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Programmable DNA scissors found for bacterial immune system

By R&D Editors | June 29, 2012

Genetic engineers and genomics researchers should
welcome the news from the Lawrence Berkeley National Laboratory (Berkeley Lab)
where an international team of scientists has discovered a new and possibly
more effective means of editing genomes. This discovery holds potentially big
implications for advanced biofuels and therapeutic drugs, as genetically
modified microorganisms, such as bacteria and fungi, are expected to play a key
role in the green chemistry production of these and other valuable chemical
products.

Jennifer Doudna, a biochemist with Berkeley Lab’s
Physical Biosciences Division and professor at the University of California
(UC) Berkeley,
helped lead the team that identified a double-RNA structure responsible for
directing a bacterial protein to cleave foreign DNA at specific nucleotide
sequences. Furthermore, the research team found that it is possible to program
the protein with a single RNA to enable cleavage of essentially any DNA
sequence.

“We’ve discovered the mechanism behind the RNA-guided
cleavage of double-stranded DNA that is central to the bacterial acquired
immunity system,” says Doudna, who holds appointments with UC Berkeley’s
Department of Molecular and Cell Biology and Department of Chemistry, and is an
investigator with the Howard Hughes Medical Institute (HHMI). “Our results
could provide genetic engineers with a new and promising alternative to
artificial enzymes for gene targeting and genome editing in bacteria and other
cell types.”

Doudna is one of two corresponding authors of a paper
in Science describing this work titled “A programmable dual RNA-guided
DNA endonuclease in adaptive bacterial immunity.” The second corresponding
author is Emmanuelle Charpentier of the Laboratory for Molecular Infection
Medicine at Sweden’s Umeå University.
Other coauthors of the paper were Martin Jinek, Krzysztof Chylinski, Ines
Fonfara, and Michael Hauer.

Bacterial and archaeon microbes face a never-ending
onslaught from viruses and invading circles of nucleic acid known as plasmids.
To survive, the microbes deploy an adaptive-type nucleic acid-based immune
system that revolves around a genetic element known as CRISPR, which stands for
Clustered Regularly Interspaced Short Palindromic Repeats. Through the
combination of CRISPRs and associated endonucleases, called CRISPR-associated—”Cas”—proteins,
bacteria, and archaeons are able to use small customized crRNA molecules (for
CRISPR-derived RNA) to target and destroy the DNA of invading viruses and
plasmids.

There are three distinct types of CRISPR/Cas
immunity systems. Doudna and her colleagues studied the Type II system which
relies exclusively upon one family of endonucleases for the targeting and
cleaving of foreign DNA, the Cas9 proteins.

“For the Type II CRISPR/Cas system, we found that
crRNA connects via base pairs with a trans-activating RNA (tracrRNA), to form a
two-RNA structure,” Doudna says. “These dual RNA molecules (tracrRNA:crRNA)
direct Cas9 proteins to introduce double-stranded DNA breaks at specific sites
targeted by the crRNA-guide sequence.”

Doudna and her colleagues demonstrated that the
dual tracrRNA:crRNA molecules can be engineered as a single RNA chimera for
site-specific DNA cleavage, opening the door to RNA-programmable genome
editing.

“Cas9 binds to the tracrRNA:crRNA complex which in
turn directs it to a specific DNA sequence through base-pairing between the crRNA
and the target DNA,” Doudna says. “Microbes use this elegant mechanism to
cleave and destroy viruses and plasmids, but for genome editing, the system
could be used to introduce targeted DNA changes into the genome.”

Doudna notes that the “beauty of CRISPR loci” is
that they can be moved around on plasmids.

“It is well-established that CRISPR systems can be
transplanted into heterologous bacterial strains,” she says. “Also, there is
evidence to suggest that CRISPR loci are horizontally transferred in nature.”

Doudna and her colleagues are now in the process of
gathering more details on how the RNA-guided cleavage reaction works and
testing whether the system will work in eukaryotic organisms including fungi,
worms, plants and human cells.

“Although we’ve not yet demonstrated genome
editing, given the mechanism we describe it is now a very real possibility,”
Doudna says.

Source: Lawrence Berkeley National Laboratory

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