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CRISPR/Cas9

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https://www.readbyqxmd.com/read/29164808/a-universal-approach-to-correct-various-hbb-gene-mutations-in-human-stem-cells-for-gene-therapy-of-beta-thalassemia-and-sickle-cell-disease
#1
Liuhong Cai, Hao Bai, Vasiliki Mahairaki, Yongxing Gao, Chaoxia He, Yanfei Wen, You-Chuan Jin, You Wang, Rachel L Pan, Armaan Qasba, Zhaohui Ye, Linzhao Cheng
Beta-thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with β-thalassemia (β-thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable...
November 21, 2017: Stem Cells Translational Medicine
https://www.readbyqxmd.com/read/29163669/current-status-and-perspectives-of-genome-editing-technology-for-microalgae
#2
REVIEW
Seungjib Jeon, Jong-Min Lim, Hyung-Gwan Lee, Sung-Eun Shin, Nam Kyu Kang, Youn-Il Park, Hee-Mock Oh, Won-Joong Jeong, Byeong-Ryool Jeong, Yong Keun Chang
Genome editing techniques are critical for manipulating genes not only to investigate their functions in biology but also to improve traits for genetic engineering in biotechnology. Genome editing has been greatly facilitated by engineered nucleases, dubbed molecular scissors, including zinc-finger nuclease (ZFN), TAL effector endonuclease (TALEN) and clustered regularly interspaced palindromic sequences (CRISPR)/Cas9. In particular, CRISPR/Cas9 has revolutionized genome editing fields with its simplicity, efficiency and accuracy compared to previous nucleases...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/29163593/osarm1-an-r2r3-myb-transcription-factor-is-involved-in-regulation-of-the-response-to-arsenic-stress-in-rice
#3
Feng-Zhu Wang, Mo-Xian Chen, Lu-Jun Yu, Li-Juan Xie, Li-Bing Yuan, Hua Qi, Ming Xiao, Wuxiu Guo, Zhe Chen, Keke Yi, Jianhua Zhang, Rongliang Qiu, Wensheng Shu, Shi Xiao, Qin-Fang Chen
Bioaccumulation of arsenic (As) in rice (Oryza sativa) increases human exposure to this toxic, carcinogenic element. Recent studies identified several As transporters, but the regulation of these transporters remains unclear. Here, we show that the rice R2R3 MYB transcription factor OsARM1 (ARSENITE-RESPONSIVE MYB1) regulates As-associated transporters genes. Treatment with As(III) induced OsARM1 transcript accumulation and an OsARM1-GFP fusion localized to the nucleus. Histochemical analysis of OsARM1pro::GUS lines indicated that OsARM1 was expressed in the phloem of vascular bundles in basal and upper nodes...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/29163459/crispr-cas9-system-as-a-valuable-genome-editing-tool-for-wine-yeasts-with-application-to-decrease-urea-production
#4
Ileana Vigentini, Marinella Gebbia, Alessandra Belotti, Roberto Foschino, Frederick P Roth
An extensive repertoire of molecular tools is available for genetic analysis in laboratory strains of S. cerevisiae. Although this has widely contributed to the interpretation of gene functionality within haploid laboratory isolates, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. Genetic engineering in industrial yeasts is undergoing major changes due to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering approaches...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/29163439/phosphomannosylation-and-the-functional-analysis-of-the-extended-candida-albicans-mnn4-like-gene-family
#5
Roberto J González-Hernández, Kai Jin, Marco J Hernández-Chávez, Diana F Díaz-Jiménez, Elías Trujillo-Esquivel, Diana M Clavijo-Giraldo, Alma K Tamez-Castrellón, Bernardo Franco, Neil A R Gow, Héctor M Mora-Montes
Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/29163048/nicotinic-acetylcholine-receptor-subtype-alpha-9-mediates-triple-negative-breast-cancers-based-on-a-spontaneous-pulmonary-metastasis-mouse-model
#6
Li-Chi Huang, Ching-Ling Lin, Jia-Zheng Qiu, Chun-Yu Lin, Kai-Wen Hsu, Ka-Wai Tam, Jung-Yu Lee, Jinn-Moon Yang, Chia-Hwa Lee
Triple-negative breast cancer (TNBC) subtype is associated with poor prognosis and a high risk of recurrence-related death in women. Despite the aggressiveness of TNBCs, targeted TNBC therapy is not yet available in the clinic. To overcome this challenge, we generated highly metastatic TNBC cells (LM) derived from metastasized lung cells via a serial spontaneous pulmonary metastasis animal model to identify targetable molecules for attenuating the progression of TNBC metastasis. Gene analysis of primary tumor (P), first-round (1LM) and second-round (2LM) metastasized lung cells revealed that mesenchymal-related genes were significantly expressed in LM cells, especially in 2LM cells...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/29162881/discrimination-of-crispr-cas9-induced-mutants-of-rice-seeds-using-near-infrared-hyperspectral-imaging
#7
Xuping Feng, Cheng Peng, Yue Chen, Xiaodan Liu, Xujun Feng, Yong He
Identifying individuals with target mutant phenotypes is a significant procedure in mutant exploitation for implementing genome editing technology in a crop breeding programme. In the present study, a rapid and non-invasive method was proposed to identify CRISPR/Cas9-induced rice mutants from their acceptor lines (huaidao-1 and nanjing46) using hyperspectral imaging in the near-infrared (NIR) range (874.41-1733.91 nm) combined with chemometric analysis. The hyperspectral imaging data were analysed using principal component analysis (PCA) for exploratory purposes, and a support vector machine (SVM) and an extreme learning machine (ELM) were applied to build discrimination models for classification...
November 21, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29162844/dna-nanomapping-using-crispr-cas9-as-a-programmable-nanoparticle
#8
Andrey Mikheikin, Anita Olsen, Kevin Leslie, Freddie Russell-Pavier, Andrew Yacoot, Loren Picco, Oliver Payton, Amir Toor, Alden Chesney, James K Gimzewski, Bud Mishra, Jason Reed
Progress in whole-genome sequencing using short-read (e.g., <150 bp), next-generation sequencing technologies has reinvigorated interest in high-resolution physical mapping to fill technical gaps that are not well addressed by sequencing. Here, we report two technical advances in DNA nanotechnology and single-molecule genomics: (1) we describe a labeling technique (CRISPR-Cas9 nanoparticles) for high-speed AFM-based physical mapping of DNA and (2) the first successful demonstration of using DVD optics to image DNA molecules with high-speed AFM...
November 21, 2017: Nature Communications
https://www.readbyqxmd.com/read/29162801/characterizing-a-thermostable-cas9-for-bacterial-genome-editing-and-silencing
#9
Ioannis Mougiakos, Prarthana Mohanraju, Elleke F Bosma, Valentijn Vrouwe, Max Finger Bou, Mihris I S Naduthodi, Alex Gussak, Rudolf B L Brinkman, Richard van Kranenburg, John van der Oost
CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure...
November 21, 2017: Nature Communications
https://www.readbyqxmd.com/read/29162721/loss-of-%C3%AE-b-crystallin-function-in-zebrafish-reveals-critical-roles-in-the-development-of-the-lens-and-stress-resistance-of-the-heart
#10
Sanjay Mishra, Shu-Yu Wu, Alexandra W Fuller, Zhen Wang, Kristie L Rose, Kevin L Schey, Hassane S Mchaourab
Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). While these mutations lead to modulation of their chaperone activity in vitro, the in vivo functions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, αBa and αBb We observed lens abnormalities in the mutant lines of both genes and the penetrance of the lens phenotype was higher in αBa than αBb mutants...
November 21, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29162699/dnajb1-prkaca-fusion-kinase-interacts-with-%C3%AE-catenin-and-the-liver-regenerative-response-to-drive-fibrolamellar-hepatocellular-carcinoma
#11
Edward R Kastenhuber, Gadi Lalazar, Shauna L Houlihan, Darjus F Tschaharganeh, Timour Baslan, Chi-Chao Chen, David Requena, Sha Tian, Benedikt Bosbach, John E Wilkinson, Sanford M Simon, Scott W Lowe
A segmental deletion resulting in DNAJB1-PRKACA gene fusion is now recognized as the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. Here we implement CRISPR-Cas9 genome editing and transposon-mediated somatic gene transfer to demonstrate that expression of either the endogenous fusion protein or a chimeric cDNA leads to the formation of indolent liver tumors in mice that closely resemble human FL-HCC...
November 21, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29162626/identification-of-isthmin-1-as-a-novel-clefting-and-craniofacial-patterning-gene-in-humans
#12
Lisa A Lansdon, Benjamin W Darbro, Aline L Petrin, Alissa M Hulstrand, Jennifer M Standley, Rachel B Brouillette, Abby Long, M Adela Mansilla, Robert A Cornell, Jeffery C Murray, Douglas W Houston, J Robert Manak
Orofacial clefts are one of the most common birth defects, affecting 1-2 per 1000 births, and have a complex etiology. High-resolution array-based comparative genomic hybridization has increased the ability to detect copy number variants that can be causative for complex diseases such as cleft lip and/or palate. Utilizing this technique on 97 non-syndromic cleft lip and palate cases and 43 cases with cleft palate only, we identified a heterozygous deletion of Isthmin 1 in one affected case, as well as a deletion in a second case which removes putative 3' regulatory information...
November 21, 2017: Genetics
https://www.readbyqxmd.com/read/29161506/multiplexed-crispr-cas9-genome-editing-and-gene-regulation-using-csy4-in-saccharomyces-cerevisiae
#13
Raphael Ferreira, Christos Skrekas, Jens Nielsen, Florian David
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has greatly accelerated the field of strain engineering. However, insufficient efforts have been made towards developing robust multiplexing tools in Saccharomyces cerevisiae. Here, we exploit the RNA processing capacity of the bacterial endoribonuclease Csy4 from Pseudomonas aeruginosa, to generate multiple gRNAs from a single transcript for genome editing and gene interference applications in S. cerevisiae. In regards to genome editing, we performed a quadruple deletion of FAA1, FAA4, POX1 and TES1 reaching 96% efficiency out of 24 colonies tested...
November 21, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29161464/increased-efficiency-of-targeted-mutagenesis-by-crispr-cas9-in-plants-using-heat-stress
#14
Chantal Le Blanc, Fei Zhang, Josefina Mendez, Yamile Lozano, Krishna Chatpar, Vivian Irish, Yannick Jacob
The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on GFP reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by ~5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment...
November 21, 2017: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/29161261/mrna-processing-in-mutant-zebrafish-lines-generated-by-chemical-and-crispr-mediated-mutagenesis-produces-unexpected-transcripts-that-escape-nonsense-mediated-decay
#15
Jennifer L Anderson, Timothy S Mulligan, Meng-Chieh Shen, Hui Wang, Catherine M Scahill, Frederick J Tan, Shao J Du, Elisabeth M Busch-Nentwich, Steven A Farber
As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes...
November 21, 2017: PLoS Genetics
https://www.readbyqxmd.com/read/29161010/emerging-approaches-for-spatiotemporal-control-of-targeted-genome-with-inducible-crispr-cas9
#16
Yuta Nihongaki, Takahiro Otabe, Moritoshi Sato
The breakthrough CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nuclease has revolutionized our ability in genome engineering. Although Cas9 is already a powerful tool for simple and efficient target endogenous gene manipulation, further engineering of Cas9 will improve the performance of Cas9, such as gene-editing efficiency and accuracy in vivo, and expand the application possibility of this Cas9 technology. The emerging inducible Cas9 methods, which can control the activity of Cas9 using an external stimulus such as chemicals and light, have the potential to provide spatiotemporal gene manipulation in user-defined cell population at a specific time and improve the accuracy of Cas9-mediated genome editing...
November 21, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/29160308/programmable-base-editing-of-a%C3%A2-t-to-g%C3%A2-c-in-genomic-dna-without-dna-cleavage
#17
Nicole M Gaudelli, Alexis C Komor, Holly A Rees, Michael S Packer, Ahmed H Badran, David I Bryson, David R Liu
The spontaneous deamination of cytosine is a major source of C•G to T•A transitions, which account for half of known human pathogenic point mutations. The ability to efficiently convert target A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. While the deamination of adenine yields inosine, which is treated as guanine by polymerases, no enzymes are known to deaminate adenine in DNA. Here we report adenine base editors (ABEs) that mediate conversion of A•T to G•C in genomic DNA...
October 25, 2017: Nature
https://www.readbyqxmd.com/read/29158838/crispr-genome-engineering-for-human-pluripotent-stem-cell-research
#18
REVIEW
Somali Chaterji, Eun Hyun Ahn, Deok-Ho Kim
The emergence of targeted and efficient genome editing technologies, such as repurposed bacterial programmable nucleases (e.g., CRISPR-Cas systems), has abetted the development of cell engineering approaches. Lessons learned from the development of RNA-interference (RNA-i) therapies can spur the translation of genome editing, such as those enabling the translation of human pluripotent stem cell engineering. In this review, we discuss the opportunities and the challenges of repurposing bacterial nucleases for genome editing, while appreciating their roles, primarily at the epigenomic granularity...
2017: Theranostics
https://www.readbyqxmd.com/read/29158600/crispr-cas9-library-screening-for-drug-target-discovery
#19
REVIEW
Morito Kurata, Kouhei Yamamoto, Branden S Moriarity, Masanobu Kitagawa, David A Largaespada
CRISPR/Cas9-based tools have rapidly developed in recent years. These include CRISPR-based gene activation (CRISPRa) or inhibition (CRISPRi), for which there are libraries. CRISPR libraries for loss of function have been widely used to identify new biological mechanisms, such as drug resistance and cell survival signals. CRISPRa is highly useful in screening for gain of functions, and CRISPRi is a more powerful tool than RNA interference (RNAi) libraries in screening for loss of functions. Positive selection using a CRISPR library can detect survival cells with specific conditions, such as drug treatment, and it can easily clarify drug resistance mechanisms...
November 20, 2017: Journal of Human Genetics
https://www.readbyqxmd.com/read/29158538/pinapl-py-a-comprehensive-web-application-for-the-analysis-of-crispr-cas9-screens
#20
Philipp N Spahn, Tyler Bath, Ryan J Weiss, Jihoon Kim, Jeffrey D Esko, Nathan E Lewis, Olivier Harismendy
Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service...
November 20, 2017: Scientific Reports
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