cleavage of the invading DNA,” says Charpentier.
“What was particularly
interesting was that the guide RNA was displaying very similar behavior to miRNA and siRNA,
the agents involved in RNA
interference in eukaryotic cells.” This finding intrigued the scientists, and
they began to read around the subject a little more. RNA interference is
the biological
process by which RNA molecules inhibit gene expression, and siRNA has been used in the past as a genetic tool to silence genes of interest,
albeit with varying degrees of
success. What excited Charpentier and her colleagues was that any molecule that was acting similar to siRNA might also have the potential to be used in a similar manner. In recent years, interest in genome editing
has mushroomed with the advent of techniques
using site-specific nucleases
such as TALENs, ZFNs and meganucleases. These technologies can introduce breaks in the DNA at specific sites, allowing for gene disruption or gene displacement but, despite being successfully used in various hosts, are held back by various limitations, of which the most significant is the time and cost
requirements of developing such
engineered, customised proteins for each desired target sequence.
specific DNA cleavage with this tool in
their own model
organisms, and it has proven to be effective in human cells, zebrafish, mice, bacteria and yeast. The potential of this system is huge,
allowing the ability to study genetics in ways that were previously impossible. For example, if there were three changes in or around a gene that might cause a disease, it would be difficult to study them directly using current methods. Now, however, one could take a cell from someone who has had their genome mapped and create an induced pluripotent stem cell
(a cell that behaves
similar to those in embryos), and then use the CRISPR-Cas system to edit the three changes. Researchers would then be able to compare this cell to a cell without those specific changes – an incredibly useful thing to be able to do in terms of creating new medicines, to give one example. Genome engineering using the CRISPR- Cas system promises to have
broad applications in synthetic biology, direct and
AT A GLANCE Project Information
Project Title: CRISPR-Cas9: Full title: Genomic scissors created from bacterial immune system
Project Objective: The project objective was to decipher the molecular mechanism by which proteins target and cleave invading genomes in CRISPR-Cas mediated bacterial adaptive immunity.
Project Duration and Timing: The project started in July 2008 and ended in July 2012: a total of 4 years. Research on the project is still continuing in Emmanuelle Charpentier’s laboratory.
“Our discovery stemmed from our interest in a family of small regulatory RNAs in bacteria” Realising the potential of their discovery,
the researchers published their findings in the journals Nature and Science, and very quickly the scientific community took notice. “The piece published in Science was the first to suggest that the CRISPR effector enzyme Cas9, guided by dual-RNAs for site-specific DNA cleavage, might be used for RNA- reprogrammable
genome editing,” says
Charpentier. “The significance of this was the idea of a system in which the RNA – relatively easy to manipulate - determined the point at which the DNA would be broken.” In bacteria, the Cas9 protein is guided by
two distinct RNA transcripts, crRNA and tracrRNA. This paper showed that this dual RNA could be reconfigured as a single-guide RNA (sgRNA), including sequences sufficient to programme Cas9 to introduce double- stranded breaks in target DNA. Numerous papers
have now been published from
researchers all over the world who were keen to see whether they could achieve site
www.projectsmagazine.eu.com
multiplexed perturbation of gene networks, and targeted ex vivo and in vivo gene therapy.
Future challenges will be to
analyse and address possible off-target effects and improve the efficiency and specificity of the system. In this regard, it will be important to compare CRISPR-Cas with existing genome-editing tools. In addition to genome editing, this approach the
offers exciting such possibilities as of
transcriptional gene silencing using an inactive Cas9, or engineering Cas9 to have new functions,
activation. The discovery and application of bacterial
systems, such as restriction
enzymes and thermostable polymerases, have revolutionised molecular biology in the past. With RNA-guided Cas9
that has the potential to reshape the genome engineering landscape in biotechnology and medicine.
55 enzymes,
bacteria now offer a versatile tool for rewriting genomic sequence
information transcriptional
Project Funding: The project was funded by the Swedish Research Council (grants K2010-57X-21436-01-3, K2013- 57X-21436-04-3 and 621-2011- 5752-LiMS) (Sweden), the Kempe Foundation (Sweden), Umeå University (Sweden), the Austrian Science Fund (grants P17238-B09 and W1207-B09) (Austria), the Austrian Agency for Research Promotion (grant 812138-SCK/KUG) (Austria), the Theodor Körner Fonds (Austria) and the European Community (FP6, BACRNAs-018618). The current research in Emmanuelle Charpentier’s laboratory is also funded by the Helmholtz Association and Humboldt Foundation in Germany.
Project Partners: Elitza Deltcheva, Krzysztof Chylinksi and Ines Fonfara (Emmanuelle Charpentier’s Laboratory, The Laboratory for Molecular Infection Medicine Sweden, Umea University, Umea, Sweden; Helmholtz Centre for Infection Research, Hannover Medical School, Germany); Martin Jinek and Jennifer Doudna (University of California, Berkeley, USA)
Contact: Tel: 49 531 61815500 Email:
emmanuelle.charpentier@
helmholtz-hzi.de Web:
www.helmholtz-hzi.de/de/ forschung/forschungsschwerpunkte/ bakterielle_und_virale_ krankheitserreger/regulation_in_der_ infektionsbiologie/e_charpentier/
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