Long ssDNA Template for Gene Editing
Although CRISPR/Cas system has revolutionized genetic manipulation, obtaining the desired mutation with high efficiency is still difficult to complete successfully. Homology directed repair (HDR) recombination was proved to be more powerful to introduce the desired mutation at double-strand break (DSB) site generated by CRISPR/Cas, than the error-prone non-homologous end joining (NHEJ) repair. In addition to the selected PAM sites, Cas9 enzyme, and guide RNA format, the type of donor DNA can significantly affect the efficiency of HDR at the time of repair. Increasing evidences indicated that long single strand DNA (ssDNA) template which contains a desired insert flanked by at least 300 bp homology arms complementary to the ends of DSB, was superior to single-stranded oligo-deoxynucleotides (ssODNs) and double strand DNA (dsDNA) for error-free and seamless insertion of exogenous genetic materials at DSB. Furthermore, the less toxicity of long ssDNA, compared to ssODNs and dsDNA, is especially of importance for genomic manipulation in primary cells, such as T cells, B cells, and stem cells etc. Although several methods were recruited to prepare long ssDNA, such as chemical synthesis, biotin magnetic bead assisted separation of long ssDNA, DNA exonuclease based digestion of long PCR products, DNA nickase-based digestion of plasmid, Taq enzyme-based asymmetric PCR (aPCR), these methods with distinct strength and weakness all require the tedious and time-consuming multiple steps along with various concern, such as not high-fidelity synthetic oligonucleotides, exonuclease side reaction, potential mutation and terminal deletion caused by Taq-based aPCR etc. In our proprietary technique of long ssDNA preparation, firstly, forward and reverse homology arm DNA fragments and insert DNA were assembled. Secondarily, the circled DNA was then amplified by rolling circle amplification (RCA). Finally, long ssDNA were obtained by enzyme unitization of the resulting DNA concatemer. All of these reactions were performed in a single tube, with minimum handle time. Using our RCA-based long ssDNA production Premix (Cat#: ss100), any length and amount of long ssDNA with highest purity can be efficiently produced at ease. The following schematic (left) describes the procedures of long ssDNA preparation and long ssDNA-mediated and site-specific gene knockin, while the right is typical ssDNA production.
In addition to gene editing, long ssDNA was also used for ssDNA sequencing, In Vitro transcription, SNP detection, DNA aptamer production, subtractive hybridization, and nuclease S1 mapping etc.
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