ssDNA in Genome Editing: Mechanisms, Applications, and Challenges

Genetic engineering has been driven forward by genome editing technologies and, of these, CRISPR-Cas platforms are among those that have inspired the field. One of the most promising materials in these technologies is single-stranded DNA (ssDNA), which has attracted great attention because it is such a useful donor template for genome editing. More recently, it was discovered that intracellular production of ssDNA can be used to continuously engineer genomes, creating new opportunities for genetic engineering. This can be especially useful for dynamic, genome-scale changes without the need for an external oligonucleotide synthesis, cutting costs and increasing efficiency in genetic studies and biomanufacturing.

What is ssDNA?

Single-stranded DNA (ssDNA) consists of a single strand of nucleotides, as opposed to double-stranded DNA (dsDNA), which forms a double helix. ssDNA plays a crucial role in various biological processes such as DNA replication, repair, and recombination. It also serves as a key intermediate in several biotechnological applications, including genome engineering and gene therapy. Unlike dsDNA, which requires unwinding and separation before recombination or editing can occur, ssDNA is more readily incorporated into the target genome, making it a powerful tool for precise genome modifications. ssDNA can also be used in the production of RNA molecules, allowing for the creation of mRNA vaccines and other RNA-based therapeutics.

ssDNA vs dsDNA. (Cohen, I.; et al, 2013)

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ssDNA Example

Synthetic ssDNA

Synthetic ssDNA refers to single-stranded DNA molecules chemically produced in the lab using solid-phase oligonucleotide synthesis. These short DNA sequences, typically a few to several hundred nucleotides, are used in various applications, including genome editing, PCR, and diagnostics. Here are the Applications of synthetic ssDNA:

• Genome Editing: Synthetic ssDNA serves as a donor template for CRISPR/Cas9, enabling precise insertions, deletions, or mutations.
• PCR Primers: Short ssDNA sequences are used as primers in PCR to amplify specific DNA regions.
• Diagnostic Assays: ssDNA probes are key in molecular diagnostics like qPCR and FISH for detecting specific DNA sequences.

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In Vivo Synthesized ssDNA

In vivo synthesized ssDNA is produced inside living cells, typically from engineered plasmids or viral vectors. This method leverages cellular machinery for replication and transcription, enabling scalable production. In vivo ssDNA yields are often higher than chemically synthesized ssDNA, especially with systems like rolling-circle replication (RCR). ssDNA produced inside cells integrates more efficiently with cellular processes, improving outcomes in applications such as homologous recombination and gene integration. Here are the Applications of in vivo synthesized ssDNA:

• Continuous Genome Engineering: In vivo ssDNA supports repeated genome editing cycles in bacteria, mammalian, or yeast cells, aiding large-scale biotechnological applications.
• Gene Therapy: In vivo ssDNA enables direct integration of therapeutic genes into patient cells for streamlined gene therapy.

Circular ssDNA

Circular ssDNA (covalently closed circular ssDNA) forms a continuous loop, offering greater stability than linear ssDNA due to its covalently linked ends, which prevent exonuclease degradation. This stability makes it ideal for long-term storage and continuous genome engineering. Circular ssDNA can also serve as a substrate for rolling-circle replication, enabling rapid production of multiple ssDNA copies from a single template. Here are the Applications of circular ssDNA:

• High-Yield ssDNA Production: Circular ssDNA in engineered bacterial systems using RCR enables large-scale, long-term ssDNA production.
• Genome Editing: Circular ssDNA serves as a donor template in genome editing, especially with CRISPR-based tools requiring ssDNA for homology-directed repair.

Linear ssDNA

Linear ssDNA consists of single-stranded molecules with distinct 5' and 3' ends, often derived from circular ssDNA templates or chemically synthesized. It offers precise control over sequence length and insertion points. The free ends of linear ssDNA are critical for applications like homologous recombination and genome integration, making it commonly used as a donor template in genome editing. Here are the applications of linear ssDNA:

• Homologous Recombination: Linear ssDNA is used in CRISPR-Cas9 genome editing for precise insertion or modification, aiding alignment and integration at the target locus.
• Mutagenesis: Linear ssDNA introduces specific mutations or deletions, enabling precise genetic engineering.
• Gene Synthesis: Linear ssDNA serves as a template for synthesizing large genes or synthetic pathways, providing control over sequence composition.

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Intracellular Production of ssDNA

The intracellular production of single-stranded DNA (ssDNA) has emerged as a pivotal process in modern biotechnology and genetic engineering. This method leverages the natural biological systems within cells to generate ssDNA continuously and efficiently, making it an invaluable tool for large-scale and long-term genetic modification projects. By harnessing cellular machinery, researchers can generate significant quantities of ssDNA for a variety of applications, including gene editing, gene therapy, synthetic biology, and bio-manufacturing.

Rolling Circle Replication Mechanism

Rolling-circle replication is the most widely used method for intracellular ssDNA production. It is a natural process used by certain viruses and plasmids to replicate their genomes. In this method, a circular DNA molecule (plasmid) is replicated by an initiator protein, creating multiple copies of ssDNA in a continuous manner.

• Process: RCR begins when a single-strand nick is introduced into the circular DNA molecule by a replicative helicase or initiator protein. This nick causes the DNA to "roll" around the template, displacing the newly synthesized strand. The displaced strand forms the ssDNA. This process is continuous, meaning that many ssDNA molecules can be produced from a single circular template.
• Plasmid-based Systems: Researchers can insert a rolling-circle replicon into bacterial or mammalian cells. This replicon is engineered to initiate rolling-circle replication within the cell, generating multiple ssDNA molecules. These plasmids typically include elements that allow for efficient replication and high yield of ssDNA.
• Examples: Common plasmids used for RCR-based ssDNA production include pC194, which has been used in bacterial systems, and various viral vectors that utilize RCR for high-efficiency DNA replication.

What is Genome Editing?

Genome editing refers to the process of introducing targeted genetic modifications into the DNA of living organisms. This can involve inserting, deleting, or altering specific sequences of nucleotides to achieve desired traits or to study the effects of genetic changes. The CRISPR-Cas9 system has emerged as one of the most powerful tools for genome editing, allowing for precise modifications in a wide range of organisms. In this system, a guide RNA directs the Cas9 nuclease to a specific target sequence in the genome, where it induces a double-strand break. This break can then be repaired using a donor template, such as ssDNA, to insert or modify genetic material.

CRISPR Genome Editing

Genome editing has undergone a dramatic transformation with the advent of CRISPR-Cas9 technology, a revolutionary tool that allows for precise, targeted alterations to the DNA of living organisms. Since its discovery, CRISPR-Cas9 has redefined genetic research, enabling applications ranging from basic science and agricultural improvements to potential therapies for genetic diseases. With its efficiency, cost-effectiveness, and simplicity, CRISPR-Cas9 has become the method of choice for genome engineering, making previously complex tasks in genetics accessible and practical.

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ssDNA in Genome Editing

Single-stranded DNA (ssDNA) has become an indispensable tool in the realm of genome editing, especially in technologies like CRISPR-Cas9. Its role in genome editing spans beyond simple template usage; it is pivotal for precise genetic modifications, facilitating the introduction of small-scale mutations, gene insertions, deletions, and large sequence alterations with high accuracy. ssDNA's ability to efficiently participate in homologous recombination (HR) makes it an ideal candidate for correcting or modifying genomic sequences at the targeted location.

Mechanisms of ssDNA-Mediated Genome Editing

Genome editing via ssDNA typically utilizes the mechanism of homologous recombination (HR). In this context, ssDNA serves as a repair template during the cellular repair of a double-strand break (DSB) induced by genome editing tools like CRISPR-Cas9. The process unfolds in several stages:

• Cas9-Mediated DNA Cleavage: The CRISPR-Cas9 system creates a double-strand break at a pre-determined genomic location. This cleavage activates the cell's DNA repair machinery, which typically attempts to fix the break using one of two pathways. (1) Non-homologous end joining (NHEJ), which is error-prone and can result in insertions or deletions (indels). (2) Homologous recombination (HR), which is a more accurate repair mechanism that requires a homologous DNA template to guide the repair.
• Incorporation of ssDNA Template: When ssDNA is introduced into the cell following the Cas9-induced break, it acts as the donor template for the HR repair process. The ssDNA carries a sequence identical to the region of the target genome surrounding the break, but it also contains the desired edit—be it a mutation, insertion, or modification. The cell's repair machinery uses the ssDNA template to perform a highly accurate repair, incorporating the desired genetic modification into the target locus. Since ssDNA can be designed to closely match the target sequence, it ensures precise integration of the desired changes without the errors that are often associated with NHEJ.
• Precision of ssDNA-Mediated Modifications: One of the significant advantages of using ssDNA over other DNA forms, such as double-stranded DNA (dsDNA), is that it enables more efficient base-pairing during the repair process. ssDNA can be designed to precisely match the sequence on the homologous chromosome, allowing for the precise insertion of single-base mutations, small deletions, or even the integration of larger genetic elements like reporter genes or resistance markers.

The ability to employ ssDNA in HR-based genome editing vastly improves the precision of genetic modifications. Unlike dsDNA, which may have more complex secondary structures or be subject to partial degradation, ssDNA is straightforward and efficiently incorporated into the genome, especially when it is designed as a circular or linear construct that optimizes its stability and uptake by cells.

ssDNA in CRISPR Genome Editing

The CRISPR-Cas9 system has transformed the landscape of genome editing by enabling precise cuts in DNA and facilitating the incorporation of desired genetic modifications. ssDNA plays a critical role in enhancing the efficiency of CRISPR-based editing by acting as a donor template during the repair phase. The efficiency of HDR (homology-directed repair), the pathway responsible for accurate genome editing, is highly dependent on the donor template used.

• Template Design and Efficiency: ssDNA templates are designed to carry specific genetic changes, such as point mutations or insertions, offering a clean and efficient method for genome editing. Unlike plasmids or dsDNA, ssDNA minimizes undesired sequences or competition for integration.
• CRISPR-Cas9 Enhancements: Recent advances in CRISPR include base editing, which uses a modified Cas9 enzyme (e.g., dCas9) with a deaminase to introduce point mutations without double-strand breaks. Paired with ssDNA, this method enables precise edits at the base level with high efficiency.
• Dual-Guide RNA Systems: Dual-guide RNA systems induce staggered double-strand breaks, and the resulting gap is repaired using ssDNA, enabling the insertion of large genetic elements or correction of larger regions. This approach enhances precision and reduces off-target effects.

Advantages of ssDNA in Genome Editing

• Higher Efficiency of Integration: ssDNA is generally more efficiently integrated into the genome during HR compared to dsDNA. The single-stranded nature of the donor template ensures that it is immediately accessible for homology-based repair, whereas dsDNA may need to be unwound or processed before it can be utilized.
• Lower Risk of Unintended Modifications: ssDNA minimizes the risk of random integrations and off-target effects commonly associated with larger dsDNA templates. The targeted nature of ssDNA-based repair ensures that the genetic modification occurs precisely at the desired site.
• Reduced Risk of Degradation: ssDNA, particularly when synthesized in circular forms or protected by stabilizing agents, is more resistant to exonucleolytic degradation compared to dsDNA. This makes ssDNA a more reliable tool in genome editing applications, especially in systems where the DNA is expressed over long periods or in high-copy numbers.

Challenges of ssDNA in Genome Editing

Despite the many advantages of using ssDNA for genome editing, there are several challenges that researchers must consider to maximize its potential:

• Delivery Efficiency: The efficient delivery of ssDNA into target cells remains a critical factor in the success of genome editing applications. While viral vectors, nanoparticles, and electroporation have been successfully used to introduce ssDNA into mammalian cells, the efficiency of these methods can vary depending on the cell type and experimental conditions.
• Targeting Specific Genomic Loci: Achieving precise delivery to specific genomic loci can be challenging, particularly in mammalian cells. While CRISPR-Cas9 can target specific regions of the genome, the efficient integration of ssDNA requires careful design to ensure that the repair template is aligned with the target locus.
• Off-Target Effects: Despite improvements in CRISPR technology, off-target effects remain a concern in genome editing. ssDNA-based repair mechanisms are generally more specific than NHEJ, but researchers must still account for potential off-target edits, especially when using longer ssDNA templates or complex genetic modifications.

The ability to leverage ssDNA in genome editing holds great promise for therapeutic applications, particularly in the context of gene therapy. By using CRISPR-Cas9 to introduce precise edits into the genome and ssDNA as the donor template, researchers can correct genetic mutations at the root cause of many genetic disorders. Conditions such as cystic fibrosis, sickle cell anemia, and muscular dystrophy could potentially be addressed through precise genetic modifications facilitated by ssDNA.

Reference

1. Cohen, I.; et al. Microtubule attachment and spindle assembly checkpoint signaling at the kinetochore. Nature Reviews Molecular Cell Biology. 2013, 14(1): 25-37.