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Optimizing Donor DNA for Enhanced CRISPR Genome Editing

Posted by Guest Blogger on Mar 24, 2016 10:30:00 AM

This post was contributed by guest blogger Chris Richardson, a Postdoctoral Researcher in Jacob Corn’s lab.

CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The interplay between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes Cas9 extremely effective as a genome editing reagent. This interplay falls into two broad categories and thus, causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining (NHEJ) pathway disrupt target gene sequences (thus inactivating genes), while breaks repaired by homology directed repair (HDR) pathways can modify the sequence of a gene (thus altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently than NHEJ and the efficiency of these editing reactions is low. Understanding the biological cause of this repair bias is a fascinating (and yet unanswered) question. Our recent paper (Richardson et al 2016) revealed some of the biophysical parameters that can influence the HDR/NHEJ decision.

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Topics: Genome Engineering, CRISPR

Modulate the Activity of 17 Signaling Pathways with One Kit!

Posted by Kendall Morgan on Mar 22, 2016 10:30:00 AM

When cancers are treated with drugs designed to hit them right where it hurts, the effects are often remarkable but fleeting.

“What’s been shown by others is that, in a relatively short amount of time, cancers become resistant to drugs, particularly targeted therapies,” said Kris Wood of Duke University Medical Center. “While we do not yet have a comprehensive understanding of how cancers become resistant, an emerging theme is that they do so by activating signaling pathways controlling properties like growth, survival, and differentiation.”

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Topics: Plasmid Technology, Cancer, Plasmid Kits

Minigenomes - a Safe Way to Study Dangerous Viruses

Posted by Guest Blogger on Mar 17, 2016 10:30:00 AM

This post was contributed by guest blogger Tessa Cressey.

The highly pathogenic Ebola virus belongs to the group of nonsegmented negative sense RNA viruses, along with other viruses that cause disease in humans such as measles, mumps, and rabies. Research on Ebola virus has been limited, in part, due to the necessity for working with this virus under the highest biosafety level conditions, BSL-4. In this regard, minigenome systems, such as the one developed for Zaire ebolavirus (EBOV) are extremely useful, allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4).

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Topics: Viral Vectors

Suggest A Plasmid You'd Like to Find at Addgene

Posted by Tyler Ford on Mar 16, 2016 1:30:00 PM


Have you ever been reading a paper and thought to yourself, “Oh! This plasmid would be perfect for my next experiment!” but were unable to find the plasmid at Addgene? Well, we’re here to help with our Suggest a Plasmid Campaign.

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Topics: Scientific Sharing, Inside Addgene

Tips for Titering Your Lentiviral Preps

Posted by Meghan Rego on Mar 15, 2016 10:30:00 AM


The day has arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested your lentivirus. Now it’s time to move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure to move a project forward, transductions should not be rushed into. Before you start any transduction, you should always titer your virus - that is determine the amount of virus you actually have in your prep. Taking time to properly titer your virus will not only ensure that your infection is designed in the best possible way but it may also save you time in the long run. Read on for an overview of the titering options and the benefits and drawbacks of different methods (for comprehensive protocols for all of the methods discussed here refer to
Kutner et. al.).

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Topics: Techniques, Viral Vectors

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