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Joel McDade

Joel R. McDade is a Business Development Manager at Addgene whose interests include Genome Editing and Science Education.

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The PAM Requirement and Expanding CRISPR Beyond SpCas9

Posted by Joel McDade on Aug 20, 2020 10:30:00 AM

Originally published Nov 12, 2015 and last updated Aug 20, 2020.

Cas9 can be used to modify any desired genomic target provided that (1) the sequence is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide PAM sequence serves as a binding signal for Cas9 and this sequence is a strict requirement for Cas9-mediated DNA cleavage.

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Topics: CRISPR, Cas Proteins

CRISPR 101: Targeting RNA with Cas13a (C2c2)

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

This post was updated on Jul 27, 2020.

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. 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, Cas Proteins

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

Genome engineering using Cas9/gRNA Ribonucleoproteins (RNPs)

Posted by Joel McDade on Apr 21, 2016 10:30:00 AM

CRISPR has quickly become the preferred system for genome engineering due to its simplicity, as it requires only Cas9 and a guide RNA (gRNA).  Choosing the correct method to deliver both Cas9 and gRNAs to your target cells is absolutely critical as failure to adequately express either component will result in a failed experiment.  In our previous blog post entitled “CRISPR 101 - Mammalian Expression Systems and Delivery Methods” we provided a general overview of the most common ways in which you can deliver Cas9 and gRNAs to your target cells and discussed a few key advantages and disadvantages of each method. In this blog post, we will go into greater detail about why and how Cas9/gRNA Ribonucleoprotein complexes (Cas9 RNPs) are being used for genome engineering experiments and provide a general framework for getting started with Cas9 RNPs in your research.

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Topics: CRISPR, CRISPR Expression Systems and Delivery Methods

Components of CRISPR/Cas9

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

Updated Mar 26, 2020.

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, xCas9 is a variant with increased PAM flexibiliy and eSpCas9/SpCas9-HF1 have increased targeting specificity).

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Topics: CRISPR, CRISPR 101

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