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Lun Cui, Antoine Vigouroux, François Rousset, Hugo Varet, Varun Khanna, David Bikard
High-throughput CRISPR-Cas9 screens have recently emerged as powerful tools to decipher gene functions and genetic interactions. Here we use a genome-wide library of guide RNAs to direct the catalytically dead Cas9 (dCas9) to block gene transcription in Escherichia coli. Using a machine-learning approach, we reveal that guide RNAs sharing specific 5-nucleotide seed sequences can produce strong fitness defects or even kill E. coli regardless of the other 15 nucleotides of guide sequence. This effect occurs at high dCas9 concentrations and can be alleviated by tuning the expression of dCas9 while maintaining strong on-target repression...
May 15, 2018: Nature Communications
Glennis A Logsdon, Ben E Black
Major advances in gene-editing technologies have enabled the rapid dissection of proteins in complex biological systems, facilitating biological experiments to complement biochemical studies with purified components. In this editorial, we highlight CRISPR/Cas9-based strategies to rapidly manipulate endogenous genes - strategies that have already transformed functional studies of proteins in metazoan systems. We further describe emerging tools using a catalytically dead version of Cas9 (dCas9) that do not cleave DNA, but can alter gene expression and/or local chromatin states, edit single nucleotide bases, and permit the visualization of specific genomic loci...
May 15, 2018: Biochemical Journal
Charis L Himeda, Takako I Jones, Ching-Man Virbasius, Lihua Julie Zhu, Michael R Green, Peter L Jones
Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic de-repression of the disease locus, leading to pathogenic misexpression of the DUX4 gene in skeletal muscle. While the factors and pathways involved in normal repression of the FSHD locus in healthy cells have been well characterized, very little is known about those responsible for the aberrant activation of DUX4-fl in FSHD myocytes. Reasoning that DUX4-fl activators might represent useful targets for small molecule inhibition, we performed a highly targeted, candidate-based screen of epigenetic regulators in primary FSHD myocytes...
April 26, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Tina Lebar, Roman Jerala
Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence...
2018: Methods in Molecular Biology
Samuel A Myers, Jason Wright, Ryan Peckner, Brian T Kalish, Feng Zhang, Steven A Carr
Regulation of gene expression is primarily controlled by changes in the proteins that occupy genes' regulatory elements. We developed genomic locus proteomics (GLoPro), in which we combine CRISPR-based genome targeting, proximity labeling, and quantitative proteomics to discover proteins associated with a specific genomic locus in native cellular contexts.
May 7, 2018: Nature Methods
Xin D Gao, Li-Chun Tu, Aamir Mir, Tomás Rodriguez, Yuehe Ding, John Leszyk, Job Dekker, Scott A Shaffer, Lihua Julie Zhu, Scot A Wolfe, Erik J Sontheimer
Mapping proteomic composition at distinct genomic loci in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. C-BERST enables the high-throughput identification of proteins associated with specific sequences, thereby facilitating annotation of these factors and their roles.
May 7, 2018: Nature Methods
Cory Schwartz, Nicholas Curtis, Ann-Kathrin Löbs, Ian Wheeldon
The yeast Yarrowia lipolytica has been widely studied for its ability to synthesize and accumulate intracellular lipids to high levels. Recent studies have identified native genes that enable growth on biomass-derived sugars, but these genes are not sufficiently expressed to facilitate robust metabolism. In this work, we developed a CRISPR-dCas9 activation (CRISPRa) system in Y. lipolytica and used it to activate native β-glucosidase expression to support growth on cellobiose. A series of different transcriptional activators were compared for their effectiveness in Y...
May 5, 2018: Biotechnology Journal
Ghada Mkannez, Valérie Gagné-Ouellet, Mohamed Jalloul Nsaibia, Marie-Chloé Boulanger, Mickael Rosa, Deborah Argaud, Fayez Hadji, Nathalie Gaudreault, Gabrielle Rhéaume, Luigi Bouchard, Yohan Bossé, Patrick Mathieu
Aims: Calcific aortic valve disease (CAVD) is characterized by the osteogenic transition of valve interstitial cells (VICs). In CAVD, lysophosphatidic acid (LysoPA), a lipid mediator with potent osteogenic activity, is produced in the aortic valve (AV) and is degraded by membrane-associated phospholipid phosphatases (PLPPs). We thus hypothesized that a dysregulation of PLPPs could participate to the osteogenic reprograming of VICs during CAVD. Methods and Results: The expression of PLPPs was examined in human control and mineralized AVs and comprehensive analyses were performed to document the gene regulation and impact of PLPPs on the osteogenic transition of VICs...
May 2, 2018: Cardiovascular Research
Xiaotian Wu, Shiqi Mao, Yantao Yang, Muaz N Rushdi, Christopher J Krueger, Antony K Chen
The clustered regularly interspersed short palindromic repeat (CRISPR) gene-editing system has been repurposed for live-cell genomic imaging, but existing approaches rely on fluorescent protein reporters, making sensitive and continuous imaging difficult. Here, we present a fluorophore-based live-cell genomic imaging system that consists of a nuclease-deactivated mutant of the Cas9 protein (dCas9), a molecular beacon (MB), and an engineered single-guide RNA (sgRNA) harboring a unique MB target sequence (sgRNA-MTS), termed CRISPR/MB...
April 30, 2018: Nucleic Acids Research
Sungyeon Jang, Sungho Jang, Gyoo Yeol Jung
CRISPR interference (CRISPRi) is widely utilized for regulation of target gene expression by repressing transcription. Simple design rules for the single guide RNA (sgRNA) and multiplexity won this method immense popularity. However, quantitative control of the expression levels at varying degrees in a dynamic manner using CRISPRi has been regarded difficult. To deal with this limitation, Fontana et al. [1] modulated the expression levels of the components of CRISPRi, the deactivated Cas9 (dCas9) and the sgRNAs, using various constitutive or inducible promoters...
May 1, 2018: Biotechnology Journal
Rugile Stanyte, Johannes Nuebler, Claudia Blaukopf, Rudolf Hoefler, Roman Stocsits, Jan-Michael Peters, Daniel W Gerlich
Faithful genome transmission in dividing cells requires that the two copies of each chromosome's DNA package into separate but physically linked sister chromatids. The linkage between sister chromatids is mediated by cohesin, yet where sister chromatids are linked and how they resolve during cell cycle progression has remained unclear. In this study, we investigated sister chromatid organization in live human cells using dCas9-mEGFP labeling of endogenous genomic loci. We detected substantial sister locus separation during G2 phase irrespective of the proximity to cohesin enrichment sites...
April 25, 2018: Journal of Cell Biology
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
Matthew Deaner, Allison Holzman, Hal S Alper
Metabolic engineering typically utilizes a suboptimal step-wise gene target optimization approach to parse a highly connected and regulated cellular metabolism. While the endonuclease-null CRISPR/Cas system has enabled gene expression perturbations without genetic modification, it has been mostly limited to small sets of gene targets in eukaryotes due to inefficient methods to assemble and express large sgRNA operons. In this work, we develop a TEF1p-tRNA expression system and demonstrate that the use of tRNAs as splicing elements flanking sgRNAs provides higher efficiency than both Pol III and ribozyme-based expression across a variety of single sgRNA and multiplexed contexts...
April 16, 2018: Biotechnology Journal
Jingxuan Wang, Peng Zhao, Ying Li, Lida Xu, Pingfang Tian
BACKGROUND: Klebsiella pneumoniae is a promising industrial species for bioproduction of bulk chemicals such as 1,3-propanediol, 2,3-butanediol and 3-hydroxypropionic acid (3-HP). However, lactic acid is a troublesome by-product when optimizing for 3-HP production. Therefore, it is highly desirable to minimize lactic acid. RESULTS: Here, we show that lactic acid synthesis can be largely blocked by an engineered CRISPR interference (CRISPRi) system in K. pneumoniae...
April 5, 2018: Microbial Cell Factories
Suhyung Cho, Jongoh Shin, Byung-Kwan Cho
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome...
April 5, 2018: International Journal of Molecular Sciences
Johan Vad-Nielsen, Anders Lade Nielsen, Yonglun Luo
When studying complex interconnected regulatory networks, effective methods for simultaneously manipulating multiple genes expression are paramount. Previously, we have developed a simple method for generation of an all-in-one CRISPR gRNA expression array. We here present a Golden Gate Assembly-based system of synergistic activation mediator (SAM) compatible CRISPR/dCas9 gRNA expression array for the simultaneous activation of multiple genes. Using this system, we demonstrated the simultaneous activation of the transcription factors, TWIST, SNAIL, SLUG, and ZEB1 a human breast cancer cell line...
March 26, 2018: Journal of Biotechnology
Francesca Ceroni, Alice Boo, Simone Furini, Thomas E Gorochowski, Olivier Borkowski, Yaseen N Ladak, Ali R Awan, Charlie Gilbert, Guy-Bart Stan, Tom Ellis
Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct...
March 26, 2018: Nature Methods
Ya Feng Cui, Yun Qin Yan, Dan Liu, Yu Sheng Pang, Jiang Wu, Shu Feng Li, Hui Li Tong
C2C12 murine myoblasts are a common model for studying muscle differentiation. Platelet endothelial aggregation receptor-1 (PEAR1), an epidermal growth factor repeat-containing transmembrane receptor, is known to participate in platelet contact-induced activation. In the present study, we demonstrated that PEAR1 is involved in the differentiation of C2C12 murine myoblasts. Western blotting and immunofluorescence staining were used to determine PEAR1 expression and localization during C2C12 cell differentiation...
March 22, 2018: Experimental Cell Research
Yu Hong, Guangqing Lu, Jinzhi Duan, Wenjing Liu, Yu Zhang
CRISPR/dCas9 binds precisely to defined genomic sequences through targeting of guide RNA (gRNA) sequences. In vivo imaging of genomic loci can be achieved by recruiting fluorescent proteins using either dCas9 or gRNA. We thoroughly validate and compare the effectiveness and specificity of several dCas9/gRNA genome labeling systems. Surprisingly, we discover that in the gRNA-labeling strategies, accumulation of tagged gRNA transcripts leads to non-specific labeling foci. Furthermore, we develop novel bimolecular fluorescence complementation (BIFC) methods that combine the advantages of both dCas9-labeling and gRNA-labeling strategies...
March 22, 2018: Genome Biology
Nicholas J Morse, James M Wagner, Kevin Reed, Madan R Gopal, Lars Lauffer, Hal S Alper
Efficient guide RNA expression often limits CRISPR-Cas9 implementation in new hosts. To address this limitation in fungal systems, we demonstrate the utility of a T7 polymerase system to effectively express sgRNAs. Initially, we developed a methodology in Saccharomyces cerevisiae using a modified version of the T7 P266L mutant polymerase with an SV40 nuclear localization signal to allow guide RNA expression immediately downstream of a T7 promoter. To improve targeting efficiency, guide RNA design was found to be tolerant to three mismatches or up to three additional bases appended to the 5' end...
March 22, 2018: ACS Synthetic Biology
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