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Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

Posted by Mary Gearing on Jun 2, 2015 2:06:00 PM

In the short time since its development, CRISPR/Cas9 genome editing has been used to study the effect of gene knockout in vivo and in vitro, as well as to insert targeted mutations through homologous recombination. To further increase the utility of CRISPR/Cas9, it will be necessary to improve its multiplexing capacity. Multiplexing is key due to the natural redundancy of biological pathways;  to observe a phenotype, the modification of multiple genes is often necessary.

Guide RNAs (gRNAs) are commonly packaged in 400-500 bp cassettes containing the RNA pol III promoter, gRNA and pol III terminator. These relatively large cassettes (considering the gRNA itself is ~100 bases) limit the number of gRNAs that can be packaged together in a single vector. In addition, the pol III promoter is relatively weak, and low expression of gRNAs from these constructs could lower genome editing efficiency.

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Topics: Plasmid Technology, Genome Engineering, CRISPR

Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

Posted by Guest Blogger on May 22, 2015 12:58:12 PM

This post was contributed by Kevin Esvelt, a Wyss Technology Development Fellow at the Wyss Institute and Harvard Medical School.

Scientists making transgenic organisms with Cas9 should be aware of the potential hazards of creating “gene drives” capable of spreading through wild populations. Whereas most genomic changes impose a fitness cost and are eliminated by natural selection, gene drives distort inheritance in their favor and consequently can spread even when costly.

If even a single organism carrying a synthetic gene drive were to escape the laboratory, the drive could eventually spread through the entire wild population with unpredictable ecological effects. Because the consequences of such a mistake would necessarily extend far beyond the laboratory and seriously damage public trust in scientists, experiments involving potential gene drives should be conducted with extreme caution.

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Topics: Plasmid Technology, Lab Tips, CRISPR, CRISPR 101

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

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