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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

An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

Posted by Guest Blogger on Jan 27, 2015 10:13:47 AM

This post was contributed by Jordan Ward who is a postdoctoral fellow at UCSF.

Emerging CRISPR/Cas9 editing technologies have transformed the palette of experiments possible in a wide range of organisms and cell lines. In C. elegans, one of the model organisms which I use to study gene regulation during developmental processes, CRISPR/Cas9 allows us to knock out sequences and introduce mutations and epitopes with unprecedented ease. In the last year, several advances in C. elegans genome editing using CRISPR/Cas9 have emerged, which I will describe below. These new C. elegans approaches rapidly enrich for editing events without the need for any selective marker to remain in the edited animal. To my knowledge these approaches have not yet been extended to other organisms/cell lines, though it is likely that many aspects will broadly improve editing efficiency.

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Topics: Plasmid Technology, Genome Engineering, CRISPR

Addgene @ Keystone: Thoughts on Precision Genome Engineering and Synbio

Posted by Eric J. Perkins on Jan 15, 2015 8:50:00 AM

It's been about 14 years since I last attended a Keystone Meeting – far too long. Holding these meetings in relatively isolated resorts creates a sense of comradery with fellow attendees from the moment you arrive. Getting off the plane in Bozeman Sunday night, it was easy to spot meeting participants. They were the ones holding poster tubes (or as our baffled flight attendant called them, "long, skinny things") and generally exuding a very-tired-but-very-excited attitude. Riding up to the resort in the shuttle, our driver regaled us with tales of back country skiing, fly fishing, and local grizzly bear attacks. He described one such recent attack as "hilarious". Welcome to Montana!

Though sadly I will not be attending the entire meeting, Monday's talks alone were worth the trip. Dr. Dana Carroll's excellent keynote address was the first of 19 talks given over the course of the day. His talk, which focused on the history of genome engineering from ZFNs through TALENs and CRISPR-Cas nucleases, provided important context for the rest of the day. He was followed by three of the biggest names in the CRISPR-Cas9 field – Jennifer Doudna, Feng Zhang, and Keith Joung. All Addgene depositors! Addgene was mentioned specifically in Dr. Zhang's introduction. His willingness to share reagents so freely with the academic community has clearly made a huge impact on this field.

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Topics: Genome Engineering, Scientific Sharing, Synthetic Biology, CRISPR

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