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https://www.readbyqxmd.com/read/28062688/piggybac-mediates-efficient-in-vivo-crispr-library-screening-for-tumorigenesis-in-mice
#1
Chunlong Xu, Xiaolan Qi, Xuguang Du, Huiying Zou, Fei Gao, Tao Feng, Hengxing Lu, Shenglan Li, Xiaomeng An, Lijun Zhang, Yuanyuan Wu, Ying Liu, Ning Li, Mario R Capecchi, Sen Wu
CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported...
January 6, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28053097/subcellular-localization-of-hiv-1-gag-pol-mrnas-regulates-sites-of-virion-assembly
#2
Jordan T Becker, Nathan M Sherer
: HIV-1 full-length, unspliced RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells...
January 4, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28034301/mechanistic-differences-between-hiv-1-and-siv-nucleocapsid-proteins-and-cross-species-hiv-1-genomic-rna-recognition
#3
Klara Post, Erik D Olson, M Nabuan Naufer, Robert J Gorelick, Ioulia Rouzina, Mark C Williams, Karin Musier-Forsyth, Judith G Levin
BACKGROUND: The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC...
December 29, 2016: Retrovirology
https://www.readbyqxmd.com/read/28030843/targeted-delivery-of-crispr-cas9-to-prostate-cancer-by-modified-grna-using-a-flexible-aptamer-cationic-liposome
#4
Shuai Zhen, Y Takahashi, S Narita, Yi-Chen Yang, Xu Li
The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand...
December 21, 2016: Oncotarget
https://www.readbyqxmd.com/read/28011935/ttll12-inhibits-the-activation-of-cellular-antiviral-signaling-through-interaction-with-visa-mavs
#5
Lin-Gao Ju, Yuan Zhu, Pin-Ji Lei, Dong Yan, Kun Zhu, Xiang Wang, Qing-Lan Li, Xue-Jing Li, Jian-Wen Chen, Lian-Yun Li, Min Wu
Upon virus infection, host cells use retinoic-acid-inducible geneI I (RIG-I)-like receptors to recognize viral RNA and activate type I IFN expression. To investigate the role of protein methylation in the antiviral signaling pathway, we screened all the SET domain-containing proteins and identified TTLL12 as a negative regulator of RIG-I signaling. TTLL12 contains SET and TTL domains, which are predicted to have lysine methyltransferase and tubulin tyrosine ligase activities, respectively. Exogenous expression of TTLL12 represses IFN-β expression induced by Sendai virus...
December 23, 2016: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/28009832/nmr-studies-of-the-structure-and-function-of-the-hiv-1-5-leader
#6
REVIEW
Sarah C Keane, Michael F Summers
The 5'-leader of the human immunodeficiency virus type 1 (HIV-1) genome plays several critical roles during viral replication, including differentially establishing mRNA versus genomic RNA (gRNA) fates. As observed for proteins, the function of the RNA is tightly regulated by its structure, and a common paradigm has been that genome function is temporally modulated by structural changes in the 5'-leader. Over the past 30 years, combinations of nucleotide reactivity mapping experiments with biochemistry, mutagenesis, and phylogenetic studies have provided clues regarding the secondary structures of stretches of residues within the leader that adopt functionally discrete domains...
December 21, 2016: Viruses
https://www.readbyqxmd.com/read/27983963/simultaneous-paralogue-knockout-using-a-crispr-concatemer-in-mouse-small-intestinal-organoids
#7
Amanda Andersson-Rolf, Alessandra Merenda, Roxana C Mustata, Taibo Li, Sabine Dietmann, Bon-Kyoung Koo
Approaches based on genetic modification have been invaluable for investigating a wide array of biological processes, with gain- and loss-of-function approaches frequently used to investigate gene function. However, the presence of paralogues, and hence possible genetic compensation, for many genes necessitates the knockout (KO) of all paralogous genes in order to observe clear phenotypic change. CRISPR technology, the most recently described tool for gene editing, can generate KOs with unprecedented ease and speed and has been used in adult stem cell-derived organoids for single gene knockout, gene knock-in and gene correction...
October 27, 2016: Developmental Biology
https://www.readbyqxmd.com/read/27974196/a-combinatorial-crispr-cas9-attack-on-hiv-1-dna-extinguishes-all-infectious-provirus-in-infected-t-cell-cultures
#8
Gang Wang, Na Zhao, Ben Berkhout, Atze T Das
Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Recent studies have found that the guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attack on this proviral DNA. Although potent inhibition of virus replication was reported, HIV-1 can escape from a single antiviral gRNA by mutation of the target sequence. Here, we demonstrate that combinations of two antiviral gRNAs delay viral escape, and identify two gRNA combinations that durably block virus replication...
December 13, 2016: Cell Reports
https://www.readbyqxmd.com/read/27936040/a-high-throughput-strategy-for-dissecting-mammalian-genetic-interactions
#9
Victoria B Stockman, Lila Ghamsari, Gorka Lasso, Barry Honig, Sagi D Shapira, Harris H Wang
Comprehensive delineation of complex cellular networks requires high-throughput interrogation of genetic interactions. To address this challenge, we describe the development of a multiplex combinatorial strategy to assess pairwise genetic interactions using CRISPR-Cas9 genome editing and next-generation sequencing. We characterize the performance of combinatorial genome editing and analysis using different promoter and gRNA designs and identified regions of the chimeric RNA that are compatible with next-generation sequencing preparation and quantification...
2016: PloS One
https://www.readbyqxmd.com/read/27929521/efficient-footprint-free-human-ipsc-genome-editing-by-consolidation-of-cas9-crispr-and-piggybac-technologies
#10
Gang Wang, Luhan Yang, Dennis Grishin, Xavier Rios, Lillian Y Ye, Yong Hu, Kai Li, Donghui Zhang, George M Church, William T Pu
Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27905467/direct-identification-of-antibiotic-resistance-genes-on-single-plasmid-molecules-using-crispr-cas9-in-combination-with-optical-dna-mapping
#11
Vilhelm Müller, Fredrika Rajer, Karolin Frykholm, Lena K Nyberg, Saair Quaderi, Joachim Fritzsche, Erik Kristiansson, Tobias Ambjörnsson, Linus Sandegren, Fredrik Westerlund
Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene...
December 1, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27890617/polycistronic-trna-and-crispr-guide-rna-enables-highly-efficient-multiplexed-genome-engineering-in-human-cells
#12
Fengping Dong, Kabin Xie, Yueying Chen, Yinong Yang, Yingwei Mao
CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic variants in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNAs) need to be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and increased insertion size lower the efficiency to make a construct...
January 22, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27866656/an-analysis-of-possible-off-target-effects-following-cas9-crispr-targeted-deletions-of-neuropeptide-gene-enhancers-from-the-mouse-genome
#13
Elizabeth Anne Hay, Abdulla Razak Khalaf, Pietro Marini, Andrew Brown, Karyn Heath, Darrin Sheppard, Alasdair MacKenzie
We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome...
November 4, 2016: Neuropeptides
https://www.readbyqxmd.com/read/27852925/anticodon-like-binding-of-the-hiv-1-trna-like-element-to-human-lysyl-trna-synthetase
#14
Sheng Liu, Roopa Comandur, Christopher P Jones, Pearl Tsang, Karin Musier-Forsyth
A critical step in the HIV-1 lifecycle involves reverse transcription of the viral genomic RNA (gRNA). Human tRNA(Lys3) serves as a primer for transcription initiation and is selectively enriched in virus particles. Human lysyl-tRNA synthetase (hLysRS) is also packaged into virions. Recently, a tRNA-like element (TLE) within the HIV-1 gRNA was shown to mimic the global tRNA fold and bind competitively to hLysRS, suggesting a mechanism of tRNA targeting to the primer binding site (PBS) and release from the synthetase...
December 2016: RNA
https://www.readbyqxmd.com/read/27848933/genome-editing-in-maize-directed-by-crispr-cas9-ribonucleoprotein-complexes
#15
Sergei Svitashev, Christine Schwartz, Brian Lenderts, Joshua K Young, A Mark Cigan
Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR-Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9-gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles...
November 16, 2016: Nature Communications
https://www.readbyqxmd.com/read/27845387/adenoviral-vectors-encoding-crispr-cas9-multiplexes-rescue-dystrophin-synthesis-in-unselected-populations-of-dmd-muscle-cells
#16
Ignazio Maggio, Jin Liu, Josephine M Janssen, Xiaoyu Chen, Manuel A F V Gonçalves
Mutations disrupting the reading frame of the ~2.4 Mb dystrophin-encoding DMD gene cause a fatal X-linked muscle-wasting disorder called Duchenne muscular dystrophy (DMD). Genome editing based on paired RNA-guided nucleases (RGNs) from CRISPR/Cas9 systems has been proposed for permanently repairing faulty DMD loci. However, such multiplexing strategies require the development and testing of delivery systems capable of introducing the various gene editing tools into target cells. Here, we investigated the suitability of adenoviral vectors (AdVs) for multiplexed DMD editing by packaging in single vector particles expression units encoding the Streptococcus pyogenes Cas9 nuclease and sequence-specific gRNA pairs...
November 15, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27845164/enhanced-crispr-cas9-mediated-precise-genome-editing-by-improved-design-and-delivery-of-grna-cas9-nuclease-and-donor-dna
#17
Xiquan Liang, Jason Potter, Shantanu Kumar, Namritha Ravinder, Jonathan D Chesnut
While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA...
January 10, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/27842490/coralina-a-universal-method-for-the-generation-of-grna-libraries-for-crispr-based-screening
#18
Anna Köferle, Karolina Worf, Christopher Breunig, Valentin Baumann, Javier Herrero, Maximilian Wiesbeck, Lukas H Hutter, Magdalena Götz, Christiane Fuchs, Stephan Beck, Stefan H Stricker
BACKGROUND: The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. RESULTS: Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens...
November 14, 2016: BMC Genomics
https://www.readbyqxmd.com/read/27834588/generation-of-ggta1-mutant-pigs-by-direct-pronuclear-microinjection-of-crispr-cas9-plasmid-vectors
#19
Chin-Kai Chuang, Chien-Hong Chen, Chung-Ling Huang, Yu-Hsiu Su, Shu-Hui Peng, Tai-Yun Lin, Hao-Chih Tai, Tien-Shuh Yang, Ching-Fu Tu
This study was conducted to confirm that 1-site and 4-site ppU6-GGTA1-gRNA CRISPR vectors together with the pCX-Flag2-NLS1-Cas9-NLS2 plasmid can both generate KO pigs by direct pronuclear microinjection. In total, 41 and 53 fertilized eggs were microinjected on 1-site and 4-site strategies, respectively. The 1-site construction generated a litter of 8 piglets, and 2 were mono-allelic mutant (mMt). The injection of 4-site constructions resulted in one biallelic mutant (bMt) and one mMt piglet in a litter of 7...
November 11, 2016: Animal Biotechnology
https://www.readbyqxmd.com/read/27832146/ribozyme-mediated-grna-generation-for-in-vitro-and-in-vivo-crispr-cas9-mutagenesis
#20
Raymond Teck Ho Lee, Ashley Shu Mei Ng, Philip W Ingham
CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target...
2016: PloS One
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