Scientists Successfully Edit the Genes of Nature’s Master Manipulators

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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a revolutionary gene editing technology that allows scientists to make precise changes to the DNA of living organisms. Scientists are now using it to engineer the viruses that evolved to engineer bacteria.

Researchers are utilizing the gene-editing technology CRISPR to modify the viruses that have evolved to engineer bacteria.

CRISPR, the revolutionary gene-editing tool, is making waves in the scientific community once more with its potential to edit the genomes of viruses that infect bacteria.

Led by CRISPR pioneers Jennifer Doudna and Jill Banfield, a team has used a rare form of CRISPR to engineer custom bacteriophages, a development that could aid in the treatment of drug-resistant infections and allow researchers to control microbiomes without the use of antibiotics. The research, published in Nature Microbiologyrepresents a significant achievement as the engineering of bacteriophages has long been a challenge for the scientific community.

“Bacteriophages are some of the most abundant and diverse biological entities on Earth. Unlike prior approaches, this editing strategy works against the tremendous 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 simply called phages, insert their genetic material into bacterial cells using a syringe-like apparatus, then hijack the protein-building machinery of their hosts in order to reproduce themselves – usually killing the bacteria in the process. (They’re harmless to other organisms, including us humans, even though electron microscopy images have revealed that they look like sinister alien spaceships.)

CRISPR-Cas is a type of immune defense mechanism that many bacteria and archaea use against phages. A CRISPR-Cas system consists of short snippets of[{” attribute=””>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 cutting them into harmless pieces, after being guided into place by the RNA.

Over millennia, the perpetual evolutionary battle between phage offense and bacterial defense forced phages to specialize. There are a lot of microbes, so there are also a lot of phages, each with unique adaptations. This astounding diversity has made phage editing difficult, including making them resistant to many forms of CRISPR, which is why the most commonly used system – CRISPR-Cas9 – doesn’t work for this application.

“Phages have many ways to evade defenses, ranging from anti-CRISPRs to just being good at repairing their own

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 diverse DNA-based phages by targeting their RNA after it has been converted from DNA by the bacteria’s own enzymes prior to protein translation.

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

First, they made segments of DNA composed of the phage sequence they wanted to create flanked by native phage sequences and put them into the phage’s target bacteria. When the phages infected the DNA-laden microbes, a small percentage of the phages reproducing inside the microbes took up the altered DNA and incorporated it into their genomes in place of the original sequence. This step is a longstanding DNA editing technique called homologous recombination. The decades-old problem in phage research is that although this step, the actual phage genome editing, works just fine, isolating and replicating the phages with the edited sequence from the larger pool of normal phages is very tricky.

This is where the CRISPR-Cas13 comes in. In step two, the scientists engineered another strain of host-microbe to contain a CRISPR-Cas13 system that senses and defends against the normal phage genome sequence. When the phages made in step one were exposed to the 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 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, which ranged from enormous multi-gene deletions all the way down to precise replacements of a single amino

The study was was funded by the Department of Energy Microbial Community Analysis & Functional Evaluation in Soils (m-CAFES) Scientific Focus Area.