Cas9 is used in genome editing to cut DNA at a specific location identified by a CRISPR guide RNA that binds to the target site. However, Cas9 can sometimes cut DNA in places where the sequence is similar to the target: these are called off-target effects, and can have serious implications, disrupting the function of otherwise healthy genes.
'An unintended cut at the wrong place in the human genome can have profound consequences,' said co-author Dr Michael Boettcher, of the Medical Faculty of the Martin Luther University, Germany. 'That is why we need a more specific system.'
The new research, published in Nature Chemical Biology, tackled this imprecision by investigating how Cas9 interacts with the guide RNA that directs the enzyme to the DNA target site.
The study uncovered a group of amino acid residues in the Cas9 'bridge helix', an intricate network that plays a critical role in guiding the enzyme to its target. These residues help form a stable loop, vital for Cas9 activity. The guide RNA bound to the Cas9 enzyme pairs with the complementary strand of the DNA target sequence whilst displacing the second DNA strand. Thus, cutting both strands of DNA.
The researchers went on to create new Cas9 variants by changing these amino acid residues in cells of the bacteria e-coli and discovered that they cut less frequently at off-target sites. It was further discovered that one of these variants also proved to increase the genome-editing specificity of Cas9 in human cells.
'Our results provide a new basis for further optimisation of CRISPR-Cas9,' said corresponding author Professor Emmanuelle Charpentier, director of the Max Planck Unit, Germany. 'They demonstrate the need to gain more knowledge about the biochemistry of CRISPR-Cas systems to further improve them.'