Recombineering technique may overcome barriers in bacterial engineering

Genetic engineering of bacteria has transformed contemporary medicine, from bacterial-based insulin that prevents the use of animal pancreases to a deeper insight into infectious diseases and enhanced treatments.

But despite these advances, serious restrictions continue to remain that limit the progress in various other areas.

Image Credit: Anna Kazantseva/Pond5.

Recombineering, or recombination-mediated genetic engineering, is a decades-old bacterial engineering method that allows researchers to rarely swap DNA pieces of their choice for the areas of the bacterial genome.

Unfortunately, this versatile and valuable approach has not been used extensively because it was mostly limited to Escherichia coli—the laboratory rat of the bacterial world—and a few other species of bacteria.

Now researchers from the Blavatnik Institute at Harvard Medical School and the Biological Research Center in Szeged, Hungary, have developed a novel genetic engineering technique that could super-charge recombineering and largely open the bacterial world to this underutilized method.

A report describing the researchers’ method was published on May 28^th, 2020, in the PNAS journal.

The researchers have developed a high-throughput screening technique to search for the most efficient proteins that function as the recombineering engines. These proteins referred to as SSAPs, are located inside phages-viruses that infect bacteria.

The researchers used the new approach and eventually identified two proteins that seem to be specifically promising. This method helps to screen over 200 SSAPs.

One of the proteins increased the efficiency of single-spot edits of the bacterial genome by two-fold. It also enhanced the potential to carry out multiplex editing by 10 times—rendering multiple edits genome-wide simultaneously.

The other protein allowed efficient recombineering in the human pathogen called Pseudomonas aeruginosa. This pathogen is a common cause of fatal hospital-acquired infections for which there has been a scarcity of excellent genetic tools for a long time.

Recombineering will be a very critical tool that will augment our DNA writing and editing capabilities in the future, and this is an important step in improving the efficiency and reach of the technology.”

Timothy Wannier, Study First Author and Research Associate, Department of Genetics, Lab of George Church, Harvard Medical School

Wannier is also the Robert Winthrop Professor of Genetics at Harvard Medical School.

According to the researchers, earlier genetic engineering methods, such as CRISPR Cas9-based gene-editing, have not been suitable for bacteria because these techniques involved “cutting and pasting” the DNA.

This is because bacteria are different from multicellular organisms and do not have the machinery to repair double-stranded DNA breaks precisely and efficiently, and hence DNA cutting can considerably impede the stability of the bacterial genome, added Wannier. The benefit of the recombineering technique is that it functions without having to cut the DNA.

Rather, the recombineering technique involves stealthy edits into the genome during the reproduction of bacteria. Bacteria typically reproduce by dividing into two.

At the time of that process, a single strand of their double-stranded circular DNA chromosomes migrates to every daughter cell together with a new second strand that develops during the earlier stage of fission.

The raw materials used for recombineering are short—around 90 base strands of DNA that are made to order. Except for edits in the center of the strand, each strand is identical to a sequence in the genome. When the second strands of the daughter cells grow, these short strands move into place and efficiently incorporate the edits into their genomes.

Among various potential applications, edits could be made to intervene with a gene to localize its function or, otherwise, to increase the production of valuable bacterial products. SSAPs regulate the attachment and correct placement of the short strand inside the new, growing half of the daughter chromosome.

Recombineering might allow the replacement of a naturally occurring bacterial amino acid—that is, the building blocks of proteins—with an artificial one. Wannier added that among other things, doing so may allow the use of bacteria for environmental cleaning of various contaminants, including oil spills, which rely on the survival of these artificial amino acids.

This means the modified bacteria may be easily annihilated as soon as the work is completed to avoid the risks of discharging the engineered microbes into the environment.

The bacteria would require artificial amino acid supplements to survive, meaning that they are preprogrammed to perish without the artificial feed stock.”

Timothy Wannier, Study First Author and Research Associate, Department of Genetics, Lab of George Church, Harvard Medical School

A version of recombineering, known as multiplex automated genome engineering (MAGE), could considerably increase the advantages of the technique. The major advantage of MAGE is its potential to perform multiple edits all through the genome in a single shot.

John Aach, a lecturer in genetics at Harvard Medical School, stated that MAGE could result in progress in projects that require reengineering of whole metabolic pathways. Case in point are large-scale attempts to design microbes to convert wood waste into liquid fuels.

Aach added, “Many investigator-years’ effort in that quest have made great progress, even if they have not yet produced market-competitive products.”

Such efforts need testing of several combinations of edits, he added.

Aach continued, “We have found that using MAGE with a library of DNA sequences is a very good way of finding the combinations that optimize pathways.”

A recent successor of recombineering called directed evolution with random genomic mutations (DIvERGE), shows promise in fighting against infectious diseases and may pave the way for addressing antibiotic resistance.

DIvERGE can expedite natural bacterial evolution by introducing random mutations into the genome. Akos Nyerges explained that this helps scientists to quickly discover the changes that could emerge naturally in harmful bacteria, possibly making them impervious to the antibiotic treatment.

Improvements in recombineering will allow researchers to more quickly test how bacterial populations can gain resistance to new antibacterial drugs, helping researchers to identify less resistance-prone antibiotics.”

Akos Nyerges, Research Fellow, Department of Genetics, Lab of George Church, Harvard Medical School

Earlier, Nyerges was working at the Biological Research Center of the Hungarian Academy of Sciences.

The researchers added that recombineering will usher in a whole new realm of applications that would be difficult to predict at this moment.

“The new method greatly improves our ability to modify bacteria. If we could modify a letter here and there in the past, the new approach is akin to editing words all over a book, and doing so opens up the scientific imagination in a way that was not previously possible,” Wannier concluded.