NOVEL HIGH-THROUGHPUT ASSAYS FOR FUNCTIONAL CHARACTERIZATION OF GENOME EDITING OUTCOMES

Abstract

Introduction: Genome editing technologies, notably CRISPR-Cas systems, have revolutionized functional genomics and precision medicine by allowing for precise genetic modifications. However, there are limitations of conventional validation methods that hinder accurate and large-scale characterization of genome editing outcomes.
Objective: The goal of this study was to develop and evaluate novel high-throughput assay platforms for rapid and functional characterization of genome editing outcomes in mammalian cells.

Methods: We evaluated editing efficiency, mutation frequency, off-target effects and cellular responses following CRISPR-mediated genome editing using multiplexed next-generation sequencing, fluorescence-based reporter assays, RNA-sequencing and single-cell transcriptomics. Comparative functional analysis was performed in HEK293 and stem cell models.

Findings: The developed high-throughput platforms achieved over 95% of editing detection sensitivity and around 92% of off-target identification accuracy. Functional assays showed that the DNA repair outcomes of CRISPR-Cas9, base editing and prime editing systems differ from each other. Single-cell sequencing enhanced detection of rare editing events and cellular heterogeneity . Fluorescence assays provided rapid real-time screening with improved scalability.

Conclusion: The results show that integrated high-throughput functional assays enable efficient, scalable and accurate characterization of genome editing outcomes. These platforms have great potential to improve the validation of therapeutic genome editing, the assessment of genomic safety, and the clinical translation.