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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

Sleeping Beauty Awakens for Genome Engineering

Posted by Emma Markham on Jun 30, 2015 10:00:00 AM

Transposons are sequences of DNA that can move around in a genome. In a laboratory setting, transposons can be used to both introduce genes into an organism’s genome (see figure) and to disrupt endogenous genes at the site of insertion. In both of these cases, transposons combine the advantages of viruses and naked DNA while eliminating some of the drawbacks. Specifically, viruses are able to infect and replicate in host cells, but they are susceptible to cells’ defense mechanisms. The use of non-viral vectors, like transposons, avoids many, though not all, of these defenses. For some applications of genome engineering - such as certain forms of gene therapy - avoiding the use of viruses is also important for social and regulatory reasons.

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Topics: Plasmid Technology, Genome Engineering

Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

Posted by Mary Gearing on Jun 2, 2015 2:06:00 PM

In the short time since its development, CRISPR/Cas9 genome editing has been used to study the effect of gene knockout in vivo and in vitro, as well as to insert targeted mutations through homologous recombination. To further increase the utility of CRISPR/Cas9, it will be necessary to improve its multiplexing capacity. Multiplexing is key due to the natural redundancy of biological pathways;  to observe a phenotype, the modification of multiple genes is often necessary.

Guide RNAs (gRNAs) are commonly packaged in 400-500 bp cassettes containing the RNA pol III promoter, gRNA and pol III terminator. These relatively large cassettes (considering the gRNA itself is ~100 bases) limit the number of gRNAs that can be packaged together in a single vector. In addition, the pol III promoter is relatively weak, and low expression of gRNAs from these constructs could lower genome editing efficiency.

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Topics: Plasmid Technology, Genome Engineering, CRISPR

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