Decoding the Cuts: A New Tool for Precise Gene Editing Analysis

Gene editing tools like Cas9 act as molecular scissors, but understanding exactly how they slice through the genome remains a complex challenge. A newly developed method called BreakTag provides a scalable solution for characterising these programmable nucleases with high precision. Unlike standard checks, BreakTag offers an unbiased look at nuclease activity, specifically identifying ‘off-target’ cuts where the editor snips the wrong section of DNA.

Crucially, the method analyses the ‘scission profile’, distinguishing between blunt and staggered double-strand breaks. This detail is vital because the shape of the cut is mechanistically linked to how the cell repairs the damage, influencing the final genetic outcome. The protocol combines genomic DNA digestion with next-generation sequencing and data analysis via a tool named BreakInspectoR.

To further enhance predictive power, the team introduced XGScission, a machine learning model trained on BreakTag data. This model can forecast the relative frequency of cut types at entirely new sequences. With the entire workflow taking approximately three days, researchers can now rapidly assess and optimise the behaviour of engineered Cas variants.