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Michael J Ziller, Juan A Ortega, Katharina A Quinlan, David P Santos, Hongcang Gu, Eric J Martin, Christina Galonska, Ramona Pop, Susanne Maidl, Alba Di Pardo, Mei Huang, Herbert Y Meltzer, Andreas Gnirke, C J Heckman, Alexander Meissner, Evangelos Kiskinis
The somatic DNA methylation (DNAme) landscape is established early in development but remains highly dynamic within focal regions that overlap with gene regulatory elements. The significance of these dynamic changes, particularly in the central nervous system, remains unresolved. Here, we utilize a powerful human embryonic stem cell differentiation model for the generation of motor neurons (MNs) in combination with genetic mutations in the de novo DNAme machinery. We quantitatively dissect the role of DNAme in directing somatic cell fate with high-resolution genome-wide bisulfite-, bulk-, and single-cell-RNA sequencing...
March 14, 2018: Cell Stem Cell
Suhyung Cho, Donghui Choe, Eunju Lee, Sun Chang Kim, Bernhard Ø Palsson, Byung-Kwan Cho
Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in Escherichia coli mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq)...
March 15, 2018: ACS Synthetic Biology
Scot P Ouellette
Chlamydia is an obligate intracellular bacterium and, as such, has significantly reduced its genome size and content. Although recent advances have allowed for transformation of C. trachomatis with an exogenous plasmid, genetic manipulation of Chlamydia remains challenging. In particular, the ability to create conditional knockouts has not been developed. This is particularly important given the likelihood that most genes within the small genome of Chlamydia may be essential. Here, I describe the feasibility of using CRISPR interference (CRISPRi) based on the catalytically inactive Cas9 variant (dCas9) of Staphylococcus aureus to inducibly, and reversibly, repress gene expression in C...
2018: Frontiers in Cellular and Infection Microbiology
Dan Gao, Fu-Sen Liang
The ability to edit specific epigenetic modifications at defined gene loci is pivotal to understand the biological function of these epigenetic marks. Here we describe a new inducible method to integrate the dCas9-based genome targeting with abscisic acid (ABA)-based chemically induced proximity (CIP) technologies to modify histone tail modifications at specific genome loci in living cells. ABA leads to rapid hetero-dimerization of the PYL and ABI proteins, which can be individually fused to dCas9 and a histone-modifying enzyme core domain...
2018: Methods in Molecular Biology
Sumiyo Morita, Takuro Horii, Izuho Hatada
DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are very important, and many epigenome editing technologies have been developed, based on zinc finger proteins, TALEs, and CRISPR/dCas9 targeting. We describe a protocol to induce and assess DNA demethylation on a target gene...
2018: Methods in Molecular Biology
Désirée Goubert, Mihály Koncz, Antal Kiss, Marianne G Rots
Epigenetic editing is a promising approach to modulate the local chromatin environment of target genes with the ultimate goal of stable gene expression reprogramming. Epigenetic editing tools minimally consist of a DNA-binding domain and an effector domain. The CRISPR/dCas9 platform, where mutations in the nuclease domains render the Cas9 protein inactive, is widely used to guide epigenetic effectors to their intended genomic loci. Its flexible nature, simple use, and relatively low cost have revolutionized the research field of epigenetic editing...
2018: Methods in Molecular Biology
Jessica A Kretzmann, Cameron W Evans, Marck Norret, Pilar Blancafort, K Swaminathan Iyer
The potential impact of CRISPR/Cas9, TALE, and zinc finger technology is immense, both with respect to their use as tools for understanding the roles and functions of the genomic elements and epigenome modifications in an endogenous context and as new methods for treatment of diseases. Application of such technologies has drawn attention, however, to the prevailing lack of effective delivery methods. Promising viral and non-viral methods both currently fall short when the efficient delivery of large plasmids or multiple plasmids is required...
2018: Methods in Molecular Biology
Rutger A F Gjaltema, Edda G Schulz
In a swift revolution, CRISPR/Cas9 has reshaped the means and ease of interrogating biological questions. Particularly, mutants that result in a nuclease-deactivated Cas9 (dCas9) provide scientists with tools to modulate transcription of genomic loci at will by targeting transcriptional effector domains. To interrogate the temporal order of events during transcriptional regulation, rapidly inducible CRISPR/dCas9 systems provide previously unmet molecular tools. In only a few years of time, numerous light and chemical-inducible switches have been applied to CRISPR/dCas9 to generate dCas9 switches...
2018: Methods in Molecular Biology
Antoine Vigouroux, Enno Oldewurtel, Lun Cui, David Bikard, Sven van Teeffelen
Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which RNA polymerase "kicks out" dCas9 from the target and completes transcription. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts...
March 8, 2018: Molecular Systems Biology
Chiahao Tsui, Carla Inouye, Michaella Levy, Andrew Lu, Laurence Florens, Michael P Washburn, Robert Tjian
Eukaryotic gene regulation is a complex process, often coordinated by the action of tens to hundreds of proteins. Although previous biochemical studies have identified many components of the basal machinery and various ancillary factors involved in gene regulation, numerous gene-specific regulators remain undiscovered. To comprehensively survey the proteome directing gene expression at a specific genomic locus of interest, we developed an in vitro nuclease-deficient Cas9 (dCas9)-targeted chromatin-based purification strategy, called "CLASP" (Cas9 locus-associated proteome), to identify and functionally test associated gene-regulatory factors...
March 5, 2018: Proceedings of the National Academy of Sciences of the United States of America
Nhung T Nguyen, Lian He, Margie Martinez-Moczygemba, Yun Huang, Yubin Zhou
Tools capable of modulating gene expression in living organisms is very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically-inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically-encoded calcium actuators with dCas9 to control gene expression...
February 28, 2018: ACS Synthetic Biology
Toshitsugu Fujita, Miyuki Yuno, Hodaka Fujii
OBJECTIVE: Previously, we developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology, which isolates specific genomic regions while preserving their molecular interactions. In enChIP, the locus of interest is tagged with engineered DNA-binding molecules such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, consisting of a catalytically inactive form of Cas9 (dCas9) and guide RNA, followed by affinity purification of the tagged locus to allow identification of associated molecules...
February 27, 2018: BMC Research Notes
Toshitsugu Fujita, Fusako Kitaura, Asami Oji, Naoki Tanigawa, Miyuki Yuno, Masahito Ikawa, Ichiro Taniuchi, Hodaka Fujii
We developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules...
February 26, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
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
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
Javier Gallego-Bartolomé, Jason Gardiner, Wanlu Liu, Ashot Papikian, Basudev Ghoshal, Hsuan Yu Kuo, Jenny Miao-Chi Zhao, David J Segal, Steven E Jacobsen
DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa , which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN ( FWA ) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype...
February 14, 2018: Proceedings of the National Academy of Sciences of the United States of America
Christina Galonska, Jocelyn Charlton, Alexandra L Mattei, Julie Donaghey, Kendell Clement, Hongcang Gu, Arman W Mohammad, Elena K Stamenova, Davide Cacchiarelli, Sven Klages, Bernd Timmermann, Tobias Cantz, Hans R Schöler, Andreas Gnirke, Michael J Ziller, Alexander Meissner
In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR-Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome...
February 9, 2018: Nature Communications
Yi Zheng, Wei Shen, Jian Zhang, Bo Yang, Yao-Nan Liu, Huihui Qi, Xia Yu, Si-Yao Lu, Yun Chen, Yu-Zhou Xu, Yun Li, Fred H Gage, Shuangli Mi, Jun Yao
CRISPR-Cas9 has been demonstrated to delete genes in postmitotic neurons. Compared to the establishment of proliferative cell lines or animal strains, it is more challenging to acquire a highly homogeneous consequence of gene editing in a stable neural network. Here we show that dCas9-based CRISPR interference (CRISPRi) can efficiently silence genes in neurons. Using a pseudotarget fishing strategy, we demonstrate that CRISPRi shows superior targeting specificity without detectable off-target activity. Furthermore, CRISPRi can achieve multiplex inactivation of genes fundamental for neurotransmitter release with high efficiency...
February 5, 2018: Nature Neuroscience
Ruth M Williams, Upeka Senanayake, Mara Artibani, Gunes Taylor, Daniel Wells, Ahmed Ashour Ahmed, Tatjana Sauka-Spengler
CRISPR-Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present an optimised, adaptable toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise novel efficient guide-RNA mini expression vectors utilising chick U6 promoters, provide a strategy for rapid somatic gene knockout and establish a protocol for evaluation of mutational penetrance by targeted next generation sequencing...
January 31, 2018: Development
Ran Peng, Ye Wang, Wan-Wan Feng, Xin-Jing Yue, Jiang-He Chen, Xiao-Zhuang Hu, Zhi-Feng Li, Duo-Hong Sheng, You-Ming Zhang, Yue-Zhong Li
BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation...
January 29, 2018: Microbial Cell Factories
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