Rolling Circle Amplification for Genetic Editing Efficiency

The patent replaces complex mechanical/instrumental systems (PCR machines, sequencers, droplet generators) with a simple optical detection system. The core substitution is replacing the mechanical amplification and detection machinery with a cell-based biosensor system that uses biological components (engineered cells expressing reporter genes) to directly report editing efficiency through optical signals that can be measured with standard microscopy or flow cytometry equipment.

The patent introduces engineered cells as intermediary biosensors that mediate between the genetic-editing reaction and the detection system. These cells contain reporter genes (such as GFP or other fluorescent proteins) whose expression levels directly correlate with editing efficiency, allowing indirect but accurate measurement without requiring direct analysis of the edited DNA sequences.

The patent performs preliminary action by pre-engineering cells with reporter genes that are constitutively expressed or inducibly expressed in response to editing events. This preliminary setup allows for real-time or near-real-time detection of editing efficiency without requiring subsequent sequencing steps, as the cells continuously report their editing status through their optical properties.

The patent substitutes the time-consuming sequencing process with rapid optical detection. Instead of extracting DNA, amplifying it through multiple PCR cycles, and sequencing it over days, the system uses live cells that emit optical signals detectable within minutes to hours, dramatically reducing the time required while maintaining measurement precision.

The patent uses engineered cells as intermediary biosensors that provide quantitative information about editing efficiency. These cells serve as a bridge between the simple optical detection system and the complex molecular events of genetic editing, translating editing outcomes into measurable optical signals with high precision while requiring only simple equipment.

The patent changes the detection parameter from direct DNA sequence analysis (which requires complex instrumentation) to optical property measurement (fluorescence intensity, cell sorting parameters) which can be measured with simple equipment. By transforming the editing efficiency information into optical signals through the cell-based reporter system, the patent achieves high measurement precision using only basic laboratory equipment.