Development of a CRISPR-Cas9 genome editing system for the extremely thermophilic bacterium Anaerocellum bescii

Deep within the world of industrial biology, there exists a group of microscopic workers that thrive where most life would instantly perish. These are extreme thermophiles, bacteria that grow best at temperatures near the boiling point of water. Among them is a particularly talented species called Anaerocellum bescii. This bacterium is a natural powerhouse, capable of breaking down tough plant fibers like wood and straw into simple sugars that can be turned into fuels and chemicals. Because it can do this work at high heat, it offers a unique advantage for manufacturing: the high temperature keeps unwanted contaminants away and speeds up chemical reactions. However, to turn this natural talent into a reliable factory, scientists need to rewrite the bacterium's internal instructions, or genome. Until now, doing this in such a heat-loving organism has been like trying to perform delicate surgery with a sledgehammer—slow, clumsy, and prone to damaging the delicate machinery of the cell.

For years, researchers have struggled to edit the genes of A. bescii because the tools they use in cooler, more common bacteria simply melt or stop working at the scorching temperatures this organism requires. The standard method involved a tedious, multi-step process of swapping DNA pieces, which often left behind unwanted genetic scars or caused the bacteria to become unstable. The scientific community needed a new approach, one that could operate effectively in the heat. In a recent study, a team at Princeton University successfully built the first precise gene-editing system for this extreme bacterium. They achieved this by borrowing a molecular tool from another heat-loving microbe, adapting it to work at 75 degrees Celsius, and using it to make clean, specific cuts in the DNA of A. bescii.

The researchers turned to a protein called Cas9, which acts like a pair of molecular scissors that can be programmed to find and cut a specific sequence of DNA. The common version of this protein, used in many labs, is made by bacteria that live at normal body temperature and would instantly fall apart in the heat of A. bescii. To solve this, the team selected a version of Cas9 from Geobacillus stearothermophilus, a bacterium that naturally lives in hot springs. This heat-stable version, known as GeoCas9, was designed to remain active even at the high temperatures where A. bescii grows. The team constructed a small, circular piece of DNA, essentially a delivery vehicle, that carried the instructions for this heat-resistant scissors and a guide that told it exactly where to cut. They introduced this vehicle into the bacteria and then lowered the culture temperature slightly to 65 degrees Celsius just long enough to let the cutting happen, before returning the bacteria to their normal hot environment.

Once the scissors made their cut, the bacteria's own repair mechanisms kicked in to fix the break. The researchers provided a template for this repair, instructing the cell to remove a specific gene and replace it with a harmless, short sequence of DNA that served as a marker. Using this method, they successfully removed two different genes from the bacterium's genome. The first gene they deleted was responsible for producing lactate, a waste product. When this gene was gone, the bacteria stopped making lactate and instead produced more acetate, a useful chemical. The second gene they removed was essential for making uracil, a building block of DNA. Without this gene, the bacteria could no longer grow unless the scientists added uracil to their food. This confirmed that the editing was precise and that the bacteria were behaving exactly as predicted by the missing instructions.

The team then demonstrated that this new system could be used repeatedly. They took the bacteria that had already lost the uracil-making gene and used the same editing tool to remove the lactate-making gene as well. This proved that the process could be stacked, allowing scientists to make multiple changes to the same organism without the genetic chaos that often plagued older methods. To show the full potential of their system, they targeted a third gene involved in making tryptophan, an amino acid. By deleting the genes for tryptophan synthase, they created a strain that could not survive without tryptophan in its food. This created a new way to select for successful experiments: if the bacteria grew without added tryptophan, it meant they had successfully taken up a new piece of DNA that restored their ability to make it.

Finally, the researchers used this new selection method to test if the bacteria could hold onto and use a new piece of DNA carrying a reporter gene. They gave the tryptophan-dependent bacteria a circular DNA vehicle that contained the missing tryptophan genes along with a gene for a heat-stable enzyme that glows when tested. The bacteria that took up this vehicle were able to grow without added tryptophan and, when tested, showed strong activity from the new enzyme. This confirmed that the new editing system not only allows for the removal of unwanted genes but also provides a robust way to add new functions to the bacterium. By establishing a reliable, heat-tolerant method for editing the genome, the researchers have opened the door to engineering A. bescii more efficiently, potentially accelerating the development of biological factories that can convert plant waste into valuable products.