Long ssDNA Template for Gene Editing

          Single-stranded DNAs (ssDNA) have been widely used in molecular biology research and molecular diagnosis, e.g. DNA sequences, DNA/RNA hybridization, SNP analyses, DNA chips , SSCP analyses , SELEX , etc. Several methods, as summarized in the following table, were recruited to prepare ssDNA, such as chemical synthesis, Taq enzyme-based asymmetric PCR (aPCR), DNA exonuclease-based digestion of long PCR products, biotin magnetic bead assisted separation of long ssDNA, DNA nickase-based digestion of plasmid. However, these methods with distinct strength and weakness all require the tedious and time-consuming multiple steps along with various concerns, such as low-fidelity synthetic oligonucleotides with terminal conjugation, exonuclease side reaction, potential mutation and terminal deletion caused by Taq-based aPCR, low efficiency, etc.

             In our proprietary technique of ssDNA preparation, Gibson Assembly and Rolling Circular Amplification (RCA) were recruited to produce ssDNA. Briefly, (1) the front and back homology arm (HA) DNA fragments containing Type IIS restriction enzyme site loop and insert DNA were assembled first. (2) The circled DNA was then amplified by rolling circle amplification (RCA). (3) Finally, ssDNA were obtained by Type IIS restriction enzyme unitization of the resulted ssDNA concatemer. Using our RCA-based approach, any length and any amount of long ssDNA with the highest purity can be efficiently produced at ease. The following schematic describes the procedure of long ssDNA preparation followed by the long ssDNA-mediated and site-specific gene knock-in in the CRISPR/Cas system.

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Fig 1. Schematic describing the procedure of long ssDNA preparation and its application for CRISPR/Cas9 HDR. The front and back fragments homologous to the target genes, the knock-in DNA fragment, were prepared by PCR separately. All fragments have at least 20 bp overlap to each other for efficient assembly in the following reaction. After assembly (circulation), the ssDNA Premix was added to extend the ssDNA synthesis. The prepared long ssDNA was then used for CRISPR/Cas9 HDR.