Time-lapsed images show how magnesium ions coordinate double-stranded breaks in CRISPR-Cas9

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Gene-editing technology known as CRISPR has revolutionized agriculture, health research and more.

Published in research Nature is the catalystFlorida State University scientists have produced the first high-resolution, time-lapse images of magnesium ions interacting with the CRISPR-Cas9 enzyme as it cuts strands of DNA, providing clear evidence that magnesium plays a role in both breaking and closing chemical bonds. Simultaneous cutting of DNA.

If you’re cutting a gene, you don’t want to keep a single strand of broken DNA, because the cell can easily repair it without editing. You want to break both strands. You need two cut firings at once. Magnesium plays a role in that, and we’ve seen how it works.”

Hong Li, Professor of Chemistry and Biochemistry and Director of the Institute of Molecular Biophysics

CRISPR-Cas9 is the most widely used tool for genetic manipulation. The technology uses a recombinant enzyme to bind to DNA, allowing changes to be made at specific locations in a genome.

Scientists know that magnesium plays a role in this process, but exactly how has been unclear, and no one has been able to capture close-up time-lapse images of the process. Using a slower version of CRISPR-Cas9, this study shows that magnesium ions at the center of the catalytic reaction hold the key to near-simultaneous cutting.

“I think a lot of times in science, even though you can isolate something, you want proof,” Lee said. “For example, with magnesium everyone knows you need it, but without seeing it in action, it’s not a complete science, right? You don’t have the same level of understanding of how it works.”

The researchers used the cryo-electron microscope at FSU’s Biological Sciences Imaging Resource, which can produce images with near-atomic resolution to observe the metal ions and other atoms at work within the CRISPR-Cas9 enzyme. This allowed them to collect data that not only confirmed their earlier hypotheses but also led to surprising discoveries about how magnesium modulates double-separation breaks.

CRISPR debuted in gene editing in 2013, and since then, scientists have worked to improve its reliability and expand its applicability to a wide variety of organisms and cell types.

“By changing the active sites -; sets of ‘scissors’ that cut target and non-target DNA strands -; we can affect Cas9’s ability to use alternative metals for cutting,” said Mitchell Roth, doctoral candidate and co-author of the paper. “There’s still a lot to explore with CRISPR.”

Understanding how each component affects enzyme function gives scientists insight into what new knowledge and avenues can be used for research. Lee and his team plan further research to investigate how CRISPR-Cas9 can be repurposed for other purposes.

The paper was co-authored by former postdoctoral researchers Anuska Das and Jay Rai, doctoral candidate Yuerong Shu, graduate student Megan L. Medina and former graduate student McKenzie R. Barakat, all of FSU.

This research was supported by the National Institutes of Health.


Journal Reference:

Das, A., etc. (2023). Coupled catalytic conditions and the role of metal coordination in Cas9. Nature is the catalyst. doi.org/10.1038/s41929-023-01031-1.

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