December 5, 2023

Most of you have heard of CRISPR/Cas9, the gene-editing technique that has revolutionized biomedical research. Now, researchers have discovered a new way to use the technology that could improve its editing efficiency and provide a new way to repair DNA, adding another tool to the gene-editing toolbox.

CRISPR/Cas9 technology was adapted from bacteria, a naturally occurring genome editing system, for immune defense. When bacteria are infected by a virus, they “cut out” a small piece of the virus’s DNA and insert it into themselves in a specific arrangement called a CRISPR array. This means that the virus can be identified at a later date and can be targeted for destruction if it invades bacteria again.

Gene editing in humans relies on the enzyme Cas9, which “snips out” segments of DNA under the direction of CRISPR. The removed section can be replaced with a similar (homologous) but improved DNA template through a process called homology-directed repair, which initiates the cell’s natural DNA repair machinery. Viruses—modified so that they don’t cause disease—are commonly used to deliver template DNA to the nucleus because they can enter cells efficiently.

Now, researchers at UC Santa Barbara have developed a nonviral delivery system that increases the efficiency of CRISPR/Cas9’s gene-editing capabilities and dramatically improves homology-directed repair.

Viruses for gene editing purposes are expensive, difficult to scale, and potentially toxic to cells. The researchers therefore set their sights on developing an alternative delivery method that would add interstrand crosslinks to homology-directed repair templates.

The separation of the two helical strands of DNA is critical for cellular processes such as replication and transcription. Interstrand crosslinks (ICLs) are toxic DNA lesions that link these strands together, inhibiting dissociation, and thus transcription and replication. Many cancer chemotherapy treatments produce ICLs, which prevent cancer cells from replicating.

The researchers found that adding ICLs to the damage caused by the homology-directed repair template actually increased the likelihood of successful gene editing and stimulated cellular repair.

“Essentially, what we’ve done is we’ve taken this template DNA and destroyed it,” said Chris Richardson, the study’s corresponding author. “In fact, we’ve broken it in the worst way I can think of. The phone doesn’t say, ‘Hey, this is trash; let me throw it away.’ The cell actually says, ‘Hey, this look Looks good; let me insert it into my genome.’”

They found that the use of ICLs increased gene editing activity by as much as three-fold compared to non-crosslinked controls. As editing activity increased, the researchers expected to see more errors; instead, they found no increase in mutation frequency.

“What we think is happening is that the cells detect and try to repair the damaged DNA that we’ve added this crosslink to,” Richardson said. “In doing so, it delays the passage of the cell through a checkpoint that would normally stop the recombination process. So by extending the time it takes for the cell to recombine, it makes it more likely that the edit will be completed.”

Recombination is the process by which segments of DNA are broken and repaired (recombined) to produce new versions of the DNA sequence (alleles).

The researchers say their new gene-editing method will help develop more effective disease models in laboratory settings, opening the door to better clinical and therapeutic interventions.

“We can more efficiently knock out genes and insert things into the genome to study systems outside of the human body in a laboratory setting,” said Hannah Ghasemi, first author of the study.

The study was published in the journal nature biotechnology.

source: University of California, Santa Barbara