Friday, 25 August 2017

How CRISPR proteins find their target

In addition to the Cas9 protein that bacteria use to bind and snip DNA, bacteria have other Cas proteins that know where to insert that viral DNA into the CRISPR region to remember which viruses have attacked and mount a defense. A research team has discovered how these proteins -- Cas1 and Cas2 -- locate and insert the viral DNA, and it relies on the flexibility of these enzymes and the shape of the DNA.

These proteins, which were recently used to encode a movie in the CRISPR regions of bacterial genomes, rely on the unique flexibility of the CRISPR DNA to recognize it as the site where viral DNA should be inserted, ensuring that "memories" of prior viral infections are properly stored.

The structures reveal that a third protein, IHF, binds near the insertion site and bends the DNA into a U-shape, allowing Cas1-Cas2 to bind both parts of the DNA simultaneously. The lead authors, graduate student Addison Wright and postdoctoral fellow Jun-Jie Liu, along with co-authors Gavin Knott, Kevin Doxzen and Eva Nogales, discovered that the reaction requires that the target DNA bend and partly unwind, something that only occurs at the proper target.

CRISPR systems are a bacterial immune system that allows bacteria to adapt and defend against the viruses that infect them. CRISPR stands for clustered regularly interspaced short palindromic repeats and refers to the unique region of DNA where snippets of viral DNA are stored for future reference, allowing the cell to recognize any virus that tries to re-infect. The viral DNA alternates with the "short palindromic repeats," which serve as the recognition signal to direct Cas1-Cas2 to add new viral sequences.

Specific recognition of these repeats by Cas1-Cas2 restricts integration of viral DNA to the CRISPR array, allowing it to be used for immunity and avoiding the potentially fatal effects of inserting viral DNA in the wrong place, Wright said.

While many DNA-binding proteins directly "read out" the nucleotides of their recognition sequence, Cas1-Cas2 recognize the CRISPR repeat through more indirect means: its shape and flexibility. In addition to coding for proteins, the nucleotide sequence of a stretch of DNA also determines the molecule's physical properties, with some sequences acting as flexible hinges and others forming rigid rods. The sequence of the CRISPR repeat allows it to bend and flex in just the right way to be bound by Cas1-Cas2, allowing the proteins to recognize their target by shape.

The discovery of how Cas1-Cas2 recognize their target opens the door for modification of the proteins themselves. By tweaking the proteins, researchers might be able to redirect them to sequences other than the CRISPR repeat and expand their application into organisms without their own CRISPR locus.


Addison V. Wright, Jun-Jie Liu, Gavin J. Knott, Kevin W. Doxzen, Eva Nogales, Jennifer A. Doudna. Structures of the CRISPR genome integration complex. Science, July 2017 DOI: 10.1126/science.aao0679

Posted by Dr. Tim Sandle

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