ssDNA Template in Genome Editing: Mechanisms and Applications

Single-stranded DNA (ssDNA) templates are essential in genome editing, providing precise instructions for modifying genetic sequences. They are widely used in CRISPR-based homology-directed repair (HDR) to introduce specific mutations or correct genetic defects with high accuracy. Their efficiency and versatility make them indispensable tools in molecular biology and genetic engineering.

Understanding ssDNA templates and their role in genome modification is crucial for advancing gene therapy, functional genomics, and disease modeling.

Basic Structure And Composition

Single-stranded DNA (ssDNA) consists of a linear sequence of nucleotides, each containing a phosphate group, a deoxyribose sugar, and one of four nitrogenous bases: adenine (A), thymine (T), cytosine (C), or guanine (G). Unlike double-stranded DNA (dsDNA), which forms a helical structure through complementary base pairing, ssDNA remains a single polymeric strand, making it more flexible and prone to forming secondary structures such as hairpins or loops. These structural variations affect stability and interaction with cellular machinery, particularly in genome editing, where precise base pairing is crucial for HDR.

The length of ssDNA templates varies depending on the intended modification. Short ssDNA oligonucleotides, typically 30 to 200 nucleotides long, are used for introducing point mutations or small insertions and deletions. Longer templates, extending up to several kilobases, facilitate more extensive genomic alterations, such as gene knock-ins or large sequence replacements. While shorter templates integrate more easily, they may lack sufficient homology arms for stable recombination. Longer templates ensure greater sequence fidelity but pose challenges in synthesis and delivery.

Chemical modifications enhance ssDNA stability and functionality. Phosphorothioate linkages improve resistance to exonuclease degradation, while locked nucleic acids (LNAs) and 2′-O-methyl modifications enhance hybridization specificity and reduce off-target effects. These modifications help counteract degradation by cellular nucleases, improving editing efficiency. Additionally, optimizing sequence design helps minimize secondary structures that could interfere with HDR.

Methods For Synthesis

Generating ssDNA templates involves multiple approaches tailored to length, purity, and application. Chemical synthesis is the most common method for short ssDNA oligonucleotides, typically up to 200 nucleotides. This process uses solid-phase phosphoramidite chemistry, where nucleotides are sequentially added to a growing chain anchored to a solid support. Automated synthesizers ensure high fidelity, though chemical synthesis becomes inefficient for longer sequences due to cumulative errors and incomplete elongation.

For longer sequences, enzymatic methods provide viable alternatives. Asymmetric PCR preferentially amplifies a single DNA strand by using an excess of one primer, making it useful for generating ssDNA templates in the 200–1000 nucleotide range. However, optimizing primer ratios and thermal cycling conditions is necessary to prevent double-stranded byproducts. Another approach, exonuclease digestion, selectively degrades one strand from a double-stranded DNA precursor. Lambda exonuclease, which targets the 5′-phosphorylated strand, is commonly used to produce high-purity ssDNA suitable for genome editing.

For large ssDNA constructs beyond a kilobase, in vitro transcription followed by reverse transcription is an effective method. RNA transcripts generated from a DNA template using T7 RNA polymerase are reverse-transcribed into ssDNA, with the RNA strand subsequently degraded. This strategy enables high-yield production but requires careful sequence design to prevent secondary structures that could impede transcription efficiency.

Mechanisms In Genome Editing

Single-stranded DNA (ssDNA) templates enable precise genome modifications through homology-directed repair (HDR). When introduced into cells alongside programmable nucleases like CRISPR-Cas9, they provide homologous sequences that guide repair machinery to incorporate desired genetic changes. The efficiency of this process depends on template length, homology arm design, and intracellular stability. Unlike double-stranded templates, ssDNA reduces the risk of random insertions or genomic rearrangements, making it a preferred choice for precision editing.

Following a CRISPR-Cas9-induced double-strand break (DSB), the cell’s repair pathways determine the modification outcome. HDR, which occurs predominantly during the S and G2 phases of the cell cycle, uses ssDNA templates for high-fidelity sequence incorporation. Complementary homology arms flanking the desired edit facilitate accurate base pairing, guiding DNA polymerase and ligase to integrate the new sequence. Proteins like RAD51 enhance strand invasion, improving recombination efficiency, though competition with the more error-prone non-homologous end joining (NHEJ) pathway can reduce overall success.

The structural properties of ssDNA templates influence repair dynamics. Secondary structures such as hairpins can hinder accessibility to repair machinery, reducing HDR efficiency. Researchers optimize template design by incorporating phosphorothioate modifications or adjusting homology arm symmetry to improve hybridization. Delivery methods, including electroporation and lipid-based transfection, enhance ssDNA uptake in various cell types. Intracellular nucleases degrade ssDNA, but chemical modifications and protective carrier molecules help extend template lifespan.

Role In DNA Repair Pathways

Single-stranded DNA (ssDNA) templates guide error-free genomic repair by serving as donor strands in homology-directed repair (HDR). Cellular repair proteins, including recombinases like RAD51, facilitate strand invasion and annealing, ensuring high-fidelity correction of double-strand breaks (DSBs), particularly in dividing cells where HDR is most active.

Beyond HDR, ssDNA influences other repair mechanisms such as single-strand annealing (SSA) and microhomology-mediated end joining (MMEJ). SSA aligns homologous regions flanking a break, leading to sequence deletions between repeats. Though inherently mutagenic, ssDNA templates can leverage SSA’s homology-driven repair to introduce precise modifications. Similarly, MMEJ relies on short microhomology sequences for alignment before ligation, and ssDNA templates can enhance accuracy in small insertions or deletions.

Applications In Primary Cell Cultures

Single-stranded DNA (ssDNA) templates enable precise genome modifications in primary cell cultures, which retain their original phenotype and provide physiologically relevant models for studying gene function and disease mechanisms. Unlike immortalized cell lines, primary cells have limited proliferative capacity and are sensitive to transfection, making efficient ssDNA delivery crucial for high-fidelity genome edits.

One significant application is the correction of genetic mutations in patient-derived cells. In hematopoietic stem cells, ssDNA templates have been used to correct mutations associated with sickle cell disease and β-thalassemia. Electroporation-mediated ssDNA delivery combined with CRISPR-Cas9 has achieved efficient gene correction while maintaining cell viability. Similarly, in induced pluripotent stem cells (iPSCs), ssDNA-mediated editing has introduced disease-associated mutations for studying conditions like cystic fibrosis and Duchenne muscular dystrophy, providing accurate models for therapeutic testing.

Beyond disease correction, ssDNA templates play a role in engineering primary cells for specialized functions. In immunotherapy, T cells can be modified to express chimeric antigen receptors (CARs) with precise sequence modifications introduced via ssDNA-mediated HDR. This ensures that engineered cells maintain their intended function without unintended genomic alterations. In organoid research, ssDNA templates facilitate lineage-specific genetic marker introduction, enabling precise tracking of cellular differentiation and tissue development. These applications highlight the versatility of ssDNA templates in advancing personalized medicine and functional genomics.