Researchers develop quality assurance tool for genome editing

Boston Children's HospitalFeb 9 2015

A research team at Boston Children's Hospital's Program in Cellular and Molecular Medicine (PCMM) has developed a quality assurance tool for scientists using genome-editing technologies like the increasingly popular CRISPR. The assay, called high throughput genome translocation sequencing (HTGTS), rapidly gauges—for any given gene—these technologies' accuracy and their risk for causing potentially dangerous genomic collateral damage.

The assay's ability to provide such information, the team notes in a paper in the February issue of Nature Biotechnology, could help genome-editing technologies safely mature from powerful laboratory tools into equally powerful therapeutic ones.

Genome-editing technologies—namely, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR—use combinations of enzymes and targeted "guide" sequences to cut DNA at precise locations in a cell's genome. However, the enzymes can also make "off-target" cuts at unintended sites, depending on how accurately a given guide sequence targets the enzymes to a gene of interest.

In addition, when genome-editing enzymes cut DNA, they essentially break the chromosome the target gene sits on. Ideally, the resulting pieces re-attach at the break site. However, they may also attach to broken pieces from other chromosomes—especially if the enzyme has made off-target cuts—creating chromosome translocations. Sometimes those translocations are physically unstable, causing a chromosome to break again, or disrupt the cell in ways that promote cancerous growth.

Researchers currently use sequence-based algorithms to assess enzyme/guide sequence combinations' off-target risk, but those algorithms rely on limited data. They also cannot reveal probable translocations. "The algorithms are getting better," says study lead author Richard Frock, PhD, a fellow in the laboratory of study senior author and PCMM director Frederick Alt, PhD. "But you still worry about the one rare, off-target effect that's not predicted but falls in a coding region and totally debilitates a gene."

"What you'd like to have," Alt explains, "is a really sensitive and easy assay that lets you sort through a number of target/enzyme combinations and look genome-wide to see the frequency of off-target cuts and patterns of translocation, and understand their possible effects on the cell."

His laboratory's HTGTS assay—adapted from a method originally developed to study chromosome breaks in cancer and antibody development—allows researchers to do just that: reveal how likely it is that a given combination of enzyme and target will cause off-target cuts, and whether an enzyme/target combination could result in problematic translocations.

Alt thinks the HTGTS assay will help expand the use of gene-editing enzymes and support their development as clinical tools.

"The assay gives researchers as much information as possible to run their experiments for a given purpose," he says. "It shows how, by using the right levels of enzyme, you can balance on- and off-target breaks, and allows researchers to find which enzymes work best and, if needed, to modify them to work better. It will help the field do what it wants to do."