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Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

Posted by Guest Blogger on Apr 7, 2015 12:21:00 PM

This post was contributed by Adam Chin-Fatt, a Ph.D. student at the University of Western Ontario. Adam summarizes Zalatan JG, et al.'s recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds." Adam has also created a video to help scientists visualize the concepts discussed in the paper.

The transcriptional control of multiple loci is deftly coordinated by the eukaryotic cell for the execution of many complex cellular behaviors, such as differentiation or metabolism. Our attempts to manipulate these cellular behaviors often fall short with the generation of various flux imbalances. The conventional approach has typically been to either systematically delete/overexpress endogenous genes or to introduce heterologous genes, but the trend of research has shifted in recent years toward tinkering with regulatory networks and multiplex gene control. However, these approaches are often met with the challenges of regulatory bottlenecks and their scope is limited by the lack of well characterized inducible promoters. Far removed from the bio-industry’s vision of ‘biofactories’, most successes in metabolic engineering have been limited to the overexpression of various metabolites in Escherichia coli or Saccharomyces cerevisiae with few techniques that are easily transferrable across host species or metabolic pathways. A new study takes us one step closer to the vision of metabolic biofactories by demonstrating the use of CRISPR-based RNA scaffolds to mimic natural transcriptional programs on multiple genes.

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Topics: Plasmid Technology, Genome Engineering, Synthetic Biology, CRISPR

Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens

Posted by Kendall Morgan on Feb 24, 2015 2:50:00 PM

CRISPR technology has changed how scientists edit and control genes, but according to the Broad Institute's Silvana Konermann, the first generation of CRISPR-Cas9 plasmids were not designed with gene activation in mind. “We had not managed to create a system to allow us to reliably activate essentially any gene,” she says. The technical leap from mutating and deactivating a gene or genes to selectively activating them with the CRISPR system was a large one.  The question for her then was this: Can you engineer CRISPR-Cas9 activators that work well enough on any gene that they could be used by people with little bioengineering expertise?

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Topics: Plasmid Technology, CRISPR, pooled libraries

An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

Posted by Guest Blogger on Jan 27, 2015 10:13:47 AM

This post was contributed by Jordan Ward who is a postdoctoral fellow at UCSF.

Emerging CRISPR/Cas9 editing technologies have transformed the palette of experiments possible in a wide range of organisms and cell lines. In C. elegans, one of the model organisms which I use to study gene regulation during developmental processes, CRISPR/Cas9 allows us to knock out sequences and introduce mutations and epitopes with unprecedented ease. In the last year, several advances in C. elegans genome editing using CRISPR/Cas9 have emerged, which I will describe below. These new C. elegans approaches rapidly enrich for editing events without the need for any selective marker to remain in the edited animal. To my knowledge these approaches have not yet been extended to other organisms/cell lines, though it is likely that many aspects will broadly improve editing efficiency.

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Topics: Plasmid Technology, Genome Engineering, CRISPR

Plasmids 101: Cre-lox

Posted by A Max Juchheim on Jan 13, 2015 10:47:00 AM

In previous posts for our Plasmids 101 series, we examined a number of important plasmid elements – promoters, origins of replication, protein tags, and antibiotic resistance markers (just to name a few). In this edition, we’re going to take a look at a very interesting tool that can be used for creating (excuse the pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells, and/or tissue-specific cell types: Cre-lox recombination.

What is Cre-lox?

The Cre-lox system is a technology that can be used to induce site-specific recombination events. The system consists of two components derived from the P1 bacteriophage: the Cre recombinase and a loxP recognition site. The P1 bacteriophage uses these components as part of its natural viral lifecycle, and researchers have adapted the components for use in genome manipulation.

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Topics: Plasmid Technology, Plasmid Elements, Plasmids 101

Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

Posted by Larissa Haliw on Dec 2, 2014 2:23:00 PM

Hodaka Fujii, M.D., Ph.D., is an Associate Professor at Osaka University. The Fujii lab specializes in developing novel technologies to analyze molecular mechanisms of genome functions such as epigenetic regulation and transcription by using locus-specific chromatin immunoprecipitation (locus-specific ChIP). These methods consist of insertional chromatin immunoprecipitation (iChIP) and engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), both developed in the lab. In June 2014, Dr. Fujii joined Addgene's Advisory Board. 

Addgene: Your lab has worked extensively with enChIP systems. Can you describe this technology and its advantages?

Fujii: In the last several years, my lab has been working on development of technologies for biochemical analysis of genome functions such as transcription and epigenetic regulation. To elucidate molecular mechanisms of regulation of genome functions, we need to identify molecules associated with specific genomic regions of interest in a non-biased manner. To achieve this goal, it is necessary to isolate specific genomic regions while retaining molecular interactions.

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Topics: Plasmid Technology, Interview, Investigator Feature

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