Hi, Tide.

Bill Reid, Ph.D. Postdoctoral Researcher, Institute for Bioscience and Biotechnology Research, University of Maryland College Park

Bill Reid, Ph.D. Postdoctoral Researcher, Institute for Bioscience and Biotechnology Research, University of Maryland College Park

The use of sequence-targeting nucleases (TALENs, CRISPR-Cas9, zinc finger nucleases) to introduce small insertions/deletions into the genome of an organism is an effective means of generating specific mutants.

Although these technologies are effective, they still require testing and optimization to determine the ideal reaction conditions, notably the testing of sgRNAs for the CRISPR-Cas9 system to identify suitable guides for DNA cleavage.

The generation of small deletions targeted by sgRNAs in the CRISPR-Cas9 system occurs due to cleavage of the DNA followed by imperfect repair via the non-homologous end joining pathway, which results in a “pool” of deletions at the cut site.

Cel 1 and T7 endonucleases can recognize local mismatches and cleave DNA at that site.

Cel 1 and T7 endonucleases can recognize local mismatches and cleave DNA at that site.

Several techniques are available for assessing the efficacy of sgRNAs, such as the Surveyor Cel-1 and T7 nuclease assays, as well as high resolution melt curve analysis. These approaches provide robust semi-quantitative information about the generated deletions, but provide little quantitative information regarding the deletion length or abundance.

As a method to characterize the small deletions generated during genome editing, Brinkman et al (2014) developed a technique they call “TIDE” (Tracking of Indels by DEcomposition). In TIDE analysis, PCR amplicons spanning the targeted deletion of control and nuclease-treated DNA are sequenced using Sanger sequencing, and the resulting chromatograms are compared to each other.

If deletion events have occurred in the nuclease-treated sample, the Sanger chromatogram will display sequence “decay”, occurring when the sequence trace reaches the base position of the deletion event and begins to simultaneously sequence the different deletions present within the genome-edited pool. By comparing the sequence decay of the treated sample to the control sample, TIDE can not only confirm the efficacy of a sgRNA, but can also estimate the frequency of deletion size/insertion event.

Sanger Sequencing Chromatogram showing "Sequence Decay"

Sanger Sequencing Chromatogram showing “Sequence Decay”

The TIDE approach is a convenient way to characterize the pool of mutants generated via genome editing, and is a great approach for characterizing knock-outs requiring a specific deletion, particularly for clonal cell lines, and also serves as an alternative strategy for testing the efficacy of sgRNAs.

TIDE: Tracking of Indels by DEcomposition

TIDE: Tracking of Indels by DEcomposition

NKI

A website interface for data analysis is hosted by the Netherlands Cancer Institute and is available at tide.nki.nl.

 

Brinkman, Eva K., Chen, Tao, Amendola, Mario, van Steensel, Bas (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucl. Acids Res. 2014 Oct 09; 10.1093/nar/gku936

Recent Related Posts on Technology Topics:

 CRISPR/Cas9 for RNA Cleavage

Guide RNA Design & Off-Target Analysis: A Tool for Non-Model Organisms 

“CHOPCHOP”ing with CRISPRs and TALENs: A New Web Resource for Genome Editing

Facebooktwittergoogle_plusredditpinterestlinkedinmailFacebooktwittergoogle_plusredditpinterestlinkedinmail

Post a Comment

Your email address will not be published. Required fields are marked *