The research was funded through an Exploration Project from the Agencia Nacional de Investigación y Desarrollo (ANID), awarded in the first call of this innovative instrument.

The project, led by scientists Dr. Simón Beard and Dr. Raquel Quatrini, used gene-editing tools (CRISPR/Cas) encapsulated in lipid nanoparticles to create a high-precision system to eliminate acid-generating bacteria in mining waste accumulation sites without affecting other native microorganisms present in the area.

An acidic legacy

Chile is the world's leading copper producer, with an estimated production of 5,5 million tons of fine copper in 2024. This activity has generated hundreds of environmental liabilities, such as the more than 800 tailings dams recorded across the country.

When these mining wastes are exposed to air and moisture, they can generate acid mine drainage. This phenomenon is characterized by affecting surface waters in contact with these materials, making them highly toxic, primarily due to two factors:

  • High acidity: Water progressively acidifies over time, with pH values potentially dropping as low as 0,5.

  • High concentration of heavy metals: along with the drop in pH, elements such as arsenic, lead, cadmium, and copper are solubilized, which are toxic to living beings.

The primary cause of this environmental disaster is biological. A group of *acidophilic* and *lithotrophic* microorganisms—meaning they thrive in acidic environments and are capable of literally “eating” rocks—oxidize the minerals present in tailings, generating sulfuric acid and releasing toxic metals into the environment.

As Dr. Simon Beard, an academic at Universidad San Sebastian and researcher at the Centro Basal Ciencia & Vida, explains, "we seek to contribute from science with innovative solutions to face the environmental challenges of mining activity. From our experience in the molecular microbiology of acidophilic bacteria, we wanted to explore the development of next-generation molecular tools based on gene editing, while also taking on the challenge of obtaining vehicle formulations for the delivery of these tools in acidic environments."

He adds that "a distinctive feature of our research lies in the incorporation of *synthetic biology* principles into the design of these molecular tools. We are very satisfied with the results, as we developed a platform of genetic *parts and devices* that we can combine and reuse to adapt the functionality and specificity of our tools with great ease."

For her part, Dr. Raquel Quatrini, principal investigator and alternate director of the Centro Basal Ciencia & Vida, and academic of the Biochemistry program at the Faculty of Sciences of Universidad San Sebastian, states, "the possibility of selectively intervening in microbial communities represents a central challenge in current biotechnology, with implications ranging from human health to sustainable mining. My research group seeks to understand the rules governing the organization of microbial communities involved in bioleaching and to develop tools that allow directing their functions, promoting beneficial processes or mitigating their negative impacts, such as acid mine drainage. Although still in the experimental validation stage, the results generated in this project bring us one step closer in that direction."

CRISPR and liposomes

The research team wondered if it was possible to use their knowledge of these microbial communities to selectively inhibit only the bacteria responsible for this problem without affecting the entire community. To this end, a strategy based on more than one layer of selectivity was tested, both in the delivery of the molecular scissors and in their cutting specificity within the target bacterium's genome.

This proposal originates from two cutting-edge technologies:

  1. CRISPR/Cas molecular scissors: The project uses the famous gene-editing tool CRISPR/Cas. It functions as a “molecular scissor,” composed of a protein that cuts DNA and an RNA guide that directs it to a specific site in the genome. The team's goal is to design guides that target DNA sequences exclusive to acid-generating bacteria, thereby ensuring the scissors destroy their genetic material without affecting the rest of the bacterial community, nor plants, fish, or animals.

  2. A lipid nanoparticle vehicle (liposomes): For these scissors to work, they must survive the extreme environment of the drainage and enter the bacteria. To achieve this, the team explores the use of liposomes, which function as “soap bubbles” that encapsulate the CRISPR tools, protect them from acidity, and facilitate their fusion with the target bacteria, which can also be functionalized for targeted delivery.

Catalina López, a Master's student in Molecular Biomedicine at Universidad San Sebastian, explains that "what we did was design molecular scissors based on CRISPR/Cas systems with precise instructions on where they have to cut in the DNA of the bacteria of interest. Another aspect of the development was evaluating different *promoters* to regulate the amount of this molecular scissor, in order to obtain a genetic *doser* that allows us to increase or decrease the intensity of the system, preventing unwanted or non-specific effects resulting from high expression levels. We then tested all the components in the laboratory to ensure that our tool only cut in the right place and under the desired conditions, confirming that the tool was specific, safe, and reliable before moving on to its encapsulation and delivery to demonstrate its functioning in more complex systems."