Cryo-EM reveals Cas9’s new role in bacterial immune memory

Researchers at Vilnius University’s Life Sciences Center (VU LSC) have discovered a surprising new function for Cas9, the famed CRISPR-Cas ‘gene scissors’, showing it plays a key role in how bacteria acquire immunity against viruses. The findings [1], published in Molecular Cell, reveal that Cas9 doesn’t just cut DNA - it helps select and integrate viral DNA fragments into the bacterial genome, forming the basis of immune memory.

Until now, Cas9’s role was thought to be limited to cleaving viral DNA during infection. VU LSC scientists, including PhD student Ugnė Gaižauskaitė and Dr Giedrius Sasnauskas, found that Cas9 teams up with the proteins Cas1, Cas2, and Csn2 to form a ‘supercomplex’. This molecular machine selects the viral DNA fragment - the future spacer - and orchestrates its integration into the CRISPR locus, giving bacteria a record of past viral invaders.

“This shows that Cas9 is multitasking - defending the cell and shaping bacterial memory,” said Gaižauskaitė. “We can now see how the same protein performs distinct functions with remarkable precision.”

The breakthrough relied on cutting-edge cryogenic electron microscopy (cryo-EM) using VU LSC’s Glacios Cryo-TEM microscope, one of Lithuania’s most advanced instruments, valued at €2.5 million. Through cryo-EM, the team resolved 11 distinct CRISPR-Cas protein structures, including three variants of the supercomplex, revealing the intricate choreography of more than ten protein and nucleic acid components at near-atomic resolution.

Dr Sasnauskas added: “Observing these complexes at this level allowed us to pinpoint how Csn2 assembles the supercomplex onto viral DNA - a previously unknown step in CRISPR adaptation.”

Beyond fundamental microbiology, these insights could influence the next generation of gene-editing technologies, making Cas9-based tools even more precise and versatile. Understanding its natural functions also opens possibilities for innovative applications in information storage and synthetic biology.

With this discovery, VU LSC not only expands our knowledge of bacterial immunity but also highlights how advanced microscopy techniques like cryo-EM are crucial for uncovering molecular secrets that can reshape biotechnology.

More information online

1. Structural Insights Into Cas9-Mediated Prespacer Selection in CRISPR-Cas Adaptation published in Molecular Cell