By Bio-IT World Staff
February 7, 2014 | A new study of the Cas9 protein, used in both natural and artificial CRISPR gene editing complexes, reveals its crystal structure and new information on its partnership with gRNA to target specific DNA sequences for cleaving. Cas9 is the key enzyme in the CRISPR system, making double-strand cuts in native DNA – allowing genetic engineers to remove sequences from the genome, and even introduce new sequences to replace them. The protein uses gRNA (guide RNA) to target particular regions of the genome, but the physical processes by which gRNA, Cas9 and DNA interact are not yet fully understood.
In a paper published yesterday in Science, co-senior authors Jennifer Doudna and Eva Nogales, of the Howard Hughes Medical Institute at UC Berkeley, demonstrate the crystal structure of two distantly-related Cas9 proteins – one from the bacterium Streptococcus pyogenes, and one from Actinomyces naeslundii – using x-ray crystallography. While these two versions of Cas9 differ greatly in size and various structural domains, the newly obtained images show that their active sites, where gRNA bonds to the protein, are highly conserved across species. The researchers went on to obtain new images, through electron microscopy against a stained background, of the Cas9 proteins reacting with either just gRNA, or both gRNA and complementary DNA sequences. This second round of imaging revealed that gRNA changes the 3D structure of Cas9, opening a cleft between two active lobes of the protein which are then able to bind to DNA. This shows for the first time that gRNA does not only direct Cas9 to the appropriate DNA sequence to cut, but also activates the enzyme, which remains dormant in the absence of gRNA.
These insights into which sites on the Cas9 protein are functionally essential, and how it works with gRNA to cleave DNA, may help biological engineers modify CRISPR-Cas9 systems for use in particular gene editing applications.
In addition to her academic research into the CRISPR complex, Doudna is also a founding member of Editas, a company seeking to harness CRISPR-Cas9 for therapeutic use against genetic diseases in humans. (See, "Gene Therapy's Next Generation.")