How to Edit the Genes of Nature’s Master Manipulators

CRISPR, the Nobel Prize-winning gene-editing technology, is poised to have a profound impact on the fields of microbiology and medicine once again.

A team led by CRISPR pioneer Jennifer Doudna and her long-time collaborator Jill Banfield have developed a clever tool to edit the genomes of bacteria-infecting viruses called bacteriophages using a rare form of CRISPR. The ability to easily engineer custom-designed phages, which has long eluded the research community, could help researchers control microbiomes without antibiotics or harsh chemicals, and treat dangerous drug-resistant infections. An article describing the work was recently published in microbiology of nature.

“Bacteriophages are some of the most abundant and diverse biological entities on Earth. Unlike previous approaches, this editing strategy works against the tremendous genetic diversity of bacteriophages,” said first author Benjamin Adler, a postdoctoral fellow at the Doudna’s laboratory. “There are so many exciting directions here – discovery is literally at your fingertips.”

Bacteriophages, also called simply phages, insert their genetic material into bacterial cells using a syringe-like apparatus, then hijack their hosts’ protein-building machinery to reproduce, usually killing the bacteria in the process. (They are harmless to other organisms, including us humans, although electron microscopy images have revealed that they look like sinister alien spacecraft.)

CRISPR-Cas is a type of immune defense mechanism that many bacteria and archaea use against phages. A CRISPR-Cas system consists of short RNA fragments that are complementary to the phage gene sequences, allowing the microbe to recognize when invasive genetic material has been inserted, and scissors-like enzymes that neutralize the phage genes by cut them into harmless pieces. after being guided into place by RNA.

For millennia, the perpetual evolutionary battle between phage attack and bacterial defense forced phages to specialize. There are many microbes, so there are also many phages, each with unique adaptations. This staggering diversity has made phage editing difficult, even making them resistant to many forms of CRISPR, which is why the most widely used system, CRISPR-Cas9, does not work for this application.

“Phages have many ways of evading defenses, from anti-CRISPR to being good at repairing their own DNA,” Adler said. “So, in a sense, the adaptations encoded in phage genomes that make them so good at manipulating microbes are exactly the same reason why it has been so difficult to develop a general-purpose tool for editing their genomes.”

Project leaders Doudna and Banfield have developed numerous CRISPR-based tools together since they first collaborated on early CRISPR research in 2008. That work, conducted at the Lawrence Berkeley National Laboratory (Berkeley Lab), was cited by the committee. of the Nobel Prize when Doudna and her other collaborator, Emmanuelle Charpentier, received the prize in 2020.

The Berkeley Lab and UC Berkeley team of researchers from Doudna and Banfield were studying the properties of a rare form of CRISPR called CRISPR-Cas13 (derived from a bacterium commonly found in the human mouth) when they discovered that this version of the defense works against a wide variety of phages.

The potency of CRISPR-Cas13 in fighting phages was unexpected given that few microbes use it, Adler explained. The scientists were doubly surprised because all the phages it defeated in the tests infect using double-stranded DNA, but the CRISPR-Cas13 system only targets and cuts single-stranded viral RNA.

Like other types of viruses, some phages have DNA-based genomes, and others have RNA-based genomes. However, all known viruses use RNA to express their genes. The CRISPR-Cas13 system effectively neutralized nine different DNA phages infecting strains of E.coli, however, they have almost no similarity between their genomes.

According to co-author and phage expert Vivek Mutalik, a staff scientist in Berkeley Lab’s Biosciences Area, these findings indicate that the CRISPR system can defend itself against various DNA-based phages by targeting their RNA after it has been converted from DNA by the bacterium itself. enzymes before protein translation.

Next, the team demonstrated that the system can be used to edit phage genomes rather than simply hack them defensively.

First, they made segments of DNA made up of the phage sequence they wanted to create flanked by native phage sequences and put them into the phage’s target bacterium. When phages infected microbes loaded with DNA, a small percentage of the phages that reproduced inside the microbes took the altered DNA and incorporated it into their genomes instead of the original sequence.

This step is a long-standing DNA editing technique called homologous recombination. The decades-old problem in phage research is that although this step, actual editing of the phage genome, works well, isolating and replicating sequence-edited phages from the larger pool of normal phages is very complicated.

This is where CRISPR-Cas13 comes into play. In step two, the scientists engineered another strain of host microbe to contain a CRISPR-Cas13 system that detects and defends against the normal phage genome sequence. When the phages created in step one were exposed to second-round hosts, the phages with the original sequence were defeated by the CRISPR defense system, but the small number of edited phages were able to evade it. They survived and replicated.

Experiments with three unrelated E. coli phages showed an astonishing success rate: more than 99% of the phages produced in the two-step processes contained edits, ranging from massive multiple gene deletions to precise single amino acid replacements. .

“In my opinion, this work on phage engineering is one of the major milestones in phage biology,” Mutalik said. “As phages impact microbial ecology, evolution, population dynamics, and virulence, fluid engineering of bacteria and their phages has profound implications for fundamental science, but also has the potential to make a real difference in all aspects of science.” aspects of the bioeconomy. In addition to human health, this phage engineering capability will affect everything from biomanufacturing and agriculture to food production.”

Encouraged by their initial results, the scientists are currently working to expand the CRISPR system for use in more types of phage, starting with those that affect soil microbial communities. They are also using it as a tool to explore the genetic mysteries within phage genomes. Who knows what other amazing tools and technologies can be inspired by the spoils of the microscopic war between bacteria and viruses?