Gene Editing: From Innovation to Application

Gene editing is rapidly evolving, and its relevance across biotechnology and medicine continues to expand. As these tools advance, so does the need for reliable ways to measure editing efficiency and identify off‑target effects. Here, we review today’s major gene-editing technologies and explain how understanding their edit outcomes can help you choose appropriate downstream measurement methods, including high‑precision approaches such as Droplet Digital™ PCR (ddPCR™) technology.

Gene editing is revolutionizing biotechnology and accelerating drug discovery, synthetic biology, and the development of novel therapeutic strategies by allowing scientists to precisely alter DNA sequences within living cells and organisms. Established and emerging gene-editing technologies can facilitate various desirable therapeutic outcomes, including correcting genetic alterations that cause disease, investigating gene function, and developing personalized therapies. While these technologies are readily available and enable targeted manipulation of genomic DNA, quantification of gene-editing efficiency remains a time- and resource-intensive challenge. Samples must also be screened for off-target effects or edits occurring at unintended sites. The DNA repair processes at on- and off-target sites can also result in insertions or deletions, as well as larger structural variations (SVs; e.g., duplications, inversions, large deletions, and translocations) that interfere with gene expression or cause significant genotoxicity¹. Developed for the absolute quantification of nucleic acids, Droplet Digital PCR technology is an ultrasensitive method that addresses key challenges in gene-editing workflows by enabling detection of low-frequency genome-editing events and quality-control optimization through a rapid, low-cost workflow.

Gene-Editing Technologies

Modern gene editing utilizes engineered nuclease tools that precisely target DNA sequences and harness cellular DNA repair pathways to achieve the desired modification. Early tools such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) use engineered enzymes composed of a non-specific DNA endonuclease domain fused with a sequence-specific DNA-binding domain to induce targeted double-strand breaks (DSBs)². Once the DNA is cut, the cell repairs the damage through either error-prone non-homologous end joining (NHEJ), which can disrupt genes, or homology-directed repair (HDR), which relies on the availability of a homologous sequence as a repair template and can introduce insertions, deletions, or mutations. However, ZFNs and TALENs have notable limitations, as designing and generating these custom fusion proteins can be technically complex and laborious. The development of the CRISPR-Cas9 system, in which a guide RNA directs the Cas9 enzyme to a specific genomic location through base pairing, led to its rapid adoption and refinement as a genome-editing technology. While Cas9 generates DSBs and relies on the same DNA repair mechanisms as ZFNs and TALENs, this system offers several advantages, including its ability to be easily reprogrammed to target any sequence or numerous sites through multiplexing³. Newer tools derived from CRISPR-Cas9 aim to avoid the creation of DSBs². Base editors consist of a catalytically altered Cas9 nickase coupled with a deaminase that chemically converts one nucleotide into another, enabling the precise introduction of specific point mutations. Prime editing technology further expands the possibilities in gene editing by coupling a Cas9 nickase with a reverse transcriptase (RT) and by using a guide RNA fused to an RT template and a primer-binding site (PBS). The complementary DNA strand is nicked and extended by RT upon its hybridization to the PBS; the RT-template sequence is then introduced to the target site. This approach allows short DNA sequences to be inserted, deleted, or replaced. Prime editors continue to evolve with the identification of Cas9 modifications and incorporation of RNA template-stabilizing proteins that dramatically reduce error rates⁴.

Applications of Gene Editing

Gene editing has the potential to drive vast and impactful advancements in medicine. By allowing precise modification of the genetic causes of disease, these technologies have broad applications in the development of novel therapies across a wide range of conditions, including inherited disorders, cancers, and infectious diseases. Importantly, gene editing has bolstered the development of treatments tailored to individual patients and designed to address rare disorders. This year, a personalized, CRISPR-based gene-editing platform was successfully implemented in a human patient to treat carbamoyl phosphate synthetase 1 (CPS1) deficiency, a rare and life-threatening genetic disease. CPS1 deficiency hampers the processing of byproducts from protein metabolism in the liver, leading to the toxic accumulation of ammonia in the body, which typically presents in patients during the neonatal period⁵. In this trial, the patient received two doses of the therapy at approximately 7 and 8 months of age, after which dietary protein intake could be increased while medication to control ammonia levels was reduced⁶. No serious adverse events occurred, suggesting that this platform could be adapted to treat other genetic disorders, although long-term safety and efficacy still need to be evaluated. A first-in-human phase 1 clinical trial of a CRISPR-Cas9 gene-editing therapy was recently completed in patients with progressive, metastatic gastrointestinal epithelial cancer⁷. By targeting and deactivating the CISH gene, which encodes an intracellular immune checkpoint protein, in tumor-infiltrating lymphocytes (TILs), this therapy may promote antitumor activity. In this study, 12 patients received an infusion of autologous, modified TILs. Disease was stable in six (50%) and four (33%) patients at 28 and 56 days, respectively. Notably, one patient with disease refractory to other immunotherapies had a complete response, which was ongoing for >21 months. These results suggest that CRISPR-Cas9-mediated editing of a novel gene target in TILs may provide future therapeutic options for patients with advanced metastatic disease resistant to currently available immune checkpoint inhibitors.

Challenges in Measuring Editing Efficiency

While gene editing is expanding therapeutic possibilities, its efficiency can be limited by factors such as cell type, particularly primary cells or induced pluripotent stem cells (iPSCs). Editing efficiency in these cell types can fall below 5%, requiring highly sensitive techniques to measure editing events. Traditional methods, such as Sanger sequencing and gel-based methods, fall short in identifying low-frequency editing events in genetically heterogeneous cell populations (bulletin 6712). Although next-generation sequencing (NGS) is a sensitive alternative, it presents significant challenges in terms of protocol complexity and cost. Further, sample preparation for NGS can inadvertently introduce biases that impact the detection of edited alleles, which may skew the results. Addressing these limitations is essential for optimizing gene-editing protocols and advancing therapeutic applications.

ddPCR Technology and Synergy with Gene Editing

Droplet Digital PCR technology is a rapid, ultrasensitive method designed for the absolute quantification of nucleic acids. The sensitivity and precision of ddPCR technology are achieved by partitioning a sample into approximately 20,000 nanoliter-sized droplets, each of which serves as an individual PCR reaction. The application of Poisson statistics enables absolute nucleic acid quantification without the need for a standard curve (bulletin 6407). Additionally, the ddPCR workflow is simple, cost-effective, and offers high-throughput capabilities. Pairing ddPCR technology with gene-editing protocols provides an accurate and convenient method for validating the success and efficiency of target sequence modifications. For example, ddPCR technology has been used as a screening tool for CRISPR-Cas9-engineered knock-in reporter cell lines for dopaminergic (DA) neurons derived from human iPSCs⁸. The cell lines were engineered to express enhanced green fluorescent protein (eGFP) under the control of an endogenous promoter for tyrosine hydroxylase, a common marker of DA neurons. A ddPCR strategy was designed to detect both eGFP insertion and identify successful HDR events, in which the assay spanned the genomic region upstream of the homology sequence that was cloned and fused with eGFP. Thus, amplification occurred only in instances of recombination at the correct genomic locus. In addition, ddPCR technology can be used to investigate the functions of genes and to evaluate specific insertions or deletions or SVs over time, such as in modified cells undergoing clonal expansion¹. This method can be employed to assess the potential for oncogenic transformation of edited cells. Further, due to the partitioning of each sample into thousands of individual reactions, ddPCR technology may be more resistant to interference from PCR inhibitors compared with traditional real-time quantitative PCR (qPCR), making it more advantageous in the analysis of complex clinical samples for applications such as cancer research and viral load monitoring⁹.

Conclusion

The integration of gene editing and ddPCR technology accelerates innovation in biotechnology and medicine by addressing critical challenges in the analysis of genetic modifications, particularly in primary cell types and other complex samples. As an ultrasensitive, accurate, and convenient method for the absolute quantification of nucleic acids, ddPCR technology enables the detection of low-frequency genome-editing events and validation of target sequence modifications via a rapid, low-cost workflow, which is essential for optimizing gene-editing protocols.

References

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Trafton A (2025). A more precise way to edit the genome. MIT News. https://news.mit.edu/2025/more-precise-way-edit-genome-0917, accessed March 25, 2026.
Noori M et al. (2024). Carbamoly-phosphate synthetase 1 (CPS1) deficiency: A tertiary center retrospective cohort study and literature review. Mol Genet Metab Rep 41, 101156.
Musunuru K et al. (2025). Patient-Specific In Vivo Gene Editing to Treat a Rare Genetic Disease. N Engl J Med 392, 2235–2243.
Lou E et al. (2025). Targeting the intracellular immune checkpoint CISH with CRISPR-Cas9-edited T cells in patients with metastatic colorectal cancer: a first-in-human, single-centre, phase 1 trial. Lancet Oncol 26, 559–570.
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Gene Editing: Navigating the Path from Innovation to Application