang, BC and Yun, JY and Kim, ST and Shin, Y and Ryu, J and Choi, M and Woo, JW and Kim, JS},
title = {Precision genome engineering through adenine base editing in plants.},
journal = {Nature plants},
volume = {4},
number = {7},
pages = {427-431},
doi = {10.1038/s41477-018-0178-x},
pmid = {29867128},
issn = {2055-0278},
mesh = {*Adenine ; Arabidopsis/genetics ; Brassica napus/genetics ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome, Plant/*genetics ; Plants, Genetically Modified ; Protoplasts ; },
abstract = {The recent development of adenine base editors (ABEs) has enabled efficient and precise A-to-G base conversions in higher eukaryotic cells. Here, we show that plant-compatible ABE systems can be successfully applied to protoplasts of Arabidopsis thaliana and Brassica napus through transient transfection, and to individual plants through Agrobacterium-mediated transformation to obtain organisms with desired phenotypes. Targeted, precise A-to-G substitutions generated a single amino acid change in the FT protein or mis-splicing of the PDS3 RNA transcript, and we could thereby obtain transgenic plants with late-flowering and albino phenotypes, respectively. Our results provide 'proof of concept' for in planta ABE applications that can lead to induced neo-functionalization or altered mRNA splicing, opening up new avenues for plant genome engineering and biotechnology.},
}

*Adenine
Arabidopsis/genetics
Brassica napus/genetics
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Gene Editing/*methods
Genetic Engineering/*methods
Genome, Plant/*genetics
Plants, Genetically Modified
Protoplasts

@article {pmid29615404,
year = {2018},
author = {Stelling, A and Hashwah, H and Bertram, K and Manz, MG and Tzankov, A and Müller, A},
title = {The tumor suppressive TGF-β/SMAD1/S1PR2 signaling axis is recurrently inactivated in diffuse large B-cell lymphoma.},
journal = {Blood},
volume = {131},
number = {20},
pages = {2235-2246},
doi = {10.1182/blood-2017-10-810630},
pmid = {29615404},
issn = {1528-0020},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Disease Models, Animal ; Gene Targeting ; Germinal Center/pathology ; Humans ; Lymphoma, Large B-Cell, Diffuse/*metabolism ; Mice ; Mice, Knockout ; Models, Biological ; Receptors, Lysosphingolipid/*metabolism ; *Signal Transduction ; Smad1 Protein/*metabolism ; Transforming Growth Factor beta/*metabolism ; },
abstract = {The sphingosine-1-phosphate receptor S1PR2 and its downstream signaling pathway are commonly silenced in diffuse large B-cell lymphoma (DLBCL), either by mutational inactivation or through negative regulation by the oncogenic transcription factor FOXP1. In this study, we examined the upstream regulators of S1PR2 expression and have newly identified the transforming growth factor-β (TGF-β)/TGF-βR2/SMAD1 axis as critically involved in S1PR2 transcriptional activation. Phosphorylated SMAD1 directly binds to regulatory elements in the S1PR2 locus as assessed by chromatin immunoprecipitation, and the CRISPR-mediated genomic editing of S1PR2, SMAD1, or TGFBR2 in DLBCL cell lines renders cells unresponsive to TGF-β-induced apoptosis. DLBCL clones lacking any 1 of the 3 factors have a clear growth advantage in vitro, as well as in subcutaneous xenotransplantation models, and in a novel model of orthotopic growth of DLBCL cells in the spleens and bone marrow of MISTRG mice expressing various human cytokines. The loss of S1pr2 induces hyperproliferation of the germinal center (GC) B-cell compartment of immunized mice and accelerates MYC-driven lymphomagenesis in spontaneous and serial transplantation models. The specific loss of Tgfbr2 in murine GC B-cell phenocopies the effects of S1pr2 loss on GC B-cell hyperproliferation. Finally, we show that SMAD1 expression is aberrantly downregulated in >85% of analyzed DLBCL patients. The combined results uncover an important novel tumor suppressive function of the TGF-β/TGF-βR2/SMAD1/S1PR2 axis in DLBCL, and show that DLBCL cells have evolved to inactivate the pathway at the level of SMAD1 expression.},
}

Animals
Apoptosis
CRISPR-Cas Systems
Cell Line, Tumor
Cell Proliferation
Disease Models, Animal
Gene Targeting
Germinal Center/pathology
Humans
Lymphoma, Large B-Cell, Diffuse/*metabolism
Mice
Mice, Knockout
Models, Biological
Receptors, Lysosphingolipid/*metabolism
*Signal Transduction
Smad1 Protein/*metabolism
Transforming Growth Factor beta/*metabolism

@article {pmid29483683,
year = {2018},
author = {Kim, JS},
title = {Precision genome engineering through adenine and cytosine base editing.},
journal = {Nature plants},
volume = {4},
number = {3},
pages = {148-151},
doi = {10.1038/s41477-018-0115-z},
pmid = {29483683},
issn = {2055-0278},
mesh = {*Adenine ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Cytosine ; Gene Editing/*methods ; Genetic Engineering/*methods ; },
abstract = {Adenine base editors (ABEs), composed of an engineered deaminase and a catalytically impaired CRISPR-Cas9 variant, are powerful new tools for targeted base editing in cells and organisms. Together with cytosine base editors (CBEs), ABEs enable single-nucleotide conversions cleanly, efficiently and reversibly without double-stranded DNA cleavage, advancing genome editing in a new dimension.},
}

*Adenine
CRISPR-Associated Protein 9
*CRISPR-Cas Systems
*Cytosine
Gene Editing/*methods
Genetic Engineering/*methods

@article {pmid29455189,
year = {2018},
author = {Fiala, C and Diamandis, EP},
title = {Is it time to abandon the Nobel Prize?.},
journal = {Clinical chemistry and laboratory medicine},
volume = {56},
number = {8},
pages = {1196-1197},
doi = {10.1515/cclm-2018-0027},
pmid = {29455189},
issn = {1437-4331},
mesh = {CRISPR-Cas Systems ; Humans ; *Nobel Prize ; Research Personnel/*psychology ; },
}

CRISPR-Cas Systems
Humans
*Nobel Prize
Research Personnel/*psychology

@article {pmid31106371,
year = {2019},
author = {Labun, K and Montague, TG and Krause, M and Torres Cleuren, YN and Tjeldnes, H and Valen, E},
title = {CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkz365},
pmid = {31106371},
issn = {1362-4962},
abstract = {The CRISPR-Cas system is a powerful genome editing tool that functions in a diverse array of organisms and cell types. The technology was initially developed to induce targeted mutations in DNA, but CRISPR-Cas has now been adapted to target nucleic acids for a range of purposes. CHOPCHOP is a web tool for identifying CRISPR-Cas single guide RNA (sgRNA) targets. In this major update of CHOPCHOP, we expand our toolbox beyond knockouts. We introduce functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions. We incorporate new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes. Finally, we expand our results page visualization to reveal alternative isoforms and downstream ATG sites, which will aid users in avoiding the expression of truncated proteins. The CHOPCHOP web tool now supports over 200 genomes and we have released a command-line script for running larger jobs and handling unsupported genomes. CHOPCHOP v3 can be found at https://chopchop.cbu.uib.no.},
}

@article {pmid30894433,
year = {2019},
author = {Otoupal, PB and Ito, M and Arkin, AP and Magnuson, JK and Gladden, JM and Skerker, JM},
title = {Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides.},
journal = {mSphere},
volume = {4},
number = {2},
pages = {},
doi = {10.1128/mSphere.00099-19},
pmid = {30894433},
issn = {2379-5042},
mesh = {Basidiomycota/drug effects/*genetics ; *CRISPR-Cas Systems ; Drug Resistance, Fungal ; Fungal Proteins/genetics ; Gene Editing/*methods ; *Genome, Fungal ; Streptothricins/pharmacology ; },
abstract = {Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae Significant advancements in the past few years have bolstered R. toruloides' engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism.IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast's production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.},
}

Basidiomycota/drug effects/*genetics
*CRISPR-Cas Systems
Drug Resistance, Fungal
Fungal Proteins/genetics
Gene Editing/*methods
*Genome, Fungal
Streptothricins/pharmacology

@article {pmid30894429,
year = {2019},
author = {Myrbråten, IS and Wiull, K and Salehian, Z and Håvarstein, LS and Straume, D and Mathiesen, G and Kjos, M},
title = {CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in Lactobacillus plantarum.},
journal = {mSphere},
volume = {4},
number = {2},
pages = {},
doi = {10.1128/mSphere.00007-19},
pmid = {30894429},
issn = {2379-5042},
mesh = {Bacterial Proteins/genetics ; *CRISPR-Cas Systems ; Cell Wall/enzymology/genetics ; DNA Replication ; Gene Knockdown Techniques/*methods ; *Genes, Essential ; *Genes, cdc ; Lactobacillus plantarum/*genetics ; Phenotype ; Plasmids/genetics ; RNA Interference ; },
abstract = {Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium.IMPORTANCEL. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.},
}

Bacterial Proteins/genetics
*CRISPR-Cas Systems
Cell Wall/enzymology/genetics
DNA Replication
Gene Knockdown Techniques/*methods
*Genes, Essential
*Genes, cdc
Lactobacillus plantarum/*genetics
Phenotype
Plasmids/genetics
RNA Interference

@article {pmid30867327,
year = {2019},
author = {Lombardi, L and Oliveira-Pacheco, J and Butler, G},
title = {Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species.},
journal = {mSphere},
volume = {4},
number = {2},
pages = {},
doi = {10.1128/mSphere.00125-19},
pmid = {30867327},
issn = {2379-5042},
mesh = {*CRISPR-Cas Systems ; Candida/*genetics ; Candida tropicalis/genetics ; DNA Barcoding, Taxonomic ; DNA End-Joining Repair ; Gene Editing/methods ; Genetic Vectors ; Genome, Fungal ; Mutation ; Plasmids/*genetics ; Streptothricins/pharmacology ; },
abstract = {Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalisIMPORTANCECandida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis We have also constructed a separate system for gene editing in C. tropicalis.},
}

*CRISPR-Cas Systems
Candida/*genetics
Candida tropicalis/genetics
DNA Barcoding, Taxonomic
DNA End-Joining Repair
Gene Editing/methods
Genetic Vectors
Genome, Fungal
Mutation
Plasmids/*genetics
Streptothricins/pharmacology

@article {pmid30850537,
year = {2019},
author = {Wong, E and Liao, GP and Chang, JC and Xu, P and Li, YM and Greengard, P},
title = {GSAP modulates γ-secretase specificity by inducing conformational change in PS1.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {13},
pages = {6385-6390},
doi = {10.1073/pnas.1820160116},
pmid = {30850537},
issn = {1091-6490},
support = {R01 AG061350/AG/NIA NIH HHS/United States ; R01 NS096275/NS/NINDS NIH HHS/United States ; RF1 AG057593/AG/NIA NIH HHS/United States ; R01 AG047781/AG/NIA NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Alzheimer Disease/*metabolism ; Amyloid Precursor Protein Secretases/*metabolism ; CRISPR-Cas Systems ; Catalytic Domain ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Kinetics ; Peptide Fragments/metabolism ; Presenilin-1/*chemistry ; Proteins/genetics/*metabolism ; Receptor, Notch1 ; },
abstract = {The mechanism by which γ-secretase activating protein (GSAP) regulates γ-secretase activity has not yet been elucidated. Here, we show that knockout of GSAP in cultured cells directly reduces γ-secretase activity for Aβ production, but not for Notch1 cleavage, suggesting that GSAP may induce a conformational change contributing to the specificity of γ-secretase. Furthermore, using an active-site-directed photoprobe with double cross-linking moieties, we demonstrate that GSAP modifies the orientation and/or distance of the PS1 N-terminal fragment and the PS1 C-terminal fragment, a region containing the active site of γ-secretase. This work offers insight into how GSAP regulates γ-secretase specificity.},
}

Alzheimer Disease/*metabolism
Amyloid Precursor Protein Secretases/*metabolism
CRISPR-Cas Systems
Catalytic Domain
Gene Knockdown Techniques
HEK293 Cells
Humans
Kinetics
Peptide Fragments/metabolism
Presenilin-1/*chemistry
Proteins/genetics/*metabolism
Receptor, Notch1

@article {pmid30727954,
year = {2019},
author = {Boettcher, M and Covarrubias, S and Biton, A and Blau, J and Wang, H and Zaitlen, N and McManus, MT},
title = {Tracing cellular heterogeneity in pooled genetic screens via multi-level barcoding.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {107},
doi = {10.1186/s12864-019-5480-0},
pmid = {30727954},
issn = {1471-2164},
support = {U01CA168370//National Institutes of Health/ ; 1U01MH105028//National Institutes of Health/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Testing/*methods ; Humans ; Jurkat Cells ; Molecular Typing/*methods ; *RNA, Guide ; },
abstract = {BACKGROUND: While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells.

RESULTS: Here we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA.

CONCLUSION: Using our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.},
}

*CRISPR-Cas Systems
*Gene Editing
Genetic Testing/*methods
Humans
Jurkat Cells
Molecular Typing/*methods
*RNA, Guide

@article {pmid30696716,
year = {2019},
author = {Vicencio, J and Martínez-Fernández, C and Serrat, X and Cerón, J},
title = {Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans.},
journal = {Genetics},
volume = {211},
number = {4},
pages = {1143-1154},
doi = {10.1534/genetics.119.301965},
pmid = {30696716},
issn = {1943-2631},
mesh = {Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Gene Editing/methods ; *Genes, Reporter ; Green Fluorescent Proteins/*genetics/metabolism ; },
abstract = {CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (≤200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.},
}

Animals
*CRISPR-Cas Systems
Caenorhabditis elegans/*genetics
Gene Editing/methods
*Genes, Reporter
Green Fluorescent Proteins/*genetics/metabolism

@article {pmid30568241,
year = {2019},
author = {Cornel, MC and Howard, HC and Lim, D and Bonham, VL and Wartiovaara, K},
title = {Moving towards a cure in genetics: what is needed to bring somatic gene therapy to the clinic?.},
journal = {European journal of human genetics : EJHG},
volume = {27},
number = {3},
pages = {484-487},
doi = {10.1038/s41431-018-0309-x},
pmid = {30568241},
issn = {1476-5438},
mesh = {CRISPR-Cas Systems ; Clinical Trials as Topic ; *Congresses as Topic ; Gene Editing/methods ; Genetic Therapy/*methods ; Genetics, Medical/*methods ; Humans ; },
abstract = {Clinical trials using somatic gene editing (e.g., CRISPR-Cas9) have started in Europe and the United States and may provide safe and effective treatment and cure, not only for cancers but also for some monogenic conditions. In a workshop at the 2018 European Human Genetics Conference, the challenges of bringing somatic gene editing therapies to the clinic were discussed. The regulatory process needs to be considered early in the clinical development pathway to produce the data necessary to support the approval by the European Medicines Agency. The roles and responsibilities for geneticists may include counselling to explain the treatment possibilities and safety interpretation.},
}

CRISPR-Cas Systems
Clinical Trials as Topic
*Congresses as Topic
Gene Editing/methods
Genetic Therapy/*methods
Genetics, Medical/*methods
Humans

@article {pmid30367555,
year = {2019},
author = {Xu, R and Qin, R and Li, H and Li, J and Yang, J and Wei, P},
title = {Enhanced genome editing in rice using single transcript unit CRISPR-LbCpf1 systems.},
journal = {Plant biotechnology journal},
volume = {17},
number = {3},
pages = {553-555},
doi = {10.1111/pbi.13028},
pmid = {30367555},
issn = {1467-7652},
support = {2016ZX08010-002-008//Genetically Modified Breeding Major Projects/International ; 31501239//National Natural Science Foundation of China/International ; 31701405//National Natural Science Foundation of China/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; Clostridiales/genetics ; Gene Editing/*methods ; Genome, Plant/genetics ; Oryza/*genetics ; },
}

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

@article {pmid30160362,
year = {2019},
author = {Miao, J and Yang, Z and Zhang, D and Wang, Y and Xu, M and Zhou, L and Wang, J and Wu, S and Yao, Y and Du, X and Gu, F and Gong, Z and Gu, M and Liang, G and Zhou, Y},
title = {Mutation of RGG2, which encodes a type B heterotrimeric G protein γ subunit, increases grain size and yield production in rice.},
journal = {Plant biotechnology journal},
volume = {17},
number = {3},
pages = {650-664},
doi = {10.1111/pbi.13005},
pmid = {30160362},
issn = {1467-7652},
support = {BK20161335//Natural Science Foundation of Jiangsu Province/International ; 2016YFD0100400//National Key Research and Development Programme/International ; 15KJA210003//University Science Research Project of Jiangsu Province/International ; 2016M601899//China Postdoctoral Science Foundation/International ; },
mesh = {CRISPR-Cas Systems ; Edible Grain/genetics/*growth & development ; GTP-Binding Protein gamma Subunits/*genetics/physiology ; Gene Editing/methods ; Gene Expression Regulation, Plant ; Mutation/genetics ; Oryza/*genetics/growth & development ; Plant Proteins/*genetics/physiology ; Plants, Genetically Modified/genetics/growth & development ; },
abstract = {Heterotrimeric G proteins, which consist of Gα , Gβ and Gγ subunits, function as molecular switches that regulate a wide range of developmental processes in plants. In this study, we characterised the function of rice RGG2, which encodes a type B Gγ subunit, in regulating grain size and yield production. The expression levels of RGG2 were significantly higher than those of other rice Gγ -encoding genes in all tissues tested, suggesting that RGG2 plays essential roles in rice growth and development. By regulating cell expansion, overexpression of RGG2 in Nipponbare (NIP) led to reduced plant height and decreased grain size. By contrast, two mutants generated by the clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system in the Zhenshan 97 (ZS97) background, zrgg2-1 and zrgg2-2, exhibited enhanced growth, including elongated internodes, increased 1000-grain weight and plant biomass and enhanced grain yield per plant (+11.8% and 16.0%, respectively). These results demonstrate that RGG2 acts as a negative regulator of plant growth and organ size in rice. By measuring the length of the second leaf sheath after gibberellin (GA3) treatment and the GA-induced α-amylase activity of seeds, we found that RGG2 is also involved in GA signalling. In summary, we propose that RGG2 may regulate grain and organ size via the GA pathway and that manipulation of RGG2 may provide a novel strategy for rice grain yield enhancement.},
}

CRISPR-Cas Systems
Edible Grain/genetics/*growth & development
GTP-Binding Protein gamma Subunits/*genetics/physiology
Gene Editing/methods
Gene Expression Regulation, Plant
Mutation/genetics
Oryza/*genetics/growth & development
Plant Proteins/*genetics/physiology
Plants, Genetically Modified/genetics/growth & development

@article {pmid29961753,
year = {2019},
author = {Teng, Y and Luo, M and Yu, T and Chen, L and Huang, Q and Chen, S and Xie, L and Zeng, Y and Luo, F and Xiong, H and Liu, Y and Hou, W and Feng, Y},
title = {CRISPR/Cas9-mediated deletion of miR-146a enhances antiviral response in HIV-1 infected cells.},
journal = {Genes and immunity},
volume = {20},
number = {4},
pages = {327-337},
doi = {10.1038/s41435-018-0036-x},
pmid = {29961753},
issn = {1476-5470},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cytokines/metabolism ; *Gene Deletion ; HEK293 Cells ; HIV-1/*physiology ; Host-Pathogen Interactions ; Humans ; MicroRNAs/*genetics ; *Virus Replication ; },
abstract = {The human immunodeficiency virus type 1 (HIV-1) causes persistent infection in human and induces miR-146a expression in infected cells. miR-146a represses the innate immune response by inhibiting the expression of TRAF6 and IRAK1 genes, thus negatively controls the NF-κB-related cytokines and interferon stimulated genes. Here we reported that lentiviral CRISPR/Cas9 system was highly efficient in introducing mutations in the precursor miR-146a genomic sequences, resulting in a loss of miR-146a expression and function. miR-146a ablation led to increasing cytokines production in LPS-stimulated A549 cells. Moreover, miR-146a knockout in HIV-1 infected MT2 cells markedly increased the expression of cytokines and HIV-1 restriction factors and reversed T cell exhaustion markers expression, thus influencing HIV-1 replication. Our study indicates that lentiviral CRISPR/Cas9-mediated gene editing is an effective approach to abrogate miR-146a expression, which consequently inhibits HIV-1 replication as well as proviral reactivation by enhancing the expression of cytokines and HIV-1 restriction factors.},
}

CRISPR-Cas Systems
Cell Line, Tumor
Cytokines/metabolism
*Gene Deletion
HEK293 Cells
HIV-1/*physiology
Host-Pathogen Interactions
Humans
MicroRNAs/*genetics
*Virus Replication

@article {pmid29736029,
year = {2018},
author = {Smith, PA},
title = {The best Cas scenario.},
journal = {Nature medicine},
volume = {24},
number = {5},
pages = {528-530},
doi = {10.1038/s41591-018-0038-2},
pmid = {29736029},
issn = {1546-170X},
mesh = {Algorithms ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; },
}

Algorithms
CRISPR-Associated Proteins/*metabolism
CRISPR-Cas Systems/genetics
Gene Editing
Humans

@article {pmid29661912,
year = {2018},
author = {McHugh, T and Gluszek, AA and Welburn, JPI},
title = {Microtubule end tethering of a processive kinesin-8 motor Kif18b is required for spindle positioning.},
journal = {The Journal of cell biology},
volume = {217},
number = {7},
pages = {2403-2416},
pmid = {29661912},
issn = {1540-8140},
support = {//Wellcome Trust/United Kingdom ; 12840//Cancer Research UK/United Kingdom ; 207430//Wellcome Trust/United Kingdom ; 203149//Wellcome Trust/United Kingdom ; },
mesh = {Anaphase/genetics ; CRISPR-Cas Systems/genetics ; Cell Division/genetics ; HeLa Cells ; Humans ; Kinesin/*genetics ; Microtubule-Associated Proteins/*genetics ; Microtubules/genetics/metabolism ; Mitosis/*genetics ; Protein Binding/genetics ; Spindle Apparatus/genetics ; },
abstract = {Mitotic spindle positioning specifies the plane of cell division during anaphase. Spindle orientation and positioning are therefore critical to ensure symmetric division in mitosis and asymmetric division during development. The control of astral microtubule length plays an essential role in positioning the spindle. In this study, using gene knockout, we show that the kinesin-8 Kif18b controls microtubule length to center the mitotic spindle at metaphase. Using in vitro reconstitution, we reveal that Kif18b is a highly processive plus end-directed motor that uses a C-terminal nonmotor microtubule-binding region to accumulate at growing microtubule plus ends. This region is regulated by phosphorylation to spatially control Kif18b accumulation at plus ends and is essential for Kif18b-dependent spindle positioning and regulation of microtubule length. Finally, we demonstrate that Kif18b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work reveals that Kif18b uses its motile properties to reach microtubule ends, where it regulates astral microtubule length to ensure spindle centering.},
}

Anaphase/genetics
CRISPR-Cas Systems/genetics
Cell Division/genetics
HeLa Cells
Humans
Kinesin/*genetics
Microtubule-Associated Proteins/*genetics
Microtubules/genetics/metabolism
Mitosis/*genetics
Protein Binding/genetics
Spindle Apparatus/genetics

@article {pmid29641323,
year = {2018},
author = {Shen, S and Sanchez, ME and Blomenkamp, K and Corcoran, EM and Marco, E and Yudkoff, CJ and Jiang, H and Teckman, JH and Bumcrot, D and Albright, CF},
title = {Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with Genome Editing in Transgenic Mice.},
journal = {Human gene therapy},
volume = {29},
number = {8},
pages = {861-873},
doi = {10.1089/hum.2017.227},
pmid = {29641323},
issn = {1557-7422},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems/*genetics ; Dependovirus/genetics ; Disease Models, Animal ; Gene Editing ; *Genetic Therapy ; Genetic Vectors/therapeutic use ; Hepatocytes/pathology ; Homozygote ; Humans ; Liver/metabolism/pathology ; Mice ; Mice, Transgenic/genetics ; Mutation ; alpha 1-Antitrypsin/*genetics ; alpha 1-Antitrypsin Deficiency/genetics/pathology/*therapy ; },
abstract = {Alpha-1 antitrypsin deficiency (AATD) is a hereditary liver disease caused by mutations in the SERPINA1 serine protease inhibitor gene. Most severe patients are homozygous for PiZ alleles (PiZZ; amino acid E324K), which lead to protein aggregates in hepatocytes and reduced circulating levels of AAT. The liver aggregates typically lead to fibrosis, cirrhosis, and hepatocellular carcinoma, and the reduced circulating AAT levels can lead to emphysema and chronic obstructive pulmonary diseases. In this study, two CRISPR/Cas9 gene editing approaches were used to decrease liver aggregates and increase systemic AAT-M levels in the PiZ transgenic mouse. In the first approach, AAT expression in hepatocytes was reduced more than 98% following the systemic delivery of AAV8-CRISPR targeting exon 2 of hSERPINA1, leading to reduced aggregates in hepatocytes. In the second approach, a second adeno-associated virus, which provided the donor template to correct the Z mutation, was also administered. These treated mice had reduced AAT expression (> 98%) and a low level (5%) of wildtype AAT-M mRNA. Taken together, this study shows that CRISPR gene editing can efficiently reduce liver expression of AAT-Z and restore modest levels of wildtype AAT-M in a mouse model of AATD, raising the possibility of CRISPR gene editing therapeutic for AATD.},
}

Alleles
Animals
CRISPR-Cas Systems/*genetics
Dependovirus/genetics
Disease Models, Animal
Gene Editing
*Genetic Therapy
Genetic Vectors/therapeutic use
Hepatocytes/pathology
Homozygote
Humans
Liver/metabolism/pathology
Mice
Mice, Transgenic/genetics
Mutation
alpha 1-Antitrypsin/*genetics
alpha 1-Antitrypsin Deficiency/genetics/pathology/*therapy

@article {pmid29597895,
year = {2018},
author = {Song, CQ and Wang, D and Jiang, T and O'Connor, K and Tang, Q and Cai, L and Li, X and Weng, Z and Yin, H and Gao, G and Mueller, C and Flotte, TR and Xue, W},
title = {In Vivo Genome Editing Partially Restores Alpha1-Antitrypsin in a Murine Model of AAT Deficiency.},
journal = {Human gene therapy},
volume = {29},
number = {8},
pages = {853-860},
pmid = {29597895},
issn = {1557-7422},
support = {DP2 HL137167/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; T32 GM107000/GM/NIGMS NIH HHS/United States ; R01 DK098252/DK/NIDDK NIH HHS/United States ; R24 OD018259/OD/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Dependovirus/genetics ; Disease Models, Animal ; Gene Editing ; *Genetic Therapy ; Genetic Vectors/therapeutic use ; Humans ; Mice ; Mice, Transgenic ; Mutation ; alpha 1-Antitrypsin/*genetics/therapeutic use ; alpha 1-Antitrypsin Deficiency/genetics/pathology/*therapy ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) genome editing holds promise in the treatment of genetic diseases that currently lack effective long-term therapies. Patients with alpha-1 antitrypsin (AAT) deficiency develop progressive lung disease due to the loss of AAT's antiprotease function and liver disease due to a toxic gain of function of the common mutant allele. However, it remains unknown whether CRISPR-mediated AAT correction in the liver, where AAT is primarily expressed, can correct either or both defects. Here we show that AAV delivery of CRISPR can effectively correct Z-AAT mutation in the liver of a transgenic mouse model. Specifically, we co-injected two AAVs: one expressing Cas9 and another encoding an AAT guide RNA and homology-directed repair template. In both neonatal and adult mice, this treatment partially restored M-AAT in the serum. Furthermore, deep sequencing confirmed both indel mutations and precise gene correction in the liver, permitting careful analysis of gene editing events in vivo. This study demonstrates a proof of concept for the application of CRISPR-Cas9 technology to correct AAT mutations in vivo and validates continued exploration of this approach for the treatment of patients with AAT deficiency.},
}

Animals
CRISPR-Cas Systems/*genetics
Dependovirus/genetics
Disease Models, Animal
Gene Editing
*Genetic Therapy
Genetic Vectors/therapeutic use
Humans
Mice
Mice, Transgenic
Mutation
alpha 1-Antitrypsin/*genetics/therapeutic use
alpha 1-Antitrypsin Deficiency/genetics/pathology/*therapy

@article {pmid31102655,
year = {2019},
author = {Rahman, S and Datta, M and Kim, J and Jan, AT},
title = {CRISPR/Cas: An intriguing genomic editing tool with prospects in treating neurodegenerative diseases.},
journal = {Seminars in cell & developmental biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.semcdb.2019.05.014},
pmid = {31102655},
issn = {1096-3634},
abstract = {The CRISPR/Cas genome editing tool has led to a revolution in biological research. Its ability to target multiple genomic loci simultaneously allows its application in gene function and genomic manipulation studies. Its involvement in the sequence specific gene editing in different backgrounds has changed the scenario of treating genetic disease. By unravelling the mysteries behind complex neuronal circuits, it not the only paved way in understanding the pathogenesis of the disease but helped in the development of large animal models of different neuronal diseases; thereby opened the gateways of successfully treating different neuronal disorders. This review explored the possibility of using of CRISPR/Cas in engineering DNA at the embryonic stage, as well as during the functioning of different cell types in the brain, to delineate implications related to the use of this super-specialized genome editing tool to overcome various neurodegenerative diseases that arise as a result of genetic mutations.},
}

@article {pmid31100311,
year = {2019},
author = {Li, M and Xie, H and Liu, Y and Xia, C and Cun, X and Long, Y and Chen, X and Deng, M and Guo, R and Zhang, Z and He, Q},
title = {Knockdown of hypoxia-inducible factor-1 alpha by tumor targeted delivery of CRISPR/Cas9 system suppressed the metastasis of pancreatic cancer.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jconrel.2019.05.019},
pmid = {31100311},
issn = {1873-4995},
abstract = {The hypoxic tumor microenvironment of pancreatic cancer contributes to the progression and metastasis of tumor cells. Downregulation of hypoxia-inducible factor-1α (HIF-1α) with CRISPR/Cas9 is a promising approach to modulate tumor microenvironment and inhibit tumor metastasis. However, the in vivo delivery of CRISPR/Cas9 system remains a challenge. In the present manuscript, a tumor targeted lipid-based CRISPR/Cas9 delivery system was developed to suppress HIF-1α. Plasmids encoding Cas9 and HIF-1α-targeting sgRNA were successfully constructed and coencapsulated in R8-dGR peptide modified cationic liposome with PTX. R8-dGR-Lip exhibited enhanced BxPC-3 cell targeting and deep penetration into tumor spheroids. R8-dGR-Lip/PTX/pHIF-1α successfully downregulated HIF-1α and its downstream molecules VEGF and MMP-9, leading to enhanced antimetastatic effects. Besides, the blockade of HIF-1α also promoted the cytotoxicity of PTX on BxPC-3 cell lines. Compared with pegylated liposomes, R8-dGR-Lip enhanced the distribution in tumor tissues. The targeted delivery of CRISPR/Cas9-HIF-1α system and PTX significantly inhibited tumor growth. More importantly, inhibition of HIF-1α suppressed the metastasis of pancreatic cancer and prolonged survival time. Since CRISPR/Cas 9-HIF-1α hardly affected HIF-1α expression in normal hepatic cells, the designed R8-dGR-Lip/PTX/pHIF-1α did not induce severe toxicity in vivo. This strategy broadened the in vivo application of CRISPR/Cas9 system. Downregulation of HIF-1α may be a feasible approach for antimetastatic therapy.},
}

@article {pmid31099770,
year = {2019},
author = {Rozov, SM and Deineko, EV},
title = {[Strategies for Optimizing Recombinant Protein Synthesis in Plant Cells: Classical Approaches and New Directions].},
journal = {Molekuliarnaia biologiia},
volume = {53},
number = {2},
pages = {179-199},
doi = {10.1134/S0026898419020149},
pmid = {31099770},
issn = {0026-8984},
abstract = {At present, pharmacologically significant proteins are synthesized in different expression systems, from bacterial to mammalian and insect cell cultures. The plant expression systems (especially suspension cell culture) combine the simplicity and low cost of bacterial systems with the ability to perform eukaryotic-type posttranslational protein modifications. A low (compared with bacterial systems) yield of the target recombinant protein is one of the shortcomings of the plant expression systems. In this review, methods, developed over the past two decades, to increase the level of recombinant gene expression and methods to prevent silencing, caused by a random insertion of the target gene into heterochromatin region, are considered. The emergence of CRISPR/Cas technologies led to the creation of a new approach to increase the gene expression level, directional insertion of "pharmaceutical" protein genes in specific, knowingly transcriptionally active genome regions. The plant cell housekeeping gene loci, actively expressed throughout the interphase, are these regions. The organization of some housekeeping genes, most promising for transferring recombinant protein genes in their loci, is considered in detail.},
}

@article {pmid31099553,
year = {2019},
author = {Wilbie, D and Walther, J and Mastrobattista, E},
title = {Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing.},
journal = {Accounts of chemical research},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.accounts.9b00106},
pmid = {31099553},
issn = {1520-4898},
abstract = {The discovery of CRISPR/Cas has revolutionized the field of genome editing. CRIPSR/Cas components are part of the bacterial immune system and are able to induce double-strand DNA breaks in the genome, which are resolved by endogenous DNA repair mechanisms. The most relevant of these are the error-prone nonhomologous end joining and homology directed repair pathways. The former can lead to gene knockout by introduction of insertions and deletions at the cut site, while the latter can be used for gene correction based on a provided repair template. In this Account, we focus on the delivery aspects of CRISPR/Cas for therapeutic applications in vivo. Safe and effective delivery of the CRISPR/Cas components into the nucleus of affected cells is essential for therapeutic gene editing. These components can be delivered in several formats, such as pDNA, viral vectors, or ribonuclear complexes. In the ideal case, the delivery system should address the current limitations of CRISPR gene editing, which are (1) lack of targeting specific tissues or cells, (2) the inability to enter cells, (3) activation of the immune system, and (4) off-target events. To circumvent most of these problems, initial therapeutic applications of CRISPR/Cas were performed on cells ex vivo via classical methods (e.g., microinjection or electroporation) and novel methods (e.g., TRIAMF and iTOP). Ideal candidates for such methods are, for example, hematopoietic cells, but not all tissue types are suited for ex vivo manipulation. For direct in vivo application, however, delivery systems are needed that can target the CRISPR/Cas components to specific tissues or cells in the human body, without causing immune activation or causing high frequencies of off-target effects. Viral systems have been used as a first resort to transduce cells in vivo. These systems suffer from problems related to packaging constraints, immunogenicity, and longevity of Cas expression, which favors off-target events. Viral vectors are as such not the best choice for direct in vivo delivery of CRISPR/Cas. Synthetic vectors can deliver nucleic acids as well, without the innate disadvantages of viral vectors. They can be classed into lipid, polymeric, and inorganic particles, all of which have been reported in the literature. The advantage of synthetic systems is that they can deliver the CRISPR/Cas system also as a preformed ribonucleoprotein complex. The transient nature of this approach favors low frequencies of off-target events and minimizes the window of immune activation. Moreover, from a pharmaceutical perspective, synthetic delivery systems are much easier to scale up for clinical use compared to viral vectors and can be chemically functionalized with ligands to obtain target cell specificity. The first preclinical results with lipid nanoparticles delivering CRISPR/Cas either as mRNA or ribonucleoproteins are very promising. The goal is translating these CRISPR/Cas therapeutics to a clinical setting as well. Taken together, these current trends seem to favor the use of sgRNA/Cas ribonucleoprotein complexes delivered in vivo by synthetic particles.},
}

@article {pmid31098740,
year = {2019},
author = {Luz, ACO and da Silva, JMA and Rezende, AM and de Barros, MPS and Leal-Balbino, TC},
title = {Analysis of direct repeats and spacers of CRISPR/Cas systems type I-F in Brazilian clinical strains of Pseudomonas aeruginosa.},
journal = {Molecular genetics and genomics : MGG},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00438-019-01575-7},
pmid = {31098740},
issn = {1617-4623},
support = {APQ-0836-2.02/16//Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco/ ; 404016/2016-2//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; },
abstract = {CRISPR/Cas is an adaptive immune system found in prokaryotes, with the main function of protecting these cells from invasion and possible death by mobile genetic elements. Pseudomonas aeruginosa is considered a model for type I-F CRISPR/Cas system studies. However, its CRISPR loci characteristics have not yet been thoroughly described, and its function has not yet been fully unraveled. The aims of this study were to find the frequency of the system in Brazilian clinical isolates; to identify the loci sequence, its spacer diversity and its origins; as well as to propose a unified spacer library to aid in future structural studies of the CRISPR loci of P. aeruginosa. We investigated types I-F and I-E gene markers to establish CRISPR/Cas typing, and observed two strains harboring both systems simultaneously, a very rare feature. Through amplification and sequencing of CRISPR loci related to type I-F system, we describe polymorphisms in DRs and 350 spacers, of which 97 are new. The spacers that were identified had their possible organisms or proteins of origin identified. Spacer arrays were grouped in five different CRISPR patterns and the plasticity was inferred by rearrangements in spacer arrays. Here, we perform the first detailed and focused description of CRISPR/Cas elements in Brazilian clinical strains of P. aeruginosa. Our findings reflect active and highly diverse CRISPR loci, and we suggest that CRISPR/Cas may also pose as a transcriptional regulatory mechanism. The structural and diversity features described here can provide insights into the function of CRISPR/Cas in this pathogen and help guide the development of new therapeutic strategies.},
}

@article {pmid31096876,
year = {2019},
author = {Pan, S and Li, Q and Deng, L and Jiang, S and Jin, X and Peng, N and Liang, Y and She, Q and Li, Y},
title = {A seed motif for target RNA capture enables efficient immune defense by a Type III-B CRISPR-Cas system.},
journal = {RNA biology},
volume = {},
number = {},
pages = {},
doi = {10.1080/15476286.2019.1618693},
pmid = {31096876},
issn = {1555-8584},
abstract = {CRISPR-Cas systems provide an adaptive defense against foreign nucleic acids guided by small RNAs (crRNAs) in archaea and bacteria. The Type III CRISPR systems are reported to carry RNase, RNA-activated DNase and cyclic oligoadenylate (cOA) synthetase activity, and are significantly different from other CRISPR systems. However, detailed features of target recognition, which are essential for enhancing target specificity remain unknown in Type III CRISPR systems. Here, we show that the Type III-B Cmr-α system in S. islandicus generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3'-end of crRNA or target truncation greatly influence the target capture and cleavage by the Cmr-α effector complex. Furthermore, we found that cleavage at the tag-proximal site on the target RNA by the Cmr-α RNP complex is delayed relative to the other sites, which probably provides Cas10 more time to function as a guard against invaders. Using a mutagenesis assay in vivo, we discovered that a seed motif located at the tag-distal region of the crRNA is required by Cmr1α for target RNA capture by the Cmr-α system thereby enhancing target specificity and efficiency. These findings further refine the model for immune defense of Type III-B CRISPR-Cas system, commencing on capture, cleavage and regulation.},
}

@article {pmid30951856,
year = {2019},
author = {Skakic, A and Andjelkovic, M and Tosic, N and Klaassen, K and Djordjevic, M and Pavlovic, S and Stojiljkovic, M},
title = {CRISPR/Cas9 genome editing of SLC37A4 gene elucidates the role of molecular markers of endoplasmic reticulum stress and apoptosis in renal involvement in glycogen storage disease type Ib.},
journal = {Gene},
volume = {703},
number = {},
pages = {17-25},
doi = {10.1016/j.gene.2019.04.002},
pmid = {30951856},
issn = {1879-0038},
mesh = {Antiporters/*genetics ; Apoptosis ; CRISPR-Cas Systems ; Cell Line ; Endoplasmic Reticulum Stress ; Gene Editing/*methods ; Genetic Markers ; Glycogen Storage Disease Type I/complications/*genetics ; Humans ; Kidney Diseases/*etiology/genetics ; Models, Biological ; Monosaccharide Transport Proteins/*genetics ; Mutation ; *Point Mutation ; Unfolded Protein Response ; },
abstract = {Glycogen storage disease type Ib (GSD Ib) is an autosomal recessive disorder, caused by a deficiency of ubiquitously expressed SLC37A4 protein. Deficiency of SLC37A4 leads to abnormal storage of glycogen in the liver and kidneys, resulting in long-term complications of renal disease and hepatocellular adenomas, whose mechanisms are poorly understood. Molecular markers of the adaptive responses to the metabolic stress caused by a deficiency of SLC37A4, such as markers related to the endoplasmic reticulum (ER) stress and unfolded protein response (UPR), have not been extensively studied. The aim of this study was to investigate the expression of molecular markers of the UPR response and apoptosis related to a deficiency of SLC37A4 in kidney cells. For that purpose, we intended to establish a human kidney cell model system for GSD Ib. The novel variant c.248G>A, found in GSD Ib patients, was introduced into the Flp-In™T-REx™-293 cell line using CRISPR/Cas9-mediated precise gene editing method, resulting in significant decrease of SLC37A4 gene expression. In this model system we used RT-qPCR analysis to investigate the expression of molecular markers of the UPR response (ATF4, DDIT3, HSPA5, and XBP1s) and apoptosis (BCL2, BAX). We demonstrated that under chronic metabolic stress conditions caused by SLC37A4 deficiency, the ER stress-induced UPR was triggered, resulting in suppression of the UPR molecular markers and cell survival promotion (decreased expression levels of ATF4, DDIT3, HSPA5, with the exception of XBP1s). However, persistent metabolic stress overrides an adaptation and induces apoptosis through increased expression of pro-apoptotic markers (decreased ratio of BCL2/BAX genes). We established a cellular model system characterized by a deficiency of SLC37A4, which presents pathological manifestations of GSD Ib in the kidney. Expression analysis in a novel model system supports the hypothesis that renal dysfunction in the GSD Ib is partly due to the ER stress and increased apoptosis.},
}

Antiporters/*genetics
Apoptosis
CRISPR-Cas Systems
Cell Line
Endoplasmic Reticulum Stress
Gene Editing/*methods
Genetic Markers
Glycogen Storage Disease Type I/complications/*genetics
Humans
Kidney Diseases/*etiology/genetics
Models, Biological
Monosaccharide Transport Proteins/*genetics
Mutation
*Point Mutation
Unfolded Protein Response

@article {pmid30951565,
year = {2019},
author = {Chang, SJ and Jin, SC and Jiao, X and Galán, JE},
title = {Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen.},
journal = {PLoS pathogens},
volume = {15},
number = {4},
pages = {e1007704},
doi = {10.1371/journal.ppat.1007704},
pmid = {30951565},
issn = {1553-7374},
mesh = {Bacterial Proteins/antagonists & inhibitors/genetics/*metabolism ; Bacterial Toxins/*metabolism ; Biological Transport ; CRISPR-Cas Systems ; *Endoplasmic Reticulum-Associated Degradation ; HeLa Cells ; Humans ; Intracellular Space/metabolism ; Protein Binding ; Salmonella typhi/genetics/*pathogenicity ; Typhoid Fever/genetics/metabolism/*microbiology ; },
abstract = {Typhoid toxin is a virulence factor for Salmonella Typhi and Paratyphi, the cause of typhoid fever in humans. This toxin has a unique architecture in that its pentameric B subunit, made of PltB, is linked to two enzymatic A subunits, the ADP ribosyl transferase PltA and the deoxyribonuclease CdtB. Typhoid toxin is uniquely adapted to humans, recognizing surface glycoprotein sialoglycans terminated in acetyl neuraminic acid, which are preferentially expressed by human cells. The transport pathway to its cellular targets followed by typhoid toxin after receptor binding is currently unknown. Through a genome-wide CRISPR/Cas9-mediated screen we have characterized the mechanisms by which typhoid toxin is transported within human cells. We found that typhoid toxin hijacks specific elements of the retrograde transport and endoplasmic reticulum-associated degradation machineries to reach its subcellular destination within target cells. Our study reveals unique and common features in the transport mechanisms of bacterial toxins that could serve as the bases for the development of novel anti-toxin therapeutic strategies.},
}

Bacterial Proteins/antagonists & inhibitors/genetics/*metabolism
Bacterial Toxins/*metabolism
Biological Transport
CRISPR-Cas Systems
*Endoplasmic Reticulum-Associated Degradation
HeLa Cells
Humans
Intracellular Space/metabolism
Protein Binding
Salmonella typhi/genetics/*pathogenicity
Typhoid Fever/genetics/metabolism/*microbiology

@article {pmid30898719,
year = {2019},
author = {Ryu, N and Kim, MA and Choi, DG and Kim, YR and Sonn, JK and Lee, KY and Kim, UK},
title = {CRISPR/Cas9-mediated genome editing of splicing mutation causing congenital hearing loss.},
journal = {Gene},
volume = {703},
number = {},
pages = {83-90},
doi = {10.1016/j.gene.2019.03.020},
pmid = {30898719},
issn = {1879-0038},
mesh = {Animals ; Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Exons ; Gene Editing ; Gene Targeting/*methods ; Genetic Therapy ; Hearing Loss/*congenital/genetics ; Humans ; Mice ; Staphylococcus aureus/*metabolism ; Sulfate Transporters/*genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has ushered in a new era of gene therapy. In this study, we aimed to demonstrate precise CRISPR/Cas9-mediated genome editing of the splicing mutation c.919-2A > G in intron 7 of the SLC26A4 gene, which is the second most common causative gene of congenital hearing loss. We designed candidate single-guide RNAs (sgRNAs) aimed to direct the targeting of Staphylococcus aureus Cas9 to either exon 7 or exon 8 of SLC26A4. Several of the designed sgRNAs showed targeting activity, with average indel efficiencies ranging from approximately 14% to 25%. The usage of dual sgRNAs delivered both into Neuro2a cells and primary mouse embryonic fibroblasts resulted in the successful removal of large genomic fragments within the target locus. We subsequently evaluated genome editing in the presence of artificial donor templates to induce precise target modification via homology-directed repair. Using this approach, two different donor plasmids successfully introduced silent mutations within the c.919-2A region of Slc26a4 without evident off-target activities. Overall, these results indicate that CRISPR/Cas9-mediated correction of mutations in the Slc26a4 gene is a feasible therapeutic option for restoration of hearing loss.},
}

Animals
Bacterial Proteins/metabolism
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems
Cell Line
Exons
Gene Editing
Gene Targeting/*methods
Genetic Therapy
Hearing Loss/*congenital/genetics
Humans
Mice
Staphylococcus aureus/*metabolism
Sulfate Transporters/*genetics

@article {pmid30604066,
year = {2019},
author = {Xu, WH and Liang, DY and Wang, Q and Shen, J and Liu, QH and Peng, YB},
title = {Knockdown of KDM2A inhibits proliferation associated with TGF-β expression in HEK293T cell.},
journal = {Molecular and cellular biochemistry},
volume = {456},
number = {1-2},
pages = {95-104},
doi = {10.1007/s11010-018-03493-5},
pmid = {30604066},
issn = {1573-4919},
support = {31371307//National Natural Science Foundation of China/ ; 30900816//the National Natural Science Foundation of China/ ; },
mesh = {CRISPR-Cas Systems ; *Cell Proliferation ; F-Box Proteins/genetics/*metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Jumonji Domain-Containing Histone Demethylases/genetics/*metabolism ; Pyrazoles/pharmacology ; Pyrroles/pharmacology ; Transforming Growth Factor beta/antagonists & inhibitors/genetics/*metabolism ; },
abstract = {Lysine-specific demethylase 2A (KDM2A, also known as JHDM1A or FBXL11) plays an important role in regulating cell proliferation. However, the mechanisms on KDM2A controlling cell proliferation are varied among cell types, even controversial conclusions have been drawn. In order to elucidate the functions and underlying mechanisms for KDM2A controlling cell proliferation and apoptosis, we screened a KDM2A knockout HEK293T cell lines by CRISPR-Cas9 to illustrate the effects of KDM2A on both biological process. The results indicate that knocking down expression of KDM2A can significantly weaken HEK293T cell proliferation. The cell cycle analysis via flow cytometry demonstrate that knockdown expression of KDM2A will lead more cells arrested at G2/M phase. Through the RNA-seq analysis of the differential expressed genes between KDM2A knockdown HEK293T cells and wild type, we screened out that TGF-β pathway was significantly downregulated in KDM2A knockdown cells, which indicates that TGF-β signaling pathway might be the downstream target of KDM2A to regulate cell proliferation. When the KDM2A knockdown HEK293T cells were transient-transfected with KDM2A overexpression plasmid or treated by TGF-β agonist hydrochloride, the cell proliferation levels can be partial or completely rescued. However, the TGF-β inhibitor LY2109761 can significantly inhibit the KDM2A WT cells proliferation, but not the KDM2A knockdown HEK293T cells. Taken together, these findings suggested that KDM2A might be a key regulator of cell proliferation and cell cycle via impacting TGF-β signaling pathway.},
}

CRISPR-Cas Systems
*Cell Proliferation
F-Box Proteins/genetics/*metabolism
Gene Knockdown Techniques
HEK293 Cells
Humans
Jumonji Domain-Containing Histone Demethylases/genetics/*metabolism
Pyrazoles/pharmacology
Pyrroles/pharmacology
Transforming Growth Factor beta/antagonists & inhibitors/genetics/*metabolism

@article {pmid30379982,
year = {2018},
author = {Liu, W and Zhang, Y and Li, S and Li, Y},
title = {Rapid and efficient CRISPR-mediated genome editing with cloning-free method.},
journal = {Acta biochimica et biophysica Sinica},
volume = {50},
number = {11},
pages = {1173-1175},
doi = {10.1093/abbs/gmy107},
pmid = {30379982},
issn = {1745-7270},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Humans ; },
}

CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
Gene Editing/*methods
Humans

@article {pmid30312651,
year = {2019},
author = {Graham, GV and Conlon, JM and Abdel-Wahab, YH and Flatt, PR},
title = {Glucagon-like peptides-1 from phylogenetically ancient fish show potent anti-diabetic activities by acting as dual GLP1R and GCGR agonists.},
journal = {Molecular and cellular endocrinology},
volume = {480},
number = {},
pages = {54-64},
doi = {10.1016/j.mce.2018.10.011},
pmid = {30312651},
issn = {1872-8057},
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Cricetinae ; Cricetulus ; Cyclic AMP/metabolism ; Fishes/*metabolism ; Glucagon-Like Peptide 1/administration & dosage/chemistry/*pharmacology ; Glucagon-Like Peptide-1 Receptor/*antagonists & inhibitors/metabolism ; HEK293 Cells ; Humans ; Hypoglycemic Agents/*pharmacology ; Insulin Secretion/drug effects ; Mice ; *Phylogeny ; Rats ; Receptors, Glucagon/*agonists/metabolism ; },
abstract = {Glucagon-like peptides-1 (GLP-1)from phylogenetically ancient fish (lamprey, dogfish, ratfish, paddlefish and bowfin) and from a teleost, the rainbow trout produced concentration-dependent stimulations of insulin release from clonal β-cells and isolated mouse islets. Lamprey and paddlefish GLP-1 were the most potent and effective. Incubation of BRIN-BD11 cells with GLP-1 receptor (GLP1R) antagonist, exendin-4 (9-39) attenuated insulinotropic activity of all peptides whereas glucagon receptor (GCGR) antagonist [des-His1,Pro4,Glu9] glucagon amide significantly decreased the activities of lamprey and paddlefish GLP-1 only. The GIP receptor antagonist GIP (6-30) Cex-K40 [Pal] attenuated the activity of bowfin GLP-1. All peptides (1 μM) produced significant increases in cAMP concentration in CHL cells transfected with GLP1R but only lamprey and paddlefish GLP-1 stimulated cAMP production in HEK293 cells transfected with GCGR. Intraperitoneal administration of lamprey and paddlefish GLP-1 (25 nmol/kg body weight) in mice produced significant decreases in blood glucose and increased insulin concentrations comparable to the effects of human GLP-1. Lamprey and paddlefish GLP-1 display potent insulinotropic activity in vitro and glucose-lowering activity in vivo that is mediated through GLP1R and GCGR so that these peptides may constitute templates for design of new antidiabetic drugs.},
}

Amino Acid Sequence
Animals
CRISPR-Cas Systems/genetics
Cell Line
Cricetinae
Cricetulus
Cyclic AMP/metabolism
Fishes/*metabolism
Glucagon-Like Peptide 1/administration & dosage/chemistry/*pharmacology
Glucagon-Like Peptide-1 Receptor/*antagonists & inhibitors/metabolism
HEK293 Cells
Humans
Hypoglycemic Agents/*pharmacology
Insulin Secretion/drug effects
Mice
*Phylogeny
Rats
Receptors, Glucagon/*agonists/metabolism

@article {pmid29913057,
year = {2018},
author = {McCormick, S},
title = {Red fruit, orange fruit, orange fruit, red fruit: genome editing in tomato.},
journal = {The Plant journal : for cell and molecular biology},
volume = {95},
number = {1},
pages = {3-4},
doi = {10.1111/tpj.13967},
pmid = {29913057},
issn = {1365-313X},
mesh = {CRISPR-Cas Systems ; Fruit ; Gene Editing ; Gene Targeting ; Lycopersicon esculentum/*genetics ; Replicon ; },
}

CRISPR-Cas Systems
Fruit
Gene Editing
Gene Targeting
Lycopersicon esculentum/*genetics
Replicon

@article {pmid28963182,
year = {2017},
author = {McNally, EM},
title = {Gene Editing for the Heart: Correcting Dystrophin Mutations.},
journal = {Circulation research},
volume = {121},
number = {8},
pages = {896-898},
pmid = {28963182},
issn = {1524-4571},
support = {R01 HL061322/HL/NHLBI NIH HHS/United States ; R01 HL128075/HL/NHLBI NIH HHS/United States ; U54 AR052646/AR/NIAMS NIH HHS/United States ; UL1 TR001422/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Dystrophin/*genetics ; *Gene Editing ; Mice ; Mutation ; },
}

Animals
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Dystrophin/*genetics
*Gene Editing
Mice
Mutation

@article {pmid31095294,
year = {2019},
author = {Radovčić, M and Čulo, A and Ivančić-Baće, I},
title = {Cas3-stimulated runaway replication of modified ColE1 plasmids in Escherichia coli is temperature dependent.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnz106},
pmid = {31095294},
issn = {1574-6968},
abstract = {The CRISPR-Cas system constitutes an adaptive immunity system of prokaryotes against mobile genetic elements using a crRNA-mediated interference mechanism. In Type I CRISPR-Cas systems, crRNA guided by a Cascade complex recognises the matching target DNA and promotes an R-loop formation, RNA-DNA hybrid. The helicase-nuclease Cas3 protein is then recruited to the Cascade/R-loop complex where it nicks and degrades DNA. The Cas3 activity in CRISPR-Cas immunity is reduced in Δhns cells at 37°C for unknown reasons. Cas3 can also influence regulation of plasmid replication and promote uncontrolled ('runaway') replication of ColE1 plasmids independently of other CRISPR-Cas components, requiring only its helicase activity. In this work we wanted to test whether Cas3-stimulated uncontrolled plasmid replication is affected by the temperature in Δhns and/or ΔhtpG mutants. We found that Cas3-stimulated uncontrolled plasmid replication occurs only at 37°C, irrespective of the genotype of the analysed mutants, and dependent on Cas3 helicase function. We also found that plasmid replication was strongly reduced by the hns mutation at 30°C and that Cas3 could interfere with T4 phage replication at both incubation temperatures.},
}

@article {pmid31094569,
year = {2019},
author = {Wang, Y and Li, X and Osmundson, T and Shi, L and Yan, H},
title = {Comparative Genomic Analysis of a Multidrug-Resistant Listeria monocytogenes ST477 Isolate.},
journal = {Foodborne pathogens and disease},
volume = {},
number = {},
pages = {},
doi = {10.1089/fpd.2018.2611},
pmid = {31094569},
issn = {1556-7125},
abstract = {Listeria monocytogenes is an opportunistic human foodborne pathogen that causes severe infections with high hospitalization and fatality rates. Clonal complex 9 (CC9) contains a large number of sequence types (STs) and is one of the predominant clones distributed worldwide. However, genetic characteristics of ST477 isolates, which also belong to CC9, have never been examined, and little is known about the detail genomic traits of this food-associated clone. In this study, we sequenced and constructed the whole-genome sequence of an ST477 isolate from a frozen food sample in China and compared it with 58 previously sequenced genomes of 25 human-associated, 5 animal, and 27 food isolates consisting of 6 CC9 and 52 other clones. Phylogenetic analysis revealed that the ST477 clustered with three Canadian ST9 isolates. All phylogeny revealed that CC9 isolates involved in this study consistently possessed the invasion-related gene vip. Mobile genetic elements (MGEs), resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system were elucidated among CC9 isolates. Our ST477 isolate contained a Tn554-like transposon, carrying five arsenical-resistance genes (arsA-arsD, arsR), which was exclusively identified in the CC9 background. Compared with the ST477 genome, three Canadian ST9 isolates shared nonsynonymous nucleotide substitutions in the condensin complex gene smc and cell surface protein genes ftsA and essC. Our findings preliminarily indicate that the extraordinary success of CC9 clone in colonization of different geographical regions is likely due to conserved features harboring MGEs, functional virulence and resistance genes. ST477 and three ST9 genomes are closely related and the distinct differences between them consist primarily of changes in genes involved in multiplication and invasion, which may contribute to the prevalence of ST9 isolates in food and food processing environment.},
}

@article {pmid31009976,
year = {2019},
author = {Assimos, DG},
title = {Re: CRISPR/Cas9-Mediated Glycolate Oxidase Disruption is an Efficacious and Safe Treatment for Primary Hyperoxaluria Type I.},
journal = {The Journal of urology},
volume = {201},
number = {5},
pages = {853-854},
doi = {10.1097/01.JU.0000554109.94248.01},
pmid = {31009976},
issn = {1527-3792},
mesh = {Alcohol Oxidoreductases/genetics ; Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Hyperoxaluria, Primary/*genetics ; },
}

Alcohol Oxidoreductases/genetics
Animals
CRISPR-Cas Systems
Disease Models, Animal
Hyperoxaluria, Primary/*genetics

@article {pmid30819928,
year = {2019},
author = {Zuo, E and Sun, Y and Wei, W and Yuan, T and Ying, W and Sun, H and Yuan, L and Steinmetz, LM and Li, Y and Yang, H},
title = {Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.},
journal = {Science (New York, N.Y.)},
volume = {364},
number = {6437},
pages = {289-292},
doi = {10.1126/science.aav9973},
pmid = {30819928},
issn = {1095-9203},
mesh = {Animals ; *Blastomeres ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Cytosine ; DNA Mutational Analysis ; Embryo, Mammalian ; Female ; Gene Editing/*methods ; Genome-Wide Association Study ; Male ; Mice ; Mice, Inbred C57BL ; Mutation ; *Polymorphism, Single Nucleotide ; },
abstract = {Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.},
}

Animals
*Blastomeres
*CRISPR-Associated Protein 9
*CRISPR-Cas Systems
*Cytosine
DNA Mutational Analysis
Embryo, Mammalian
Female
Gene Editing/*methods
Genome-Wide Association Study
Male
Mice
Mice, Inbred C57BL
Mutation
*Polymorphism, Single Nucleotide

@article {pmid30816887,
year = {2019},
author = {Graul, AI and Dulsat, C and Pina, P and Cruces, E and Tracy, M},
title = {The year's new drugs and biologics 2018: Part II - News that shaped the industry in 2018.},
journal = {Drugs of today (Barcelona, Spain : 1998)},
volume = {55},
number = {2},
pages = {131-160},
doi = {10.1358/dot.2019.55.2.2974962},
pmid = {30816887},
issn = {1699-3993},
mesh = {*Biological Products ; CRISPR-Cas Systems ; *Drug Approval ; Drug Development ; Drug Industry/*trends ; Hemorrhagic Fever, Ebola ; Orphan Drug Production ; },
abstract = {This eagle's-eye overview of the drug industry in 2018 provides insight into some of last year's top stories, including a large and still growing outbreak of Ebola in the Democratic Republic of the Congo, as well as the potential uses (and abuses) of CRISPR technology. As in previous years, we also review orphan drug development, new regulatory agency-supported programs such as Priority Medicines Scheme (PRIME) and Sakigake, pipeline attrition, and pharma/biotech mergers and acquisitions of note. Finally, we take a glimpse into the crystal ball to anticipate the new drugs that will be approved in 2019.},
}

*Biological Products
CRISPR-Cas Systems
*Drug Approval
Drug Development
Drug Industry/*trends
Hemorrhagic Fever, Ebola
Orphan Drug Production

@article {pmid30621179,
year = {2019},
author = {Xu, CL and Ruan, MZC and Mahajan, VB and Tsang, SH},
title = {Viral Delivery Systems for CRISPR.},
journal = {Viruses},
volume = {11},
number = {1},
pages = {},
doi = {10.3390/v11010028},
pmid = {30621179},
issn = {1999-4915},
support = {P30EY019007/NH/NIH HHS/United States ; R01EY018213/NH/NIH HHS/United States ; R01EY024698/NH/NIH HHS/United States ; R01EY026682/NH/NIH HHS/United States ; R21AG050437/NH/NIH HHS/United States ; R24EY027285/NH/NIH HHS/United States ; 5P30CA013696/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/methods ; Gene Transfer Techniques ; Genetic Engineering/*methods ; Genetic Therapy ; *Genetic Vectors ; Genome ; Humans ; Mice ; Models, Animal ; Viruses/*genetics ; },
abstract = {The frontiers of precision medicine have been revolutionized by the development of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 as an editing tool. CRISPR/Cas9 has been used to develop animal models, understand disease mechanisms, and validate treatment targets. In addition, it is regarded as an effective tool for genome surgery when combined with viral delivery vectors. In this article, we will explore the various viral mechanisms for delivering CRISPR/Cas9 into tissues and cells, as well as the benefits and drawbacks of each method. We will also review the history and recent development of CRISPR and viral vectors and discuss their applications as a powerful tool in furthering our exploration of disease mechanisms and therapies.},
}

Animals
*CRISPR-Cas Systems
Gene Editing/methods
Gene Transfer Techniques
Genetic Engineering/*methods
Genetic Therapy
*Genetic Vectors
Genome
Humans
Mice
Models, Animal
Viruses/*genetics

@article {pmid30482929,
year = {2018},
author = {Cyranoski, D and Ledford, H},
title = {Genome-edited baby claim provokes international outcry.},
journal = {Nature},
volume = {563},
number = {7733},
pages = {607-608},
doi = {10.1038/d41586-018-07545-0},
pmid = {30482929},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/genetics ; China ; Embryo Research/*ethics/*legislation & jurisprudence ; Female ; Gene Editing/*ethics/*legislation & jurisprudence ; HIV/physiology ; HIV Infections/genetics/prevention & control ; Humans ; Infant, Newborn ; *Internationality ; Male ; Practice Guidelines as Topic ; Punishment ; Receptors, CCR5/deficiency/genetics/metabolism ; Receptors, CXCR4/genetics/metabolism ; *Scientific Misconduct ; Twins/*genetics ; Virus Internalization ; },
}

CRISPR-Cas Systems/genetics
China
Embryo Research/*ethics/*legislation & jurisprudence
Female
Gene Editing/*ethics/*legislation & jurisprudence
HIV/physiology
HIV Infections/genetics/prevention & control
Humans
Infant, Newborn
*Internationality
Male
Practice Guidelines as Topic
Punishment
Receptors, CCR5/deficiency/genetics/metabolism
Receptors, CXCR4/genetics/metabolism
*Scientific Misconduct
Twins/*genetics
Virus Internalization

@article {pmid30287096,
year = {2018},
author = {Wienert, B and Martyn, GE and Funnell, APW and Quinlan, KGR and Crossley, M},
title = {Wake-up Sleepy Gene: Reactivating Fetal Globin for β-Hemoglobinopathies.},
journal = {Trends in genetics : TIG},
volume = {34},
number = {12},
pages = {927-940},
doi = {10.1016/j.tig.2018.09.004},
pmid = {30287096},
issn = {0168-9525},
mesh = {Adult ; CRISPR-Cas Systems/genetics ; Gene Editing/trends ; Genetic Therapy/*trends ; Hemoglobinopathies/blood/*genetics/pathology ; Humans ; Mutation ; beta-Globins/*genetics/therapeutic use ; gamma-Globins/*genetics/therapeutic use ; },
abstract = {Disorders in hemoglobin (hemoglobinopathies) were the first monogenic diseases to be characterized and remain among the most common and best understood genetic conditions. Moreover, the study of the β-globin locus provides a textbook example of developmental gene regulation. The fetal γ-globin genes (HBG1/HBG2) are ordinarily silenced around birth, whereupon their expression is replaced by the adult β-globin genes (HBB primarily and HBD). Over 50 years ago it was recognized that mutations that cause lifelong persistence of fetal γ-globin expression ameliorate the debilitating effects of mutations in β-globin. Since then, research has focused on therapeutically reactivating the fetal γ-globin genes. Here, we summarize recent discoveries, focusing on the influence of genome editing technologies, including CRISPR-Cas9, and emerging gene therapy approaches.},
}

Adult
CRISPR-Cas Systems/genetics
Gene Editing/trends
Genetic Therapy/*trends
Hemoglobinopathies/blood/*genetics/pathology
Humans
Mutation
beta-Globins/*genetics/therapeutic use
gamma-Globins/*genetics/therapeutic use

@article {pmid30107443,
year = {2018},
author = {Sasaki-Honda, M and Jonouchi, T and Arai, M and Hotta, A and Mitsuhashi, S and Nishino, I and Matsuda, R and Sakurai, H},
title = {A patient-derived iPSC model revealed oxidative stress increases facioscapulohumeral muscular dystrophy-causative DUX4.},
journal = {Human molecular genetics},
volume = {27},
number = {23},
pages = {4024-4035},
pmid = {30107443},
issn = {1460-2083},
mesh = {CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Chromosomal Proteins, Non-Histone/*genetics ; Chromosomes, Human, Pair 4/genetics ; DNA Damage/genetics ; Gene Expression Regulation ; Homeodomain Proteins/*genetics ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Muscle Cells/metabolism/pathology ; Muscle, Skeletal/metabolism/pathology ; Muscular Dystrophy, Facioscapulohumeral/*genetics/metabolism/pathology ; Mutation ; Oxidative Stress/genetics ; },
abstract = {Double homeobox 4 (DUX4), the causative gene of facioscapulohumeral muscular dystrophy (FSHD), is ectopically expressed in the skeletal muscle cells of FSHD patients because of chromatin relaxation at 4q35. The diminished heterochromatic state at 4q35 is caused by either large genome contractions [FSHD type 1 (FSHD1)] or mutations in genes encoding chromatin regulators, such as SMCHD1 [FSHD type 2 (FSHD2)]. However, the mechanism by which DUX4 expression is regulated remains largely unknown. Here, using a myocyte model developed from patient-derived induced pluripotent stem cells, we determined that DUX4 expression was increased by oxidative stress (OS), a common environmental stress in skeletal muscle, in both FSHD1 and FSHD2 myocytes. We generated FSHD2-derived isogenic control clones with SMCHD1 mutation corrected by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) and homologous recombination and found in the myocytes obtained from these clones that DUX4 basal expression and the OS-induced upregulation were markedly suppressed due to an increase in the heterochromatic state at 4q35. We further found that DNA damage response (DDR) was involved in OS-induced DUX4 increase and identified ataxia-telangiectasia mutated, a DDR regulator, as a mediator of this effect. Our results suggest that the relaxed chromatin state in FSHD muscle cells permits aberrant access of OS-induced DDR signaling, thus increasing DUX4 expression. These results suggest OS could represent an environmental risk factor that promotes FSHD progression.},
}

CRISPR-Cas Systems/genetics
Chromatin/genetics
Chromosomal Proteins, Non-Histone/*genetics
Chromosomes, Human, Pair 4/genetics
DNA Damage/genetics
Gene Expression Regulation
Homeodomain Proteins/*genetics
Humans
Induced Pluripotent Stem Cells/*metabolism
Muscle Cells/metabolism/pathology
Muscle, Skeletal/metabolism/pathology
Muscular Dystrophy, Facioscapulohumeral/*genetics/metabolism/pathology
Mutation
Oxidative Stress/genetics

@article {pmid29860374,
year = {2018},
author = {Mans, R and Wijsman, M and Daran-Lapujade, P and Daran, JM},
title = {A protocol for introduction of multiple genetic modifications in Saccharomyces cerevisiae using CRISPR/Cas9.},
journal = {FEMS yeast research},
volume = {18},
number = {7},
pages = {},
pmid = {29860374},
issn = {1567-1364},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Deletion ; Gene Editing/*methods ; Gene Expression ; Genome, Fungal/genetics ; Mutagenesis, Site-Directed ; Plasmids/genetics ; RNA, Guide/genetics/metabolism ; Saccharomyces cerevisiae/*genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics ; Transformation, Genetic ; },
abstract = {Here, two methods are described for efficient genetic modification of Saccharomyces cerevisiae using CRISPR/Cas9. The first method enables the modification of a single genetic locus using in vivo assembly of a guide RNA (gRNA) expression plasmid without the need for prior cloning. A second method using in vitro assembled plasmids that could contain up to two gRNAs was used to simultaneously introduce up to six genetic modifications (e.g. six gene deletions) in a single transformation step by transforming up to three gRNA expression plasmids simultaneously. The method is not only suitable for gene deletion but is also applicable for in vivo site-directed mutagenesis and integration of multiple DNA fragments in a single locus. In all cases, the strain transformed with the gRNA expression plasmids was equipped with a genomic integration of Spcas9, leading to strong and constitutive expression of SpCas9. The protocols detailed here have been streamlined to be executed by virtually any yeast molecular geneticist.},
}

CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems
Gene Deletion
Gene Editing/*methods
Gene Expression
Genome, Fungal/genetics
Mutagenesis, Site-Directed
Plasmids/genetics
RNA, Guide/genetics/metabolism
Saccharomyces cerevisiae/*genetics/metabolism
Saccharomyces cerevisiae Proteins/genetics
Transformation, Genetic

@article {pmid29723654,
year = {2018},
author = {Carmona-Aldana, F and Zampedri, C and Suaste-Olmos, F and Murillo-de-Ozores, A and Guerrero, G and Arzate-Mejía, R and Maldonado, E and Navarro, RE and Chimal-Monroy, J and Recillas-Targa, F},
title = {CTCF knockout reveals an essential role for this protein during the zebrafish development.},
journal = {Mechanisms of development},
volume = {154},
number = {},
pages = {51-59},
doi = {10.1016/j.mod.2018.04.006},
pmid = {29723654},
issn = {1872-6356},
mesh = {Animals ; Apoptosis/genetics ; CCCTC-Binding Factor/*genetics ; CRISPR-Cas Systems/genetics ; Cell Survival/genetics ; Chromatin/genetics ; Embryonic Development/*genetics ; Gene Knockout Techniques/methods ; Humans ; RNA, Messenger/genetics ; Zebrafish ; },
abstract = {Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.},
}

Animals
Apoptosis/genetics
CCCTC-Binding Factor/*genetics
CRISPR-Cas Systems/genetics
Cell Survival/genetics
Chromatin/genetics
Embryonic Development/*genetics
Gene Knockout Techniques/methods
Humans
RNA, Messenger/genetics
Zebrafish

@article {pmid29116883,
year = {2018},
author = {Khanal, C and McGawley, EC and Overstreet, C and Stetina, SR},
title = {The Elusive Search for Reniform Nematode Resistance in Cotton.},
journal = {Phytopathology},
volume = {108},
number = {5},
pages = {532-541},
doi = {10.1094/PHYTO-09-17-0320-RVW},
pmid = {29116883},
issn = {0031-949X},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Resistance/*genetics ; Gossypium/genetics/*parasitology ; Plant Breeding ; Plant Diseases/*parasitology ; RNA, Double-Stranded/genetics ; *Tylenchoidea ; },
abstract = {The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) has emerged as the most important plant-parasitic nematode of cotton in the United States cotton belt. Success in the development of reniform nematode-resistant upland cotton cultivars (Gossypium hirsutum L.) has not been realized despite over three decades of breeding efforts. Research approaches ranging from conventional breeding to triple species hybrids to marker-assisted selection have been employed to introgress reniform nematode resistance from other species of cotton into upland cultivars. Reniform nematode-resistant breeding lines derived from G. longicalyx were developed in 2007. However, these breeding lines displayed stunting symptoms and a hypersensitive response to reniform nematode infection. Subsequent breeding efforts focused on G. barbadense, G. aridum, G. armoreanum, and other species that have a high level of resistance to reniform nematode. Marker-assisted selection has greatly improved screening of reniform nematode-resistant lines. The use of advanced molecular techniques such as CRISPER-Cas9 systems and alternative ways such as delivery of suitable "cry" proteins and specific double-stranded RNA to nematodes will assist in developing resistant cultivars of cotton. In spite of the efforts of cotton breeders and nematologists, successes are limited only to the development of reniform nematode-resistant breeding lines. In this article, we provide an overview of the approaches employed to develop reniform nematode-resistant upland cotton cultivars in the past, progress to date, major obstacles, and some promising future research activity.},
}

Animals
CRISPR-Cas Systems
Disease Resistance/*genetics
Gossypium/genetics/*parasitology
Plant Breeding
Plant Diseases/*parasitology
RNA, Double-Stranded/genetics
*Tylenchoidea

@article {pmid28596164,
year = {2017},
author = {Fu, Y and Shaw, RM},
title = {CASAAV Technology to Examine Regulators of Heart Failure: Cause or Effect.},
journal = {Circulation research},
volume = {120},
number = {12},
pages = {1846-1848},
pmid = {28596164},
issn = {1524-4571},
support = {11GRNT7030001/AHA/American Heart Association-American Stroke Association/United States ; 13EIA14480016/AHA/American Heart Association-American Stroke Association/United States ; R01 HL094414/HL/NHLBI NIH HHS/United States ; R01 HL138577/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Heart Failure ; Humans ; *Myocytes, Cardiac ; },
}

CRISPR-Cas Systems
*Heart Failure
Humans
*Myocytes, Cardiac

@article {pmid28355532,
year = {2017},
author = {Coté, AJ and Raj, A},
title = {FISHing Out the Details of CRISPR Genome Tracks.},
journal = {Biophysical journal},
volume = {112},
number = {6},
pages = {1045-1046},
pmid = {28355532},
issn = {1542-0086},
support = {U01 HL129998/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Genomics ; },
}

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

@article {pmid31090220,
year = {2019},
author = {Yeo, WL and Heng, E and Tan, LL and Lim, YW and Lim, YH and Hoon, S and Zhao, H and Zhang, MM and Wong, FT},
title = {Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.27021},
pmid = {31090220},
issn = {1097-0290},
abstract = {Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes streptomycetes has enabled high efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. Novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes, enabling efficient homology directed repair (HDR)-mediated knock-in and deletion. In strains where spCas9 was non-functional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria. This article is protected by copyright. All rights reserved.},
}

@article {pmid31089700,
year = {2019},
author = {Makarova, KS and Gao, L and Zhang, F and Koonin, EV},
title = {Unexpected connections between type VI-B CRISPR-Cas systems, bacterial natural competence, ubiquitin signaling network and DNA modification through a distinct family of membrane proteins.},
journal = {FEMS microbiology letters},
volume = {366},
number = {8},
pages = {},
doi = {10.1093/femsle/fnz088},
pmid = {31089700},
issn = {1574-6968},
abstract = {In addition to core Cas proteins, CRISPR-Cas loci often encode ancillary proteins that modulate the activity of the respective effectors in interference. Subtype VI-B1 CRISPR-Cas systems encode the Csx27 protein that down-regulates the activity of Cas13b when the type VI-B locus is expressed in Escherichia coli. We show that Csx27 belongs to an expansive family of proteins that contain four predicted transmembrane helices and are typically encoded in predicted operons with components of the bacterial natural transformation machinery, multidomain proteins that consist of components of the ubiquitin signaling system and proteins containing the ligand-binding WYL domain and a helix-turn-helix domain. The Csx27 family proteins are predicted to form membrane channels for ssDNA that might comprise the core of a putative novel, Ub-regulated system for DNA uptake and, possibly, degradation. In addition to these associations, a distinct subfamily of the Csx27 family appears to be a part of a novel, membrane-associated system for DNA modification. In Bacteroidetes, subtype VI-B1 systems might degrade nascent transcripts of foreign DNA in conjunction with its uptake by the bacterial cell. These predictions suggest several experimental directions for the study of type VI CRISPR-Cas systems and distinct mechanisms of foreign DNA uptake and degradation in bacteria.},
}

@article {pmid31087001,
year = {2019},
author = {Adiego-Pérez, B and Randazzo, P and Daran, JM and Verwaal, R and Roubos, JA and Daran-Lapujade, P and van der Oost, J},
title = {Multiplex Genome Editing of Microorganisms Using CRISPR-Cas.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnz086},
pmid = {31087001},
issn = {1574-6968},
abstract = {Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing.},
}

@article {pmid31086829,
year = {2019},
author = {Burgsdorf, I and Handley, KM and Bar-Shalom, R and Erwin, PM and Steindler, L},
title = {Life at Home and on the Roam: Genomic Adaptions Reflect the Dual Lifestyle of an Intracellular, Facultative Symbiont.},
journal = {mSystems},
volume = {4},
number = {4},
pages = {},
doi = {10.1128/mSystems.00057-19},
pmid = {31086829},
issn = {2379-5077},
abstract = {"Candidatus Synechococcus feldmannii" is a facultative intracellular symbiont of the Atlanto-Mediterranean sponge Petrosia ficiformis. Genomic information of sponge-associated cyanobacteria derives thus far from the obligate and extracellular symbiont "Candidatus Synechococcus spongiarum." Here we utilized a differential methylation-based approach for bacterial DNA enrichment combined with metagenomics to obtain the first draft genomes of "Ca. Synechococcus feldmannii." By comparative genomics, we revealed that some genomic features (e.g., iron transport mediated by siderophores, eukaryotic-like proteins, and defense mechanisms, like CRISPR-Cas [clustered regularly interspaced short palindromic repeats-associated proteins]) are unique to both symbiont types and absent or rare in the genomes of taxonomically related free-living cyanobacteria. These genomic features likely enable life under the conditions found inside the sponge host. Interestingly, there are many genomic features that are shared by "Ca. Synechococcus feldmannii" and free-living cyanobacteria, while they are absent in the obligate symbiont "Ca. Synechococcus spongiarum." These include genes related to cell surface structures, genetic regulation, and responses to environmental stress, as well as the composition of photosynthetic genes and DNA metabolism. We speculate that the presence of these genes confers on "Ca. Synechococcus feldmannii" its facultative nature (i.e., the ability to respond to a less stable environment when free-living). Our comparative analysis revealed that distinct genomic features depend on the nature of the symbiotic interaction: facultative and intracellular versus obligate and extracellular. IMPORTANCE Given the evolutionary position of sponges as one of the earliest phyla to depart from the metazoan stem lineage, studies on their distinct and exceptionally diverse microbial communities should yield a better understanding of the origin of animal-bacterium interactions. While genomes of several extracellular sponge symbionts have been published, the intracellular symbionts have, so far, been elusive. Here we compare the genomes of two unicellular cyanobacterial sponge symbionts that share an ancestor but followed different evolutionary paths-one became intracellular and the other extracellular. Counterintuitively, the intracellular cyanobacteria are facultative, while the extracellular ones are obligate. By sequencing the genomes of the intracellular cyanobacteria and comparing them to the genomes of the extracellular symbionts and related free-living cyanobacteria, we show how three different cyanobacterial lifestyles are reflected by adaptive genomic features.},
}

@article {pmid31086658,
year = {2019},
author = {Safari, F and Zare, K and Negahdaripour, M and Barekati-Mowahed, M and Ghasemi, Y},
title = {CRISPR Cpf1 proteins: structure, function and implications for genome editing.},
journal = {Cell & bioscience},
volume = {9},
number = {},
pages = {36},
doi = {10.1186/s13578-019-0298-7},
pmid = {31086658},
issn = {2045-3701},
abstract = {CRISPR and CRISPR-associated (Cas) protein, as components of microbial adaptive immune system, allows biologists to edit genomic DNA in a precise and specific way. CRISPR-Cas systems are classified into two main classes and six types. Cpf1 is a putative type V (class II) CRISPR effector, which can be programmed with a CRISPR RNA to bind and cleave complementary DNA targets. Cpf1 has recently emerged as an alternative for Cas9, due to its distinct features such as the ability to target T-rich motifs, no need for trans-activating crRNA, inducing a staggered double-strand break and potential for both RNA processing and DNA nuclease activity. In this review, we attempt to discuss the evolutionary origins, basic architectures, and molecular mechanisms of Cpf1 family proteins, as well as crRNA designing and delivery strategies. We will also describe the novel Cpf1 variants, which have broadened the versatility and feasibility of this system in genome editing, transcription regulation, epigenetic modulation, and base editing. Finally, we will be reviewing the recent studies on utilization of Cpf1as a molecular tool for genome editing.},
}

@article {pmid31085694,
year = {2019},
author = {McAllister, KN and Sorg, JA},
title = {CRISPR genome editing systems in the genus Clostridium: a timely advancement.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {},
doi = {10.1128/JB.00219-19},
pmid = {31085694},
issn = {1098-5530},
abstract = {The genus Clostridium is composed of bioproducers, which are important for the industrial production of chemicals, as well as pathogens, which are a significant burden to the patients and on the healthcare industry. Historically, even though these bacteria are well known and are commonly studied, the genetic tools to advance our understanding of these microbes have lagged behind other systems. New tools would continue the advancement of our understanding of clostridial physiology. The genetic tools available in several clostridia are not as refined as in other organisms and each exhibit their own drawbacks. With the advent of the repurposing of the CRISPR-Cas systems for genetic modification, the tools available for clostridia have improved significantly over the past four years. Several CRISPR-Cas tools, such as using wild-type Cas9, Cas9n, dCas9/CRISPRi and a newly studied Cpf1/Cas12a, are reported. These tools have the potential to greatly advance the study of clostridial species leading to future therapies or the enhanced production of industrially relevant compounds. Here we discuss the details of the CRISPR-Cas systems as well as the advances and current issues in the developed clostridial systems.},
}

@article {pmid31021314,
year = {2019},
author = {Lee, H and Dhingra, Y and Sashital, DG},
title = {The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
doi = {10.7554/eLife.44248},
pmid = {31021314},
issn = {2050-084X},
support = {GM115874//National Institute of General Medical Sciences/ ; },
abstract = {CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.},
}

@article {pmid29967496,
year = {2018},
author = {Jacobs, CL and Badiee, RK and Lin, MZ},
title = {StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins.},
journal = {Nature methods},
volume = {15},
number = {7},
pages = {523-526},
doi = {10.1038/s41592-018-0041-z},
pmid = {29967496},
issn = {1548-7105},
support = {P50 GM107615/GM/NIGMS NIH HHS/United States ; R01 GM098734/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Dimerization ; Gene Expression Regulation/*drug effects ; Gene Targeting ; HEK293 Cells ; HeLa Cells ; Humans ; Plasmids ; *Protein Engineering ; Protein Folding ; Viral Nonstructural Proteins/metabolism ; Zinc Fingers ; },
abstract = {Robust approaches for chemogenetic control of protein function would have many biological applications. We developed stabilizable polypeptide linkages (StaPLs) based on hepatitis C virus protease. StaPLs undergo autoproteolysis to cleave proteins by default, whereas protease inhibitors prevent cleavage and preserve protein function. We created StaPLs responsive to different clinically approved drugs to bidirectionally control transcription with zinc-finger-based effectors, and used StaPLs to create single-chain, drug-stabilizable variants of CRISPR-Cas9 and caspase-9.},
}

CRISPR-Cas Systems
Dimerization
Gene Expression Regulation/*drug effects
Gene Targeting
HEK293 Cells
HeLa Cells
Humans
Plasmids
*Protein Engineering
Protein Folding
Viral Nonstructural Proteins/metabolism
Zinc Fingers

@article {pmid29855571,
year = {2018},
author = {Marx, V},
title = {Stem cells: lineage tracing lets single cells talk about their past.},
journal = {Nature methods},
volume = {15},
number = {6},
pages = {411-414},
doi = {10.1038/s41592-018-0016-0},
pmid = {29855571},
issn = {1548-7105},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Lineage/*physiology ; DNA Barcoding, Taxonomic ; Gene Expression Regulation ; Multipotent Stem Cells/*physiology ; Single-Cell Analysis/*methods ; },
}

Animals
CRISPR-Cas Systems
Cell Lineage/*physiology
DNA Barcoding, Taxonomic
Gene Expression Regulation
Multipotent Stem Cells/*physiology
Single-Cell Analysis/*methods

@article {pmid29403034,
year = {2018},
author = {Zheng, Y and Shen, W and Zhang, J and Yang, B and Liu, YN and Qi, H and Yu, X and Lu, SY and Chen, Y and Xu, YZ and Li, Y and Gage, FH and Mi, S and Yao, J},
title = {CRISPR interference-based specific and efficient gene inactivation in the brain.},
journal = {Nature neuroscience},
volume = {21},
number = {3},
pages = {447-454},
doi = {10.1038/s41593-018-0077-5},
pmid = {29403034},
issn = {1546-1726},
mesh = {Affect/physiology ; Animals ; Brain Chemistry/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Proliferation ; Cognition/physiology ; Dentate Gyrus/metabolism ; Fear/psychology ; Gene Silencing ; Hindlimb Suspension/psychology ; Learning/physiology ; Male ; Maze Learning ; Memory/physiology ; Mice ; Mice, Inbred C57BL ; RNA Interference ; Synaptotagmin I/genetics ; },
abstract = {CRISPR-Cas9 has been demonstrated to delete genes in postmitotic neurons. Compared to the establishment of proliferative cell lines or animal strains, it is more challenging to acquire a highly homogeneous consequence of gene editing in a stable neural network. Here we show that dCas9-based CRISPR interference (CRISPRi) can efficiently silence genes in neurons. Using a pseudotarget fishing strategy, we demonstrate that CRISPRi shows superior targeting specificity without detectable off-target activity. Furthermore, CRISPRi can achieve multiplex inactivation of genes fundamental for neurotransmitter release with high efficiency. By developing conditional CRISPRi tools targeting synaptotagmin I (Syt1), we modified the excitatory to inhibitory balance in the dentate gyrus of the mouse hippocampus and found that the dentate gyrus has distinct regulatory roles in learning and affective processes in mice. We therefore recommend CRISPRi as a useful tool for more rapid investigation of gene function in the mammalian brain.},
}

Affect/physiology
Animals
Brain Chemistry/*genetics
CRISPR-Cas Systems/*genetics
Cell Proliferation
Cognition/physiology
Dentate Gyrus/metabolism
Fear/psychology
Gene Silencing
Hindlimb Suspension/psychology
Learning/physiology
Male
Maze Learning
Memory/physiology
Mice
Mice, Inbred C57BL
RNA Interference
Synaptotagmin I/genetics

@article {pmid29335603,
year = {2018},
author = {Zhou, H and Liu, J and Zhou, C and Gao, N and Rao, Z and Li, H and Hu, X and Li, C and Yao, X and Shen, X and Sun, Y and Wei, Y and Liu, F and Ying, W and Zhang, J and Tang, C and Zhang, X and Xu, H and Shi, L and Cheng, L and Huang, P and Yang, H},
title = {In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice.},
journal = {Nature neuroscience},
volume = {21},
number = {3},
pages = {440-446},
doi = {10.1038/s41593-017-0060-6},
pmid = {29335603},
issn = {1546-1726},
mesh = {Animals ; Astrocytes/physiology ; Brain Chemistry/*genetics ; CRISPR-Cas Systems/*genetics ; Female ; Male ; Mice ; Mice, Transgenic ; Nerve Tissue Proteins/metabolism ; Nervous System/metabolism ; Neurons/physiology ; Nuclear Proteins/metabolism ; Primary Cell Culture ; RNA, Long Noncoding/genetics ; Transcriptional Activation/*genetics ; },
abstract = {Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.},
}

Animals
Astrocytes/physiology
Brain Chemistry/*genetics
CRISPR-Cas Systems/*genetics
Female
Male
Mice
Mice, Transgenic
Nerve Tissue Proteins/metabolism
Nervous System/metabolism
Neurons/physiology
Nuclear Proteins/metabolism
Primary Cell Culture
RNA, Long Noncoding/genetics
Transcriptional Activation/*genetics

@article {pmid28874671,
year = {2017},
author = {Wu, J and Vilarino, M and Suzuki, K and Okamura, D and Bogliotti, YS and Park, I and Rowe, J and McNabb, B and Ross, PJ and Belmonte, JCI},
title = {CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {10487},
pmid = {28874671},
issn = {2045-2322},
support = {DP1 DK113616/DK/NIDDK NIH HHS/United States ; R25 GM056765/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Genetic Therapy/methods/*veterinary ; Homeodomain Proteins/genetics/metabolism ; Humans ; Organogenesis/*genetics ; Pancreas/embryology/*metabolism ; Pluripotent Stem Cells/metabolism/transplantation ; Stem Cell Transplantation/methods/veterinary ; Swine/embryology/*genetics ; Tissue and Organ Harvesting/methods/veterinary ; Trans-Activators/genetics/metabolism ; },
abstract = {Genome editing using programmable nucleases has revolutionized biomedical research. CRISPR-Cas9 mediated zygote genome editing enables high efficient production of knockout animals suitable for studying development and relevant human diseases. Here we report efficient disabling pancreatogenesis in pig embryos via zygotic co-delivery of Cas9 mRNA and dual sgRNAs targeting the PDX1 gene, which when combined with chimeric-competent human pluriopotent stem cells may serve as a suitable platform for the xeno-generation of human tissues and organs in pigs.},
}

Animals
*CRISPR-Cas Systems
Genetic Therapy/methods/*veterinary
Homeodomain Proteins/genetics/metabolism
Humans
Organogenesis/*genetics
Pancreas/embryology/*metabolism
Pluripotent Stem Cells/metabolism/transplantation
Stem Cell Transplantation/methods/veterinary
Swine/embryology/*genetics
Tissue and Organ Harvesting/methods/veterinary
Trans-Activators/genetics/metabolism

@article {pmid30926307,
year = {2019},
author = {Zhang, R and Wu, H and Lian, Z},
title = {Bioinformatics analysis of evolutionary characteristics and biochemical structure of FGF5 Gene in sheep.},
journal = {Gene},
volume = {702},
number = {},
pages = {123-132},
doi = {10.1016/j.gene.2019.03.040},
pmid = {30926307},
issn = {1879-0038},
mesh = {Animals ; CRISPR-Cas Systems ; Computational Biology ; Evolution, Molecular ; Fibroblast Growth Factor 4/classification ; Fibroblast Growth Factor 5/*chemistry/classification/*genetics ; Fibroblast Growth Factors/chemistry/genetics ; Humans ; Mice ; Models, Molecular ; Mutation ; Phylogeny ; Protein Conformation, beta-Strand ; Protein Multimerization ; Receptor, Fibroblast Growth Factor, Type 1/chemistry ; Sequence Alignment ; Sheep/anatomy & histology/*genetics ; Wool/anatomy & histology ; },
abstract = {Fibroblast growth factor (FGF) 5 regulates the development and periodicity of hair follicles, which can affect hair traits. Loss-of-function mutations associated with long-hair phenotypes have been described in several mammalian species. Sheep is an important economic animal, however, the evolution characterizations and biological mechanism of oFGF5 (Ovis aries FGF5) gene are still poorly understood. In this study, oFGF5 gene was obtained by resequencing the whole genome of three Dorper sheep and RACE of two Kazakh sheep FGF5. We proposed FGF5 was phylogenetically related to FGF4 family and oFGF5 clearly orthologed to goat FGF5. Six loci were found from the positive selection results of FGF5 and half of them located on signal peptide. The basically similar rates of function-altering substitutions in sheep and goat lineage and the rest of the mammalian lineage of 365 SNPs indicated that the FGF5 gene was quite conservative during evolution. Homology modeling of the oFGF5 suggested that it has a highly conserved FGF superfamily domain containing 10 β-strands. Furthermore, the protein-protein docking analysis revealed that oFGF5 have the potential to form heterodimers with oFGFR1, the predicted interaction interface of FGF5-FGFR1 heterodimer was formed mainly by residues from FGF superfamily domain. Our observations suggested the evolutionary and structural biology features of oFGF5 might be relevant to its function about hair follicle development and modulating hair growth, and we confirmed our speculation by using the FGF5 gene editing sheep produced by CRISPR/Cas9 technology.},
}

Animals
CRISPR-Cas Systems
Computational Biology
Evolution, Molecular
Fibroblast Growth Factor 4/classification
Fibroblast Growth Factor 5/*chemistry/classification/*genetics
Fibroblast Growth Factors/chemistry/genetics
Humans
Mice
Models, Molecular
Mutation
Phylogeny
Protein Conformation, beta-Strand
Protein Multimerization
Receptor, Fibroblast Growth Factor, Type 1/chemistry
Sequence Alignment
Sheep/anatomy & histology/*genetics
Wool/anatomy & histology

@article {pmid30576352,
year = {2018},
author = {Itakura, Y and Inagaki, S and Wada, H and Hayashi, S},
title = {Trynity controls epidermal barrier function and respiratory tube maturation in Drosophila by modulating apical extracellular matrix nano-patterning.},
journal = {PloS one},
volume = {13},
number = {12},
pages = {e0209058},
doi = {10.1371/journal.pone.0209058},
pmid = {30576352},
issn = {1932-6203},
mesh = {Animals ; Behavior, Animal/physiology ; CRISPR-Cas Systems/genetics ; Drosophila/growth & development/*metabolism ; Drosophila Proteins/genetics/*metabolism ; Embryo, Nonmammalian/metabolism ; Epidermis/metabolism/pathology ; Extracellular Matrix/*metabolism ; Gene Knockout Techniques ; Larva/growth & development/metabolism ; Osmolar Concentration ; Trachea/metabolism ; },
abstract = {The outer surface of insects is covered by the cuticle, which is derived from the apical extracellular matrix (aECM). The aECM is secreted by epidermal cells during embryogenesis. The aECM exhibits large variations in structure, function, and constituent molecules, reflecting the enormous diversity in insect appearances. To investigate the molecular principles of aECM organization and function, here we studied the role of a conserved aECM protein, the ZP domain protein Trynity, in Drosophila melanogaster. We first identified trynity as an essential gene for epidermal barrier function. trynity mutation caused disintegration of the outermost envelope layer of the cuticle, resulting in small-molecule leakage and in growth and molting defects. In addition, the tracheal tubules of trynity mutants showed defects in pore-like structures of the cuticle, and the mutant tracheal cells failed to absorb luminal proteins and liquid. Our findings indicated that trynity plays essential roles in organizing nano-level structures in the envelope layer of the cuticle that both restrict molecular trafficking through the epidermis and promote the massive absorption pulse in the trachea.},
}

Animals
Behavior, Animal/physiology
CRISPR-Cas Systems/genetics
Drosophila/growth & development/*metabolism
Drosophila Proteins/genetics/*metabolism
Embryo, Nonmammalian/metabolism
Epidermis/metabolism/pathology
Extracellular Matrix/*metabolism
Gene Knockout Techniques
Larva/growth & development/metabolism
Osmolar Concentration
Trachea/metabolism

@article {pmid30464262,
year = {2018},
author = {He, YJ and Meghani, K and Caron, MC and Yang, C and Ronato, DA and Bian, J and Sharma, A and Moore, J and Niraj, J and Detappe, A and Doench, JG and Legube, G and Root, DE and D'Andrea, AD and Drané, P and De, S and Konstantinopoulos, PA and Masson, JY and Chowdhury, D},
title = {DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells.},
journal = {Nature},
volume = {563},
number = {7732},
pages = {522-526},
doi = {10.1038/s41586-018-0670-5},
pmid = {30464262},
issn = {1476-4687},
mesh = {BRCA1 Protein/*deficiency/genetics ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chromosome Aberrations ; Cytoplasmic Dyneins/*metabolism ; DNA/*metabolism ; DNA Damage/drug effects ; Drug Resistance, Neoplasm/drug effects ; Female ; Gene Editing ; *Genes, BRCA1 ; Genomic Instability/drug effects ; Homologous Recombination/drug effects ; Humans ; MRE11 Homologue Protein/*metabolism ; Mutation ; Ovarian Neoplasms/genetics/pathology ; Platinum/pharmacology ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Protein Binding ; *Recombinational DNA Repair/drug effects ; Transcription Factors/metabolism ; },
abstract = {Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.},
}

BRCA1 Protein/*deficiency/genetics
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Chromosome Aberrations
Cytoplasmic Dyneins/*metabolism
DNA/*metabolism
DNA Damage/drug effects
Drug Resistance, Neoplasm/drug effects
Female
Gene Editing
*Genes, BRCA1
Genomic Instability/drug effects
Homologous Recombination/drug effects
Humans
MRE11 Homologue Protein/*metabolism
Mutation
Ovarian Neoplasms/genetics/pathology
Platinum/pharmacology
Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
Protein Binding
*Recombinational DNA Repair/drug effects
Transcription Factors/metabolism

@article {pmid30340831,
year = {2018},
author = {Li, XF and Li, SY and Dai, CM and Li, JC and Huang, DR and Wang, JY},
title = {PP2A inhibition by LB-100 protects retinal pigment epithelium cells from UV radiation via activation of AMPK signaling.},
journal = {Biochemical and biophysical research communications},
volume = {506},
number = {1},
pages = {73-80},
doi = {10.1016/j.bbrc.2018.10.077},
pmid = {30340831},
issn = {1090-2104},
mesh = {AMP-Activated Protein Kinases/*genetics/metabolism ; Animals ; Bridged Bicyclo Compounds, Heterocyclic/*pharmacology ; CRISPR-Cas Systems ; Cell Line ; Cell Survival/drug effects/radiation effects ; Enzyme Activation ; Enzyme Inhibitors/*pharmacology ; Epithelial Cells/cytology/*drug effects/metabolism/radiation effects ; Gene Editing ; Gene Expression Regulation ; Humans ; Mice ; NADP/metabolism ; Piperazines/*pharmacology ; Primary Cell Culture ; Protein Phosphatase 2/antagonists & inhibitors/*genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Retinal Pigment Epithelium/cytology/drug effects/metabolism/radiation effects ; Signal Transduction ; Sunscreening Agents/*pharmacology ; Ultraviolet Rays/adverse effects ; },
abstract = {AMP-activated protein kinase (AMPK) signaling activation can inhibit Ultra-violet (UV) radiation (UVR)-induced retinal pigment epithelium (RPE) cell injuries. LB-100 is a novel inhibitor of protein phosphatase 2A (PP2A), the AMPKα1 phosphatase. Here, our results demonstrated that LB-100 significantly inhibited UVR-induced viability reduction, cell death and apoptosis in established ARPE-19 cells and primary murine RPE cells. LB-100 activated AMPK, nicotinamide adenine dinucleotide phosphate (NADPH) and Nrf2 (NF-E2-related factor 2) signalings, inhibiting UVR-induced oxidative injuries and DNA damage in RPE cells. Conversely, AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A mutation, almost blocked LB-100-induced RPE cytoprotection against UVR. Importantly, CRISPR/Cas9-mediated PP2A knockout mimicked and nullified LB-100-induced anti-UVR activity in RPE cells. Collectively, these results show that PP2A inhibition by LB-100 protects RPE cells from UVR via activation of AMPK signaling.},
}

AMP-Activated Protein Kinases/*genetics/metabolism
Animals
Bridged Bicyclo Compounds, Heterocyclic/*pharmacology
CRISPR-Cas Systems
Cell Line
Cell Survival/drug effects/radiation effects
Enzyme Activation
Enzyme Inhibitors/*pharmacology
Epithelial Cells/cytology/*drug effects/metabolism/radiation effects
Gene Editing
Gene Expression Regulation
Humans
Mice
NADP/metabolism
Piperazines/*pharmacology
Primary Cell Culture
Protein Phosphatase 2/antagonists & inhibitors/*genetics/metabolism
RNA, Small Interfering/genetics/metabolism
Reactive Oxygen Species/metabolism
Retinal Pigment Epithelium/cytology/drug effects/metabolism/radiation effects
Signal Transduction
Sunscreening Agents/*pharmacology
Ultraviolet Rays/adverse effects

@article {pmid30336982,
year = {2018},
author = {Bardet, M and Seeholzer, T and Unterreiner, A and Woods, S and Krappmann, D and Bornancin, F},
title = {MALT1 activation by TRAF6 needs neither BCL10 nor CARD11.},
journal = {Biochemical and biophysical research communications},
volume = {506},
number = {1},
pages = {48-52},
doi = {10.1016/j.bbrc.2018.10.029},
pmid = {30336982},
issn = {1090-2104},
mesh = {B-Cell CLL-Lymphoma 10 Protein/deficiency/*genetics ; CARD Signaling Adaptor Proteins/deficiency/*genetics ; CRISPR-Cas Systems ; Enzyme Activation/drug effects ; Gene Editing/methods ; Gene Expression Profiling ; Gene Expression Regulation ; Guanylate Cyclase/deficiency/*genetics ; HEK293 Cells ; Humans ; Jurkat Cells ; Lymphocyte Activation/drug effects ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/*genetics/metabolism ; Signal Transduction ; TNF Receptor-Associated Factor 6/*genetics/metabolism ; Tetradecanoylphorbol Acetate/pharmacology ; },
abstract = {The MALT1 (Mucosa associated lymphoid tissue lymphoma translocation protein 1) paracaspase couples antigen receptors on lymphocytes to downstream signaling events. Activation of MALT1 is known to involve stimulus-dependent CBM complex formation, that is, the recruitment of BCL10-bound MALT1 to a CARD-Coiled Coil protein. Beyond this canonical, CBM-dependent mechanism of MALT1 activation, recent studies suggest that MALT1 protease activity may be triggered by alternative mechanisms. For instance, the E3-ligase TRAF6 can activate MALT1 proteolytic function and induce MALT1 auto-cleavage. However, the interplay between CBM and TRAF6 with regard to MALT1 activation has remained incompletely elucidated. Here, by generating CRISPR/Cas9-derived knock-out Jurkat T-cells, we show that TRAF6 was dispensable for CARD11/BCL10-dependent MALT1 activation upon T-cell stimulation. However, ectopically-expressed TRAF6 could induce MALT1 activity in Jurkat T-cells devoid of either CARD11 or BCL10. These data provide unequivocal evidence that TRAF6-mediated MALT1 activation does not require the upstream scaffold CARD11 or the interaction between MALT1 and BCL10. Thus, TRAF6 may be part of a previously unidentified non-canonical pathway that triggers MALT1 protease activity independently of canonical CBM signalosomes.},
}

B-Cell CLL-Lymphoma 10 Protein/deficiency/*genetics
CARD Signaling Adaptor Proteins/deficiency/*genetics
CRISPR-Cas Systems
Enzyme Activation/drug effects
Gene Editing/methods
Gene Expression Profiling
Gene Expression Regulation
Guanylate Cyclase/deficiency/*genetics
HEK293 Cells
Humans
Jurkat Cells
Lymphocyte Activation/drug effects
Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/*genetics/metabolism
Signal Transduction
TNF Receptor-Associated Factor 6/*genetics/metabolism
Tetradecanoylphorbol Acetate/pharmacology

@article {pmid29886342,
year = {2018},
author = {Payungwoung, T and Shinzawa, N and Hino, A and Nishi, T and Murata, Y and Yuda, M and Iwanaga, S},
title = {CRISPR/Cas9 system in Plasmodium falciparum using the centromere plasmid.},
journal = {Parasitology international},
volume = {67},
number = {5},
pages = {605-608},
doi = {10.1016/j.parint.2018.06.002},
pmid = {29886342},
issn = {1873-0329},
mesh = {Artemisinins/pharmacology ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Centromere/*genetics ; Drug Resistance/genetics ; Gene Editing ; *Genome, Protozoan ; Mutagenesis, Site-Directed ; Organisms, Genetically Modified ; Plasmids/genetics ; Plasmodium falciparum/drug effects/*genetics ; },
abstract = {The CRISPR/Cas9 nuclease system is a powerful method to genetically modify the human malarial parasite, Plasmodium falciparum. Currently, this method is carried out by co-transfection with two plasmids, one containing the Cas9 nuclease gene, and another encoding the sgRNA and the donor template DNA. However, the efficiency of modification is currently low owing to the low frequency of these plasmids in the parasites. To improve the CRISPR/Cas9 nuclease system for P. falciparum, we developed a novel method using the transgenic parasite, PfCAS9, which stably expresses the Cas9 nuclease using the centromere plasmid. To examine the efficiency of genetic modification using the PfCAS9 parasite, we performed site-directed mutagenesis of kelch13 gene, which is considered to be involved in artemisinin resistance. Our results demonstrated that the targeted mutation could be introduced with almost 100% efficiency when the transfected PfCAS9 parasites were treated with two drugs to maintain both the centromere plasmid containing the Cas9 nuclease and the plasmid having the sgRNA. Therefore, the PfCAS9 parasite is a useful parasite line for the genetic modification of P. falciparum.},
}

Artemisinins/pharmacology
CRISPR-Associated Protein 9/*genetics
*CRISPR-Cas Systems
Centromere/*genetics
Drug Resistance/genetics
Gene Editing
*Genome, Protozoan
Mutagenesis, Site-Directed
Organisms, Genetically Modified
Plasmids/genetics
Plasmodium falciparum/drug effects/*genetics

@article {pmid29740169,
year = {2018},
author = {Remacha, L and Currás-Freixes, M and Torres-Ruiz, R and Schiavi, F and Torres-Pérez, R and Calsina, B and Letón, R and Comino-Méndez, I and Roldán-Romero, JM and Montero-Conde, C and Santos, M and Pérez, LI and Pita, G and Alonso, MR and Honrado, E and Pedrinaci, S and Crespo-Facorro, B and Percesepe, A and Falcioni, M and Rodríguez-Perales, S and Korpershoek, E and Ramón-Maiques, S and Opocher, G and Rodríguez-Antona, C and Robledo, M and Cascón, A},
title = {Gain-of-function mutations in DNMT3A in patients with paraganglioma.},
journal = {Genetics in medicine : official journal of the American College of Medical Genetics},
volume = {20},
number = {12},
pages = {1644-1651},
doi = {10.1038/s41436-018-0003-y},
pmid = {29740169},
issn = {1530-0366},
mesh = {Adrenal Gland Neoplasms/*genetics/pathology ; Adult ; CRISPR-Cas Systems/genetics ; DNA (Cytosine-5-)-Methyltransferases/*genetics ; DNA Methylation ; Female ; Gain of Function Mutation ; Genetic Predisposition to Disease ; Genotype ; Germ-Line Mutation/genetics ; Humans ; Male ; Paraganglioma/*genetics/pathology ; Pheochromocytoma/*genetics/pathology ; Whole Exome Sequencing ; },
abstract = {PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors.

METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed.

RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma.

CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.},
}

Adrenal Gland Neoplasms/*genetics/pathology
Adult
CRISPR-Cas Systems/genetics
DNA (Cytosine-5-)-Methyltransferases/*genetics
DNA Methylation
Female
Gain of Function Mutation
Genetic Predisposition to Disease
Genotype
Germ-Line Mutation/genetics
Humans
Male
Paraganglioma/*genetics/pathology
Pheochromocytoma/*genetics/pathology
Whole Exome Sequencing

@article {pmid29519807,
year = {2018},
author = {Antoniani, C and Meneghini, V and Lattanzi, A and Felix, T and Romano, O and Magrin, E and Weber, L and Pavani, G and El Hoss, S and Kurita, R and Nakamura, Y and Cradick, TJ and Lundberg, AS and Porteus, M and Amendola, M and El Nemer, W and Cavazzana, M and Mavilio, F and Miccio, A},
title = {Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human β-globin locus.},
journal = {Blood},
volume = {131},
number = {17},
pages = {1960-1973},
doi = {10.1182/blood-2017-10-811505},
pmid = {29519807},
issn = {1528-0020},
mesh = {*Anemia, Sickle Cell/genetics/metabolism/pathology/therapy ; *CRISPR-Cas Systems ; Cell Line ; *Fetal Hemoglobin/biosynthesis/genetics ; *Gene Editing ; *Genetic Loci ; Hematopoietic Stem Cells/*metabolism/pathology ; Humans ; beta-Globins/*genetics/metabolism ; },
abstract = {Naturally occurring, large deletions in the β-globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and β-thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the δ- and β-globin genes and a putative γ-δ intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in γ-globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the β-globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of γ-globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of β-hemoglobinopathies.},
}

*Anemia, Sickle Cell/genetics/metabolism/pathology/therapy
*CRISPR-Cas Systems
Cell Line
*Fetal Hemoglobin/biosynthesis/genetics
*Gene Editing
*Genetic Loci
Hematopoietic Stem Cells/*metabolism/pathology
Humans
beta-Globins/*genetics/metabolism

@article {pmid29463564,
year = {2018},
author = {Gallipoli, P and Giotopoulos, G and Tzelepis, K and Costa, ASH and Vohra, S and Medina-Perez, P and Basheer, F and Marando, L and Di Lisio, L and Dias, JML and Yun, H and Sasca, D and Horton, SJ and Vassiliou, G and Frezza, C and Huntly, BJP},
title = {Glutaminolysis is a metabolic dependency in FLT3ITD acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition.},
journal = {Blood},
volume = {131},
number = {15},
pages = {1639-1653},
pmid = {29463564},
issn = {1528-0020},
support = {MC_UU_12022/6//Medical Research Council/United Kingdom ; 14-1069//Worldwide Cancer Research/United Kingdom ; MR/M010392/1//Medical Research Council/United Kingdom ; //Wellcome Trust/United Kingdom ; MC_PC_12009//Medical Research Council/United Kingdom ; G1000288//Medical Research Council/United Kingdom ; 109967/Z/15/Z//Wellcome Trust/United Kingdom ; C56179/A21617//Cancer Research UK/United Kingdom ; },
mesh = {CRISPR-Cas Systems ; Enzyme Activation/drug effects/genetics ; Genome-Wide Association Study ; Glutamine/genetics/*metabolism ; Humans ; K562 Cells ; *Leukemia, Myeloid, Acute/drug therapy/enzymology/genetics ; *Mutation ; Protein Kinase Inhibitors/*pharmacology ; THP-1 Cells ; *fms-Like Tyrosine Kinase 3/antagonists & inhibitors/genetics/metabolism ; },
abstract = {FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation-driven leukemias.},
}

CRISPR-Cas Systems
Enzyme Activation/drug effects/genetics
Genome-Wide Association Study
Glutamine/genetics/*metabolism
Humans
K562 Cells
*Leukemia, Myeloid, Acute/drug therapy/enzymology/genetics
*Mutation
Protein Kinase Inhibitors/*pharmacology
THP-1 Cells
*fms-Like Tyrosine Kinase 3/antagonists & inhibitors/genetics/metabolism

@article {pmid28557982,
year = {2017},
author = {Park, J and Childs, L and Kim, D and Hwang, GH and Kim, S and Kim, ST and Kim, JS and Bae, S},
title = {Digenome-seq web tool for profiling CRISPR specificity.},
journal = {Nature methods},
volume = {14},
number = {6},
pages = {548-549},
pmid = {28557982},
issn = {1548-7105},
mesh = {Algorithms ; CRISPR-Associated Proteins/*genetics ; CRISPR-Cas Systems/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; High-Throughput Nucleotide Sequencing/*methods ; Internet ; Sensitivity and Specificity ; },
}

Algorithms
CRISPR-Associated Proteins/*genetics
CRISPR-Cas Systems/*genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
High-Throughput Nucleotide Sequencing/*methods
Internet
Sensitivity and Specificity

@article {pmid31080012,
year = {2019},
author = {Wilkinson, M and Drabavicius, G and Silanskas, A and Gasiunas, G and Siksnys, V and Wigley, DB},
title = {Structure of the DNA-Bound Spacer Capture Complex of a Type II CRISPR-Cas System.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2019.04.020},
pmid = {31080012},
issn = {1097-4164},
abstract = {CRISPR and associated Cas proteins function as an adaptive immune system in prokaryotes to combat bacteriophage infection. During the immunization step, new spacers are acquired by the CRISPR machinery, but the molecular mechanism of spacer capture remains enigmatic. We show that the Cas9, Cas1, Cas2, and Csn2 proteins of a Streptococcus thermophilus type II-A CRISPR-Cas system form a complex and provide cryoelectron microscopy (cryo-EM) structures of three different assemblies. The predominant form, with the stoichiometry Cas18-Cas24-Csn28, referred to as monomer, contains ∼30 bp duplex DNA bound along a central channel. A minor species, termed a dimer, comprises two monomers that sandwich a further eight Cas1 and four Cas2 subunits and contains two DNA ∼30-bp duplexes within the channel. A filamentous form also comprises Cas18-Cas24-Csn28 units (typically 2-6) but with a different Cas1-Cas2 interface between them and a continuous DNA duplex running along a central channel.},
}

@article {pmid31036811,
year = {2019},
author = {Hirano, S and Abudayyeh, OO and Gootenberg, JS and Horii, T and Ishitani, R and Hatada, I and Zhang, F and Nishimasu, H and Nureki, O},
title = {Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {1968},
doi = {10.1038/s41467-019-09741-6},
pmid = {31036811},
issn = {2041-1723},
support = {DP1 MH100706/MH/NIMH NIH HHS/United States ; R01 DK097768/DK/NIDDK NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*chemistry/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Corynebacterium diphtheriae/enzymology/metabolism ; Crystallography, X-Ray ; DNA Cleavage ; DNA Restriction-Modification Enzymes/*chemistry/*metabolism ; HEK293 Cells ; Humans ; Hydrogen Bonding ; },
abstract = {The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets bearing a protospacer adjacent motif (PAM) and complementarity to an RNA guide. Unlike other Cas9 orthologs, Corynebacterium diphtheriae Cas9 (CdCas9) recognizes the promiscuous NNRHHHY PAM. However, the CdCas9-mediated PAM recognition mechanism remains unknown. Here, we report the crystal structure of CdCas9 in complex with the guide RNA and its target DNA at 2.9 Å resolution. The structure reveals that CdCas9 recognizes the NNRHHHY PAM via a combination of van der Waals interactions and base-specific hydrogen bonds. Moreover, we find that CdCas9 exhibits robust DNA cleavage activity with the optimal 22-nucleotide length guide RNAs. Our findings highlight the mechanistic diversity of the PAM recognition by Cas9 orthologs, and provide a basis for the further engineering of the CRISPR-Cas9 genome-editor nucleases.},
}

CRISPR-Associated Protein 9/*chemistry/*metabolism
CRISPR-Cas Systems
Cell Line
Corynebacterium diphtheriae/enzymology/metabolism
Crystallography, X-Ray
DNA Cleavage
DNA Restriction-Modification Enzymes/*chemistry/*metabolism
HEK293 Cells
Humans
Hydrogen Bonding

@article {pmid30898720,
year = {2019},
author = {Herai, RH},
title = {Avoiding the off-target effects of CRISPR/cas9 system is still a challenging accomplishment for genetic transformation.},
journal = {Gene},
volume = {700},
number = {},
pages = {176-178},
doi = {10.1016/j.gene.2019.03.019},
pmid = {30898720},
issn = {1879-0038},
mesh = {CRISPR-Cas Systems ; Gene Editing/ethics/*methods ; Genome, Human ; Humans ; RNA, Guide ; *Transformation, Genetic ; },
abstract = {The recent disclosure of a human embryo subjected to a genetic transformation using the CRISPR/cas9 system give rise to several concerns on ethical questions about its uncontrolled use in humans. Although CRISPR/cas9 has demonstrated its efficiency, this system still lacks the capability to avoid the introduction of undesirable mutations through the target genome. In this Letter, we present several undesirable impacts that CRISPR/cas9 system have in the genetic transformation of the human genome. We briefly discuss, using the very recent literature from distinct high impact journals, the main concerns related to CRISPR/cas9 to deal with off-target effects and how the research community has treated it.},
}

CRISPR-Cas Systems
Gene Editing/ethics/*methods
Genome, Human
Humans
RNA, Guide
*Transformation, Genetic

@article {pmid30778240,
year = {2019},
author = {Beyret, E and Liao, HK and Yamamoto, M and Hernandez-Benitez, R and Fu, Y and Erikson, G and Reddy, P and Izpisua Belmonte, JC},
title = {Single-dose CRISPR-Cas9 therapy extends lifespan of mice with Hutchinson-Gilford progeria syndrome.},
journal = {Nature medicine},
volume = {25},
number = {3},
pages = {419-422},
doi = {10.1038/s41591-019-0343-4},
pmid = {30778240},
issn = {1546-170X},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Genetic Therapy/*methods ; Lamin Type A/*genetics/metabolism ; *Longevity ; Mice ; Mutation ; Progeria/*genetics ; Protein Isoforms/genetics/metabolism ; },
abstract = {Hutchinson-Gilford progeria syndrome (HGPS) is a rare lethal genetic disorder characterized by symptoms reminiscent of accelerated aging. The major underlying genetic cause is a substitution mutation in the gene coding for lamin A, causing the production of a toxic isoform called progerin. Here we show that reduction of lamin A/progerin by a single-dose systemic administration of adeno-associated virus-delivered CRISPR-Cas9 components suppresses HGPS in a mouse model.},
}

Animals
*CRISPR-Cas Systems
Disease Models, Animal
Genetic Therapy/*methods
Lamin Type A/*genetics/metabolism
*Longevity
Mice
Mutation
Progeria/*genetics
Protein Isoforms/genetics/metabolism

@article {pmid30778239,
year = {2019},
author = {Santiago-Fernández, O and Osorio, FG and Quesada, V and Rodríguez, F and Basso, S and Maeso, D and Rolas, L and Barkaway, A and Nourshargh, S and Folgueras, AR and Freije, JMP and López-Otín, C},
title = {Development of a CRISPR/Cas9-based therapy for Hutchinson-Gilford progeria syndrome.},
journal = {Nature medicine},
volume = {25},
number = {3},
pages = {423-426},
doi = {10.1038/s41591-018-0338-6},
pmid = {30778239},
issn = {1546-170X},
mesh = {Animals ; *CRISPR-Cas Systems ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Lamin Type A/*genetics/metabolism ; Mice ; Point Mutation ; Progeria/genetics/*therapy ; },
abstract = {CRISPR/Cas9-based therapies hold considerable promise for the treatment of genetic diseases. Among these, Hutchinson-Gilford progeria syndrome, caused by a point mutation in the LMNA gene, stands out as a potential candidate. Here, we explore the efficacy of a CRISPR/Cas9-based approach that reverts several alterations in Hutchinson-Gilford progeria syndrome cells and mice by introducing frameshift mutations in the LMNA gene.},
}

Animals
*CRISPR-Cas Systems
Genetic Therapy/*methods
HEK293 Cells
Humans
Lamin Type A/*genetics/metabolism
Mice
Point Mutation
Progeria/genetics/*therapy

@article {pmid30778238,
year = {2019},
author = {Nelson, CE and Wu, Y and Gemberling, MP and Oliver, ML and Waller, MA and Bohning, JD and Robinson-Hamm, JN and Bulaklak, K and Castellanos Rivera, RM and Collier, JH and Asokan, A and Gersbach, CA},
title = {Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy.},
journal = {Nature medicine},
volume = {25},
number = {3},
pages = {427-432},
doi = {10.1038/s41591-019-0344-3},
pmid = {30778238},
issn = {1546-170X},
support = {DP2 OD008586/OD/NIH HHS/United States ; K99 EB023979/EB/NIBIB NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems/*genetics/immunology ; Dependovirus ; Disease Models, Animal ; Dystrophin/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors ; Immunity, Cellular/immunology ; Immunity, Humoral/immunology ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne/genetics/*therapy ; },
abstract = {Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy1-6, however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice7; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study.},
}

Animals
Animals, Newborn
CRISPR-Cas Systems/*genetics/immunology
Dependovirus
Disease Models, Animal
Dystrophin/genetics
Gene Editing/*methods
Genetic Therapy/methods
Genetic Vectors
Immunity, Cellular/immunology
Immunity, Humoral/immunology
Mice
Mice, Inbred mdx
Muscular Dystrophy, Duchenne/genetics/*therapy

@article {pmid30504875,
year = {2018},
author = {Hsu, JY and Fulco, CP and Cole, MA and Canver, MC and Pellin, D and Sher, F and Farouni, R and Clement, K and Guo, JA and Biasco, L and Orkin, SH and Engreitz, JM and Lander, ES and Joung, JK and Bauer, DE and Pinello, L},
title = {CRISPR-SURF: discovering regulatory elements by deconvolution of CRISPR tiling screen data.},
journal = {Nature methods},
volume = {15},
number = {12},
pages = {992-993},
doi = {10.1038/s41592-018-0225-6},
pmid = {30504875},
issn = {1548-7105},
support = {DP2 HL137300/HL/NHLBI NIH HHS/United States ; K08 DK093705/DK/NIDDK NIH HHS/United States ; R03 DK109232/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Gene Expression Regulation ; Genetic Engineering/*methods ; Genome, Human ; Genomics/*methods ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; *Regulatory Sequences, Nucleic Acid ; },
}

*CRISPR-Cas Systems
Gene Expression Regulation
Genetic Engineering/*methods
Genome, Human
Genomics/*methods
High-Throughput Nucleotide Sequencing/*methods
Humans
*Regulatory Sequences, Nucleic Acid

@article {pmid30504872,
year = {2018},
author = {Ikeda, K and Uchida, N and Nishimura, T and White, J and Martin, RM and Nakauchi, H and Sebastiano, V and Weinberg, KI and Porteus, MH},
title = {Efficient scarless genome editing in human pluripotent stem cells.},
journal = {Nature methods},
volume = {15},
number = {12},
pages = {1045-1047},
doi = {10.1038/s41592-018-0212-y},
pmid = {30504872},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; Embryonic Stem Cells/cytology/*metabolism ; *Gene Editing ; Gene Expression Regulation ; *Genome, Human ; *Homologous Recombination ; Humans ; Pluripotent Stem Cells/cytology/*metabolism ; RNA, Small Interfering/*genetics ; },
abstract = {Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.},
}

*CRISPR-Cas Systems
Embryonic Stem Cells/cytology/*metabolism
*Gene Editing
Gene Expression Regulation
*Genome, Human
*Homologous Recombination
Humans
Pluripotent Stem Cells/cytology/*metabolism
RNA, Small Interfering/*genetics

@article {pmid30504868,
year = {2018},
author = {Rusk, N},
title = {Recording transcriptional activity.},
journal = {Nature methods},
volume = {15},
number = {12},
pages = {999},
doi = {10.1038/s41592-018-0242-5},
pmid = {30504868},
issn = {1548-7105},
mesh = {*CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/*methods ; Humans ; RNA/*analysis/*genetics ; *Transcription, Genetic ; },
}