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Anti-CRISPRs: Switching Off CRISPR-Cas9

Posted by Beth Kenkel on May 23, 2017 10:30:00 AM

CRISPR-Cas9 technology is constantly evolving. Variants of Cas9 can be used for genome editingactivating gene expression, repressing gene expression, and much more. But there’s one thing that’s been missing: a way to shut off Cas9’s activity after it’s been turned on. The concern is that the longer Cas9 remains active in a cell, the greater chances there are for off-target edits to occur. Although methods to switch on Cas9 activity using light or drugs have been developed, the field lacked an “off-switch” for Cas9.

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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

CRISPR-Cas9: Tips for Optimizing sgRNA Activity

Posted by Guest Blogger on Feb 19, 2016 10:18:31 AM

 This post was contributed by John Doench of the Broad Institute.

For more infomation on gRNA design, see our post: How to Design Your gRNA for CRISPR Genome Editing

Whether designing a small number of sgRNAs for a gene of interest, or an entire library of sgRNAs to cover a genome, the ease of programing the CRISPR system presents an embarrassment of riches of potential sgRNAs. How to decide between them? By taking into account both on-target efficacy and the potential for off-target activity, experiments utilizing CRISPR technology can provide a straightforward means of determining loss-of-function phenotypes for any gene of interest.

Predicting sgRNA efficacy

We have recently examined sequence features that enhance on-target activity of sgRNAs by creating all possible sgRNAs for a panel of genes and assessing, by flow cytometry, which sequences led to complete protein knockout (1).

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Topics: CRISPR, CRISPR 101, CRISPR gRNAs

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, CRISPR 101

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