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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

Lentiviral CRISPR Libraries Enable Genome-Scale, Knockout Screening

Posted by Kendall Morgan on Feb 20, 2014 11:57:00 AM

It’s clear that CRISPR-Cas9 technology has really changed the game for anyone looking to quickly and easily manipulate specific genes. But what if you want to study genes all across the genome? Two new human lentiviral CRISPR library systems described in companion papers in Science in December were developed as an answer to that question. Additionally, a concurrent Nature Biotechnology article describes the development of a mouse lentiviral CRISPR library. 

“This enables you to do customized genetic modification on a scale that was really not possible before,” said the Broad Institute's Ophir Shalem. “Whole regions of the genome which were not accessible before are now accessible using this technology.”

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Topics: Plasmid Technology, Genome Engineering, CRISPR, pooled libraries

Quick, Versatile Plant Transgenesis with GreenGate Plasmids

Posted by Kendall Morgan on Jan 23, 2014 10:23:00 AM

A few years ago, researchers introduced what’s been called a major breakthrough in cloning technology with the Golden Gate method. For the first time, it was possible to quickly and efficiently assemble a large number of building blocks with just two or three inexpensive enzymes. Now, a new toolkit - aptly named GreenGate – offers all of those same advantages to researchers working in plant model systems.

“The cloning is all done in vitro; it’s no different in mouse, human or plants,” explained Jan Lohmann of Heidelberg University. “What is different is the plasmids you use to bring this into your target organism. We have designed a Golden Gate system based on the daily needs of an advanced molecular plant science lab.”

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Topics: Plasmid Technology, Genome Engineering, Synthetic Biology, Plasmid Kits

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