How a Precision Gene-Editing Tool Addresses CRIPSR’s Teething Issues

A new age of genetic medicine is here. Since the mid-2010s, when biotech companies first began commercializing CRISPR-Cas9, historic milestones after historic milestones have flown by. Patents have been granted. Regulatory approvals have been issued. Clinical trials have demonstrated the potential of life-changing new medicines. And this is just the beginning.

But amid all the fervor and history-making lies a few teething issues.

Namely, even with the improved specificity CRISPR-Cas9 has brought to gene-editing, accidental, off-target effects still happen. This margin for mistakes has raised concerns about the safety and efficacy of any gene therapy that arises from the much-hyped technology.

Dr. Simon Reed isn’t too concerned, however.

During a Technology Networks webinar last year – “Measuring Precision in Gene-Editing Systems for Cell and Gene Therapy” – Reed explained how INDUCE-seq, a polymerase chain reaction (PCR)-free technology developed by his company, Broken String Biosciences, can map off-target effects during genetic editing. The innovation, he says, could transform the way scientists measure and address genomic breaks in gene-editing systems.

The margin of CRISPR

“They’re not perfect,” Reed said, regarding the efficacy of CRISPR-Cas9 technologies. “They have this propensity for it to go off-target.”

“And what we mean by off-target is that they can direct the nucleases that perform the gene-editing step at other locations in the genome,” he told the Technology Networks audience back in 2024.

Waving his cursor over a diagram in his PowerPoint presentation, Reed attempted to elucidate how these kinds of off-target errors occur.

“So, this is the point in the genome where we want to make the intended edit, and it will target the nuclease,” he said, indicating to the slide.

Credit: Broken String Biosciences.

“You may have heard about the Cas9 nucleases, which introduces the break into the genome. Really, this is the first step of the editing process. And then the next stage is using the cells’ natural repair systems to repair that break and make the editing change to correct the genetic defect.”

“Now the issue is that, whilst this is very good, there may be other locations in the genome which are not intended that can also be hit by these systems.”

Given that the human genome consists of over three billion base pairs of DNA, Reed likened the search for these off-target effects to looking for a needle in a haystack. Existing tools to find errors often rely on PCR amplification, which can introduce biases and distort quantitative measurements of these breaks.

INDUCE-seq, though, is something new altogether.

Introducing INDUCE-seq

Developed by Broken String Biosciences, INDUCE-seq is “a PCR-free method that provides an unbiased, whole-genome approach to identifying editing-induced breaks with remarkable precision,” according to Reed.

“First of all, it’s a platform technology that is capable of mapping both on- and off-target induced breaks in the genome on a whole genome scale,” he added, “using next generation sequencing-based technologies.”

According to Reed and Broken String Biosciences, the technology offers:

• A PCR-free process
• Whole genome coverage
• Broad compatibility: Applicable across various cell types, including primary cells, induced pluripotent stem cells (iPSCs) and cell lines.
• High sensitivity: Detects breaks at low recurrence rates, ensuring even rare events are identified.
• Platform versatility: Works with multiple nuclease-based editing systems, including CRISPR, prime editing and others.

Reed explained that INDUCE-seq operates in three key steps.

“There are three elements to this,” he told the Technology Networks audience. “Stage one is the labeling of the breaks that are present inside the genome, inside the intact cell. We put the cells into a 96-well plate, and we immobilize them on that surface and then permeabilize the cells, then we add in the essentially modified p5 adapters, which are part of the next generation sequencing process.”

“These p5 adapters label the naturally occurring breaks inside the cell at the time of the labeling process,” he added. “They may be breaks that are induced by your gene-editing system, but they can also be breaks that are naturally occurring.”

Next comes DNA extraction. Labeled DNA is extracted and sequenced using next generation sequencing technology, providing a direct readout of any breaks.

The last step is when the data are analyzed. The INDUCE-seq bioinformatics tools map any breaks to their genomic locations, distinguishing between naturally occurring and editing-induced events through a unique feature called break recurrency.

“This is actually very important,” Reed stressed, “because the relationship between the number of breaks at the on-target to some of these off-target breaks here really help these companies that are developing these types of medicines to really understand the off-target effects, where those off-targets might exist and what effect that might have.”

Editing the future

To test INDUCE-seq’s efficacy, Reed and his colleagues at Broken String Biosciences enrolled the technology in a pilot study with the Health and Environmental Sciences Institute (HESI).

Two independent industry partners – AstraZeneca and Novartis – first conducted CRISPR-Cas9-based gene-editing of five well-studied genetic targets using two different cell types. INDUCE-seq was then used to assess for on- and off-target breaks in the genome. The genetic changes at these locations were subsequently measured using error-corrected sequencing using Duplex-seq4, which allows for sensitive detection of mutations.

By the end of the study, the two pharmaceutical companies found that INDUCE-seq reproducibly detected on-target and off-target editing in different cell types.

The positive findings have certainly buoyed Broken String’s ambitions for its new tool.

“We think this is going to be important in the future,” Reed said. “INDUCE-seq can be used to optimize the gene-editing protocols, making them safer by design.”

The company’s next steps include incorporating artificial intelligence into INDUCE’s processes.

"We’re beginning to train algorithms to distinguish naturally occurring breaks from induced ones, which could transform how we predict and mitigate off-target risks,” Reeves added.