Modularized CRISPR/dCas9 Effector Toolkit for Target-Specific Gene Regulation

Agne, Michael; Blank, Ilona; Emhardt, Alica J.; Gäbelein, Christoph G.; Gawlas, Fenja; Gillich, Nadine; Gonschorek, Patrick; Juretschke, Thomas J; Krämer, Stefan; Louis, Natalie; Müller, Anne; Rudorf, Alina; Schäfer, Lisa M; Scheidmann, Manuel C.; Schmunk, Lisa; Schwenk, Philipp; Stammnitz, Maximilian R; Warmer, Philipp; Weber, Wilfried; Fischer, Adrian; Kaufmann, Beate; Wagner, Hanna J.; Radziwill, Gerald

doi:10.1021/sb500035y

Cited by 33 publications

(16 citation statements)

References 9 publications

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“…For example, rAAV-delivered Cas9 expression in vivo has the potential to last for years and even an entire lifetime in recipients. In addition, the catalytically dead Cas9 (dCas9) fused with effector protein domains has been shown to either activate or silence specific gene expression at the transcriptional level, representing a versatile approach to regulating gene expression[35][36][37] . This usually requires the continuing presence of dCas9-effector to exert a stable gene regulatory effect.…”

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confidence: 99%

Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses

et al. 2015

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CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9.

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confidence: 99%

Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses

et al. 2015

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“…It took a while before this study was followed by ZF-targeting the HER2/neu gene in cancer [66] and even in vivo by targeting the murine Fosb gene [67•]. Similarly, authors have fused a TALE, targeting the E - Cadherin gene, and dCas9, in combination with sgRNAs to target VEGF - A , to the SET domain of the histone methyltransferase G9a and demonstrated that this approach is effective in repressing genes, as seen with ZFPs [68, 69]. In the meantime, Zinc Fingers were also exploited in the first DNA methylation targeting studies by fusion to the catalytic domains of DNA methyltransferases Dnmt3a or including a fusion between Dnmt3a and Dnmt3L, which catalyze the de novo methylation of DNA.…”

Section: Epigenetic Repressionmentioning

confidence: 99%

Epigenetic Editing: On the Verge of Reprogramming Gene Expression at Will

Cano-Rodríguez

Rots

2016

Curr Genet Med Rep

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Genome targeting has quickly developed as one of the most promising fields in science. By using programmable DNA-binding platforms and nucleases, scientists are now able to accurately edit the genome. These DNA-binding tools have recently also been applied to engineer the epigenome for gene expression modulation. Such epigenetic editing constructs have firmly demonstrated the causal role of epigenetics in instructing gene expression. Another focus of epigenome engineering is to understand the order of events of chromatin remodeling in gene expression regulation. Groundbreaking approaches in this field are beginning to yield novel insights into the function of individual chromatin marks in the context of maintaining cellular phenotype and regulating transient gene expression changes. This review focuses on recent advances in the field of epigenetic editing and highlights its promise for sustained gene expression reprogramming.

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“…The procedure is easy and fast (a single cloning step, virus production, and transduction), worked with 70% to 100% efficiency (depending on the assay-T7 endonuclease assay or sequencing), and was specific for the target site. To this end, the endonuclease activity of Cas9 is disabled through the insertion of point mutations, and the mutant Cas9 is fused to transcriptional activator or repressor domains (e.g., Agne et al, 2014). Here, simultaneous single-strand breaks (nicks) at opposite strands of the DNA target site using a mutant version of Cas9 and appropriately spaced guide RNAs create double-stranded breaks while increasing the specificity for cleavage.…”

Section: Of the Human Fbxl3 Locus Consists Of A 20-nt Guide Sequence mentioning

confidence: 99%

Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells

2015

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Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms.

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Modularized CRISPR/dCas9 Effector Toolkit for Target-Specific Gene Regulation

Cited by 33 publications

References 9 publications

Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses

Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses

Epigenetic Editing: On the Verge of Reprogramming Gene Expression at Will

Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells

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