[2012-06-29] For bacteria, snipping apart DNA that bears certain signature sequences is a defense mechanism. For scientists working in the lab, the same strategy can be a powerful research tool. With a newly discovered component of an adaptive bacterial immune system, scientists have identified a targeted method of slicing DNA that they say can be easily customized for a variety of applications in the lab.
Tools that snip apart DNA strands in defined locations are essential for editing genomes in the laboratory to study or alter gene function. To target the specific site in the genome they are interested in, researchers often have to design and produce a protein that will recognize and bind to that particular DNA sequence, a laborious and time-consuming process.
This could change. In a paper published online June 28, 2012, in the journal Science, Emmanuelle Charpentier, former group leader at the MFPL, University of Vienna, now located at the MIMS, Umea University and her collaborator Howard Hughes Medical Institute investigator Jennifer Doudna at the University of California, Berkeley report the discovery of an RNA-based complex used by bacteria to guide the DNA-cutting enzyme Cas9 to specific sites in the genomes of viruses and other invaders, thus silencing their genes. From this bacterial complex, Charpentier's lab, her graduate student Krzysztof Chylinski located at MFPL, University of Vienna and their colleagues in Berkeley, Doudna and Martin Jinek, an HHMI Research Specialist in Doudna's lab, have crafted a system with which an easily programmable guide RNA can be used in the lab to direct Cas9 to cleave double-stranded DNA at a desired target sequence.
"The system can be directed to any selected site. Because the guide RNA contains both the structure required for Cas9 binding and a separate guide sequence that can base pair with DNA, we can program Cas9 to cleave a specific site by simply changing the guide sequence. This system offers a straightforward way to cleave any desired site in a genome, which could be used to introduce new genetic information by coupling it to well-known cellular DNA recombination mechanisms."
Charpentier, a microbiologist, is interested in the molecular mechanisms by which RNA can influence gene expression. The discovery of the RNA-programmable DNA cleaving enzyme stems from a collaboration that she initiated last year with Jennifer Doudna at the University of California, Berkeley. Both labs were studying different aspects of RNA-based defensive systems in bacteria that recognize and destroy the genomes of invading viruses and plasmids.
First described in the late 1980s, the system is called CRISPR, for Clustered Regularly Interspaced Short Palindromic Repeats. In response to a viral infection or plasmid transformation, bits of the invader's DNA – known as proto-spacers – are integrated into the host chromosome. The captured sequences are transcribed and processed to form short crRNAs, which serve as RNA recognition elements that bind to corresponding sequences in foreign DNA. Guided by the RNAs, proteins known as Cas (CRISPR-associated) then move in and attack the invaders, cleaving their DNA and silencing them.
Researchers studying CRISPR systems in various bacteria had found that in most cases a single crRNA joins with a large, multi-protein complex to attack viruses and plasmids. However, Charpentier's lab had discovered previously that in a CRISPR subset in Streptococcus pyogenes, a human pathogen, crRNAs could only be produced in the presence of a second, trans-activating crRNA (tracrRNA) (Nature, 2011, 471(7340): 602-607). In addition, the S. pyogenes and related CRISPR systems require just a single protein, Cas9, for immunity to viruses targeted by crRNAs.
Charpentier's lab worked with the Doudna lab to investigate the molecular mechanism by which Cas9 and crRNAs function in this bacterial immune system. Martin Jinek in Doudna's lab succeeded in purifying the Cas9 protein, and working with that sample, Krzysztof Chylinski in Charpentier's lab, showed that Cas9 needed both crRNA and tracrRNA to guide and execute its attack.
The groups then decided to test whether they could link these two RNAs into a single, chimeric RNA molecule. Combining the elements of the crRNA and tracrRNA that were necessary for Cas binding and DNA target recognition into a single molecule would make the system easier to manipulate for laboratory use. It worked: the result was a DNA-cleaving enzyme that can be programmed with a single RNA molecule to cleave specific DNA sites.
The next steps, Charpentier and Doudna say, are to test the single-RNA construct along with Cas9 to find out whether the RNA-programmed enzyme works in eukaryotic organisms such as worms, fishes, plants and human cells. If that is successful, they anticipate many practical applications of the tool. For biotechnology efforts ranging from engineering biofuel-producing microorganisms to enabling cell-based medical therapies, "having a simple and inexpensive tool for genome editing available will be very important" they say.
(Courtesy of HHMI)
The Laboratory for Molecular Infection Medicine Sweden, MIMS, is the Swedish node of the Nordic EMBL Partnership for Molecular Medicine. The institute is dedicated to research on the molecular mechanisms of infections and the development of new antimicrobial strategies. MIMS is part of the research consortium Umeå Centre for Microbial Research, UCMR (http://www.ucmr.umu.se).
The Howard Hughes Medical Institute, a nonprofit medical research organization that ranks as one of the nation's largest philanthropies, plays a powerful role in advancing biomedical research and science education in the United States. Founded in 1953 by aviator and industrialist Howard R. Hughes, HHMI is headquartered in Chevy Chase, Maryland, and employs more than 3,000 individuals across the United States. Visit: http://rna.berkeley.edu
The Max F. Perutz Laboratories is a joint-venture of the University of Vienna and the Medical University of Vienna at the Campus Vienna Biocenter with research groups working on various aspects of Molecular Biology. Visit: http://www.mfpl.ac.at.
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A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Martin Jinek*,Krzysztof Chylinski*,Ines Fonfara, Michael Hauer, Jennifer A. Doudna Emmanuelle Charpentier (*equal contribution), Science DOI: 10.1126/science.1225829
Programmable DNA scissors: A double-RNA structure in the bacterial immune system has been discovered that directs Cas9 protein to cleave and destroy invading DNA at specific nucleotide sequences. This same dual RNA structure should be programmable for genome editing. (Image by H. Adam Steinberg, artforscience.com).
Read the paper in Scienc Published Online June 28 2012
Press release in Swedish from Umeå University
Web page of Emmanuelle Charpentier's lab