Researchers at the Life Sciences Center of Vilnius University have discovered how the bacterial protein Cas9, known as the CRISPR-Cas gene scissors, helps bacteria integrate DNA fragments from infecting viruses, or bacteriophages, into their genomes, allowing them to develop resistance to viral attacks.
The team's findings have been published in the journal Molecular Cell.
One of the researchers, Dr Sasnauskas, said that CRISPR-Cas systems are among the most studied bacterial defence mechanisms against viruses (bacteriophages).
Their function is typically divided into three stages: adaptation, the formation of virus-targeting RNA and interference.
"Most discussions focus on the final stage, when a Cas protein recognises and cleaves viral DNA," he stated.
"However, the first step—adaptation—is no less important, as this is when the bacterium initially acquires information about the invader."
During adaptation, a roughly 30-base pair fragment of viral DNA—called a spacer—is inserted into a specific region of the bacterial genome known as the CRISPR locus.
This enables the bacterium to 'remember' its attacker. Subsequently, CRISPR RNA (crRNA) is transcribed from this region and together with Cas proteins, it forms a surveillance complex.
"If the same virus infects the cell again, this complex recognises the matching nucleic acid sequence and cleaves it," he added.
This is essentially a form of bacterial immune memory that enables rapid and precise recognition of previously encountered viruses.
From basic biology to future technologies
"Until now, Cas9 has primarily been associated with DNA cleavage."
"Our research shows that this protein also plays an active role in the early stage of the bacterial immune response – in the selection and acquisition of new genetic memory elements," noted PhD student Ugnė Gaižauskaitė.
"The study revealed that, together with Cas1-Cas2 (the proteins responsible for inserting DNA fragments) and an auxiliary protein Csn2, Cas9 forms what researchers describe as a 'supercomplex'."
"This protein–nucleic acid complex selects an appropriate fragment of viral DNA—the future spacer—and facilitates its integration into the CRISPR locus," she explained.
According to Ugnė Gaižauskaitė, the findings provide new insight into how Cas9 has evolved to perform multiple functions.
We see that the same protein can serve different purposes: both defending against viruses and contributing to the formation of immune memory.
Why this matters
Significant insights were gained from cryogenic electron microscopy (cryo-EM) studies using the Glacios Cryo-TEM microscope at VU LSC, a highly advanced instrument valued at €2.5m.
Researchers identified 11 distinct structures of the CRISPR-Cas protein complex, including three supercomplex variants.
Each structure comprises more than ten components, including proteins and nucleic acids.
This analysis led to a detailed mechanism for selecting and integrating new spacers and revealed a previously unknown function of the ring-shaped protein Csn2.
"It turns out that Csn2 helps assemble all supercomplex components onto the viral DNA fragment," said Dr Sasnauskas.
The structural data allowed us to observe this process at near-atomic resolution. This made it possible to describe individual components as well as to understand how they function as a coordinated system.
This work advances the field's understanding of how bacterial immune systems function and reveals new aspects of CRISPR-Cas biology.
Fundamental research into natural molecular mechanisms often lays the groundwork for innovative biotechnological applications.
The Cas9 protein has already revolutionised the field of genome engineering.
A deeper understanding of how it operates within natural bacterial systems may open new avenues for developing even more precise and versatile gene-editing tools, as well as advancing information storage technologies based on CRISPR spacer integration.