High resolution images of gene-editing in action
Image: Structural arrangements of Cas9 enzyme [Zhu et al]
In a breakthrough for gene-editing, researchers have used cryo-EM to capture high-resolution, 3D images of the Cas9 enzyme in the process of precisely cutting DNA strands.
The latest results will help researchers develop modified versions of the enzyme that can more efficiently and precisely alter targeted genes, providing a more efficient version of gene-editing tool CRISPR.
An image illustrating how the Cas9 protein rearranges itself (indicated by the arrow) to enable the ‘scissoring’ action for cutting DNA. [Zhu et al]
“It is exciting to be able to see at such a high level of detail how Cas9 actually works to cut and edit DNA strands," says University of British Colombia researcher, Professor Sriram Subramaniam, who led the cryo-EM studies.
"These images provide us with invaluable information to improve the efficiency of the gene-editing process so that we can hopefully correct disease-causing DNA mutations more quickly and precisely in the future," he adds.
CRISPR is a gene-editing tool that allows scientists to cut out unwanted genes or genetic material from DNA or add a desired sequence within a gene to change its function or regulate its activity.
CRISPR uses an enzyme called Cas9 that acts like scissors that cut a specific DNA sequence.
Once cuts are made on either side of the DNA, the cell initiates repair systems that rejoin the two ends of the DNA strands back together.
CRISPR-associated protein Cas9 (white) from Staphylococcus aureus based on Protein Database ID 5AXW. [Thomas Splettstoesser]
To better understand the sequence of events involved in the process, Subramaniam and colleagues used cryo-EM technology to image the Cas9 enzyme at work.
The images provide unprecedented glimpses of the stepwise molecular motions that occur in the course of DNA cutting by Cas9, including a snapshot of the cut DNA still attached to the enzyme immediately before release.
"One of the main hurdles preventing the development of better gene-editing tools using Cas9 is that we didn't have any images of it actually cutting DNA," says Dr Miljan Simonovic, University of Illinois. "But now we have a much clearer picture, and we even see how the major domains of the enzyme move during reaction and this may be an important target for modification."
Images revealed Cas9 trapped in three distinct structural arrangements while bound to nucleic acids.
Most remarkably, in two of the three states, the target DNA was cleaved but the enzyme remained bound to it, allowing this final step of the biochemical reaction sequence to be observed with high resolution.
"We discovered several new things about how Cas9 interacts with DNA and how it operates. One of the most interesting is how several enzyme domains move in concert and cycle between ordered and disordered states during reaction,” says Simonovic.
“This feature of the enzyme in which some domains are stable while others seem to be in a process of rapidly moving between different conformational states before settling down has not been seen nor predicted before," he adds. "This new information could be used to modulate how Cas9 processes targeted DNA and could help the design of better genome-editing tools."
Research is published in Nature Structural and Molecular Biology.