How to edit the genes of nature’s master manipulators

aRepresentation of the network graph of E. coliphages and their relatives. Nodes represent phage genomes connected by edges if they share significant similarity as determined by vContact2 (protein similarity). Nodes are shaded red when used as E are classified. Coliphage and blue, if only they bear resemblance. Nodes are shaded black when scored for sensitivity to LbuCas13a. b, EOP experiments for Cas13a targeting an early or late transcript. EOP values ​​represent the average of three biological replicates for a single crRNA compared to an RFP-targeted negative control cRNA. Photo credit: Nature Microbiology (2022). DOI: 10.1038/s41564-022-01258-x” width=”800″ height=”530″/>

Comparison of LbuCas13a anti-phage activity across the dsDNA-E. coli phage phylogeny. aNetwork diagram representation E. coli phages and their relatives. Nodes represent phage genomes connected by edges if they share significant similarity as determined by vContact2 (protein similarity). Nodes are shaded red if classified as E. coli Phage and blue, if only they show similarity. Nodes are shaded black when scored for sensitivity to LbuCas13a. b, EOP experiments for Cas13a designed to target an early or late transcript. EOP values ​​represent the average of three biological replicates for a single crRNA compared to an RFP-targeted negative control cRNA. Recognition: natural microbiology (2022). DOI: 10.1038/s41564-022-01258-x

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

A team led by CRISPR pioneer Jennifer Doudna and her longtime 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 create custom phages — something that 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 natural microbiology.

“Bacteriophages are among the most abundant and diverse biological entities on earth. In contrast to previous approaches, this editing strategy counteracts the enormous genetic diversity of bacteriophages,” said first author Benjamin Adler, a postdoctoral fellow in Doudna’s lab. “There are so many exciting directions here – discovery is literally at our fingertips.”

Bacteriophages, also known simply as phages, introduce their genetic material into bacterial cells with a syringe-like device, and then hijack their hosts’ protein-making machinery to reproduce – usually killing the bacteria in the process. (They are harmless to other organisms, including us humans, although electron microscopy images have shown them to look like eerie alien spaceships.)

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

Over millennia, the ongoing evolutionary struggle between phage attack and bacterial defense forced phages to specialize. There are many microbes, so there are many phages, each with unique adaptations. This amazing diversity has made phage editing difficult, including resistance to many forms of CRISPR, which is why the most commonly used system – CRISPR-Cas9 – does not work for this application.

“Phages have many ways to evade defenses, from anti-CRISPRs to their ability to repair their own DNA,” Adler said. “In a sense, the adaptations encoded in phage genomes that make them so good at manipulating microbes are exactly the same reason why it was so difficult to develop a general-purpose tool to manipulate their genomes.”

Project leaders Doudna and Banfield have co-developed numerous CRISPR-based tools since first collaborating on an early investigation of CRISPR in 2008. This work – performed at Lawrence Berkeley National Laboratory (Berkeley Lab) – was cited by the Nobel Prize Committee when Doudna and her other collaborator, Emmanuelle Charpentier, received the 2020 prize.

Doudna and Banfield’s team of researchers from Berkeley Lab and UC Berkeley 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 system against a wide range of phages.

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

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

According to co-author and phage expert Vivek Mutalik, a research associate in the Biosciences Area at Berkeley Lab, these results suggest that the CRISPR system can defend itself against various DNA-based phages by targeting their RNA after they from the DNA of the bacteria itself has been transformed by enzymes prior to protein translation.

Next, the team demonstrated that the system can be used to edit phage genomes instead of just chopping them up defensively.

First, they made segments of DNA consisting of the phage sequence they wanted to generate flanked by native phage sequences and inserted them into the phage’s target bacteria. When the phage infected the DNA-laden microbes, a small percentage of the phage that reproduced inside the microbes took up the altered DNA and incorporated it into their genome in place of the original sequence.

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

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

Experiments with three unrelated E. coli phages showed an amazing success rate: more than 99% of the phages produced in the two-step processes contained the changes, which ranged from enormous deletions of multiple genes to the precise replacement of a single amino acid.

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

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

More information:
Benjamin A. Adler et al., Broadband CRISPR-Cas13a Enables Efficient Editing of the Phage Genome, natural microbiology (2022). DOI: 10.1038/s41564-022-01258-x

Provided by Lawrence Berkeley National Laboratory

Citation: How to edit the genes of nature’s master manipulators (2022, December 5), retrieved December 5, 2022 from

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