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https://www.readbyqxmd.com/read/27905063/efficient-gene-targeting-in-mouse-zygotes-mediated-by-crispr-cas9-protein
#1
Chris J Jung, Junli Zhang, Elizabeth Trenchard, Kent C Lloyd, David B West, Barry Rosen, Pieter J de Jong
The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9-FokI...
November 30, 2016: Transgenic Research
https://www.readbyqxmd.com/read/27903907/chromatin-binding-of-gcn5-in-drosophila-is-largely-mediated-by-cp190
#2
Tamer Ali, Marcus Krüger, Sabin Bhuju, Michael Jarek, Marek Bartkuhn, Rainer Renkawitz
Centrosomal 190 kDa protein (CP190) is a promoter binding factor, mediates long-range interactions in the context of enhancer-promoter contacts and in chromosomal domain formation. All Drosophila insulator proteins bind CP190 suggesting a crucial role in insulator function. CP190 has major effects on chromatin, such as depletion of nucleosomes, high nucleosomal turnover and prevention of heterochromatin expansion. Here, we searched for enzymes, which might be involved in CP190 mediated chromatin changes. Eighty percent of the genomic binding sites of the histone acetyltransferase Gcn5 are colocalizing with CP190 binding...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899645/efficient-targeted-dna-methylation-with-chimeric-dcas9-dnmt3a-dnmt3l-methyltransferase
#3
Peter Stepper, Goran Kungulovski, Renata Z Jurkowska, Tamir Chandra, Felix Krueger, Richard Reinhardt, Wolf Reik, Albert Jeltsch, Tomasz P Jurkowski
DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899566/asymmetric-positioning-of-cas1-2-complex-and-integration-host-factor-induced-dna-bending-guide-the-unidirectional-homing-of-protospacer-in-crispr-cas-type-i-e-system
#4
K N R Yoganand, R Sivathanu, Siddharth Nimkar, B Anand
CRISPR-Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27846887/crispr-interference-crispri-for-gene-regulation-and-succinate-production-in-cyanobacterium-s-elongatus-pcc-7942
#5
Chun-Hung Huang, Claire R Shen, Hung Li, Li-Yu Sung, Meng-Ying Wu, Yu-Chen Hu
BACKGROUND: Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence-specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various procaryotic cells. RESULTS: To employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes expressing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome...
November 15, 2016: Microbial Cell Factories
https://www.readbyqxmd.com/read/27842593/easy-regulation-of-metabolic-flux-in-escherichia-coli-using-an-endogenous-type-i-e-crispr-cas-system
#6
Yizhao Chang, Tianyuan Su, Qingsheng Qi, Quanfeng Liang
BACKGROUND: Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid. RESULTS: By knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defense (Cascade), we constructed a native CRISPRi system in E...
November 15, 2016: Microbial Cell Factories
https://www.readbyqxmd.com/read/27832543/using-an-inducible-crispr-dcas9-krab-effector-system-to-dissect-transcriptional-regulation-in-human-embryonic-stem-cells
#7
Krishna Mohan Parsi, Erica Hennessy, Nicola Kearns, René Maehr
CRISPR-Cas9 effector systems have wide applications for the stem cell and regenerative medicine field. The ability to dissect the functional gene regulatory networks in pluripotency and potentially in differentiation intermediates of all three germ layers makes this a valuable tool for the stem cell community. Catalytically inactive Cas9 fused to transcriptional/chromatin effector domains allows for silencing or activation of a genomic region of interest. Here, we describe the application of an inducible, RNA-guided, nuclease-deficient (d) Cas9-KRAB system (adapted from Streptococcus pyogenes) to silence target gene expression in human embryonic stem cells, via KRAB repression at the promoter region...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27799472/empower-multiplex-cell-and-tissue-specific-crispr-mediated-gene-manipulation-with-self-cleaving-ribozymes-and-trna
#8
Li Xu, Lixia Zhao, Yandi Gao, Jing Xu, Renzhi Han
Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells...
October 30, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27798611/directed-evolution-using-dcas9-targeted-somatic-hypermutation-in-mammalian-cells
#9
Gaelen T Hess, Laure Frésard, Kyuho Han, Cameron H Lee, Amy Li, Karlene A Cimprich, Stephen B Montgomery, Michael C Bassik
Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously...
October 31, 2016: Nature Methods
https://www.readbyqxmd.com/read/27788303/efficient-gene-transcription-repression-in-clostridium-using-crispr-dcas9
#10
(no author information available yet)
No abstract text is available yet for this article.
December 2016: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/27786189/crispr-rna-guided-foki-nucleases-repair-a-pah-variant-in-a-phenylketonuria-model
#11
Yi Pan, Nan Shen, Sabine Jung-Klawitter, Christian Betzen, Georg F Hoffmann, Jörg D Hoheisel, Nenad Blau
The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed...
October 27, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27776111/complex-transcriptional-modulation-with-orthogonal-and-inducible-dcas9-regulators
#12
Yuchen Gao, Xin Xiong, Spencer Wong, Emeric J Charles, Wendell A Lim, Lei S Qi
The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR-dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells...
October 24, 2016: Nature Methods
https://www.readbyqxmd.com/read/27723754/targeted-aid-mediated-mutagenesis-tam-enables-efficient-genomic-diversification-in-mammalian-cells
#13
Yunqing Ma, Jiayuan Zhang, Weijie Yin, Zhenchao Zhang, Yan Song, Xing Chang
A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants...
October 10, 2016: Nature Methods
https://www.readbyqxmd.com/read/27718551/paired-design-of-dcas9-as-a-systematic-platform-for-the-detection-of-featured-nucleic-acid-sequences-in-pathogenic-strains
#14
Yihao Zhang, Long Qian, Weijia Wei, Yu Wang, Beining Wang, Pingping Lin, Wenchao Liu, Luze Xu, Xiang Li, Dongming Liu, Sida Cheng, Jiaofeng Li, Yixuan Ye, Hang Li, Xiaohan Zhang, Yiming Dong, Xuejin Zhao, Cuihua Liu, Haoqian M Zhang, Qi Ouyang, Chunbo Lou
We developed an in vitro DNA detection system using a pair of dCas9 proteins linked to split halves of luciferase. Luminescence was induced upon co-localization of the reporter pair to a ~44 bp target sequence defined by sgRNAs. We used the system to detect Mycobacterium tuberculosis DNA with high specificity and sensitivity. The re-programmability of dCas9 was further leveraged in an array design that accesses sequence information across the entire genome.
October 10, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27709568/all-in-one-crispr-cas9-foki-dcas9-vector-mediated-multiplex-genome-engineering-in-cultured-cells
#15
Tetsushi Sakuma, Takuya Sakamoto, Takashi Yamamoto
CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27694915/integration-and-exchange-of-split-dcas9-domains-for-transcriptional-controls-in-mammalian-cells
#16
Dacheng Ma, Shuguang Peng, Zhen Xie
Programmable and precise regulation of dCas9 functions in response to multiple molecular signals by using synthetic gene circuits will expand the application of the CRISPR-Cas technology. However, the application of CRISPR-Cas therapeutic circuits is still challenging due to the restrictive cargo size of existing viral delivery vehicles. Here, we construct logic AND circuits by integrating multiple split dCas9 domains, which is useful to reduce the size of synthetic circuits. In addition, we engineer sensory switches by exchanging split dCas9 domains, allowing differential regulations on one gene, or activating two different genes in response to cell-type specific microRNAs...
October 3, 2016: Nature Communications
https://www.readbyqxmd.com/read/27694625/challenges-of-crispr-cas9-applications-for-long-non-coding-rna-genes
#17
Ashish Goyal, Ksenia Myacheva, Matthias Groß, Marcel Klingenberg, Berta Duran Arqué, Sven Diederichs
The CRISPR/Cas9 system provides a revolutionary genome editing tool for all areas of molecular biology. In long non-coding RNA (lncRNA) research, the Cas9 nuclease can delete lncRNA genes or introduce RNA-destabilizing elements into their locus. The nuclease-deficient dCas9 mutant retains its RNA-dependent DNA-binding activity and can modulate gene expression when fused to transcriptional repressor or activator domains. Here, we systematically analyze whether CRISPR approaches are suitable to target lncRNAs...
September 30, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27676121/determination-of-local-chromatin-composition-by-casid
#18
Elisabeth Schmidtmann, Tobias Anton, Pascaline Rombaut, Franz Herzog, Heinrich Leonhardt
Chromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci. Subsequent streptavidin-mediated precipitation and mass spectrometry identified both known and previously unknown chromatin factors associated with repetitive telomeric, major satellite and minor satellite DNA...
September 27, 2016: Nucleus
https://www.readbyqxmd.com/read/27662091/editing-dna-methylation-in-the-mammalian-genome
#19
X Shawn Liu, Hao Wu, Xiong Ji, Yonatan Stelzer, Xuebing Wu, Szymon Czauderna, Jian Shu, Daniel Dadon, Richard A Young, Rudolf Jaenisch
Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter...
September 22, 2016: Cell
https://www.readbyqxmd.com/read/27571369/targeted-dna-demethylation-in-vivo-using-dcas9-peptide-repeat-and-scfv-tet1-catalytic-domain-fusions
#20
Sumiyo Morita, Hirofumi Noguchi, Takuro Horii, Kazuhiko Nakabayashi, Mika Kimura, Kohji Okamura, Atsuhiko Sakai, Hideyuki Nakashima, Kenichiro Hata, Kinichi Nakashima, Izuho Hatada
Despite the importance of DNA methylation in health and disease, technologies to readily manipulate methylation of specific sequences for functional analysis and therapeutic purposes are lacking. Here we adapt the previously described dCas9-SunTag for efficient, targeted demethylation of specific DNA loci. The original SunTag consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, which induces demethylation, we changed the linker length to 22 amino acids...
October 2016: Nature Biotechnology
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