Challenges and strategies for CRISPR-Cas molecular diagnostics from mechanism to clinical translation

• Comprehensive CRISPR-Cas enzyme profiling: Systematically classifies Cas9, Cas12, Cas13, and Cas14 based on their cis- and trans-cleavage mechanisms, sequence preferences, and molecular detection applications. • Multi-dimensional optimization strategies: Details advancements in Cas protein engineering, AI-driven crRNA design, sample pretreatment, reaction system refinement, and signal readout diversification to enhance sensitivity, specificity, and stability. • Broad clinical applicability: Evaluates CRISPR-based diagnostics across infectious diseases, oncology, genetic disorders, and chronic disease management, with platforms like SHERLOCK and DETECTR achieving regulatory approval. • Integrated amplification and detection: Highlights one-pot, lyophilized, and microfluidic systems that combine isothermal amplification with CRISPR detection for rapid, portable, and contamination-free workflows. • Critical challenges and future pathways: Identifies key hurdles in sensitivity-specificity balance, standardization, cost, and ethics, while proposing engineering and regulatory strategies to accelerate clinical translation. CRISPR-Cas systems offer transformative potential for molecular diagnostics, overcoming limitations in portability, multiplexing, and point-of-care applicability inherent in conventional methods like PCR. This review systematically examines CRISPR-Cas detection technologies. It classifies key Cas enzymes based on their distinct cis- and trans-cleavage mechanisms and sequence preferences, outlining their roles in detecting nucleic acid and non-nucleic acid targets. Comprehensive strategies for optimizing detection performance are detailed, including Cas protein engineering for high-fidelity variants, crRNA engineering using artificial Intelligence design and structural modifications, sample pretreatment methods for target enrichment, and reaction system refinements integrating isothermal amplification and digital partitioning. Signal enhancement strategies enable diverse readouts through fluorescence, electrochemistry, lateral flow, and smartphone-based detection, while stability improvements involve lyophilization and thermostable engineering. The diverse applications evaluated span infectious disease diagnosis for pathogens like SARS-CoV-2 and antimicrobial resistance, precision oncology including liquid biopsy, genetic disease screening for SNPs and aneuploidy, and chronic/immune disease management for biomarkers such as cytokines. Platforms like SHERLOCK and DETECTR have achieved regulatory approvals. The review critically analyzes challenges for enhancing efficiency, including balancing sensitivity and specificity, integrating workflows, establishing standardized protocols, ensuring reproducibility, reducing costs, and addressing ethical considerations. These analyses establish a foundation for the clinical translation and commercialization of CRISPR-Cas molecular diagnostics.