Interstrand Cross Links Improve Non-Viral Gene Editing

University of California (UC) Santa Barbara researchers have added to the ever-growing genome-editing toolbox. In Nature Biotechnology, they publish a method that improves CRISPR/Cas9 editing efficiency without using viral material.

The CRISPR-Cas9 toolbox

In just over a decade, CRISPR/Cas9 genome-editing has dramatically changed the landscape of scientific research, offering new approaches to study, model and treat human disease, enhance crop production and even mitigate the effects of climate change. It has also evolved, with new CRISPR-based approaches yielding higher efficiencies and less off-target effects published frequently. 

The CRISPR-Cas9 method harnesses a defense technique first discovered in archaea by Professor Francisco Mojica, and later in bacteria. In bacteria, the CRISPR system is used to protect against viral invaders. The bacteria “cut” a fragment of an invading virus’ genetic material using enzymes – called Cas nucleases – which can bind to and create double strand breaks (DSBs) in the viral DNA.

This genetic material is then stored within the bacterial genome where it serves as a “genetic memory”. If the invader returns, a complex known as a guide RNA (gRNA) guides a type of Cas called Cas9 to the genetic sequence in the viral DNA, where Cas9 creates a double-stranded break. In bacteria, this prevents the virus from replicating because it lacks a DNA repair system.

Harnessing this system for gene editing, scientists can engineer the guide RNA to target a DNA sequence of their choice. When Cas9 creates a double stranded break in DNA, the cell works to try and repair the DNA. There are two key types of DNA repair adopted in genome-editing approaches: non-homologous end joining – NHEJ – and homology-directed repair – HDR.

These systems can be used to insert new genes or genetic fragments, which require the delivery of a DNA template encoding the new sequences or fragments to be incorporated. Picture this template DNA as a cargo that relies on a delivery system to enter the nucleus. Viruses are often used, but they have their shortcomings such as potential toxicity and difficulty with scale-up. Non-viral mechanisms are more attractive as they have reduced costs and are more scalable, but their efficacy has been limited.

Improving non-viral gene editing

Scientists – including the laboratory of assistant professor Chris Richardson at UC Santa Barbara – are exploring different ways to increase HDR efficiency using non-viral approaches. “Improved non-viral gene editing would be a powerful approach to unraveling DNA repair mechanisms, a useful laboratory technique and a promising strategy for the treatment of a multitude of diseases,” Richardson and colleagues write.

In the new paper, the researchers used non-viral approaches for delivering the template DNA, but with an added twist: they introduced interstrand crosslinks (ICLs) to the template. Ironically, damaging the DNA has favorable outcomes for HDR. “ICLs added to an HDR template – which we refer to as xHDRTs – dramatically improve editing rates in nonviral gene editing workflows in a dose-dependent manner,” the researchers write.

Testing the effect of damaging the DNA template in human cell lines, stem cell lines and T cells, the researchers found the approach stimulates HDR by threefold, without increasing the number of mutations or adversely impacting the outcome of the repair process. “Basically, what we’ve done is taken this template DNA and damaged it,” Richardson says. “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’.” The result, they say, is a highly efficient and minimally error-prone nonviral system of gene editing.

Applications in ex vivo manufacturing of cell therapies 

At this stage, the researchers can’t say with certainty why this approach stimulates the cell’s natural repair mechanism, but they have a hypothesis: “What we think happens is that the cell detects and tries to repair the damaged DNA that we’ve added this crosslink to,” Richardson says. “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.”

The team propose that their method is best suited for ex vivo gene editing applications, where it could find applications in the manufacturing of cell therapies with further developments.

“We can more effectively knock down genes and insert things into genomes to study systems outside of the human body in a lab setting,” says Hannah Ghasemi, graduate student in the Richardson lab and lead author of the study.

Reference: Ghasemi HI, Bacal J, Yoon AC, et al. Interstrand crosslinking of homologous repair template DNA enhances gene editing in human cells. Nat Biotechnol. 2023. doi: 10.1038/s41587-022-01654-y

This article is a rework of a press release issued by the University of California Santa Barbara. Material has been edited for length and content.