CRISPR GENome and epigenome engineering improves loss-of-function genetic-screening approaches.

CRISPR-Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR gene and epigenome engineering (CRISPRgenee), which uses two specific single guide RNAs (sgRNAs) to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgenee outperforms conventional CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of epithelial-to-mesenchymal transition (EMT) and impairs neuronal differentiation in a human induced pluripotent stem cell (iPSC) model. CRISPRgenee exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko screens, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgenee increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens.