The Guide-it Long ssDNA Production System v2 enables on-demand production of long single-stranded DNA (ssDNA) up to 5,000 nt in length for use as a repair template in knockin experiments involving CRISPR/Cas9 technology or other genome editing tools. This kit provides a simple and fast method for converting a dsDNA PCR product of any sequence into ssDNA via selective digestion of either the sense or the antisense strand. Each kit allows for the preparation of 50 ssDNA templates.

Although ssDNA templates have been shown to provide several important advantages over dsDNA templates for precise genome editing applications utilizing homology directed repair (HDR) (Roth et al., 2018), the use of long ssDNA has been limited due to the difficulty and cost of producing it. The Guide-it Long ssDNA Production System v2 is designed to make the benefits of ssDNA templates accessible to all researchers by providing an efficient, economical method for obtaining ssDNA on demand.

Benefits of using ssDNA as a template over dsDNA:

• Significantly reduced random and off-target integration, resulting in improved gene editing efficiency and lower likelihood of experimental artefacts
• Minimal cytotoxicity, enabling recovery of larger populations of edited cells
• Negligible transgene expression from nonintegrated templates, allowing for easier identification of correctly edited clones