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https://www.readbyqxmd.com/read/28931002/enhanced-proofreading-governs-crispr-cas9-targeting-accuracy
#1
Janice S Chen, Yavuz S Dagdas, Benjamin P Kleinstiver, Moira M Welch, Alexander A Sousa, Lucas B Harrington, Samuel H Sternberg, J Keith Joung, Ahmet Yildiz, Jennifer A Doudna
The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing(1-4). High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown(5-9). Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state(10) when bound to mismatched targets...
September 20, 2017: Nature
https://www.readbyqxmd.com/read/28930690/the-self-inactivating-kamicas9-system-for-the-editing-of-cns-disease-genes
#2
Nicolas Merienne, Gabriel Vachey, Lucie de Longprez, Cécile Meunier, Virginie Zimmer, Guillaume Perriard, Mathieu Canales, Amandine Mathias, Lucas Herrgott, Tim Beltraminelli, Axelle Maulet, Thomas Dequesne, Catherine Pythoud, Maria Rey, Luc Pellerin, Emmanuel Brouillet, Anselme L Perrier, Renaud du Pasquier, Nicole Déglon
Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease...
September 19, 2017: Cell Reports
https://www.readbyqxmd.com/read/28928381/crispr-cas9-induced-targeted-mutagenesis-and-gene-replacement-to-generate-long-shelf-life-tomato-lines
#3
Qing-Hui Yu, Baike Wang, Ning Li, Yaping Tang, Shengbao Yang, Tao Yang, Juan Xu, Chunmiao Guo, Peng Yan, Qiang Wang, Patiguli Asmutola
Quickly and precisely gain genetically enhanced breeding elites with value-adding performance traits is desired by the crop breeders all the time. The present of gene editing technologies, especially the CRISPR/Cas9 system with the capacities of efficiency, versatility and multiplexing provides a reasonable expectation towards breeding goals. For exploiting possible application to accelerate the speed of process at breeding by CRISPR/Cas9 technology, in this study, the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system transformation method was used for obtaining tomato ALC gene mutagenesis and replacement, in absence and presence of the homologous repair template...
September 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28928209/combining-comprehensive-analysis-of-off-site-lambda-phage-integration-with-a-crispr-based-means-of-characterizing-downstream-physiology
#4
Yu Tanouchi, Markus W Covert
During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by targeting over 300 unique sites in the host Escherichia coli genome. Moreover, these integration events can have important phenotypic consequences for the host, including changes in cell motility and increased antibiotic resistance...
September 19, 2017: MBio
https://www.readbyqxmd.com/read/28928041/enhancing-crispr-cas9-mediated-homology-directed-repair-in-mammalian-cells-by-expressing-saccharomyces-cerevisiae-rad52
#5
Simin Shao, Chonghua Ren, Zhongtian Liu, Yichun Bai, Zhilong Chen, Zehui Wei, Xin Wang, Zhiying Zhang, Kun Xu
Precise genome editing with desired point mutations can be generated by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance for gene function study, gene therapy and animal breeding. However, HDR efficiency is inherently low and improvements are necessitated. Herein, we determined that the HDR efficiency could be enhanced by expressing Rad52, a gene that is involved in the homologous recombination process. Both the Rad52 co-expression and Rad52-Cas9 fusion strategies yielded approximately 3-fold increase in HDR during the surrogate reporter assays in human HEK293T cells, as well as in the genome editing assays...
September 16, 2017: International Journal of Biochemistry & Cell Biology
https://www.readbyqxmd.com/read/28927827/crispr-cas9-mediated-blos2-knockout-resulting-in-disappearance-of-yellow-strips-and-white-spots-on-the-larval-integument-in-spodoptera-litura
#6
Guan-Heng Zhu, Ying-Chuan Peng, Mei-Yan Zheng, Xiao-Qing Zhang, Jia-Bin Sun, Yongping Huang, Shuang-Lin Dong
The custom-design bacterial CRISPR/Cas9 system has been recently used in some insects, indicating a powerful technique for studies on gene function and transgenic insects. However, its use in lepidopteran pests is scarce. Here, we reported a CRISPR/Cas9 system mediated mutagenesis of biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene in a noctuid pest Spodoptera litura. A fragment of SlitBLOS2 was identified by analyzing a S. litura transcriptome database by local basic BLAST, and the full length cDNA was acquired by RACE strategy...
September 16, 2017: Journal of Insect Physiology
https://www.readbyqxmd.com/read/28926338/melk-is-not-necessary-for-the-proliferation-of-basal-like-breast-cancer-cells
#7
Hai-Tsang Huang, Hyuk-Soo Seo, Tinghu Zhang, Yubao Wang, Baishan Jiang, Qing Li, Dennis L Buckley, Behnam Nabet, Justin M Roberts, Joshiawa Paulk, Shiva Dastjerdi, Georg E Winter, Hilary McLauchlan, Jennifer Moran, James E Bradner, Michael J Eck, Sirano Dhe-Paganon, Jean J Zhao, Nathanael S Gray
Thorough preclinical target validation is essential for the success of drug discovery efforts. In this study, we combined chemical and genetic perturbants, including the development of a novel selective maternal embryonic leucine zipper kinase (MELK) inhibitor HTH-01-091, CRISPR/Cas9-mediated MELK knockout, a novel chemical-induced protein degradation strategy, RNA interference and CRISPR interference to validate MELK as a therapeutic target in basal-like breast cancers (BBC). In common culture conditions, we found that small molecule inhibition, genetic deletion, or acute depletion of MELK did not significantly affect cellular growth...
September 19, 2017: ELife
https://www.readbyqxmd.com/read/28926145/a-crispr-reimagining-new-twists-and-turns-of-crispr-beyond-the-genome-engineering-revolution
#8
Robert J Plummer, Yi Guo, Ying Peng
Despite its explosive applications in genome engineering, CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) has been developed into a versatile tool beyond its well-known nuclease function. In this prospect article, we summarize a few exciting "off-label" applications of CRISPR including manipulating DNA sequences, visualizing chromosomal loci in living cells, and modulating transcription and chromatin structures. These novel applications will likely elevate CRISPR tools into yet another level of sophistication and diversity, leading to many more exciting cell biological discoveries...
September 19, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/28924386/alkbh4-and-atrn-act-maternally-to-regulate-zebrafish-epiboly
#9
Qingrui Sun, Xingfeng Liu, Bo Gong, Di Wu, Anming Meng, Shunji Jia
During embryonic gastrulation, coordinated cell movements occur to bring cells to their correct position. Among them, epiboly produces the first distinct morphological changes, which is essential for the early development of zebrafish. Despite its fundamental importance, little is known to understand the underlying molecular mechanisms. By generating maternal mutant lines with CRISPR/Cas9 technology and using morpholino knockdown strategy, we showed that maternal Alkbh4 depletion leads to severe epiboly defects in zebrafish...
2017: International Journal of Biological Sciences
https://www.readbyqxmd.com/read/28923954/macroevolutionary-shifts-of-wnta-function-potentiate-butterfly-wing-pattern-diversity
#10
Anyi Mazo-Vargas, Carolina Concha, Luca Livraghi, Darli Massardo, Richard W R Wallbank, Linlin Zhang, Joseph D Papador, Daniel Martinez-Najera, Chris D Jiggins, Marcus R Kronforst, Casper J Breuker, Robert D Reed, Nipam H Patel, W Owen McMillan, Arnaud Martin
Butterfly wing patterns provide a rich comparative framework to study how morphological complexity develops and evolves. Here we used CRISPR/Cas9 somatic mutagenesis to test a patterning role for WntA, a signaling ligand gene previously identified as a hotspot of shape-tuning alleles involved in wing mimicry. We show that WntA loss-of-function causes multiple modifications of pattern elements in seven nymphalid butterfly species. In three butterflies with a conserved wing-pattern arrangement, WntA is necessary for the induction of stripe-like patterns known as symmetry systems and acquired a novel eyespot activator role specific to Vanessa forewings...
September 18, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28923944/single-master-regulatory-gene-coordinates-the-evolution-and-development-of-butterfly-color-and-iridescence
#11
Linlin Zhang, Anyi Mazo-Vargas, Robert D Reed
The optix gene has been implicated in butterfly wing pattern adaptation by genetic association, mapping, and expression studies. The actual developmental function of this gene has remained unclear, however. Here we used CRISPR/Cas9 genome editing to show that optix plays a fundamental role in nymphalid butterfly wing pattern development, where it is required for determination of all chromatic coloration. optix knockouts in four species show complete replacement of color pigments with melanins, with corresponding changes in pigment-related gene expression, resulting in black and gray butterflies...
September 18, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28923848/body-shape-and-coloration-of-silkworm-larvae-are-influenced-by-a-novel-cuticular-protein
#12
Gao Xiong, Xiaoling Tong, Tingting Gai, Chunlin Li, Liang Qiao, Antónia Monteiro, Hai Hu, Minjin Han, Xin Ding, Songyuan Wu, Zhonghuai Xiang, Cheng Lu, Fangyin Dai
The genetic basis of body shape and coloration patterns on caterpillars is often assumed to be regulated separately, but it is possible that common molecules affect both types of trait simultaneously. Here we examine the genetic basis of a spontaneous cuticle defect in silkworm, where larvae exhibit a bamboo-like body shape and also decreased pigmentation. We performed linkage mapping and mutation screening to determine the gene product that affects body shape and coloration simultaneously. In these mutant larvae we identified a null mutation in BmorCPH24, a gene encoding a cuticular protein with low complexity sequence...
September 18, 2017: Genetics
https://www.readbyqxmd.com/read/28923495/crispr-cas9-engineering-of-adult-mouse-liver-demonstrates-that-the-dnajb1-prkaca-gene-fusion-is-sufficient-to-induce-tumors-resembling-fibrolamellar-hepatocellular-carcinoma
#13
Lars H Engelholm, Anjum Riaz, Denise Serra, Frederik Dagnæs-Hansen, Jens V Johansen, Eric Santoni-Rugiu, Steen H Hansen, Francesco Niola, Morten Frödin
BACKGROUND & AIMS: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis...
September 15, 2017: Gastroenterology
https://www.readbyqxmd.com/read/28923089/dna-epigenome-editing-using-crispr-cas-suntag-directed-dnmt3a
#14
Yung-Hsin Huang, Jianzhong Su, Yong Lei, Lorenzo Brunetti, Michael C Gundry, Xiaotian Zhang, Mira Jeong, Wei Li, Margaret A Goodell
BACKGROUND: DNA methylation has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression. RESULTS: Here, we utilize nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltransferase 3A (DNMT3A) (dCas9-SunTag-DNMT3A) to amplify the local DNMT3A concentration to methylate genomic sites of interest...
September 18, 2017: Genome Biology
https://www.readbyqxmd.com/read/28922955/comparison-of-zfns-versus-crispr-specific-nucleases-for-genome-edition-of-the-wiskott-aldrich-syndrome-locus
#15
Alejandra Gutierrez-Guerrero, Sabina Sanchez-Hernandez, Giuseppe Galvani, Javier Pinedo-Gomez, Almudena Sanchez-Gilabert, Rocio Martin-Guerra, Marien Cobo, Philip Gregory, Michael Holmes, Karim Benabdellah, Francisco Martin
Primary immunodeficiencies (PID), including Wiskott-Aldrich syndrome (WAS), are a main target for genome editing (GE) strategies using specific nucleases (SNs) since a small number of corrected hematopoietic stem cells (HSCs) could cure patients. In this work, we have designed different WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific Zinc Finger Nucleases (ZFNs) using K562 cells as a cellular model and plasmid nucleofection or integrative-deficient Lentiviral Vectors (IDLVs) for delivery...
September 19, 2017: Human Gene Therapy
https://www.readbyqxmd.com/read/28921815/low-gluten-non-transgenic-wheat-engineered-with-crispr-cas9
#16
Susana Sánchez-León, Javier Gil-Humanes, Carmen V Ozuna, María J Giménez, Carolina Sousa, Daniel F Voytas, Francisco Barro
Celiac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley, and rye. The -gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33-mer is the main immunodominant peptide in celiac patients. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33-mer in the -gliadin genes. Twenty-one mutant lines were generated, all showing strong reduction in -gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%...
September 18, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28920889/therapies-targeting-dna-and-rna-in-huntington-s-disease
#17
REVIEW
Edward J Wild, Sarah J Tabrizi
No disease-slowing treatment exists for Huntington's disease, but its monogenic inheritance makes it an appealing candidate for the development of therapies targeting processes close to its genetic cause. Huntington's disease is caused by CAG repeat expansions in the HTT gene, which encodes the huntingtin protein; development of therapies to target HTT transcription and the translation of its mRNA is therefore an area of intense investigation. Huntingtin-lowering strategies include antisense oligonucleotides and RNA interference targeting mRNA, and zinc finger transcriptional repressors and CRISPR-Cas9 methods aiming to reduce transcription by targeting DNA...
October 2017: Lancet Neurology
https://www.readbyqxmd.com/read/28919449/genome-editing-in-fishes-and-their-applications
#18
Bo Zhu, Wei Ge
There have been revolutionary progresses in genome engineering in the past few years. The newly-emerged genome editing technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats associated with Cas9 (CRISPR/Cas9) have enabled biological scientists to perform efficient and precise targeted genome editing in different species. Fish represent the largest group of vertebrates with many species having values for both scientific research and aquaculture industry...
September 14, 2017: General and Comparative Endocrinology
https://www.readbyqxmd.com/read/28919077/engineering-quantitative-trait-variation-for-crop-improvement-by-genome-editing
#19
Daniel Rodríguez-Leal, Zachary H Lemmon, Jarrett Man, Madelaine E Bartlett, Zachary B Lippman
Major advances in crop yields are needed in the coming decades. However, plant breeding is currently limited by incremental improvements in quantitative traits that often rely on laborious selection of rare naturally occurring mutations in gene-regulatory regions. Here, we demonstrate that CRISPR/Cas9 genome editing of promoters generates diverse cis-regulatory alleles that provide beneficial quantitative variation for breeding. We devised a simple genetic scheme, which exploits trans-generational heritability of Cas9 activity in heterozygous loss-of-function mutant backgrounds, to rapidly evaluate the phenotypic impact of numerous promoter variants for genes regulating three major productivity traits in tomato: fruit size, inflorescence branching, and plant architecture...
September 13, 2017: Cell
https://www.readbyqxmd.com/read/28918562/crispr-cas9-genome-editing-in-wheat
#20
Dongjin Kim, Burcu Alptekin, Hikmet Budak
Genome editing has been a long-term challenge for molecular biology research, particularly for plants possess complex genome. The recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile tool for genome editing which enables editing of multiple genes based on the guidance of small RNAs. Even though the efficiency of CRISPR/Cas9 system has been shown with several studies from diploid plants, its application remains a challenge for plants with polyploid and complex genome...
September 16, 2017: Functional & Integrative Genomics
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