Advances in CRISPR genome-editing

7 October 2014

Scientists at the Broad Institute have created a new ‘Cas9 mouse’ model, simplifying the application of the CRISPR-Cas9 system for in vivo genome editing experiments.

The CRISPR/Cas9 genome-editing technique published in 2013 and patented earlier this year is a novel form of artificial genome editing. It allows scientists to investigate the role of any specific gene in the development of disease by turning it on, turning it off or altering it and monitoring the effects. 

The original CRISPR/Cas9 genome-editing system requires injecting both Cas9 (a ‘cleaving’ enzyme that cuts DNA) and guide RNA (which directs Cas9 to the target DNA sequence) into cells. However, in the new system the mouse is equipped with Cas9 so only the guide RNA needs to be injected. Whilst the original system could be used to test the effects of mutations in vitro, the challenges of delivering the Cas9 enzyme in vivo were an obstacle now effectively removed in the ‘Cas9 mouse’ model.

The Cas9 mouse model is particularly useful in cases such as cancer where mutations in more than one gene are crucial to the disease process, allowing rapid analysis. Researchers have already used it to model mutations associated with lung adenocarcinoma. 

Feng Zhang, assistant professor at the McGovern Institute of Brain Research at MIT and senior co-author of the Cell paper reporting the new mouse model, said: “The goal in developing the mouse was to empower researchers so that they can more rapidly screen through the long list of genes that have been implicated in disease and normal biological processes.” The ‘Cas9 mouse’ is being made available to the entire scientific community on request.

CRISPR genome-editing is also driving new work against infectious disease. Two new Nature Biotechnology studies have used the system to tackle antibiotic-resistant bacteria.

The first study used it to tackle bacteria which contained genes for antibiotic resistance. The researchers created guide RNA for CRISPR genome-editing that targeted the gene for the NDM-1 enzyme, which allows bacteria to resist a broad range of beta-lactam antibiotics. In doing so, they killed 99% of NDM-1 carrying bacteria, whereas beta-lactam antibiotics did not induce any significant killing. They also showed that the CRISPR system could be used to target specific bacteria for removal from diverse bacterial communities using their genetic signatures. The researchers are now testing the approach on mice.    

In a separate study, researchers at the Rockefeller University have used CRISPR genome-editing to selectively kill antibiotic-resistant bacteria from a mixed population by targeting the bacterial plasmids that carry antibiotic resistance genes for destruction by Cas9. In the same way, targeting plasmids harbouring tetracycline resistance in Staphylocuccus aureus cells not only restored tetracycline sensitivity but also effectively ‘immunised’ other S.aureus cells, since the incorporation of Cas9 prevented uptake of resistance carrying plasmids. Lead researcher Luciano Marraffini warned that the system needs improvement to become less discriminating before it can be developed as a new class of antibiotics.

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