CRISPR 101: Mammalian Expression Systems and Delivery Methods

Posted by Nicole Waxmonsky on Sep 24, 2015 10:30:00 AM


CRISPR technology has been widely adopted for genome editing purposes for numerous reasons including that it's cheaper, faster, and easier than prior editing techniques. With more and more CRISPR tools being published each month, you may be considering using CRISPR for your next experiment. In this blog post we’ll provide an overview of some CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.

 

 

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Topics: Genome Engineering, CRISPR, CRISPR 101

CRISPR 101: Validating Your Genome Edit

Posted by Melina Fan on Jul 30, 2015 10:30:00 AM

You’ve created your gRNA expression construct and used Cas9 to introduce it into your target cells. Hooray! You’re ready to begin reading out data, right? Almost. In this blog post we’ll explain how to verify that your cells were appropriately edited. We’ll also cover the basic techniques for detecting insertion, deletion, and mutation events.

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Topics: Genome Engineering, CRISPR, CRISPR 101

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

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

CRISPR 101: Homology Directed Repair

Posted by Chari Cortez on Mar 12, 2015 1:48:00 PM

DNA lesions are defined as sites of structural or base-pairing damage of DNA. Perhaps the most nocuous type of lesion results from breakage of both DNA strands – a double-strand break (DSB) – as repair of DSBs is paramount for genome stability. DSBs can be caused by intracellular factors such as nucleases and reactive oxygen species, or external forces such as ionizing radiation and ultraviolet light; however, these types of breaks occur randomly and unpredictably. To provide some control over the location of the DNA break, scientists have engineered plasmid-based systems that can target and cut DNA at specified sites. Regardless of what causes the DSB, the repair mechanisms function in the same way.

In this post, we will describe the general mechanism of homology directed repair with a focus on repairing breaks engineered in the lab for genome modification purposes.

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Topics: Genome Engineering, CRISPR, CRISPR 101

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