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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

Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome Editing to Primary Cells

Posted by Kendall Morgan on Sep 30, 2014 4:50:49 PM

Researchers have shown that it is possible to deliver RNA-guided CRISPR/Cas9 nuclease complexes  using adenoviral vectors (AdVs), to a wide range of human cells, including mesenchymal stem cells, and in a rather straightforward manner. These adenoviral CRISPR/Cas9 genome editing tools developed and demonstrated by Manuel Gonçalves and his colleagues at Leiden University Medical Center are now available at Addgene along with a description of their experimental protocol. The three plasmids which have been deposited to Addgene are: pAdSh.PGK.Cas9, pAdSh.U6.gRNAS1, pAdSh.U6.gRNAGFP.

“Although AdVs are being deployed for delivering zinc-finger nucleases into human cells, we think they are still underused in the emerging field of genome editing,” Gonçalves said. “In contrast, AdVs are extensively being explored for genetic vaccination and oncolytic approaches. In genome editing, they are not used much, but we do think they have a very bright future.”

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Topics: Genome Engineering, CRISPR

CRISPR-Cas9 FAQs Answered!

Posted by Caroline LaManna on Mar 13, 2014 12:08:00 PM

As Kendall mentioned in Tuesday's blog post, keeping up with the newest CRISPR technologies and their applications can be exhausting. A quick search for "CRISPR", short-hand for Clustered Regularly Interspaced Short Palindromic Repeats, in Pubmed returned 728 articles (3/12/2014). With so many options for CRISPR plasmid tools and numerous experimental design decisions to make, it makes sense that scientists, many of whom are venturing into genome editing for the first time, have lots of questions.

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Topics: Plasmid Technology, Genome Engineering, Lab Tips, CRISPR

Keeping Up With CRISPR/Cas9

Posted by Kendall Morgan on Mar 11, 2014 1:55:37 PM

If any of you are finding it hard to keep up with the news on CRISPR, there's a pretty good reason for that. Lately, significant advances in the understanding and application of CRISPR/Cas9 technology are coming along at a fast and furious pace. In December, as we've blogged about before, there was the first direct demonstration that CRISPR's could be used to correct disease mutations, both in mice and in human cells. But that important advance was quickly followed by even splashier news: Jiahao Sha of Nanjing Medical University had successfully used the CRISPR/Cas9 system to precisely edit specific genes in monkeys.

"Our study shows that the CRISPR/Cas9 system enables simultaneous disruption of two target genes in one step without producing off-target mutations," Sha was quoted as saying when the news first broke. "Considering that many human diseases are caused by genetic abnormalities, targeted genetic modification in monkeys is invaluable for the generation of human disease models."

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Topics: Plasmid Technology, Genome Engineering, CRISPR

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