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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

Cpf1: A New Tool for CRISPR Genome Editing

Posted by Mary Gearing on Oct 14, 2015 10:30:00 AM

This post was updated on Dec 5, 2017.

In 2015, Zetsche et al. added to the CRISPR toolbox with their characterization of two Cpf1 orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain a RuvC-like endonuclease domain, but they lack Cas9’s second HNH endonuclease domain. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. In certain cases, Cpf1 may be better suited for genome editing than Cas9 - read on to learn more about Cpf1 and check out our CRISPR guide for a refresher on CRISPR/Cas9. 

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Topics: Genome Engineering, CRISPR

CRISPR 101: Mammalian Expression Systems and Delivery Methods

Posted by Nicole Waxmonsky on Sep 24, 2015 10:30:00 AM

This post was updated on Dec 4, 2017.

CRISPR technology has been widely adopted for genome editing purposes because it's cheaper, faster, and easier than prior editing techniques. More and more CRISPR tools are being published each month, making CRISPR a great choice for your next experiment!

In this blog post we’ll provide an overview of some CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.

 

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Topics: Genome Engineering, CRISPR, CRISPR 101

CRISPR 101: Validating Your Genome Edit

Posted by Melina Fan on Jul 30, 2015 10:30:00 AM

You’ve created your gRNA expression construct and used Cas9 to introduce it into your target cells. Hooray! You’re ready to begin reading out data, right? Almost. In this blog post we’ll explain how to verify that your cells were appropriately edited. We’ll also cover the basic techniques for detecting insertion, deletion, and mutation events.

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Topics: Genome Engineering, CRISPR, CRISPR 101

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