How TIDE identifies genetic variants beyond CRISPR-induced indels
When researchers think of TIDE, they think CRISPR/Cas9-induced indels. But how about when repair processes create defined DNA changes that do not look like indels at all? That is when you extend TIDE beyond indels and into model-based analysis.
In a recent study, researchers at the Institute for Research in Biomedicine Bellinzona in Switzerland investigated triplet-repeat expansions seen in several genetic conditions, including in non-dividing cells. One of these conditions, Huntington's disease, results from CAG expansions in the HTT gene. Understanding the underlying cause of these repeats and the protective factors can offer treatment opportunities beyond symptom management.
Although genetic studies have identified the modifiers that influence repeat expansions, the molecular mechanisms underlying these repeats remain largely unclear.
Senoussi and colleagues designed an elegant approach to evaluate the position and regulation of DNA cuts and the resulting repeat expansions or protective contractions. They recreated DNA-associated mispairs, loops, and nicks at different positions in controlled plasmid systems to test how repair proteins processed them. In this way, the researchers could evaluate the interactions between the proteins known to influence DNA stability under different conditions.
However, given the number of proteins being tested across several conditions, the team needed a simple, time-efficient tool to evaluate gene repeat expansions and protective contractions. Although TIDE fulfills both these requirements, the tool's specificity to indel decompositions with unpredictable sizes made it less suited for analyzing specific triplet repeat expansions.
They turned to Tracking of Insertions, Deletions, and Recombination events (a.k.a. TIDER). As an extension of TIDE, TIDER quantifies and ranks specific sequence changes rather than indel efficiency. While TIDE requires two sequencing traces (sample and control), TIDER requires three: sample, control, and reference traces with the expected gene modifications.
With TIDER's simulated reference feature, the authors could model potential insertions by simulating a reference derived from the control sequence. This allowed them to test multiple predicted outcomes and identify a dominant +12 bp insertion—consistent with the direct copying of a (CAG)4 loop into the DNA.
TIDER also helped reveal the opposing, protective pathway driven by FAN1. When FAN1 was included alongside the full protein panel, the sequencing signal from the looped strand reflected dominant removal of the (CAG)4 loop, with no corresponding modification detected on the opposite strand. This dual confirmation, revealing strand-specific outcomes consistent with expansion or contraction depending on which proteins were active, illustrates how TIDER can resolve distinct outcomes at the same genomic site and under different conditions.
In other words, two competing pathways determine the fate of a DNA loop: whether its repeats expand or contract.
"Our reconstituted reactions illuminate mechanisms underlying the instability of trinucleotide repeats and reveal biochemical reactions amenable to therapeutic interventions," the authors concluded.
Beyond repeat expansions
While the researchers employed several biochemical assays to reach these conclusions, TIDER enabled Senoussi and colleagues to obtain rapid, precise sequencing data for defined gene variants. Beyond repeat expansion studies, this exemplifies how TIDE and its related algorithms can assess predefined sequence variants.
TIDE is increasingly evolving beyond unpredictable indels and into reference-guided analyses of genetic processes, such as homology-directed repair, base editing, and mismatch repair. The applications enable users to identify what, when, and where expected genetic variants occur—even at low signals—as long as the expected variant sequence is predefined.
Are you interested in applying TIDE or other TIDE-associated tools to your sequencing data? Our team can help tailor the approach to your specific needs.