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Bio-Rad Laboratories Aims to Create Genome Editing Tools Pipeline, Starting With ddPCR


NEW YORK (GenomeWeb) – Genome editing continues to be one of the most compelling trends in biological research as investigators maintain a steady pace of studies and papers on new CRISPR-Cas systems, innovative research tools, and various uses for the technology.

But academia is certainly not alone in its enthusiasm for CRISPR — industry is joining in as various companies are either setting out to develop new tools based on the genome editing technology or find CRISPR-based applications for tools that already exist.

In June 2015, Bio-Rad Laboratories said that it had found a way to create a genome editing application for its popular droplet digital PCR (ddPCR) technology. "Droplet digital PCR is a tool that we've used for rare cancer mutation detection," Jen Berman, a scientist at Bio-Rad's Digital Biology Center, told GenomeWeb at the Festival of Genomics in Boston that year. "This is just another flavor of rare mutation detection."

The company recently materialized its vision, releasing two of several planned assays that use ddPCR to detect genome edits. The ddPCR technology increases the signal-to-noise ratio, allowing users to quantify extremely rare edits, even those with frequencies of 0.5 percent and from as little as 5 nanograms of genomic DNA. Users can expect to obtain results within one day, according to Bio-Rad.

Bio-Rad began working on the CRISPR applications for ddPCR in late 2014 and early 2015, and teamed up with Bruce Conklin at the University of California, San Francisco on the publication of a paper in Scientific Reports in March 2016 that outlined how ddPCR could be used to precisely and systematically detect both homology-directed repair (HDR) and nonhomologous end-joining (NHEJ).

"We developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome," they wrote at the time.

At the same time, Boris Fehse — a researcher at the University Medical Centre, Hamburg-Eppendorf — and his team had also latched onto the idea of using digital PCR as a validation tool for genome editing, publishing their own papers in Nucleic Acids Research in May 2015 and in Nature Protocols in February 2016.

"We work on CCR5 knockout — it is a chemokine receptor which is necessary for HIV to enter the cell," he said in an interview. "Even though there is a receptor and you can measure it on the surface of the cell, it's not expressed in all the T cells that we wanted to modify, so you never know the knockout rate of the receptor when you apply genome editing. We thought, what can we do to measure this on a gene level with high specificity and without always performing next-generation sequencing which is a bit laborious and expensive?"

Fehse and his team were already using digital PCR in the lab for other applications, and thought it might be especially useful in detecting edits made through NHEJ, which are notoriously harder to detect than edits made with HDR. "Due to the process of NHEJ, you get a new pattern. But because you don't know what it looks like, you can't just use primers like in PCR to measure this," he said. "But then we had the idea to use a probe which binds to the wild-type sequence and would not be expected to bind to the edited version and we found that it works. We published this, and we started to discuss this with people from Bio-Rad and we learned they already started to work on that, so we had a parallel development."

Once Bio-Rad's paper with the Conklin lab was published, pharma and biotech customers began deluging Bio-Rad with requests for an automated pipeline that could perform ddPCR for the detection of genome edits. "We had over 20 customers from big pharma companies with 50 different requests saying that they couldn't do this themselves," Carolyn Reifsnyder, senior marketing manager at Bio-Rad, told GenomeWeb. "Genome edit validation using digital PCR started as an application you could do on your own, but it wasn't plug-and-play. So, we decided to build an automated pipeline."

Reifsnyder describes ddPCR as a "Goldilocks" tool when it comes to detecting genome edits. "It's no surprise to know that genome edits are rare — they're hard to detect, they can be cell-type dependent, they can be locus dependent. And especially with stem cells, less than 5 percent of your genomes get edited," she said. "So, you need a way to validate what you're doing. [But] researchers had either too much or too little technology to do this validation work before digital PCR."

She described T7 endonucleases as "insensitive or finicky," said Sanger sequencing had too long of a turnaround time, and noted that next-generation sequencing — while very precise — is very expensive and not always accessible. "So digital PCR is the Goldilocks — our sensitivity, our precision, the fact that you're talking less than a day for turnaround time to results, it's relatively inexpensive. We can see things that are happening on target, and we can also look for off-target effect. It really allows for quick iteration and evaluation of your CRISPR system," Reifsnyder added. She also noted that the assays have great concordance with NGS and flow cytometry, so it fits nicely into existing workflows.

Fehse said he appreciates the technology's ability to detect edits made through both HDR and NHEJ. "In the case of HDR, it's much easier to detect because you know what you're looking for. In NHEJ, essentially each cell you're looking at might have another mutation and looks a bit different. So, if you want to measure it, you have to sequence thousands of cells and look at whether they are mutated and which pattern you see," he said. "The big advantage to digital PCR is you measure individual copies of the gene in each droplet of the digital PCR device. In each droplet you have one allele, so you look for one individual allele whether it has or doesn't have the mutation."

He also noted that ddPCR is significantly more sensitive that molecular biology assays, and far less expensive and less time consuming than NGS, without the need for sophisticated bioinformatics analysis pipelines. "You put your samples in and essentially in the end you get your results out. It's quite clear cut," Fehse said.

However, the technology does have one limitation, he added. Though ddPCR can detect genome edits, it cannot show the exact sequences that have been changed. A researcher would still need NGS in order to do that. "If you sequence all the cells, you know what all the alleles look like. Here, you just know whether it's mutated or not," Fehse said. "The sensitivity is essentially the same, but you don't see all the mutations you might with the sequencing."

If a researcher needs to know the exact nature of the edits made, this limitation could be remedied by using NGS after using ddPCR to detect whether edits were made in the first place. However, in a hypothetical clinical setting, or in clinical or preclinical studies, Fehse said, "if you have a patient treated with genome editing and you want to measure the cells every week, then it's much easier to look in a routine way with PCR [than with NGS]. In gene therapy trials which use gene editing, this [would be] an ideal approach."

Bio-Rad plans to continue releasing ddPCR-based assays for the detection of genome edits. "There are actually about seven different classes of assays and designs you can apply to the base technology," Reifsnyder said. "We launched two of them in this first release. We're already working on another third and fourth class, specifically what we call total edits — to be able to [detect HDR and NHEJ] in one well. People can do all these things today, but we don't have a canned solution for them. So, we are refining our menu to make it more plug-and-play."

The company will likely release one or two more assays in 2018, and further iterations in subsequent years. "We're seeing great adoption of the product we launched this year, so we want to wait and get a little more feedback before we release any changes to it," she added.

Further, Bio-Rad is considering how the rest of its tools could fit into a CRISPR research pipeline. "Gene editing is] a nice fit … into our oncology play, with liquid biopsy being the primary driver of our technology right now," said Reifsnyder. "We also are putting a lot of effort as a whole into a CRISPR workflow, so using not just ddPCR for validation, but also our flow cytometry and the Zoe — a fluorescent microscope that works in bright light. There are all kinds of tools, including the electroporation and other basic kinds of cell handling technology that we have. There's quite a bit in Bio-Rad's portfolio that fits into the overall CRISPR workflow."