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Yi-Hsien Chen, Shondra M Pruett-Miller
The availability of human pluripotent stem cells (hPSCs) and progress in genome engineering technology have altered the way we approach scientific research and drug development screens. Unfortunately, the procedures for genome editing of hPSCs often subject cells to harsh conditions that compromise viability: a major problem that is compounded by the innate challenge of single-cell culture. Here we describe a generally applicable workflow that supports single-cell cloning and expansion of hPSCs after genome editing and single-cell sorting...
August 4, 2018: Stem Cell Research
Terence R Flotte
No abstract text is available yet for this article.
August 2018: Human Gene Therapy
Xian Wang, Qili Zhao, Li Wang, Jun Liu, Huayan Pu, Shaorong Xie, Changhai Ru, Yu Sun
Microinjection is a widely used technique for introducing exogenous materials into cells. Many applications of microinjection, such as gene editing and drug testing, rely on the accurate control of the deposition volume. However, the deposition volume in microinjection is presently calibrated in open medium without considering the cell inner pressure effect, which we experimentally show in this paper that can induce an error between the actual deposition volume and set volume to be as large as 30%. In this work, the relationship between cell inner pressure and deposition volume was analytically modeled and experimentally validated...
August 10, 2018: Langmuir: the ACS Journal of Surfaces and Colloids
Yoshitaka Fujiki, Yutaka Yamamoto, Aiko Sueta, Mutsuko Yamamoto-Ibusuki, Lisa Goto-Yamaguchi, Mai Tomiguchi, Takashi Takeshita, Hirotaka Iwase
Background: Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3B (APOBEC3B) is a gene editing enzyme with cytidine deaminase activity and high expression of its mRNA in breast tumors have been shown to be associated with progressive cases and poor prognosis. In this study, we aimed to examine the relationship between the expression of APOBEC3B and the effect of neoadjuvant chemotherapy (NAC) using pretreatment biopsy tissue, and examined whether the expression of APOBEC3B influenced chemotherapy efficacy...
July 17, 2018: Oncotarget
Benjamin E Deverman, Bernard M Ravina, Krystof S Bankiewicz, Steven M Paul, Dinah W Y Sah
Adeno-associated viral (AAV) vectors are a rapidly emerging gene therapy platform for the treatment of neurological diseases. In preclinical studies, transgenes encoding therapeutic proteins, microRNAs, antibodies or gene-editing machinery have been successfully delivered to the central nervous system with natural or engineered viral capsids via various routes of administration. Importantly, initial clinical studies have demonstrated encouraging safety and efficacy in diseases such as Parkinson disease and spinal muscular atrophy, as well as durability of transgene expression...
August 10, 2018: Nature Reviews. Drug Discovery
Catherin Marin-Mogollon, Fiona J A van Pul, Shinya Miyazaki, Takashi Imai, Jai Ramesar, Ahmed M Salman, Beatrice M F Winkel, Ahmad Syibli Othman, Hans Kroeze, Severine Chevalley-Maurel, Arturo Reyes-Sandoval, Meta Roestenberg, Blandine Franke-Fayard, Chris J Janse, Shahid M Khan
BACKGROUND: Rodent malaria parasites where the gene encoding circumsporozoite protein (CSP) has been replaced with csp genes from the human malaria parasites, Plasmodium falciparum or Plasmodium vivax, are used as pre-clinical tools to evaluate CSP vaccines in vivo. These chimeric rodent parasites produce sporozoites in Anopheles stephensi mosquitoes that are capable of infecting rodent and human hepatocytes. The availability of chimeric P. falciparum parasites where the pfcsp gene has been replaced by the pvcsp would open up possibilities to test P...
August 9, 2018: Malaria Journal
Troy T Rohn, Nayoung Kim, Noail F Isho, Jacob M Mack
Despite a wealth of knowledge gained in the past three decades concerning the molecular underpinnings of Alzheimer's disease (AD), progress towards obtaining effective, disease modifying therapies has proven to be challenging. In this manner, numerous clinical trials targeting the production, aggregation, and toxicity of beta-amyloid, have failed to meet efficacy standards. This puts into question the beta-amyloid hypothesis and suggests that additional treatment strategies should be explored. The recent emergence of CRISPR/Cas9 gene editing as a relatively straightforward, inexpensive, and precise system has led to an increased interest of applying this technique in AD...
2018: Journal of Alzheimer's Disease and Parkinsonism
Jacqueline P Robbins, Leo Perfect, Elena M Ribe, Marcello Maresca, Adrià Dangla-Valls, Evangeline M Foster, Richard Killick, Paulina Nowosiad, Matthew J Reid, Lucia Dutan Polit, Alejo J Nevado, Daniel Ebner, Mohammad Bohlooly-Y, Noel Buckley, Menelas N Pangalos, Jack Price, Simon Lovestone
Our understanding of the molecular processes underlying Alzheimer's disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating β-amyloid (Aβ) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in Aβ processing. Here we investigate how CLU mediates Aβ-driven neurodegeneration in human induced pluripotent stem cell (iPSC)-derived neurons...
2018: Frontiers in Neuroscience
Zachary WareJoncas, Jarryd M Campbell, Gabriel Martínez-Gálvez, William A C Gendron, Michael A Barry, Peter C Harris, Caroline R Sussman, Stephen C Ekker
The expanding field of precision gene editing is empowering researchers to directly modify DNA. Gene editing is made possible using synonymous technologies: a DNA-binding platform to molecularly locate user-selected genomic sequences and an associated biochemical activity that serves as a functional editor. The advent of accessible DNA-targeting molecular systems, such as zinc-finger nucleases, transcription activator-like effectors (TALEs) and CRISPR-Cas9 gene editing systems, has unlocked the ability to target nearly any DNA sequence with nucleotide-level precision...
August 8, 2018: Nature Reviews. Nephrology
K Blighe, L DeDionisio, K A Christie, B Chawes, S Shareef, T Kakouli-Duarte, C Chao-Shern, V Harding, R S Kelly, L Castellano, J Stebbing, J A Lasky-Su, M A Nesbit, C B T Moore
The reporting of the first draft of the human genome in 2000 brought with it much hope for the future in what was felt as a paradigm shift toward improved health outcomes. Indeed, we have now mapped the majority of variation across human populations with landmark projects such as 1000 Genomes; in cancer, we have catalogued mutations across the primary carcinomas; whilst, for other diseases, we have identified the genetic variants with strongest association. Despite this, we are still awaiting the genetic revolution in healthcare to materialise and translate itself into the health benefits for which we had hoped...
August 8, 2018: BMC Genomics
Xinyi Li, Yichun Bai, Xinzhen Cheng, Peter Girgis Tawfek Kalds, Bing Sun, Yun Wu, Huijiao Lyu, Kun Xu, Zhiying Zhang
CRISPR/Cas9 has been emerging as a main player in genome editing field since its advent. However, CRISPR/Cas9-induced precise gene editing remains challenging since it requires no scar left after editing. Among the few reports regarding two-step 'pop in & out' technologies for precise gene editing, the combination of CRISPR/Cas9 with Cre/LoxP demonstrates a higher efficiency, but leaves behind a 34 base pair of tag sequence due to its inherent property. Another method utilizes piggyBac transposon for removing the selection cassette, and its disadvantage is the difficulty in controlling its random reintegration after releasing...
August 7, 2018: FEBS Journal
Hyun-Jung Kim, Antoni Barrientos
Mammalian mitochondrial ribosomes (mitoribosomes) synthesize 13 proteins, essential components of the oxidative phosphorylation system. They are linked to mitochondrial disorders, often involving cardiomyopathy. Mitoribosome biogenesis is assisted by multiple cofactors whose specific functions remain largely uncharacterized. Here, we examined the role of human MTG1, a conserved ribosome assembly guanosine triphosphatase. MTG1-silencing in human cardiomyocytes and developing zebrafish revealed early cardiovascular lesions...
July 31, 2018: Nucleic Acids Research
Christopher A Vakulskas, Daniel P Dever, Garrett R Rettig, Rolf Turk, Ashley M Jacobi, Michael A Collingwood, Nicole M Bode, Matthew S McNeill, Shuqi Yan, Joab Camarena, Ciaran M Lee, So Hyun Park, Volker Wiebking, Rasmus O Bak, Natalia Gomez-Ospina, Mara Pavel-Dinu, Wenchao Sun, Gang Bao, Matthew H Porteus, Mark A Behlke
Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing...
August 2018: Nature Medicine
Marion Manil-Ségalen, Małgorzata Łuksza, Joanne Kanaan, Véronique Marthiens, Simon I R Lane, Keith T Jones, Marie-Emilie Terret, Renata Basto, Marie-Hélène Verlhac
Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics...
August 6, 2018: Journal of Cell Biology
Talita Giacomet de Carvalho, Roselena Schuh, Gabriela Pasqualim, Felipe Matheus Pellenz, Eduardo Cremonese Filippi-Chiela, Roberto Giugliani, Guilherme Baldo, Ursula Matte
Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder (LSD). It is caused by mutations in the IDUA gene, which lead to the accumulation of the glycosaminoglycans dermatan and heparan sulfate. The CRISPR-Cas9 system is a new and powerful tool that allows gene editing at precise points of the genome, resulting in gene correction through the introduction and genomic integration of a wildtype sequence. In this study, we used the CRISPR-Cas9 genome editing technology to correct in vitro the most common mutation causing MPS I...
August 3, 2018: Gene
Peter J Castaldi, Feng Guo, Dandi Qiao, Fei Du, Zun Zar Chi Naing, Yan Li, Betty Pham, Tarjei S Mikkelsen, Michael H Cho, Edwin K Silverman, Xiaobo Zhou
RATIONALE: The identification of causal variants responsible for disease associations from genome-wide association studies (GWAS) facilitates functional understanding of the biological mechanisms by which those genetic variants influence disease susceptibility. OBJECTIVE: We aim to identify causal variants in or near the FAM13A GWAS locus associated with COPD. METHODS: We used an integrated approach featuring conditional genetic analysis, massively parallel reporter assays (MPRA), traditional reporter assays, chromatin conformation capture assays, and CRISPR-based gene editing to characterize COPD-associated regulatory variants in the FAM13A region in human bronchial epithelial cell lines...
August 4, 2018: American Journal of Respiratory and Critical Care Medicine
Amelia L Parker, Wee Siang Teo, Elvis Pandzic, Juan Jesus Vicente, Joshua A McCarroll, Linda Wordeman, Maria Kavallaris
Microtubules are highly dynamic structures that play an integral role in fundamental cellular functions. Different α- and β-tubulin isotypes are thought to confer unique dynamic properties to microtubules. The tubulin isotypes have highly conserved structures, differing mainly in their C-terminal tail sequences. However, little is known about the importance of the C-terminal tail in regulating and co-ordinating microtubule dynamics. We developed syngeneic human cell models using gene-editing to precisely modify the β-tubulin C-terminal tail region while preserving the endogenous microtubule network...
2018: Life science alliance
Lele Sun, Yanjing Gao, Yaoguang Wang, Qin Wei, Jiye Shi, Nan Chen, Di Li, Chunhai Fan
Intracellular delivery of proteins provides a direct means to manipulate cell function and probe the intracellular environment. However, direct cytoplasmic delivery of proteins suffers from limited availability of efficient toolsets, and thus remains challenging in research and therapeutic applications. Natural biological cargo delivery processes, like SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) complex mediated membrane fusion and other vesicle fusion in live cells, enable targeted delivery with high efficiency...
July 21, 2018: Chemical Science
Vibha Ahuja
In India, genetically modified organisms (GMOs) and the products thereof are regulated under the "Rules for the manufacture, use, import, export & storage of hazardous microorganisms, genetically engineered organisms or cells, 1989" (referred to as Rules, 1989) notified under the Environment (Protection) Act, 1986. These Rules are implemented by the Ministry of Environment, Forest and Climate Change, Department of Biotechnology and State Governments though six competent authorities. The Rules, 1989 are supported by series of guidelines on contained research, biologics, confined field trials, food safety assessment, environmental risk assessment etc...
2018: BMC Proceedings
James P Collins
Gene drives are systems of biased inheritance that enhance the likelihood a sequence of DNA passes between generations through sexual reproduction and potentially throughout a local population and ultimately all connected populations of a species. Gaps in our knowledge of gene drive systems prompted the US National Institutes of Health (NIH) and the Foundation for the NIH to ask the US National Academies of Sciences, Engineering, and Medicine (NASEM) to convene an expert panel to provide an independent, objective examination of what we know about gene drive systems...
2018: BMC Proceedings
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