Scientists from the Institut Pasteur, the CNRS, Université Paris Cité and the Gladstone Institute in the United States have developed a revolutionary directed evolution method known as DGRec. This technology can be used for the rapid, targeted modification of any genetic sequence directly within living cells, paving the way for major breakthroughs in biotechnology and medicine.

Directed evolution: from test tubes to living cells

Directed evolution is a method that mimics natural selection to create proteins with improved functions. Achieving directed evolution in the laboratory previously required complex and repetitive stages in vitro (with the potential of introducing errors, as is sometimes the case with PCR). The DGRec (DGR-mediated recombineering) method developed by the international multidisciplinary team dramatically simplifies the process, directly in vivo, by enabling the cell to generate its own genetic diversity, with no external intervention.

How does DGRec technology work?

The new technology is inspired by diversity-generating retroelements (DGRs), natural systems used by certain bacterial viruses (i.e. phages) to adapt to their hosts. By combining these elements with a "recombineering" technique, the scientists have created a tool that can:

• Precisely target DNA sequences composed of 50 to 200 base pairs.

• Generate large-scale mutations: up to 24 mutations can occur in a single sequence in just 48 hours.

• Avoid fatal errors: the natural biases of the enzyme used (reverse transcriptase) cause the system to favor certain mutations, minimizing the emergence of premature termination codons that stop the production of proteins and make them inactive.

Practical applications, from phages to nanobodies

The team has already demonstrated the efficacy of DGRec through several state-of-the-art applications:

1. Phage engineering: By evolving virus recognition proteins, the scientists have created phages capable of infecting resistant bacteria.

2. CRISPR optimization: The method has been used to evolve variants of the dCas9 protein.

3. Nanobody maturation: DGRec has been used to accelerate the development of nanobodies (small antibodies) with stronger affinity, essential for novel diagnostics and therapies with even greater modularity.

A universal tool

Although the technology was mainly developed in E. coli bacteria, the scientists have also shown that it can be adapted to more complex organisms like yeast, suggesting vast potential for applications in a variety of biomedical research fields.

As the study's authors explain, “DGRec offers a unique combination of programmability and mutation density, making it possible to focus on any locus of interest without modifying its native context.”

Find scientific papers published in Nature Biotechnology

Photo : Bactériophages d'Escherichia coli en microscopie électronique.© Institut Pasteur/Antoinette Ryter