How to edit the genes of nature’s master manipulators

anetwork graph view 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 in red if they are classified as one E. coliphage and blue if they only show similarities. Nodes are shaded black when assessed for sensitivity to LbuCas13a. b, EOP experiments for Cas13a designed to target an early or late transcript. EOP values ​​represent the mean of three biological replicates for a single crRNA compared to an RFP-targeting negative control crRNA. Credits: Nature Microbiology (2022). DOI: 10.1038/s41564-022-01258-x” width=”800″ height=”530″/>

Comparison of LbuCas13a anti-phage activity across dsDNA E. coli phage phylogeny. aNetwork graph view of 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 in red if they are classified as one E coli phage and blue if they only share similarity. Nodes are shaded black when assessed for sensitivity to LbuCas13a. b, EOP experiments for Cas13a designed to target an early or late transcript. EOP values ​​represent the mean of three biological replicates for a single crRNA compared to an RFP-targeting negative control crRNA. Credit: Nature 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 in the fields of microbiology and medicine.

A team led by CRISPR pioneer Jennifer Doudna and her longtime collaborator Jill Banfield has 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 phages, which has long eluded the research community, could help researchers control microbiomes without antibiotics or harsh chemicals, and treat dangerous drug-resistant infections. A paper describing the work was recently published in Nature microbiology.

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

Bacteriophages, also known simply as phages, insert their genetic material into bacterial cells using a syringe-like device and then hijack their hosts’ protein-building machinery to reproduce themselves—usually killing the bacteria. (They’re harmless to other organisms, including us humans, even though electron microscopy images have revealed they look like sinister alien starships.)

CRISPR-Cas is a type of immune defense mechanism that many bacteria and archaea use against phages. A CRISPR-Cas system consists of short stretches of RNA complementary to sequences in phage genes that allow the microbe to recognize when invasive genetic material has been introduced, and scissor-like enzymes that neutralize the phage genes by cutting them into harmless pieces after they have passed through the RNA into place.

Over millennia, the ongoing evolutionary battle 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 making them resistant to many forms of CRISPR. Therefore, the most commonly used system – CRISPR-Cas9 – does not work for this application.

“Phages have many ways to evade defenses, ranging from anti-CRISPRs to just 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 was so difficult to develop a general tool for editing their genomes.”

Project leaders Doudna and Banfield have co-developed numerous CRISPR-based tools since they first collaborated on an early study of CRISPR in 2008. That work—conducted at the Lawrence Berkeley National Laboratory (Berkeley Lab)—was named by the Nobel Prize Committee when Doudna and her other collaborator, Emmanuelle Charpentier, received the award in 2020.

Doudna and Banfield’s team from Berkeley Lab and UC Berkeley researchers were studying the properties of a rare form of CRISPR called CRISPR-Cas13 (derived from a bacteria commonly found in the human mouth) when they found that this version of the immune system works against a huge range of phages.

The phage-fighting power of CRISPR-Cas13 was unexpected given the number of microbes it uses, Adler explains. The scientists were doubly surprised because the phages it beat in testing all infect with double-stranded DNA, but the CRISPR-Cas13 system only targets single-stranded viral RNA and chops it off.

Like other types of viruses, 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, all of which infect strains of E coli, but have almost no resemblance in 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 against various DNA-based phages by targeting their RNA after it has been converted by the bacteria itself. of DNA. enzymes prior to protein translation.

Next, the team showed that the system could be used to edit phage genomes rather than just defensively chop them up.

First, they made DNA segments composed of the phage sequence they wanted to make, flanked by native phage sequences, and inserted them into the phage’s target bacteria. When the phages infected the DNA-loaded microbes, a small percentage of the phages that reproduced inside the microbes took up the altered DNA and incorporated it into their genomes in place 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 while this step, the actual phage genome editing, works fine, isolating and replicating the edited sequence phages from the larger pool of normal phages is very tricky.

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

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

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

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

More information:
Benjamin A. Adler et al, Broad spectrum CRISPR-Cas13a enables efficient phage genome editing, Nature microbiology (2022). DOI: 10.1038/s41564-022-01258-x

Provided by Lawrence Berkeley National Laboratory

Quote: How to Edit the Genes of Nature’s Master Manipulators (2022, December 5) Retrieved December 5, 2022 from https://phys.org/news/2022-12-genes-nature-master.html

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