Most have heard of CRISPR/Cas9, the gene-editing technology that’s revolutionized biomedical research. Now, researchers have added another tool to the gene-editing toolbox after discovering a new way of using the technology that improves its editing efficiency and provides a new way to repair DNA.
CRISPR/Cas9 tech was adapted from a naturally occurring genome editing system bacteria use as an immune defense. When bacteria are infected by a virus, they ‘cut off’ a small piece of the virus’s DNA and insert it into their own in a particular arrangement known as a CRISPR array. This means the virus can be recognized later and, if it re-invades the bacteria, can be targeted for destruction.
Gene editing in humans relies on the Cas9 enzyme which, guided by CRISPR, ‘snips out’ a fragment of DNA. The removed section can be replaced with a similar (homologous) but improved DNA template by a process called homology-directed repair, which initiates the cell’s natural DNA repair mechanisms. Viruses – modified so they can’t cause disease – are commonly used to deliver the template DNA to the cell’s nucleus because of their effectiveness at entering cells.
Now, researchers from UC Santa Barbara have developed a nonviral delivery system that increases the efficiency of CRISPR/Cas9’s gene-editing abilities and greatly improves homology-directed repair.
Viruses used for gene-editing purposes are expensive, hard to scale, and potentially toxic to cells. So the researchers looked at developing an alternative delivery method, adding interstrand crosslinks to the homology-directed repair template.
The separation of DNA’s two helical strands is essential for cellular processes such as replication and transcription. Interstrand crosslinks (ICLs) are toxic DNA lesions that tether these strands together, inhibiting separation and, therefore, transcription and replication. Many cancer chemotherapies create ICLs that block the replication of cancer cells.
The researchers found that the damage caused by adding ICLs to the homology-directed repair template actually improved the likelihood of gene-editing success and stimulated cellular repair.
“Basically, what we’ve done is taken this template DNA and damaged it,” said Chris Richardson, corresponding author of the study. “We’ve in fact damaged it in the most severe way I can think of. And the cell doesn’t say, ‘Hey, this is junk; let me throw it away.’ What the cell actually says is, ‘Hey, this looks great; let me stick it into my genome.’”
They found that using ICLs improved gene editing activity by up to three times compared with non-crosslinked controls. With the increased editing activity, the researchers expected to see more errors; instead, they saw no increase in the mutation frequency.
“What we think happens is that the cell detects and tries to repair the damaged DNA that we’ve added this crosslink to,” Richardson said. “And in doing so, it delays the cell past a checkpoint where it would normally stop this recombination process. And so by prolonging the amount of time that it takes the cell to do this recombination, it makes it more likely that the edits will go to completion.”
Recombination is the process by which pieces of DNA are broken and repaired (recombined) to produce new versions of DNA sequences (alleles).
The researchers say that their new method of gene editing will be useful in the laboratory setting to develop more efficient models of disease, opening to door to better clinical and therapeutic interventions.
“We can more effectively knock down genes and insert things into genomes to study systems outside of the human body in a lab setting,” said Hannah Ghasemi, lead author of the study.
The study was published in the journal Nature Biotechnology.
Source: UC Santa Barbara
