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CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

Posted by Joel McDade on Jan 19, 2016 10:57:10 AM

The advent of CRISPR/Cas9 made it easier than ever to efficiently make precise, targeted genome modifications. Cas9 has been modified to enable researchers to knock out, activate, repress or even image your favorite gene.  But, with such a wide variety of Cas9-based reagents available, how do you choose which Cas9 is right for your particular experiment?  This blog post will help familiarize you with the wide array of Cas9s available through Addgene’s repository and make it easy to select the Cas9 reagent that is right for you.

The first thing to do in any CRISPR experiment is identify what, exactly, you are trying to do.  Are you trying to permanently knock-out a gene in a cell type or organism? Are you trying to reduce expression of a particular gene without permanently modifying the genome?  Does it make more sense to try and activate at a particular locus?  What about modifying the epigenome at a particular location?  As you might expect, the answer to this question will substantially affect your decisions about which Cas9 you need for your experiment.  Below is a brief summary of a few of the common genetic manipulations one can carry out using Cas9 and the specific Cas9s that can be used for each.

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Topics: CRISPR, CRISPR 101

Your Top Requested Plasmid in 2015!

Posted by Tyler Ford on Dec 30, 2015 10:30:00 AM

We’ve dug into the data from our repository to find our most requested plasmid in 2015. In line with all of the exciting developments surrounding CRISPR genome engineering in the past year, we're excited to announce that the plasmid most requested from the Addgene repository in 2015 was...

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Topics: Fun, Inside Addgene, CRISPR, pooled libraries

Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

Posted by Tyler Ford on Dec 16, 2015 10:30:00 AM

As evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR is a ground-breaking technology that’s already had a huge impact on research and will be affecting our everyday lives very soon. Not only is CRISPR having effects on various biological disciplines, the base technology itself is constantly improving. Cas9 variants have been modified for genome editing, activating gene expression, visualizing genomic loci, and much more. Now, researchers from the Zhang, Joung, and Doudna labs have improved the on-target specificity of the Cas9 nuclease with engineered variants: eSpCas9SpCas9-HF1, & HypaCas9.

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Topics: Genome Engineering, CRISPR

The PAM Requirement and Expanding CRISPR Beyond SpCas9

Posted by Joel McDade on Nov 12, 2015 10:30:00 AM

This post was updated on Dec 5, 2017.

Diverse genomes and genomic targets require a variety of tools to engineer them effectively. Since the discovery and engineering of dCas9, the CRISPR toolbox has expanded to include a variety of natural and engineered Cas proteins. Read on to learn how these tools can be used to expand CRISPR's reach to new genomic loci. 

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Topics: Genome Engineering, CRISPR

The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

Posted by Guest Blogger on Nov 3, 2015 10:30:00 AM

This post was contributed by guest blogger Cameron MacPherson at the Institut Pasteur

CRISPR Software and the Piñata Effect

Two years ago I was a part of a group (Biology of Host-parasite Interactions, Institut Pasteur, Paris) that changed genome editing in the malaria community for the better (Nat. Biotechnol., 2014). Given the timing, it shouldn’t be a surprise that the CRISPR system was involved. Today, that same laboratory enjoys a successful edit rate of over 90% in their work editing the genome of Plasmodium falciparum (the parasite that causes malaria). I attribute their success to technical expertise, thoughtful single guide RNA (sgRNA) design, and the abnormally low GC content of the Plasmodium falciparum genome. To put this last point into perspective, the Plasmodium falciparum genome contains only 0.66 million targetable NGG PAM sites whereas the human genome has about 300 million. With such a sparsely targetable genome, off-targeting is less of a worry and on-targeting likely more efficient. 

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Topics: Genome Engineering, CRISPR

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