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Programmable RNA complex could speed genome editing in the lab
Date:6/29/2012

anisms."

Doudna, a biochemist, is interested in the molecular mechanisms by which RNA can influence gene expression. She says the discovery of an RNA-programmable DNA cleaving enzyme stems from a collaboration with Charpentier established last year. 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 had discovered that in Streptococcus pyogenes, a human pathogen, crRNAs could only be produced in the presence of a second RNA, which they called a trans-activating crRNA (tracrRNA). In addition, the S. pyogenes and related CRISPR systems require just a single protein, Cas9, for immunity to viruses targeted by crRNAs.

Doudna's lab worked with the Charpentier lab to investigate how Cas9 and crRNAs function in this bacterial immune system. Martin Jinek, an HHMI Research Specialist in Doudna's lab, succeeded in purifying the Cas9 protein. Krzysztof Chylinski, a graduate student in the Charpentier lab who is located at the Max F. Perutz Laboratories at the University of Vienna, used that sample to show that Cas9 needed both crRNA and tracrRNA to guide and execute its a
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Contact: Jennifer Michalowski
michalow@hhmi.org
301-215-8576
Howard Hughes Medical Institute
Source:Eurekalert

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