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Bacterial Endonculease Complex Is a New Tool for Precise Mammalian Genome Engineering

By BiotechDaily International staff writers
Posted on 07 Feb 2013
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A new tool based on endonucleases extracted from bacterial adaptive immune mechanisms that can be reprogrammed with customizable small, noncoding RNAs is beginning to be used to easily and specifically engineer the human genome.

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid.

The Cas9–crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites to generate site-specific nicks on opposite DNA strands. The Cas9–crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage.

The first description of genomic engineering using the CRISPR approach was published by investigators at the University of California, Berkeley (USA) in the August 17, 2012, issue of the journal Science. Two new papers by investigators at Harvard Medical School (Boston, MA, USA) appeared in the January 3, 2013, issue of Science and have established the concept of using CRISPR to modify the human and other mammalian genomes.

“Out of this somewhat obscure bacterial immune system comes a technology that has the potential to really transform the way that we work on and manipulate mammalian cells and other types of animal and plant cells,” said Dr. Jennifer Doudna, professor of molecular and cell biology and chemistry at the University of California, Berkeley. “This is a poster child for the role of basic science in making fundamental discoveries that affect human health. The ability to modify specific elements of an organism’s genes has been essential to advance our understanding of biology, including human health. However, the techniques for making these modifications in animals and humans have been a huge bottleneck in both research and the development of human therapeutics.

“Based on the feedback we have received, it is possible that this technique will completely revolutionize genome engineering in animals and plants,” said Dr. Doudna. “It is easy to program and could potentially be as powerful as the polymerase chain reaction (PCR).”

“I think this is going to be a real hit,” said Dr. George Church, professor of genetics at Harvard Medical School. “There are going to be a lot of people practicing this method because it is easier and about 100 times more compact than other techniques.”

Related Links:
University of California, Berkeley
Harvard Medical School

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Image: Left: Green actin fibers create architecture of the cell. Right: With cytochalasin D added, actin fibers disband and reform in the nuclei (Photo courtesy of the University of North Carolina).

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