Long ssDNA for knockins

CRISPR/Cas9 and other gene editing tools have been successfully used for obtaining knockout mutations, but knockin mutations have proven to be much harder to introduce. A major challenge has been the production and delivery of the repair template along with the Cas9-sgRNA ribonucleoprotein complex.

Although single-stranded DNA (ssDNA) repair templates have recently been shown to have several advantages over double-stranded DNA (dsDNA) templates, the usefulness of long ssDNA templates is limited due to the difficulty and cost of producing them. The Guide-it Long ssDNA Strandase Kit is designed to produce a long ssDNA oligo of up to 5 kb in length for use as a repair template in knockin experiments using CRISPR/Cas9 or other gene editing tools. This kit provides a simple method for converting a dsDNA PCR product into ssDNA by selectively digesting either the sense or the antisense strand. The kit contains sufficient reagents to create 50 ssDNA strands (25 pairs of sense and antisense strands).

Benefits of using ssDNA as a template over dsDNA:

• Drastically reduced tendency to randomly integrate into the genome, resulting in an improved gene editing efficiency
• Low cytotoxic response to ssDNA template delivery
• No expression from nonintegrated templates, making identification of correctly edited clones significantly easier
• NOTE: The Guide-it Long ssDNA Production System includes a NucleoSpin Gel and PCR Clean-up kit to purify the strandase reaction before using it for gene editing