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New Details about DNA CRISPRs and One That Targets RNA

 Audio Interview with Michael and Becky Terns

Microbial clustered regularly interspaced short palindromic repeats- better known as "CRISPR"-systems act in some ways like mammalian immune systems.  pyrococcusIn the case of CRISPRs, however, they enable bacteria and ar chaea to fend off phages and other forms of invasive DNA. That defense now also extends to invasive RNA molecules, report researchers at the University of Georgia (UGA). Moreover, CRISPR systems in any particular host have a nifty way of distinguishing self from foreign genetic material, thus accounting for how each system spares its own genome while taking apart invading genomes, reports another research team from Northwestern University. Sequences for CRISPR loci were found in nearly all archaeal genomes and about half the bacterial genomes sequenced so far. In general, these systems depend on low-molecularweight CRISPR RNAs (crRNAs) to perform surveillance for foreign genetic materials. These specialized crRNA molecules work with a suite of CRISPR-associated (Cas) proteins, which are encoded by corresponding Cas genes. Between the repeated sequences at CRISPR gene loci are nonrepetitive spacers, whose sequences match viruses and other foreign genetic elements. In general, CRISPR loci generate crRNAs that direct Cas proteins to seek and destroy invaders with complementary sequences.

In the heat-loving archaeon
Pyrococcus furiosus, six Cas proteins make up the RAMP module, which forms a complex with crRNAs and destroys invading RNA, according to Michael and Becky Terns and their collaborators at UGA in Athens. This complex recognizes, binds, and cleaves only complementary single-stranded RNA, but not complementary single-stranded DNA substrates, according to Michael Terns. The targeted RNA cleavage occurs at a specific site along the 3' end of crRNAs. These findings were something of a surprise because other CRISPR/Cas systems act on DNA, he points out. Details appear in the November 25, 2009 Cell (139:
945-956).

"Different organisms solve the immunity problem differently, and not all CRISPR systems function in the same way," says Rodolphe Barrangou, a senior scientist at Danisco USA, Inc. in Madison, Wis., who closely follows CRISPR developments (
Microbe, April 2009, p. 224). Some CRISPR sequences act by blocking bacterial conjugation, point out Erik Sontheimer and postdoctoral fellow Luciano Marraffini at Northwestern University in Evanston, Ill. They were the first to show in a clinical strain of Staphylococcus epidermidis
that CRISPR prevents the horizontal gene transfer of a plasmid linked to antibiotic resistance.

Those findings, described in the December 19, 2008 issue of
Science (322:
1843-1845), raised the question of why CRISPR does not target "self" DNA in bacterial chromosomes and cause autoimmune destruction. "It seemed necessary to explain how the CRISPR locus itself avoids the fate of incoming phage DNA or plasmid DNA," Sontheimer says.

Further experiments in
S. epidermidis on plasmids carrying various mutations led to an explanation. "The flanking sequences located upstream of the spacers are different, and they determine self from non-self," Marraffini says. Thus, this differential complementarity between crRNA and the DNA target directs the CRISPR machinery not to attack "self" DNA. Details appear in the January 28, 2010 Nature (463:
568-571).

These findings "prompt additional thinking about how CRISPR interference could be exploited for practical purposes," Sontheimer says. For instance, knowing that CRISPR sequences sometimes target RNA suggests to the Terns at UGA the possibility of knocking down gene expression in microbial species, following a strategy that is similar to using RNA interference to explore gene functions in mammalian cells. "You can artificially target the messenger RNA of a particular gene, then study the downstream consequences of the loss of that protein," Michael Terns speculates.

CRISPR sequences also might be used to protect valuable cultures of bacteria, including those used for making cheese and yogurt, from predatory viruses or undesirable genetic material, according to Barrangou. "There is a great risk of phage attack in large industrial fermentation processes," he says. To protect against such damage, CRISPR sequences could be engineered into starter cultures, or bacterial strains could be selected on the basis of their CRISPR robustness. "CRISPR is a hot topic with lots of interest across many countries," he says.

Carol Potera
Carol Potera is a freelance writer in Great Falls, Mont.
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