CRISPR-Cas9 Through Eyes of Two Leaders in the Field

Ben Matthews, PhD, Postdoctoral Fellow, Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, Click photo for Ben Matthews Profile

Ben Matthews, PhD, Postdoctoral Fellow, Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, Click photo for Ben Matthews Profile

In a comprehensive and definitive review published in Science, Jennifer Doudna and Emmanuelle Charpentier, two scientist who made seminal discoveries leading to the current boom in genome engineering, walk us through the (brief) history, scientific applications, and future promise of CRISPR-Cas9 [1].

Over the last few decades,the study of targeted genome engineering and adaptive immunity in bacteria were scientific fields on completely distinct paths, colliding only in late 2012 when the labs of Jennifer Doudna and Emmanuelle Charpentier reported the reprogramming of a double-strand DNA endonuclease, called Cas9.

Natural History of CRISPR. Image from: Image from 10.1126/science.1258096

Natural History of CRISPR. Image from: Image from 10.1126/science.1258096

They detailed a method by which Cas9, a component of the CRISPR-Cas class of adaptive immune systems found in wide swaths of bacteria and archaea, could be re-directed to target essentially any sequence of DNA through its interaction with a short RNA molecule known as a single guide RNA, or sgRNA [2] Cas9 can be targeted to any 17-20 nucleotide sequence in the genome, so long as it contains a short protospacer adjacent motif, or PAM, as its 3’neighbor.

The most commonly used nuclease, Cas9 from the bacteria Streptococcus pyogenes, has a 3 nucleotide PAM motif – NGG – allowing essentially unlimited flexibility in genome targeting (a GG is found once every 17bp in the Aedes aegypti genome). For this advance, Doudna and Charpentier were recently awarded the Breakthrough Prize in Life Sciences (Hollywood and Silicon Valley’s answer to the Nobel).

In just two years, CRISPR-Cas9 has largely supplanted the use of ZFNs and TALENs for targeted genome engineering, due to the convenience, cost benefit, and flexibility of targeting a nuclease by Watson-Crick RNA-DNA base-pairing as opposed to engineering of DNA-binding protein domains.

CRISPR-based technologies. Image from: Image from 10.1126/science.1258096

CRISPR-based technologies. Image from: Image from 10.1126/science.1258096

CRISPR-Cas9 has now been adapted to generate loss-of-function mutations, in vivo, in dozens of organisms ranging from bacteria to primates. It offers a particular boon to the insect research community due to the existence of microinjection and other methods to introduce reagents into the developing embryo. In the Vosshall lab, we now use CRISPR-Cas9 extensively in Aedes aegypti to generate frame-shift mutations via double-strand breaks, delete large sequences with multiple sgRNAs, and introduce exogenous sequences, such as fluorescent reporters, through homology-driven repair.

The promise of CRISPR-Cas9 goes beyond loss-of-function mutagenesis, and Doudna and Charpentier describe how protein fusions and targeted mutations of Cas9 can be used to generate single-strand DNA ‘nicks’, regulate gene expression, or perform imaging of chromosome dynamics. In addition, the adaptation of additional Cas enzymes from other species holds promise for increasing the flexibility of targeting. However, CRISPR-Cas9 cannot solve all problems associated with genome editing: at its core, it simply represents a more convenient platform for DNA targeting and still relies on host machinery for DNA damage repair, resulting, in many organisms, in a relatively low efficiency for homology-driven repair.

Applications of CRISPR-Cas9 according to Doudna and Charpentier. Image from 10.1126/science.1258096

Applications of CRISPR-Cas9 according to Doudna and Charpentier. Image from 10.1126/science.1258096

 

Finally, in what is perhaps an inevitable sign of the times, the discussion of their review veers towards the commercial and applied applications of this technology. The larger context for this is an increasingly vigorous battle over intellectual property and the commercialization of CRISPR-Cas9.[3] Thus far, those of us in academic labs, working on basic science and enjoying the flexibility and the power of CRISPR-Cas9, are immune from these machinations – may it continue to be so!

This is a nice review filled with a great timeline of the development of  genome editing technologies in parallel with a timeline of the discovery and study of CRISPR biology along with wonderfully clear and informative figures and authoritative insightful discussion.  Definitely worth reading.

About Ben Matthews

 

[1]  Doudna, J. A., & Charpentier, E. (2014). Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213), 1258096. doi:10.1126/science.1258096

[2]  Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816–821. doi:10.1126/science.1225829

[3]   http://www.huffingtonpost.com/pete-shanks/crispr-opportunities-for-_b_6295608.html

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