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Cre-ating New Methods for Site-specific Recombination in Drosophila

Posted by Mary Gearing on May 12, 2015 9:32:10 AM

Cre-lox recombination is an incredibly useful molecular biology tool, but like any biological system, it has certain drawbacks. First, the efficiency of Cre recombination varies for different constructs and cell types. Second, Cre may induce recombination at pseudo- or cryptic loxP sites (estimated to occur at a frequency of 1.2 per megabase in mammals), leading to DNA damage and developmental aberrations. In multiple systems, Cre itself, without the presence of a floxed construct, may produce a phenotype. This problem is especially stark in Drosophila, where expression of Cre from the standard UAS/GAL4 system is toxic to proliferating cells. A Cre-estrogen receptor ligand binding domain-fusion can prevent this toxicity, but with the caveat of partial rather than complete recombination. If you’re looking to use site-specific recombination in Drosophila, read on to learn about new recombinases suitable for this system.

Gerald Rubin’s lab sought to make complex genome modifications in Drosophila using multiple recombinases. To make multiple, precise genome edits, the recombinases used must have high activity and specificity with low cross-reactivity, as well as low toxicity.

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Topics: Plasmid Technology, Cre-lox, Drosophila

Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

Posted by Mary Gearing on Apr 24, 2015 10:39:00 AM

CRISPR-Cas9 genome editing may be the hot new way to manipulate gene expression, but other gene manipulation systems remain valuable to biology. Cre-lox recombination, discovered in the 1980s, is one of the most important ways to spatially and temporally control gene expression, especially in in vivo models, and new Cre-lox based technologies are still being developed today. In this post, I will highlight the evolution of the  Brainbow multicolor labeling system - a perfect example of the continued utility of Cre-lox. Check out our previous blog post, Plasmids 101: Cre-lox, if you need a quick primer on how Cre-lox recombination works.

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Topics: Plasmid Technology, Genome Engineering, Fluorescent Proteins

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

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