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
Choosing sgRNA design tools
Browse a collection of sgRNA design tools for Cas9-based genome editing experiments.
Choosing a target sequence for CRISPR/Cas9 gene editing
Learn how to design sgRNA sequences for successful gene editing.
The CRISPR/Cas9 system for targeted genome editing
Overview of CRISPR/Cas9 system for genome editing.
CRISPR/Cas9 genome editing tools
An overview of tools available for each step in a successful genome editing workflow.
Gene editing technical notes
Delivery of Cas9 and sgRNA to mammalian cells using a variety of innovative tools.
SNP engineering application note
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CRISPR/Cas9 gesicles overview
Learn about Guide-it CRISPR/Cas9 Gesicle Production System components and workflow.
CRISPR library screening webinar
Watch this webinar to learn how you can perform genome-wide lentiviral sgRNA screens easily.
Choosing an HDR template format
Watch a webinar on how to choose the right HDR template for knockin experiments.
Guide-it SNP Screening Kit FAQs
Get answers to frequently asked questions and view a video explaining the enzymatic assay.
Gene editing resources
Product finder
Use this gene editing product finder to quickly locate kits for screening, delivery, and downstream assays.
CRISPR tools and information
What is CRISPR/Cas9? Need help designing the best guide RNAs? Learn all this and more.
Genome-wide library screening
Learn how to conduct a CRISPR/Cas9 guide RNA library phenotypic screen and view data demonstrating the use of our library.
CRISPR/Cas9 knockouts
Technotes and tools used to create or study CRISPR-Cas9-mediated gene knockouts (indels).
CRISPR/Cas9 knockins
Information, technotes and tools used to create or study gene knockouts by CRISPR/Cas9 and HDR.
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