Long ssDNA for knockins
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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.
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
Overview
- Create long ssDNA donor templates up to 5 kb in length
- Fast, easy-to-use, three-step protocol
- No random integration or expression, leading to improved editing efficiency
- ssDNA donor templates are far less toxic to cells than dsDNA templates
More Information
Applications
- Donor templates for knockin experiments requiring DNA fragments >500 bp
Additional product information
Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
CRISPR/Cas9 information
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Gene editing resources
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