Modernizing Gene Editing Detection with Droplet Digital PCR

CRISPR-Cas9 gene editing technology has allowed researchers to reimagine the limits of molecular genetics. The genome is not an untouchable sacred text; instead, scientists regularly manipulate genomic DNA in the laboratory and for clinical applications. This new era, which some call the golden age of biotechnology, promises targeted solutions to genetic pathologies. Examples of this revolution include engineered CAR-T cells and dramatically reduced timelines for creating transgenic models. Innovations that use gene editing are limited only by the creativity of scientific teams and the speed at which they can screen edited samples.

Bottlenecked by Screening

The rate-limiting step in many gene editing projects is sifting through samples to find those that remain unedited. CRISPR-Cas9 is revolutionary, but it can have poor efficiency, as can other gene editing techniques. It’s a difficult biological problem, according to Tsukasa Sugo, PhD, founder of GenAhead Bio. “Poor editing efficiency is a challenge in gene-editing,” he says. “Editing quantification is a major bottleneck, and inefficient screening can waste valuable time and resources.”

One solution to this problem is Droplet Digital PCR (ddPCR) technology. “ddPCR allows us to quantify knock-in sequences with large residual template DNA accurately,” says Sugo. In the ddPCR workflow, donor DNA is partitioned into 20,000 droplets, where individual PCR reactions are conducted to provide absolute quantification of nucleic acids. This process allows for unparalleled precision and accuracy, which are critical for quantifying low-frequency editing events. Below, we explore four examples where ddPCR enabled groundbreaking work in gene editing.

CRISPR-SNIPER Improves Genome Editing Efficiency

CRISPR-Cas9 can generate knock-in models, but integration efficiency is low. In some instances, integration can be below the level detection by qPCR. A recently developed technique known as CRISPR-SNIPER quantifies knock-ins with precision and efficiency, saving precious time and money. Sugo, who pioneered the method, explains, “CRISPR-SNIPER uses highly sensitive ddPCR technology to precisely measure knock-in efficiency early in the experimental pipeline.” In gene editing situations where qPCR runs into technical limitations, such as single nucleotide exchanges and complex integrations, CRISPR-SNIPER can accurately measure integration at the cellular level.

Tracking HIV Infection with ddPCR

Droplet Digital PCR enabled scientists at Sangamo BioSciences to focus on the science instead of worrying about sequencing. First, researchers knocked out the receptor for human immunodeficiency virus (HIV) on CD4+ T cells ex vivo and then reintroduced the protected cells into patients, effectively curing them. Accurately measuring the frequency of HIV-infected cells in a patient is a critical component of their work. However, residual HIV DNA is so rare that it can evade detection by traditional qPCR. To combat this, researchers at Sangamo BioSciences turned to ddPCR assays to quantify HIV-positive DNA in patient samples. The extremely high sensitivity of ddPCR technology makes it excellent at detecting low copy number events. As a result, ddPCR assays allowed the team to confidently monitor patient HIV levels.

Developing Incision-Free Genome Editing

Most gene editing is performed using CRISPR, which cuts DNA to allow for its manipulation. While effective, breaking DNA can result in indels and may trigger an inflammatory DNA damage response. In their work published in Molecular Therapy, Kuang and colleagues describe an innovative cleavage-free genome editing technique that uses ddPCR assays to quantify genetic modification (Kuang et al. 2022). The method, known as replication interrupted template-driven DNA modification, stalls the replication fork. Donor DNA can then be inserted as a template for the exposed single-stranded lagging strand. The authors used ddPCR technology to demonstrate their concept to detect a single nucleotide conversion at the cellular level.

CAR-T Cell Design

CAR T cells specific to CD19 are highly effective against B-cell malignancies. However, traditional CAR T cells are engineered using viral vectors, which can be costly and time-consuming. Bishop and colleagues generated CAR T cells with Doggybones linear DNA vectors as an efficient alternative, incorporating the piggyBac transposon delivery system (Bishop et al. 2020). Additionally, the researchers used ddPCR assays to accurately quantify the integration copy number, giving them confidence that the cells would adhere to safety guidelines without sacrificing efficacy.

ddPCR Technology Brings Precision to Gene Editing

Across disciplines, targeted genome modification is making precision medicine a reality. However, gene editing is only as robust as its quality control. Droplet Digital PCR provides the precision, accuracy, and efficiency required to perform groundbreaking gene-editing science at industrial speed.

With ddPCR technology, scientists can let ddPCR take care of quality control sequencing and instead focus on what they do best: science.

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