U.S. researchers have discovered how Cas1-Cas2, the proteins responsible for the ability of the CRISPR immune system in bacteria to adapt to new viral infections, identify the site in the genome where they insert viral deoxyribonucleic acid (DNA) so they can recognize it later and mount an attack.
While CRISPR is short for Clustered Regularly Interspaced Short Palindromic Repeats, Cas stands for CRISPR Associated Protein.
These proteins 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.
Detailed in a paper published in the July 21 issue of journal Science, Jennifer Doudna and her colleagues reported their use of electron microscopy and X-ray crystallography, performed at the Advanced Light Source at Lawrence Berkeley National Laboratory, the Stanford Linear Accelerator Center, and the HHMI electron microscope facility at UC Berkeley, to capture structures of Cas1-Cas2 in the act of inserting viral DNA into the CRISPR region.
Doudna is a professor of molecular and cell biology at the University of California, Berkeley.
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 research group discovered that the reaction requires that the target DNA bend and partly unwind, something that occurs only at the proper target.
CRISPR immune system allows bacteria to adapt and defend against the viruses that infect them. CRISPR 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, explained UC Berkeley graduate student Addison Wright and one of the paper's lead authors.
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 and 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.