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CRISPR 101: RNA Editing with Cas13 and REPAIR

Posted by Mary Gearing on Nov 30, 2017 9:01:02 AM

Cas13 enzymes are quickly becoming major players in the CRISPR field. Just a year after Abudayyeh et al. (2016) identified Cas13a (C2c2) as a RNA-targeting CRISPR enzyme, Cox et al. have adapted Cas13b for precise RNA editing. This new system, termed REPAIR (RNA editing for programmable A to I (G) replacement) is the first CRISPR tool for RNA editing, and it displays high specificity and targeting flexibility. We’ll walk through how this tool was developed and potential ways you can use it in your research.

Find the plasmids from Cox et al. here!

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Topics: CRISPR, CRISPR 101

CRISPR 101: Targeting RNA with Cas13a (C2c2)

Posted by Joel McDade on Sep 21, 2017 10:07:21 AM

This post was updated on Nov 14, 2017.

CRISPR, and specifically Cas9 from S. pyogenes (SpCas9), is truly an exceptional genome engineering tool. It is easy to use, functional in most species, and has many applications (see a review of CRISPR applications here). That said, SpCas9 is not the only game in town, and other Cas proteins like SaCas9 and Cpf1 can circumvent the limitations associated with SpCas9. A novel protein, Cas13a (previously referred to as C2c2), has several unique properties that further expand the CRISPR toolbox. We'll cover how Cas13a was identified, the structure and function of Cas13a with a focus on what makes this molecule unique, and the various applications of Cas13a.

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Topics: CRISPR, CRISPR 101

CRISPR 101: Epigenetics and Editing the Epigenome

Posted by Mary Gearing on Feb 14, 2017 10:44:08 AM

This post was updated on Nov 29, 2017.

Epigenetic modifications are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation of the epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting the epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear. Today, targeted epigenetic modification at specific genomic loci is possible using CRISPR, and Addgene has a number of tools for this purpose!

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Topics: CRISPR, CRISPR 101

CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

Posted by Joel McDade on Oct 11, 2016 10:30:00 AM

CRISPR has taken the genome engineering world by storm owing to its ease of use and utility in a wide variety of organisms.  While much of current CRISPR research focuses on its potential applications for human medicine (1), the potential of CRISPR for genome engineering in plants is also being realized. There are a variety of reasons to consider using genome editing to change the genetic code of plants, including the development of crops with longer shelf life and the development of disease-resistant crops to increase agricultural yield (2,3). While it is certainly possible to select for desirable traits using traditional plant breeding approaches, these techniques are cumbersome, often requiring several rounds of selection to isolate plants with the phenotype of interest. Genome engineering, on the other hand, allows for targeted modification of known or suspected genes that regulate a desired phenotype.  In fact, CRISPR has already been used to engineer the genome of many plant species, including commonly used model organisms like Arabidopsis and Medicago truncatula and several crop species including potato, corn, tomato, wheat, mushroom, and rice (4). Despite the almost universal functionality of the CRISPR system in most organisms, some plant-specific changes to CRISPR components are necessary to enable genome editing in plant cells.  

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Topics: CRISPR, Plant Biology, CRISPR 101

Components of CRISPR/Cas9

Posted by Joel McDade on Feb 2, 2016 12:00:00 PM

At their most basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related Cpf1) to cut the genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After reading this post, we hope you will be caught up on much of the major CRISPR lingo and will be able to describe the functions of the various CRISPR/Cas9 components. Please note that while this post is intended to provide a general overview of CRISPR components, new Cas9 variants are being discovered all the time and the requirements of these different systems can vary (for example, read our posts on Cpf1 and eSpCas9/SpCas9-HF1 for some of the interesting properties of these exciting new nuclease tools).

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Topics: CRISPR 101

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