A Safer Way to Edit Genes?

CRISPR-Cas9 has set the research community on fire for its gene editing efficiency. But that doesn’t mean we can’t do better. Now, in a paper published in the journal PLOS ONE, researchers at the University of Miami Miller School of Medicine have shown a system used for decades in bacteria can also edit human cells. With a little optimization, this approach — called recombineering — could be a safer way to edit genes.

While CRISPR-Cas9 has been incredibly useful in the laboratory and has shown clinical potential, it’s also generating some misgivings. The CRISPR molecule homes in on a gene of interest, while Cas9 acts like molecular scissors to cut that gene. It’s the scissors part that can be worrisome.

“CRISPR-Cas is very exciting, but as someone who studies DNA break repair for a living, it gives me the heebie-jeebies,” said researcher and study co-author Richard Myers, Ph.D. “The worst type of DNA damage affects both strands. In double-stranded DNA, if one strand gets damaged, the other can be used as a template to repair it. But if both strands get damaged, then what are you going to do?”

While CRISPR-Cas9 was taken from bacterial self-defense mechanisms, recombineering retasks viral systems that help create more viruses. These enzyme packages, called Syn-Exo recombinases, have the advantage of altering the genome without cutting it apart.

During viral growth, the Exo (exonuclease) binds to DNA ends and degrades them, exposing single-stranded DNA. The Exo also delivers the Syn (synaptase) component, which wraps around the single strand — similar to a guitar string — and becomes a template to attract the desired genomic sequence.

By using single-stranded DNA directly, it’s possible to direct the DNA to a genomic target without cutting anything. When applied to bacteria, recombineering a viral synaptase and single-stranded DNA can be so efficient that nearly all cells are modified.

“This type of editing involves taking the natural process of DNA synthesis in the cell,” said Dr. Myers. “Then, the virus tricks the cell into thinking these small DNA molecules are part of the already-replicating DNA. The single-stranded pieces get stitched into the replicating DNA and get passed on to the next generation.”

Since its development in the 1990s, recombineering has been used to engineer bacteria and other organisms. Working with senior author Paul Schiller, Ph.D., and first author Melvys Valledor, Ph.D., both colleagues in the Department of Biochemistry and Molecular Biology, Dr. Myers wanted to show that the same mechanisms could be used to edit human DNA.

In their research, the team commandeered part of the human herpesvirus SynExo–ICP8. They found the ICP8 synaptase successfully targeted a human gene, just like other viral synaptases in bacteria, though at low efficiency. They also showed the beta protein from a different SynExo, traditionally used in bacterial engineering, failed to target the human gene — in fact, it inhibited targeting.

This work confirms that scientists need to use SynExos from viruses — in this case, human herpesvirus — that infect the cell types they’re trying to engineer. For example, researchers will have to use components from viruses that infect plants to succeed in plants.

Most importantly, the team showed that recombineering can succeed in human cells, setting the stage for another, safer, gene-editing tool.

“We don’t break any DNA, and we’re not disrupting anything,” said Dr. Myers. “We’re just slipping DNA between pieces of DNA, and it all gets joined together by the host replication machinery. If we’re able to reach the efficiency of CRISPR-Cas, it has the potential to edit genomes in a way that doesn’t have the collateral concerns of using risky, double-strand breaks to promote the exchange.”

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