Abstract:
Background: Breast cancer remains one of the leading causes of cancer-related mortality among
women worldwide. Early detection using specific biomarkers and synthetic biology tools can
significantly enhance diagnostic precision and therapeutic outcomes.
Objective: To develop a computationally validated CRISPR-Cas13-based biosensing system
integrated with synthetic genetic logic gates for early and accurate detection of breast cancer
biomarkers
Methods: A multi-disciplinary approach combining bioinformatics, molecular biology, and
computational modeling was employed. Key biomarkers; Human Epidermal Growth Factor
Receptor 2 (HER2), Mucin 1 (MUC1), and Epidermal Growth Factor Receptor (EGFR) were
identified through literature mining and database analysis (e.g., NCBI, TCGA). Guide RNAs
(gRNAs) were designed to specifically target mRNA sequences of these markers using CRISPR-
Cas13 technology. Gene circuits were engineered incorporating logic gates (AND, OR, and NOT),
promoters, terminators, and fluorescent reporters (e.g., GFP, RFP) to produce a signal only when
the correct biomarker combinations were detected. The circuits were computationally modeled and
simulated using COPASI for dynamic behavior, logic validation, and parameter sensitivity. In
silico simulations guided design refinement prior to in-vitro implementation. Genetic constructs
were planned for integration into a microfluidic device using electrochemical biosensing for output
signal detection.
Results: COPASI-based simulations validated the designed gene circuits, showing correct logic
gate operations and stable output signal generation in response to target biomarker inputs. The
AND gate circuit showed high specificity for concurrent HER2 and MUC1 detection, while OR
and NOT gates enabled flexible detection patterns.
Conclusions: The study presents a novel CRISPR-Cas13-based genetic circuit system,
computationally designed and validated for breast cancer detection. Integration with genetic logic
gates and biosensor interfaces sets the foundation for a precise, programmable diagnostic device.
