CRISPR transcriptional repression devices and layered circuits in mammalian cells

Kiani, Samira; Beal, Jacob; Ebrahimkhani, Mo R.; Huh, Jin; Hall, Richard; Xie, Zhen; Li, Yinqing; Weiss, Ron

doi:10.1038/nmeth.2969

Cited by 185 publications

(225 citation statements)

References 23 publications

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“…The dCas9 has been used for transcriptional activation and repression [134][135][136][137], for localizing fluorescent protein labels [138], and for recruiting histone modifying enzymes [139,140]. Other applications of Cas9 include building gene circuits [141][142][143], creating new anti-microbials [144], antivirals [145][146][147], and large-scale gain-and loss-of-function screening [148][149][150][151] (Table 1).…”

Section: Genome Editing Applicationsmentioning

confidence: 99%

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Mohanraju

Makarova

Zetsche

et al. 2016

Science

571

460

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Background: Prokaryotes have evolved multiple systems to combat invaders such as viruses and plasmids. Examples of such defence systems include receptor masking, restriction-modification (R-M systems), DNA interference (Argonaute), bacteriophage exclusion (BREX or PGL) and abortive infection, all of which act in an innate, non-specific manner. In addition, prokaryotes have evolved adaptive, heritable immune systems, i.e. clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR-associated proteins (CRISPR-Cas). Adaptive immunity is conferred by the integration of DNA sequences from an invading element into the CRISPR array (adaptation), which is transcribed into long pre-CRISPR (pre-cr) RNAs and processed into short crRNAs (expression), which guide Cas proteins to specifically degrade the cognate DNA on subsequent exposures (interference). Advances:A plethora of distinct CRISPR-Cas systems are represented in genomes of most archaea and almost half of the bacteria. The latest CRISPR-Cas classification scheme delineates two classes that are each subdivided into three types. Integration of biochemistry and molecular genetics has contributed significantly to revealing many of the unique features of the variant CRISPR-Cas types. Additionally, structural analysis and single molecule studies have further advanced our understanding of the molecular basis of CRISPR-Cas functionality. Recent progress includes relevant steps in the adaptation stage, when fragments of foreign DNA are processed and incorporated as new spacers into the CRISPR array. In addition, three novel CRISPR-Cas types (IV, V, and VI) have been identified, and in particular, the type V interference complexes have been experimentally characterized. Moreover, the ability to easily program sequence-specific DNA targeting and cleavage by CRISPRCas components, as demonstrated for Cas9 and Cpf1, allows for the application of CRISPR-Cas components as highly effective tools for genetic engineering and gene regulation in a wide range of eukaryotes and prokaryotes.The pressing issue of off-target cleavage by the Cas9 nuclease is being actively addressed using structure-guided engineering.Outlook: Although our understanding of the CRISPR-Cas system has increased tremendously over the past few years, much remains to be done. About the evolution of CRISPR-Cas systems, the continuing discovery of novel CRISPR-Cas variants will provide direct tests of the recently proposed modular scenario. The recent discovery and characterization of new CRISPR-Cas types with many unique features implies that our current knowledge has relatively limited power for predicting the functional details of distantly related variants. Hence, newly discovered CRISPR-Cas systems need to be thoroughly dissected with the aforementioned multi-disciplinary approaches to gain insight in their biological role, to unravel their molecular mechanism, and to harness their potential for biotechnology. As to the biology, key outstanding questions include the ecological roles of microbial ...

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Section: Genome Editing Applicationsmentioning

confidence: 99%

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Mohanraju

Makarova

Zetsche

et al. 2016

Science

571

460

View full text Add to dashboard Cite

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“…Bare dSpCas9:gRNA was also shown to work as a transcriptional repressor in mammalian cells. 16,41 In this case, promoter steric occupation prevents the binding of RNA Polymerase II (or III). This competition mechanisms is usually used in yeast cells.…”

Section: ããmentioning

confidence: 99%

CRISPR-Cas type II-based Synthetic Biology applications in eukaryotic cells

Marchisio

Huang

2017

RNA Biology

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The CRISPR-Cas system has rapidly reached a huge popularity as a new, powerful method for precise DNA editing and genome reengineering. In Synthetic Biology, the CRISPR-Cas type II system has inspired the construction of a novel class of RNA-based transcription factors. In their simplest form, they are made of a CRISPR RNA molecule, which targets a promoter sequence, and a deficient Cas9 (i.e. deprived of any nuclease activity) that has been fused to an activation or a repression domain. Up-and downregulation of single genes in mammalian and yeast cells have been achieved with satisfactory results. Moreover, the construction of CRISPR-based transcription factors is much simpler than the assembly of synthetic proteins such as the Transcription Activator-Like effectors. However, the feasibility of complex synthetic networks fully based on the CRISPR-dCas9 technology has still to be proved and new designs, which take into account different CRISPR types, shall be investigated.

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“…Weiss and his colleagues have also created multiple gene tweaks in a single experiment 8 , making it faster and easier to build complicated biological circuits that could, for example, convert a cell's metabolic machinery into a biofuel factory. "The most important goal of synthetic biology is to be able to program complex behaviour via the creation of these sophisticated circuits, " he says.…”

Section: Broken Scissorsmentioning

confidence: 99%

CRISPR: gene editing is just the beginning

Ledford¹

2016

Nature

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CRISPR transcriptional repression devices and layered circuits in mammalian cells

Cited by 185 publications

References 23 publications

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

CRISPR-Cas type II-based Synthetic Biology applications in eukaryotic cells

CRISPR: gene editing is just the beginning

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