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https://www.readbyqxmd.com/read/29470985/cytoplasmic-p53-contributes-to-the-removal-of-uracils-misincorporated-by-hiv-1-reverse-transcriptase
#1
Yossi Saragani, Amnon Hizi, Galia Rahav, Sara Zauch, Mary Bakhanashvili
HIV-1 reverse transcriptase (RT) in the cytoplasm of HIV-infected cells efficiently inserts the non-canonical dUTP into the proviral DNA, and extends the dU-terminated DNA. The misincorporation of dUTP leads to mutagenesis, and uracils can down-regulate viral gene expression. However, uracilation might also protect HIV DNA from auto-integration in the cytoplasm. Tumor suppressor p53 protein, exhibiting inherent 3'→5' exonuclease activity, provides a potential host-derived repair mechanism during HIV reverse transcription for the misincorporation of various wrong nucleotides, leading to both base-base mismatches and incorporated non-canonical ribonucleotides...
February 19, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29470549/genome-wide-identification-and-analysis-of-a-to-i-rna-editing-events-in-bovine-by-transcriptome-sequencing
#2
Mohammad Reza Bakhtiarizadeh, Abdolreza Salehi, Rocío Melissa Rivera
RNA editing increases the diversity of the transcriptome and proteome. Adenosine-to-inosine (A-to-I) editing is the predominant type of RNA editing in mammals and it is catalyzed by the adenosine deaminases acting on RNA (ADARs) family. Here, we used a largescale computational analysis of transcriptomic data from brain, heart, colon, lung, spleen, kidney, testes, skeletal muscle and liver, from three adult animals in order to identify RNA editing sites in bovine. We developed a computational pipeline and used a rigorous strategy to identify novel editing sites from RNA-Seq data in the absence of corresponding DNA sequence information...
2018: PloS One
https://www.readbyqxmd.com/read/29469899/commentary-programmable-base-editing-of-a%C3%A2-t-to-g%C3%A2-c-in-genomic-dna-without-dna-cleavage
#3
COMMENT
Ianis G Matsoukas
No abstract text is available yet for this article.
2018: Frontiers in Genetics
https://www.readbyqxmd.com/read/29468011/ligase-iv-inhibitor-scr7-enhances-gene-editing-directed-by-crispr-cas9-and-ssodn-in-human-cancer-cells
#4
Zheng Hu, Zhaoying Shi, Xiaogang Guo, Baishan Jiang, Guo Wang, Dixian Luo, Yonglong Chen, Yuan-Shan Zhu
Background: Precise genome editing is essential for both basic and translational research. The recently developed CRISPR/Cas9 system can specifically cleave a designated site of target gene to create a DNA double-strand break, which triggers cellular DNA repair mechanism of either inaccurate non-homologous end joining, or site-specific homologous recombination. Unfortunately, homology-directed repair (HDR) is challenging due to its very low efficiency. Herein, we focused on improving the efficiency of HDR using a combination of CRISPR/Cas9, eGFP, DNA ligase IV inhibitor SCR7, and single-stranded oligodeoxynucleotides (ssODN) in human cancer cells...
2018: Cell & Bioscience
https://www.readbyqxmd.com/read/29463653/efficient-and-scalable-precision-genome-editing-in-staphylococcus-aureus-through-conditional-recombineering-and-crispr-cas9-mediated-counterselection
#5
Kelsi Penewit, Elizabeth A Holmes, Kathyrn McLean, Mingxin Ren, Adam Waalkes, Stephen J Salipante
Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureus using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132 , derived from Enterococcus faecalis , as being capable of integrating single-stranded DNA oligonucleotides into the S...
February 20, 2018: MBio
https://www.readbyqxmd.com/read/29462720/engineering-introns-to-express-rna-guides-for-cas9-and-cpf1-mediated-multiplex-genome-editing
#6
Dan Ding, Kaiyuan Chen, Yuedan Chen, Hong Li, Kabin Xie
The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system has emerged as the revolutionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector (e.g., Cas9 and Cpf1) to a DNA target. Here, we elaborate a general strategy to simultaneously express multiple guide RNAs (gRNA) and CRISPR RNAs (crRNA) from introns of Cas9 and Cpf1. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpf1 to cleave the spliced intron which contains tRNA-gRNA polycistron or crRNA-crRNA array...
February 17, 2018: Molecular Plant
https://www.readbyqxmd.com/read/29461055/bidirectional-degradation-of-dna-cleavage-products-catalyzed-by-crispr-cas9
#7
Anthony A Stephenson, Austin T Raper, Zucai Suo
Since the initial characterization of Streptococcus pyogenes CRISPR/Cas9 as a powerful gene-editing tool, it has been widely accepted that Cas9 generates blunt-ended DNA products by concerted cleavage of the target (tDNA) and non-target (ntDNA) strands three nucleotides away from the protospacer adjacent motif (PAM) by HNH and RuvC nuclease active sites, respectively. Following initial DNA cleavage, RuvC catalyzes 3'→5' degradation of the ntDNA resulting in DNA products of various lengths. Here, we found that Cas9 selects multiple sites for initial ntDNA cleavage and preferentially generates staggered-ended DNA products containing single-nucleotide 5'-overhangs...
February 20, 2018: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29458716/selection-is-required-for-efficient-cas9-mediated-genome-editing-in-fusarium-graminearum
#8
Donald M Gardiner, Kemal Kazan
Genome engineering using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nucleases, such as Cas9 (CRISPR-associated protein 9), are revolutionising molecular biology. In this study, we established a Cas9-based genome editing system in Fusarium graminearum, a highly destructive fungal pathogen of cereal crops. Although the molecular toolkit of F. graminearum is well developed compared to other fungi, Cas9-mediated engineering offers a number of potential benefits, such as the ability to create marker free mutants in this species...
February 2018: Fungal Biology
https://www.readbyqxmd.com/read/29457665/comparative-analysis-of-lipid-mediated-crispr-cas9-genome-editing-techniques
#9
Kelsey P Ringer, Mark G Roth, Mitchell S Garey, Ted B Piorczynski, Arminda Suli, Jason M Hansen, Jonathan K Alder
CRISPR-Cas technology has revolutionized genome engineering. While Cas9 was not the first programmable endonuclease identified, its simplicity of use has driven widespread adoption in a short period of time. While CRISPR-Cas genome editing holds enormous potential for clinical applications, its use in laboratory settings for genotype-phenotype studies and genome-wide screens has led to breakthroughs in the understanding of many molecular pathways. Numerous protocols have been described for introducing CRISPR-Cas components into cells, and here we sought to simplify and optimize a protocol for genome editing using readily available and inexpensive tools...
February 19, 2018: Cell Biology International
https://www.readbyqxmd.com/read/29456084/rescue-of-fragile-x-syndrome-neurons-by-dna-methylation-editing-of-the-fmr1-gene
#10
X Shawn Liu, Hao Wu, Marine Krzisch, Xuebing Wu, John Graef, Julien Muffat, Denes Hnisz, Charles H Li, Bingbing Yuan, Chuanyun Xu, Yun Li, Dan Vershkov, Angela Cacace, Richard A Young, Rudolf Jaenisch
Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs...
February 8, 2018: Cell
https://www.readbyqxmd.com/read/29453406/crispr-cas9-a-tool-to-efficiently-increase-the-development-of-recombinant-african-swine-fever-viruses
#11
Manuel V Borca, Lauren G Holinka, Keith A Berggren, Douglas P Gladue
African swine fever virus (ASFV) causes a highly contagious disease called African swine fever. This disease is often lethal for domestic pigs, causing extensive losses for the swine industry. ASFV is a large and complex double stranded DNA virus. Currently there is no commercially available treatment or vaccine to prevent this devastating disease. Development of recombinant ASFV for producing live-attenuated vaccines or studying the involvement of specific genes in virus virulence has relied on the relatively rare event of homologous recombination in primary swine macrophages, causing difficulty to purify the recombinant virus from the wild-type parental ASFV...
February 16, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29453099/impact-of-integrated-viral-dna-on-the-goal-to-clear-hepatitis-b-surface-antigen-with-different-therapeutic-strategies
#12
REVIEW
Magnus Lindh, Gustaf E Rydell, Simon B Larsson
A hallmark of hepatitis B virus (HBV) infection is the presence of hepatitis B surface antigen (HBsAg) in the serum of patients. Sustained loss of HBV DNA and HBsAg from the blood are main goals for treatment, and considered as functional cure. It is rarely achieved with long-term nucleoside analogue treatment though, both because cccDNA, the template for viral replication, is not completely cleared, and probably also because hepatocytes with HBV DNA integrated into their chromosomes persist and continue to produce large amounts of HBsAg...
February 13, 2018: Current Opinion in Virology
https://www.readbyqxmd.com/read/29452938/harnessing-natural-dna-modifying-activities-for-editing-of-the-genome-and-epigenome
#13
REVIEW
Jamie E DeNizio, Emily K Schutsky, Kiara N Berrios, Monica Yun Liu, Rahul M Kohli
The introduction of site-specific DNA modifications to the genome or epigenome presents great opportunities for manipulating biological systems. Such changes are now possible through the combination of DNA-modifying enzymes with targeting modules, including dCas9, that can localize the enzymes to specific sites. In this review, we take a DNA modifying enzyme-centric view of recent advances. We highlight the variety of natural DNA-modifying enzymes-including DNA methyltransferases, oxygenases, deaminases, and glycosylases-that can be used for targeted editing and discuss how insights into the structure and function of these enzymes has further expanded editing potential by introducing enzyme variants with altered activities or by improving spatiotemporal control of modifications...
February 13, 2018: Current Opinion in Chemical Biology
https://www.readbyqxmd.com/read/29450416/a-g-quadruplex-motif-at-the-3-end-of-sgrnas-improves-crispr-cas9-based-genome-editing-efficiency
#14
Smita Nahar, Paras Sehgal, Mohd Azhar, Manish Rai, Amrita Singh, Sridhar Sivasubbu, Debojyoti Chakraborty, Souvik Maiti
Originating as a component of prokaryotic adaptive immunity, the type II CRISPR/Cas9 system has been repurposed for targeted genome editing in various organisms. Although Cas9 can bind and cleave DNA efficiently under in vitro conditions, its activity inside a cell can vary dramatically between targets owing to the differences between genomic loci and the availability of enough Cas9/sgRNA (single guide RNA) complex molecules for cleavage. Most methods have so far relied on Cas9 protein engineering or base modifications in the sgRNA sequence to improve CRISPR/Cas9 activity...
February 16, 2018: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/29449511/crispr-cas12a-target-binding-unleashes-indiscriminate-single-stranded-dnase-activity
#15
Janice S Chen, Enbo Ma, Lucas B Harrington, Maria Da Costa, Xinran Tian, Joel M Palefsky, Jennifer A Doudna
CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing based on its ability to generate targeted, double-stranded DNA (dsDNA) breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, non-specific ssDNase cleavage is also a property of other type V CRISPR-Cas12 enzymes...
February 15, 2018: Science
https://www.readbyqxmd.com/read/29447381/dna-mismatch-repair-and-oligonucleotide-end-protection-promote-base-pair-substitution-distal-from-a-crispr-cas9-induced-dna-break
#16
Tim Harmsen, Sjoerd Klaasen, Henri van de Vrugt, Hein Te Riele
Single-stranded oligodeoxyribonucleotide (ssODN)-mediated repair of CRISPR/Cas9-induced DNA double-strand breaks (DSB) can effectively be used to introduce small genomic alterations in a defined locus. Here, we reveal DNA mismatch repair (MMR) activity is crucial for efficient nucleotide substitution distal from the Cas9-induced DNA break when the substitution is instructed by the 3' half of the ssODN. Furthermore, protecting the ssODN 3' end with phosphorothioate linkages enhances MMR-dependent gene editing events...
February 13, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29446747/crispr-cas9-genome-editing-a-promising-tool-for-therapeutic-applications-of-induced-pluripotent-stem-cells
#17
Yanli Zhang, Danuta Sastre, Feng Wang
Induced pluripotent stem cells hold tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. The revolutionary technology for genome editing known as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system is recently recognized as a powerful tool for editing DNA at specific loci...
February 14, 2018: Current Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/29445193/uncoupling-foxo3a-mitochondrial-and-nuclear-functions-in-cancer-cells-undergoing-metabolic-stress-and-chemotherapy
#18
Valentina Celestini, Tugsan Tezil, Luciana Russo, Candida Fasano, Paola Sanese, Giovanna Forte, Alessia Peserico, Martina Lepore Signorile, Giovanna Longo, Domenico De Rasmo, Anna Signorile, Raffaella Maria Gadaleta, Natasha Scialpi, Mineko Terao, Enrico Garattini, Tiziana Cocco, Gaetano Villani, Antonio Moschetta, Valentina Grossi, Cristiano Simone
While aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrial functions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is a key determinant of cancer cell homeostasis, playing a dual role in survival/death response to metabolic stress and cancer therapeutics. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells upon nutrient shortage. Here, we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain...
February 14, 2018: Cell Death & Disease
https://www.readbyqxmd.com/read/29443028/microinjection-of-crispr-cas9-protein-into-channel-catfish-ictalurus-punctatus-embryos-for-gene-editing
#19
Ahmed Elaswad, Karim Khalil, David Cline, Patrick Page-McCaw, Wenbiao Chen, Maximilian Michel, Roger Cone, Rex Dunham
The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish...
January 20, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29442507/functional-insights-revealed-by-the-kinetic-mechanism-of-crispr-cas9
#20
Austin T Raper, Anthony A Stephenson, Zucai Suo
The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined and details of DNA cleavage poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid...
February 14, 2018: Journal of the American Chemical Society
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