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Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing

Posted by Guest Blogger on May 5, 2015 11:54:00 AM

This post was contributed by Johnna Roose. This post was originally published on Johnna's New Under The Sun Blog and is part of her larger tutorial series, A Beginner’s Guide to Writing Scientific Manuscripts.

Any scientific manuscript will require numerous other references to scientific literature to substantiate the facts upon which it builds. This means you have to become familiar with a body of literature related to the topic. Finding reliable references and sorting out what they mean is no small task. As a scientist, it is useful to make literature searching and reading a regular part of your routine. Set a goal to read a certain number of papers each week to keep up with the research in your area. When you are in ‘writing-mode’ for a grant or a scientific manuscript, the reading will likely be more intense, but it is a general good practice to keep up with the scientific literature a little bit at a time.

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Topics: Career, Science Communication

CRISPR 101: Non-Homologous End Joining

Posted by Guest Blogger on Apr 16, 2015 11:45:08 AM

This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill.

One advantage to using the CRISPR/Cas system for genome engineering is the fact that Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses. After the initial cut, the next steps in the process involve repairing chromosomal DSBs. It is important to know that cells possess two major repair pathways  Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) – and how these pathways work, as this could be relevant when planning your experiment. This blog has previously considered the HDR pathway; below we’ll discuss NHEJ, and how it impacts what happens to Cas9-mediated DSBs in the genome.

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Topics: Genome Engineering, CRISPR, CRISPR 101

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

The Developmental Studies Hybridoma Bank: Over 25 Years of Antibody Sharing

Posted by Guest Blogger on Mar 10, 2015 10:25:06 AM

The following post was contributed by the Developmental Studies Hybridoma Bank.


Antibodies are the most widely used class of protein-binding reagents. In the lab, their binding specificity allows scientists to target proteins of interest for labeling, quantification, purification, chromatin immunoprecipitation and more. The Developmental Studies Hybridoma Bank (DSHB), directed by David R. Soll, is a repository for monoclonal antibodies, distributing over 3,000 antibodies and associated hybridoma cell lines.

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Topics: Scientific Sharing

CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

Posted by Guest Blogger on Feb 18, 2015 10:09:22 AM

The following post was contributed by Daniel Bauer and Matthew Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint of the CRISPR article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized Experiments (JOVE). The video publication by Stuart Orkin and Daniel Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations behind this research.

 

Using CRISPR/Cas9 for Targeted Genomic Deletions

We were inspired to produce intrachromosomal deletions based on the experiments of Kim and colleagues using zinc finger nucleases to harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety of loci. We were pleasantly surprised by the efficiency of the method. One observation was an inverse relationship between deletion size and frequency [3].

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Topics: Genome Engineering, Lab Tips, CRISPR, Protocols

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