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https://www.readbyqxmd.com/read/29773895/crispr-cas9-system-targeting-regulatory-genes-of-hiv-1-inhibits-viral-replication-in-infected-t-cell-cultures
#1
Youdiil Ophinni, Mari Inoue, Tomohiro Kotaki, Masanori Kameoka
The CRISPR/Cas9 system provides a novel and promising tool for editing the HIV-1 proviral genome. We designed RNA-guided CRISPR/Cas9 targeting the HIV-1 regulatory genes tat and rev with guide RNAs (gRNA) selected from each gene based on CRISPR specificity and sequence conservation across six major HIV-1 subtypes. Each gRNA was cloned into lentiCRISPRv2 before co-transfection to create a lentiviral vector and transduction into target cells. CRISPR/Cas9 transduction into 293 T and HeLa cells stably expressing Tat and Rev proteins successfully abolished the expression of each protein relative to that in non-transduced and gRNA-absent vector-transduced cells...
May 17, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29771955/crispr-cas9-mediated-high-efficiency-knockout-of-the-eye-color-gene-vermillion-in-helicoverpa-zea-boddie
#2
Omaththage P Perera, Nathan S Little, Calvin A Pierce
Among various genome editing tools available for functional genomic studies, reagents based on clustered regularly interspersed palindromic repeats (CRISPR) have gained popularity due to ease and versatility. CRISPR reagents consist of ribonucleoprotein (RNP) complexes formed by combining guide RNA (gRNA) that target specific genomics regions and a CRISPR associated nuclease (Cas). The gRNA targeting specific gene sequences may be delivered as a plasmid construct that needs to be transcribed or as a synthetic RNA...
2018: PloS One
https://www.readbyqxmd.com/read/29754236/bacterial-genome-editing-with-crispr-cas9-taking-clostridium-beijerinckii-as-an-example
#3
Zhong-Tian Zhang, Pablo Jiménez-Bonilla, Seung-Oh Seo, Ting Lu, Yong-Su Jin, Hans P Blaschek, Yi Wang
CRISPR-Cas9 has been explored as a transformative genome engineering tool for many eukaryotic organisms. However, its utilization in bacteria remains limited and ineffective. This chapter, taking Clostridium beijerinckii as an example, describes the use of Streptococcus pyogenes CRISPR-Cas9 system guided by the single chimeric guide RNA (gRNA) for diverse genome-editing purposes, including chromosomal gene deletion, integration, single nucleotide modification, as well as "clean" mutant selection. The general principle is to use CRISPR-Cas9 as an efficient selection tool for the edited mutant (whose CRISPR-Cas9 target site has been disrupted through a homologous recombination event and thus can survive selection) against? the wild type background cells...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29746667/a-fluorescent-reporter-for-quantification-and-enrichment-of-dna-editing-by-apobec-cas9-or-cleavage-by-cas9-in-living-cells
#4
Amber St Martin, Daniel Salamango, Artur Serebrenik, Nadine Shaban, William L Brown, Francesco Donati, Uday Munagala, Silvestro G Conticello, Reuben S Harris
Base editing is an exciting new genome engineering technology. C-to-T mutations in genomic DNA have been achieved using ribonucleoprotein complexes comprised of rat APOBEC1 single-stranded DNA deaminase, Cas9 nickase (Cas9n), uracil DNA glycosylase inhibitor (UGI), and guide (g)RNA. Here, we report the first real-time reporter system for quantification of APOBEC-mediated base editing activity in living mammalian cells. The reporter expresses eGFP constitutively as a marker for transfection or transduction, and editing restores functionality of an upstream mCherry cassette through the simultaneous processing of two gRNA binding regions that each contain an APOBEC-preferred 5'TCA target site...
May 9, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29735370/generation-of-%C3%AE-solanine-free-hairy-roots-of-potato-by-crispr-cas9-mediated-genome-editing-of-the-st16dox-gene
#5
Masaru Nakayasu, Ryota Akiyama, Hyoung Jae Lee, Keishi Osakabe, Yuriko Osakabe, Bunta Watanabe, Yukihiro Sugimoto, Naoyuki Umemoto, Kazuki Saito, Toshiya Muranaka, Masaharu Mizutani
Potato (Solanum tuberosum) is a major food crop, while the most tissues of potato accumulates steroidal glycoalkaloids (SGAs) α-solanine and α-chaconine. Since SGAs confer a bitter taste on human and show the toxicity against various organisms, reducing the SGA content in the tubers is requisite for potato breeding. However, generation of SGA-free potato has not been achieved yet, although silencing of several SGA biosynthetic genes led a decrease in SGAs. Here, we show that the knockout of St16DOX encoding a steroid 16α-hydroxylase in SGA biosynthesis causes the complete abolition of the SGA accumulation in potato hairy roots...
April 24, 2018: Plant Physiology and Biochemistry: PPB
https://www.readbyqxmd.com/read/29728415/crispr-mediated-gene-editing-to-assess-the-roles-of-tet2-and-dnmt3a-in-clonal-hematopoiesis-and-cardiovascular-disease
#6
Soichi Sano, Kousei Ohshima, Ying Wang, Yasufumi Katanasaka, Miho Sano, Kenneth Walsh
<u>Rationale:</u> Clonal hematopoiesis has been associated with increased mortality and cardiovascular disease (CVD). This condition can arise from somatic mutations in pre-leukemic driver genes within hematopoietic stem/progenitor cells (HSPC). Approximately 40 candidate driver genes have been identified, but mutations in only one of these genes, Ten-Eleven Translocation-2 (TET2), has been shown to casually contribute to CVD in murine models. <u>Objective:</u> To develop a facile system to evaluate the disease characteristics of different clonal hematopoiesis driver genes using lentivirus vector and CRISPR/Cas9 methodology...
May 4, 2018: Circulation Research
https://www.readbyqxmd.com/read/29726937/design-principles-for-nuclease-deficient-crispr-based-transcriptional-regulators
#7
Michael K Jensen
The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant...
June 1, 2018: FEMS Yeast Research
https://www.readbyqxmd.com/read/29725684/systemic-delivery-of-crispr-cas9-with-peg-plga-nanoparticles-for-chronic-myeloid-leukemia-targeted-therapy
#8
Yang Liu, Gui Zhao, Cong-Fei Xu, Ying-Li Luo, Zi-Dong Lu, Jun Wang
Chronic myeloid leukemia (CML), which is characterized by the Philadelphia translocation, which fuses breakpoint cluster region (BCR) sequences from chromosome 22 upstream of the Abelson murine leukemia viral oncogene homolog (ABL) on chromosome 9, requires specific and efficient treatment. The CRISPR/Cas9 system, with its mechanism of specific DNA complementary recognition by engineered guide RNA (gRNA), allows the development of novel therapeutics for CML. To achieve targeted therapy of CML with the CRISPR/Cas9 system, we encapsulated a CRISPR/Cas9 plasmid (pCas9) expressing gRNA targeting the overhanging fusion region of the BCR-ABL gene (pCas9/gBCR-ABL) with poly(ethylene glycol)-b-poly(lactic acid-co-glycolic acid) (PEG-PLGA)-based cationic lipid-assisted polymeric nanoparticles (CLANs), which specifically disrupted the CML-related BCR-ABL gene while sparing the BCR and ABL genes in normal cells...
May 4, 2018: Biomaterials Science
https://www.readbyqxmd.com/read/29723758/optimization-of-lipid-assisted-nanoparticle-for-disturbing-neutrophils-related-inflammation
#9
Yang Liu, Zhi-Ting Cao, Cong-Fei Xu, Zi-Dong Lu, Ying-Li Luo, Jun Wang
Inflammation is closely related to the development of many diseases and is commonly characterized by abnormal infiltration of immune cells, especially neutrophils. The current therapeutics of inflammatory diseases give little attention to direct modulation of these diseases with respect to immune cells. Nanoparticles are applied for efficient drug delivery into the disease-related immune cells, but their performance is significantly affected by their surface properties. In this study, to optimize the properties of nanoparticles for modulating neutrophils-related inflammation, we prepared a library of poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-b-PLGA)-based cationic lipid-assisted nanoparticles (CLANs) with different surface PEG density and surface charge...
April 26, 2018: Biomaterials
https://www.readbyqxmd.com/read/29717007/selective-packaging-in-murine-coronavirus-promotes-virulence-by-limiting-type-i-interferon-responses
#10
Jeremiah Athmer, Anthony R Fehr, Matthew E Grunewald, Wen Qu, D Lori Wheeler, Kevin W Graepel, Rudragouda Channappanavar, Aimee Sekine, Dana Saud Aldabeeb, Michael Gale, Mark R Denison, Stanley Perlman
Selective packaging is a mechanism used by multiple virus families to specifically incorporate genomic RNA (gRNA) into virions and exclude other types of RNA. Lineage A betacoronaviruses incorporate a 95-bp stem-loop structure, the packaging signal (PS), into the nsp15 locus of ORF1b that is both necessary and sufficient for the packaging of RNAs. However, unlike other viral PSs, where mutations generally resulted in viral replication defects, mutation of the coronavirus (CoV) PS results in large increases in subgenomic RNA packaging with minimal effects on gRNA packaging in vitro and on viral titers...
May 1, 2018: MBio
https://www.readbyqxmd.com/read/29702666/one-step-generation-of-customizable-grna-vectors-for-multiplex-crispr-approaches-through-string-assembly-grna-cloning-stagr
#11
Christopher T Breunig, Tamara Durovic, Andrea M Neuner, Valentin Baumann, Maximilian F Wiesbeck, Anna Köferle, Magdalena Götz, Jovica Ninkovic, Stefan H Stricker
Novel applications based on the bacterial CRISPR system make genetic, genomic, transcriptional and epigenomic engineering widely accessible for the first time. A significant advantage of CRISPR over previous methods is its tremendous adaptability due to its bipartite nature. Cas9 or its engineered variants define the molecular effect, while short gRNAs determine the targeting sites. A majority of CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into single cells, either as an essential precondition, to increase responsive cell populations or to enhance phenotypic outcomes...
2018: PloS One
https://www.readbyqxmd.com/read/29700298/rna-guided-transcriptional-silencing-in-vivo-with-s-aureus-crispr-cas9-repressors
#12
Pratiksha I Thakore, Jennifer B Kwon, Christopher E Nelson, Douglas C Rouse, Matthew P Gemberling, Matthew L Oliver, Charles A Gersbach
CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB ) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels...
April 26, 2018: Nature Communications
https://www.readbyqxmd.com/read/29688335/crispr-cas9-enabled-targeted-mutagenesis-in-the-thermotolerant-methylotrophic-yeast-ogataea-thermomethanolica
#13
Chitwadee Phithakrotchanakoon, Aekkachai Puseenam, Sriwan Wongwisansri, Lily Eurwilaichitr, Supawadee Ingsriswang, Sutipa Tanapongpipat, Niran Roonsawang
Ogataea thermomethanolica TBRC656 is a thermotolerant methylotrophic yeast suitable for heterologous protein expression at various temperatures. However, the lack of efficient methods for targeted gene mutagenesis limits strain engineering in this yeast. In this study, we applied a CRISPR-Cas9 based-tool for targeted gene mutagenesis in O. thermomethanolica. The putative unfolded protein response (UPR) regulator OtHAC1, and the OtMAL1 (maltase) and OtMAL2 (maltose permease) genes involved with sucrose and maltose utilization were targeted for CRISPR-Cas9 mutagenesis...
April 23, 2018: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/29681919/efficient-gene-transfer-and-gene-editing-in-sterlet-acipenser-ruthenus
#14
Ji Chen, Wei Wang, Zhaohui Tian, Ying Dong, Tian Dong, Hua Zhu, Zuoyan Zhu, Hongxia Hu, Wei Hu
The sturgeon (Acipenseriformes) is an important farmed species because of its economical value. However, neither gene transfer nor gene editing techniques have been established in sturgeon for molecular breeding and gene functional study until now. In this study, we accomplished gene transfer and gene editing in sterlet ( Acipenser ruthenus ), which has the shortest sexual maturation period of sturgeons. The plasmid encoding enhanced green fluorescent protein (EGFP) was transferred into the embryos of sterlet at injection concentration of 100 ng/μL, under which condition high survival rate and gene transfer rate could be achieved...
2018: Frontiers in Genetics
https://www.readbyqxmd.com/read/29678175/gene-repression-via-multiplex-grna-strategy-in-y-lipolytica
#15
Jin-Lai Zhang, Yang-Zi Peng, Duo Liu, Hong Liu, Ying-Xiu Cao, Bing-Zhi Li, Chun Li, Ying-Jin Yuan
BACKGROUND: The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest...
April 20, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29644868/crispr-cas9-inhibits-multiple-steps-of-hiv-1-infection
#16
Lijuan Yin, Siqi Hu, Shan Mei, Hong Sun, Fengwen Xu, Jian Li, Weijun Zhu, Xiaoman Liu, Fei Zhao, Di Zhang, Shan Cen, Chen Liang, Fei Guo
CRISPR/Cas9 is an adaptive immune system that bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. In this study, we have tested this gene editing system in inhibiting HIV-1 infection by targeting the viral long terminal repeat and the gene coding sequences. We observed strong inhibition of HIV-1 infection by Cas9/gRNA, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA...
April 12, 2018: Human Gene Therapy
https://www.readbyqxmd.com/read/29643844/emerging-roles-of-n-6-methyladenosine-on-hiv-1-rna-metabolism-and-viral-replication
#17
REVIEW
Sebastián Riquelme-Barrios, Camila Pereira-Montecinos, Fernando Valiente-Echeverría, Ricardo Soto-Rifo
N6 -methyladenosine (m6 A) is the most abundant internal modification present in Eukaryotic mRNA. The functions of this chemical modification are mediated by m6 A-binding proteins (m6 A readers) and regulated by methyltransferases (m6 A writers) and demethylases (m6 A erasers), which together are proposed to be responsible of a new layer of post-transcriptional control of gene expression. Despite the presence of m6 A in a retroviral genome was reported more than 40 years ago, the recent development of sequencing-based technologies allowing the mapping of m6 A in a transcriptome-wide manner made it possible to identify the topology and dynamics of m6 A during replication of HIV-1 as well as other viruses...
2018: Frontiers in Microbiology
https://www.readbyqxmd.com/read/29643451/author-correction-optimised-metrics-for-crispr-ko-screens-with-second-generation-grna-libraries
#18
Swee Hoe Ong, Yilong Li, Hiroko Koike-Yusa, Kosuke Yusa
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
April 12, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29625148/genome-scale-screening-identification-of-sacas9-grnas-for-targeting-hiv-1-provirus-and-suppression-of-hiv-1-infection
#19
Qiankun Wang, Shuai Liu, Zhepeng Liu, Zunhui Ke, Chunmei Li, Xiao Yu, Shuliang Chen, Deyin Guo
The CRISPR/Cas9 gene-editing approach has been widely used in anti-HIV-1 gene therapy research. However, the major challenges facing the therapeutic application of CRISPR/Cas9 are the precise genome cleavage efficacy and efficient delivery of Cas9/gRNA specifically to the HIV-infected cells. Recently, a small size Cas9 from Staphylococcus aureus (SaCas9) has shown promise in genome editing in eukaryotic cells, suggesting a potential usage in blocking HIV-1 infection by targeting the HIV-1 genome. Here, we designed 43 guide RNAs (gRNAs) against the HIV-1 genome, thereby identifying 8 gRNAs that efficiently and specifically disrupt the target DNA by SaCas9...
April 3, 2018: Virus Research
https://www.readbyqxmd.com/read/29623074/retroviral-rna-dimerization-from-structure-to-functions
#20
REVIEW
Noé Dubois, Roland Marquet, Jean-Christophe Paillart, Serena Bernacchi
The genome of the retroviruses is a dimer composed by two homologous copies of genomic RNA (gRNA) molecules of positive polarity. The dimerization process allows two gRNA molecules to be non-covalently linked together through intermolecular base-pairing. This step is critical for the viral life cycle and is highly conserved among retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two gRNA copies into viral particles presents an important evolutionary advantage for immune system evasion and drug resistance...
2018: Frontiers in Microbiology
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