TIDE: Easy quantitative assessment of genome editing
TIDE is a cheap, simple and accurate assay to precisely determine the spectrum and frequency of targeted mutations generated in a pool of cells by genome editing tools such as CRISPR/Cas9, TALENs and ZFNs. See this paper.
TIDE requires only standard molecular biology reagents and involves three simple steps:
- One pair of standard PCR reactions.
- One pair of standard capillary ("Sanger") sequencing reactions.
- Analysis of the two resulting raw sequencing files using a dedicated sequence trace decomposition web tool . The algorithm accurately reconstructs the spectrum of indels from the sequence traces.
The web tool reports the precise identity of the mutations and their frequencies in a few intuitive graphs and tables. TIDE is more attractive than several currently available methods:
The Surveyor and T7 endonuclease I assays are enzymatic assays that detect polymorphisms (including mutations) in DNA. Yet, they do not identify the nature – nor the precise position – of the mixture of mutations. Moreover, these assays are semi-quantitative at best and suffer from serious background problems if polymorphisms are present.
Isolation and sequencing of 50-100 clonal DNA fragments to determine the frequency and spectrum of the mutations. This is much more labor-intensive and >25-50 times more expensive than TIDE.
Next Generation Sequencing is the most comprehensive method to determine the frequency and nature of mutations in a pool of cells. However, the costs are ~100-fold higher than TIDE (unless one multiplexes many samples, which is not an option for most users), and the turnaround times of NGS jobs tend to be several weeks in most institutes.
TIDE provides a solution for all of these limitations. Use it to test and optimize your genome editing tools. It's free.
Check out the paper.
Access the web tool.
To obtain R code: please contact us.