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https://www.readbyqxmd.com/read/29907766/efficient-rna-drug-delivery-using-red-blood-cell-extracellular-vesicles
#1
Waqas Muhammad Usman, Tin Chanh Pham, Yuk Yan Kwok, Luyen Tien Vu, Victor Ma, Boya Peng, Yuen San Chan, Likun Wei, Siew Mei Chin, Ajijur Azad, Alex Bai-Liang He, Anskar Y H Leung, Mengsu Yang, Ng Shyh-Chang, William C Cho, Jiahai Shi, Minh T N Le
Most of the current methods for programmable RNA drug therapies are unsuitable for the clinic due to low uptake efficiency and high cytotoxicity. Extracellular vesicles (EVs) could solve these problems because they represent a natural mode of intercellular communication. However, current cellular sources for EV production are limited in availability and safety in terms of horizontal gene transfer. One potentially ideal source could be human red blood cells (RBCs). Group O-RBCs can be used as universal donors for large-scale EV production since they are readily available in blood banks and they are devoid of DNA...
June 15, 2018: Nature Communications
https://www.readbyqxmd.com/read/29907613/a-modular-dcas9-suntag-dnmt3a-epigenome-editing-system-overcomes-pervasive-off-target-activity-of-direct-fusion-dcas9-dnmt3a-constructs
#2
Christian Pflueger, Dennis Tan, Tessa Swain, Trung Viet Nguyen, Jahnvi Pflueger, Christian Nefzger, Jose M Polo, Ethan Ford, Ryan Lister
DNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context...
June 15, 2018: Genome Research
https://www.readbyqxmd.com/read/29903034/biodiversity-and-host-parasite-cophylogeny-of-sphaerospora-sensu-stricto-cnidaria-myxozoa
#3
Sneha Patra, Pavla Bartošová-Sojková, Hana Pecková, Ivan Fiala, Edit Eszterbauer, Astrid S Holzer
BACKGROUND: Myxozoa are extremely diverse microscopic parasites belonging to the Cnidaria. Their life-cycles alternate between vertebrate and invertebrate hosts, predominantly in aquatic habitats. Members of the phylogenetically well-defined Sphaerospora (sensu stricto) clade predominantly infect the urinary system of marine and freshwater fishes and amphibians. Sphaerosporids are extraordinary due to their extremely long and unique insertions in the variable regions of their 18S and 28S rDNA genes and due to the formation of motile proliferative stages in the hosts' blood...
June 15, 2018: Parasites & Vectors
https://www.readbyqxmd.com/read/29899171/novel-approaches-for-the-treatment-of-familial-hypercholesterolemia-current-status-and-future-challenges
#4
Long Jiang, Lu-Ya Wang, Xiao-Shu Cheng
Familial hypercholesterolemia (FH) is an autosomal-dominant disorder that is characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and an increased risk of cardiovascular disease. Despite the use of high-dose statins and the recent addition of proprotein convertase subtilisin/kexin type 9 inhibitors as a treatment option, many patients with homozygous FH fail to achieve optimal reductions of LDL-c levels. Gene therapy has become one of the most promising research directions for contemporary life sciences and is a potential treatment option for FH...
June 13, 2018: Journal of Atherosclerosis and Thrombosis
https://www.readbyqxmd.com/read/29898980/two-distinct-approaches-for-crispr-cas9-mediated-gene-editing-in-cryptococcus-neoformans-and-related-species
#5
Ping Wang
Cryptococcus neoformans and related species are encapsulated basidiomycetous fungi that cause meningoencephalitis in individuals with immune deficiency. This pathogen has a tractable genetic system; however, gene disruption via electroporation remains difficult, while biolistic transformation is often limited by lack of multiple genetic markers and the high initial cost of equipment. The approach using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) has become the technology of choice for gene editing in many organisms due to its simplicity, efficiency, and versatility...
June 27, 2018: MSphere
https://www.readbyqxmd.com/read/29898387/genomic-knockout-of-two-presumed-forelimb-tbx5-enhancers-reveals-they-are-nonessential-for-limb-development
#6
Thomas J Cunningham, Joseph J Lancman, Marie Berenguer, P Duc Si Dong, Gregg Duester
A standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 forelimb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals...
June 12, 2018: Cell Reports
https://www.readbyqxmd.com/read/29893931/a-novel-%C3%AE-integrase-mediated-seamless-vector-transgenesis-platform-for-therapeutic-protein-expression
#7
Harshyaa Makhija, Suki Roy, Shawn Hoon, Farid John Ghadessy, Desmond Wong, Rahul Jaiswal, Dario Campana, Peter Dröge
Advances in stem cell engineering, gene therapy and molecular medicine often involve genome engineering at a cellular level. However, functionally large or multi transgene cassette insertion into the human genome still remains a challenge. Current practices such as random transgene integration or targeted endonuclease-based genome editing are suboptimal and might pose safety concerns. Taking this into consideration, we previously developed a transgenesis tool derived from phage λ integrase (Int) that precisely recombines large plasmid DNA into an endogenous sequence found in human Long INterspersed Elements-1 (LINE-1)...
June 11, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29892505/restriction-digest-screening-facilitates-efficient-detection-of-site-directed-mutations-introduced-by-crispr-in-c-albicans-ume6
#8
Ben A Evans, Olivia L Smith, Ethan S Pickerill, Mary K York, Kristen J P Buenconsejo, Antonio E Chambers, Douglas A Bernstein
Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons...
2018: PeerJ
https://www.readbyqxmd.com/read/29892067/crispr-cas9-genome-editing-induces-a-p53-mediated-dna-damage-response
#9
Emma Haapaniemi, Sandeep Botla, Jenna Persson, Bernhard Schmierer, Jussi Taipale
Here, we report that genome editing by CRISPR-Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR-Cas9...
June 11, 2018: Nature Medicine
https://www.readbyqxmd.com/read/29891532/crispr-base-editors-genome-editing-without-double-stranded-breaks
#10
REVIEW
Ayman Eid, Sahar Alshareef, Magdy M Mahfouz
The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci...
June 11, 2018: Biochemical Journal
https://www.readbyqxmd.com/read/29889198/enhanced-genome-editing-with-cas9-ribonucleoprotein-in-diverse-cells-and-organisms
#11
Behnom Farboud, Erin Jarvis, Theodore L Roth, Jiyung Shin, Jacob E Corn, Alexander Marson, Barbara J Meyer, Nipam H Patel, Megan L Hochstrasser
Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB)...
May 25, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29886029/speed-genome-editing-by-transient-crispr-cas9-targeting-and-large-dna-fragment-deletion
#12
Jing Luo, Liaoxun Lu, Yanrong Gu, Rong Huang, Lin Gui, Saichao Li, Xinhui Qi, Wenping Zheng, Tianzhu Chao, Qianqian Zheng, Yinming Liang, Lichen Zhang
Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells...
June 7, 2018: Journal of Biotechnology
https://www.readbyqxmd.com/read/29884745/a-role-in-apoptosis-regulation-for-rad-51-gene-of-caenorhabditis-elegans
#13
Marcello Germoglio, Adele Adamo
The evolutionary conserved RAD-51 protein is essential for homologous recombination in the germ line as well as homologous repair of DNA double strand breaks in all eukaryotic cells. In the nematode Caenorhabditis elegans the rad-51 gene is transcribed into messenger-RNAs potentially coding three alternative protein isoforms. Null rad-51 alleles display embryonic lethality, severe defects in chromosome structure and high level of germ line apoptosis. To dissect its functions, we genetically modified the C. elegans rad-51 gene by CRISPR/Cas9 genome-editing technology, obtaining a separation-of-function (sfi-) mutant allele that only disrupts the long transcript isoform...
June 8, 2018: Genetics
https://www.readbyqxmd.com/read/29884615/a-dual-sgrna-approach-for-functional-genomics-in-arabidopsis-thaliana
#14
Laurens Pauwels, Rebecca De Clercq, Jonas Goossens, Sabrina Iñigo, Clara Williams, Mily Ron, Anne Britt, Alain Goossens
Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana , gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation...
June 8, 2018: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/29879620/ctrl-alt-indel-genome-editing-to-reprogram-a-cell-in-the-clinic
#15
REVIEW
Fyodor D Urnov
Genome editing with engineered nucleases (zinc finger, TAL effector, or CRISPR/Cas9-based) enables `write' access to regulatory programs executed by primary human cells. A decade of its clinical development, along with a reduction of conventional gene therapy to medical and commercial practice, has made cell reprogramming via editing a viable clinical modality. Reviewed here are the first examples of this to enter the clinic: ex vivo edited T cells for infectious disease and cancer, and hematopoietic stem/progenitor cells for the hemoglobinopathies...
June 4, 2018: Current Opinion in Genetics & Development
https://www.readbyqxmd.com/read/29874063/target-specificity-of-cas9-nuclease-via-dna-rearrangement-regulated-by-the-rec2-domain
#16
Keewon Sung, Jinho Park, Younggyu Kim, Nam Ki Lee, Seong Keun Kim
Understanding the underlying principles for the target-specific nuclease activity of CRISPR/Cas9 is a prerequisite to minimize its off-target DNA cleavage for genome engineering applications. Here, we show that the non-catalytic REC2 domain of Cas9 nuclease plays a crucial role in off-target discrimination. Using single-molecule fluorescence methods, we investigate conformational dynamics of the non-target strand (NTS) of DNA interacting with Cas9 and find that REC2 regulates the NTS rearrangement for cleavage reaction with the help of positively-charged residues on its surface...
June 6, 2018: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29873882/japanese-society-of-medical-oncology-clinical-guidelines-molecular-testing-for-colorectal-cancer-treatment-third-edition
#17
Kentaro Yamazaki, Hiroya Taniguchi, Takayuki Yoshino, Kiwamu Akagi, Hideyuki Ishida, Hiromichi Ebi, Kaname Nakatani, Kei Muro, Yasushi Yatabe, Kensei Yamaguchi, Katsuya Tsuchihara
The Japanese Society of Medical Oncology (JSMO) previously published 2 editions of the clinical guidelines: "Japanese guidelines for testing of KRAS gene mutation in colorectal cancer" in 2008 and "Japanese Society of Medical Oncology Clinical Guidelines: RAS (KRAS/NRAS) mutation testing in colorectal cancer patients" in 2014. These guidelines have contributed to the proper use of KRAS and RAS mutation testing, respectively. Recently, clinical utility, particularly for colorectal cancer (CRC) patients with BRAF V600E mutation or DNA mismatch-repair (MMR) deficiency, has been established...
June 2018: Cancer Science
https://www.readbyqxmd.com/read/29872908/scarless-genome-editing-progress-towards-understanding-genotype-phenotype-relationships
#18
REVIEW
Gregory L Elison, Murat Acar
The ability to predict phenotype from genotype has been an elusive goal for the biological sciences for several decades. Progress decoding genotype-phenotype relationships has been hampered by the challenge of introducing precise genetic changes to specific genomic locations. Here we provide a comparative review of the major techniques that have been historically used to make genetic changes in cells as well as the development of the CRISPR technology which enabled the ability to make marker-free disruptions in endogenous genomic locations...
June 5, 2018: Current Genetics
https://www.readbyqxmd.com/read/29872041/in-vivo-base-editing-of-post-mitotic-sensory-cells
#19
Wei-Hsi Yeh, Hao Chiang, Holly A Rees, Albert S B Edge, David R Liu
Programmable nucleases can introduce precise changes to genomic DNA through homology-directed repair (HDR). Unfortunately, HDR is largely restricted to mitotic cells, and is typically accompanied by an excess of stochastic insertions and deletions (indels). Here we present an in vivo base editing strategy that addresses these limitations. We use nuclease-free base editing to install a S33F mutation in β-catenin that blocks β-catenin phosphorylation, impedes β-catenin degradation, and upregulates Wnt signaling...
June 5, 2018: Nature Communications
https://www.readbyqxmd.com/read/29871649/micmic-identification-of-dna-methylation-of-distal-regulatory-regions-with-causal-effects-on-tumorigenesis
#20
Yin Tong, Jianlong Sun, Chi Fat Wong, Qingzheng Kang, Beibei Ru, Ching Ngar Wong, April Sheila Chan, Suet Yi Leung, Jiangwen Zhang
Aberrant promoter methylation is a common mechanism for tumor suppressor inactivation in cancer. We develop a set of tools to identify genome-wide DNA methylation in distal regions with causal effect on tumorigenesis called MICMIC. Many predictions are directly validated by dCas9-based epigenetic editing to support the accuracy and efficiency of our tool. Oncogenic and lineage-specific transcription factors are shown to aberrantly shape the methylation landscape by modifying tumor-subtype core regulatory circuitry...
June 5, 2018: Genome Biology
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