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CRISPR 101: Homology Directed Repair

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

This post was updated on November 3, 2017.

DNA lesions are sites of structural or base-pairing damage of DNA. Perhaps the most harmful 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

CRISPR 101: A New Series on Genome Editing & CRISPR-Cas

Posted by Marcy Patrick on Mar 5, 2015 12:06:12 PM

I am sure by now you have heard of CRISPRs. (If not, you can get up to speed here and here and here.) With such a fast moving technology, it is sometimes hard to keep pace with the new advances let alone remember the (maybe) long forgotten details of the biological process required to effectively design and utilize these tools. We certainly understand and are here to help!

Starting next week, we'll release the first post in our newest blog series - CRISPR 101 - a companion series to our popular Plasmids 101 articles. These posts are created to educate all levels of scientists and provide a resource for some of the basic principles driving CRISPRs and genome editing technology.

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Topics: Genome Engineering, Plasmids 101, CRISPR, CRISPR 101

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

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

Trends in CRISPR and SynBio Technologies [Slideshare]

Posted by Joanne Kamens on Feb 4, 2015 10:58:00 AM

Addgenie Eric Perkins attended the recent Keystone Meeting "Precision Genome Engineering and Synthetic Biology". His reflections on the program are here. This was a great opportunity for Addgene to present our own data on plasmid deposits and distirbution for these fast moving fields. 

Addgene is a global nonprofit plasmid repository. Over 2,000 labs have deposited plasmids to Addgene and we distribute over 130,000 plasmids in 2014. Thus, we are in a unique position to observe and quantify how new technologies are being disseminated through the scientific community.

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Topics: Hot Plasmids, Genome Engineering, Inside Addgene, Synthetic Biology, CRISPR

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