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Significance Evaluation of the cleavage efficacy, including the on-target activity and off-target effects, for individual guide RNA (gRNA) in silico can help optimize application of CRISPR/Cas9 systems. Many bioinformatics models based on data-processing algorithms have been developed, but discrepancies in the identified key features that determine cleavage efficacy inhibit deep understanding of the cleavage mechanism and preclude further optimization of gRNA design. Here, we present a physical framework with rigorous free-energy analysis to bridge the gap between experimental structural studies and cleavage-efficacy evaluations. This tool simultaneously considers on-target activity and off-target effects in a unified framework, improves the prediction power in both realms for diverse spCas9 cleavage efficacy datasets, and is readily transferred to other CRISPR/Cas9 systems.

Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design