Leveraging synthetic ssDNA and dsDNA donor templates for CRISPR-mediated gene editing

Over the past decade, CRISPR/Cas9 gene editing has gone from a groundbreaking discovery to a powerful tool that’s already treating patients in clinical trials. Today, gene editing can be performed either ex vivo, where cells are taken from the patient or a donor, edited outside the body, before reinfusing back into the patient, or in vivo, where the editing tools are delivered directly into the body. Among the early success stories is CAR-T cell therapy, where T cells are engineered with chimeric antigen receptors (CARs) to target and destroy certain cancers.

Since the first approval of CRISPR-based therapeutic in 2023, Casgevy for sickle cell disease, the clinical pipeline for gene editing medicines has continued to expand beyond cancer and blood disorders (1). CRISPR-based therapeutics are now being explored for diverse applications, for example cardiovascular conditions and inherited monogenic disorders like cystic fibrosis. The field is even moving to personalized treatments, such as the first base editing therapy designed to correct a patient-specific mutation in an individual patient in early 2025 (2). So far, clinical successes have focused largely on knocking out disease-causing genes or performing single base edits.

The next generation of editing tools can insert large genes precisely where we want them in the genome using strategies such as homology directed repair (HDR). By supplying a DNA template alongside CRISPR/Cas9, researchers can achieve targeted insertion of large gene sequences, opening the door to tackle a much broader array of diseases, for example by correcting mutated genes or permanently inserting an entire transgene. Historically, these approaches were hindered by inefficient delivery, safety concerns with viral vectors and the lack of scalable, high-purity DNA template production.

At 4basebio we’re addressing these challenges head on, enabling the production of synthetic DNA HDR templates with the purity, integrity, and length required for next-generation gene therapies. Our DNA is compatible with our Hermes® nanoparticle platform, and we offer formulation services to support safe, efficient, non-viral delivery strategies.

How CRISPR/Cas9 Enables Precision Edits

Cas9 nucleases are used to generate double-stranded breaks (DSB) at specific genomic locations. A guide RNA (gRNA), co-delivered with the Cas9 nuclease, recognizes the protospacer adjacent motif (PAM) sequence of the target gene, guiding the Cas9 nuclease to induce a DSB at the desired location. This then triggers the cell to begin one of two repair mechanisms:

1. Non-homologous end joining (NHEJ), a quick error prone fix that directly ligates the ends, but is susceptible to mutations at the DSB,

2. Homology-directed repair (HDR), which uses a custom-designed DNA template to insert new genetic material exactly where the break occurred.

HDR templates are typically plasmid DNA (pDNA), double stranded DNA PCR products, or single-stranded DNA (ssDNA) and provide the necessary homologous regions flanking the DSB to guide repair.

Challenges to Donor Template Manufacture

The type of HDR template can affect the efficiency and accuracy of repair. pDNA, though widely used, may cause genomic integration of unwanted bacterial backbone sequences or instability. Additionally, HDR efficiency is generally low with circular pDNA templates. Consequently, other double stranded DNA (dsDNA) templates, such as PCR products, are often favored but are limited by size, fidelity and scalability, as well as being susceptible to degradation by exonucleases.

Despite its widespread use, the innate immunity to dsDNA in these different formats is problematic and although rare, studies show that dsDNA can integrate into the genome independent of target homology. Recently, there’s been growing interest in ssDNA templates on account of their lower toxicity, with reports of superior editing efficiency and reduced integration and off-target effects (3). However, ssDNA is typically produced by chemical synthesis methods that are limited to short lengths (<200 nt) and suffer low yields with high non-target impurities. These are not suitable for larger edits, where longer homology arms (500-1000 bp) are required and aren’t scalable beyond microgram quantities. That’s where enzymatic DNA manufacturing approaches are starting to change the game.

Enzymatic manufacture: A scalable alternative for long donor templates

To address these challenges, 4basebio has developed a fully enzymatic, cell-free method for making high-quality DNA donor templates, both double- and single-stranded, at larger scales than offered elsewhere on the market. The proprietary method offers:

• Scalability – from milligram to multigram quantities, including GMP-grade batches for dsDNA templates

• High fidelity – using proprietary enzymes with higher accuracy than PCR-based methods

• Safety and compliance – DNA is amplified from circular templates that are free of bacterial backbone elements like antibiotic resistance genes

• Long sequences – capable of producing double-stranded templates up to 20 kb and, single-stranded formats up to 10 kb

Open-ended or oeDNA®, 4basebio’s double-stranded donor templates, are generated using rolling circle amplification (RCA), which allows us to bypass bacterial fermentation and scale production without compromising on quality. Following amplification, the ends are processed to protect from exonuclease degradation, essential for stability in cell and gene therapy workflows. We support constructs ranging from 140 bp to 20 kb, including high GC-content sequences that are notoriously difficult to handle using traditional methods.

For single-stranded DNA, our proprietary method can produce donor templates up to 10 kilobases, the longest available on the market today. These ssDNA templates are free of double-stranded contaminants and are end-processed to resist degradation, just like oeDNA. We currently offer ssDNA at scales up to 1 milligram, with the option to increase scale following successful evaluation.

Why does this matter?

With a fully enzymatic, cell-free platform, 4basebio is addressing the major pain points of donor DNA manufacturing: safety, length, scalability, and fidelity. Whether you’re working on cell therapy, gene therapy, or advanced genome editing tools, our synthetic DNA platform is designed to help you get from concept to clinic faster with better control and fewer risks.

4basebio’s enzymatic DNA platform is built to support innovation at every stage from discovery to clinical trials.

1. FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease, https://www.fda.gov/news-events/press-announcements/fda-approves-first-gene-therapies-treat-patients-sickle-cell-disease (accessed June 19, 2025)

2. Musunuru, K. et al., Patient-specific in vivo gene editing to treat a rare genetic disease, N Engl J Med. 392, 2235-2243 (2025)

3. Roth, T.L et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nat. Lett. 559, 405–409 (2018).