Analytical dissection of CRISPR-Cas9-mediated gene editing fidelity: integrating next-generation bioanalytical platforms for precision immunotherapeutics and rare disease correction

Abstract

The clinical translation of CRISPR- Cas9 therapeutics requires rigorous and biologically grounded evaluation of genome editing fidelity that extends beyond conventional measures of on-target efficiency and canonical off-target detection. Existing fidelity assessment strategies often capture limited aspects of editing outcomes and do not fully account for context-dependent biochemical, epigenetic, and cellular heterogeneity that can influence therapeutic safety and efficacy. This review focuses on analytical and translational frameworks for CRISPR fidelity assessment, with emphasis on the strengths and limitations of current bioanalytical platforms. Recent advances integrating orthogonal technologies including digital droplet PCR, genome-wide off-target mapping approaches (e.g., CIRCLE-seq and GUIDE-seq), and single-cell proteogenomic profiling enable more comprehensive characterization of editing outcomes across molecular and functional dimensions. Applications in edited T cells targeting immune checkpoints and in hematopoietic stem cell correction models illustrate how multi-layered analyses can reveal context-dependent variability and previously undetected off-target effects. Emerging approaches, including integrative scoring frameworks and AI-assisted analytical models, are discussed in an exploratory context, with emphasis on current limitations, validation requirements, and challenges in standardization. In addition, ongoing efforts toward minimally invasive monitoring strategies are considered with respect to their potential, as well as their current constraints. Despite significant progress, major challenges remain in assay harmonization, cross-platform comparability, and translation to clinically actionable frameworks. Accordingly, CRISPR fidelity is best understood as a multi-dimensional biological phenotype that requires careful, context-specific evaluation. This perspective provides a balanced foundation for advancing genome editing toward safer and more reliable therapeutic applications.