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| The ability to genetically change bacteria is the key to researching the microbial world. Image Credit: Braňo |
Scientists from Würzburg and Braunschweig have developed a new approach that enables more efficient processing of bacterial genomes.
The ability to genetically change bacteria is the key to researching the microbial world. Genome editing - i.e. processing the genome such as DNA - is essential in order to develop new antibiotics and to use bacteria as miniature factories for the sustainable production of chemicals, materials and therapeutics. Tools based on the CRISPR gene scissors have proven helpful here because they make it possible to change different bacteria quickly, easily and reliably.
The underlying technology requires CRISPR ribonucleic acid (crRNA), which serves as a "lead RNA". It helps to control certain regions of a genome for targeted DNA cleavage. Proteins involved in homologous recombination - a natural process of exchanging genetic material between chromosomes - then insert the designed "repair template" to create a processed sequence of the DNA strand.
Remove stumbling blocks
In a current study, which is now in the journal Nature Communications In collaboration with the Helmholtz Center for Infection Research (HZI) in Braunschweig, researchers at the Würzburg Helmholtz Institute for RNA-based Infection Research (HIRI) are devoting themselves to a central challenge in genome processing in bacteria.
"CRISPR-based genome editing has become a widely used molecular biological technology, but there is a remarkable stumbling block," said HIRI department head Chase Beisel, who led the study. “During their exponential growth, bacteria multiply their genome several times in a cell cycle to keep up with cell division. By cutting the DNA, the CRISPR gene scissors lead to the premature death of the cell. As a result, editing requires effective recombination and high transformation efficiency, which is not the case with most bacterial strains - not even those that are relevant to human diseases and industrial biotechnology,” explains Beisel.
An apparently paradoxical approach
Daphne Collias, postdoc in the Beisel laboratory at the Helmholtz Institute in Würzburg, is the first author of the study and explains the results: “We discovered that a weakening of the cutting activity of CRISPR enables the cell to use the cut DNA with the template provided for the to repair homologous recombination. As a result, we were able to advance homologous recombination and get many more surviving cells."
The scientists developed a whole range of approaches that can reduce DNA cutting activity. They use various formats for ribonucleic acid, which controls cutting through the protein Cas9. They also tested Cas9 versions that cut less efficiently, reduced the expression of the lead RNA, and implemented disruptive structures and sequence mutations in the lead RNA to dissuade them from the DNA target.
"We call the modified lead RNAs 'weakened lead RNAs' or atgRNAs because they represent a flexible means of realizing CRISPR-controlled genome processing," reports Collias. “Not every one of our approaches was able to advance DNA editing, although we could usually find at least one for each editing setup."
Perspectives
To demonstrate the principle of action, the Chase Beisel team and the laboratory of Till Strowig, head of department at the HZI, have joined forces to develop genome processing in various bacterial strains Klebsiella to improve. In a multi-resistant strain, they were able to eliminate resistance to the antibiotic ampicillin by editing.
The new genetic processing approach can advance basic research on bacteria related to human health and diseases. Edited bacteria could also be used as therapeutic probiotics or as production hosts for therapeutics in the future.
Funding: The study was carried out by the Joint Programming Initiative on Antimicrobial Resistance (JPI-AMR) and funded by the Federal Ministry of Education and Research (BMBF).
Published in journal: Nature Communications
Source/Credit: University of Würzburg
Reference Number: bio020923_04