Αlpha¹ Review in Progress

Gene knockout experiments are essential for dissecting gene function, and CRISPR has made targeted gene disruption more accessible than ever. Single-cell CRISPR screening enables the construction of rich genetic perturbation landscapes, facilitating the identification of genes whose perturbation strongly reshapes cellular states. However, due to the nonlinear dependencies within gene networks, identifying the most impactful tangible genes remains challenging. Existing virtual knockout methods estimate downstream effects of single-gene deletions but do not evaluate whether such perturbations disrupt global information flow or compromise cellular robustness. To address this limitation, we adapt a perturbation-response framework originally developed for protein structural dynamics to identify gene modules most susceptible to perturbation. We introduce the single-cell Perturbation Impact Index (scPII), a data-driven metric derived from gene regulatory networks that quantifies system-level responses to gene perturbations, without using any CRISPR screening information. Our results demonstrate that scPII effectively identifies genes whose perturbation has the greatest system-wide impact. Analysis of single-cell RNA-seq data from the Cancer Cell Line Encyclopedia revealed a strong correlation between scPII-derived gene impact scores and gene effect scores from genome-wide CRISPR screens. These findings indicate that scPII provides a robust metric for quantifying gene knockout effects. More broadly, integrating perturbation response scanning with gene regulatory networks offers a powerful framework for advancing single-cell data analytics in biomedical research.