CRISPR GENome and Epigenome Engineering (CRISPRgenee) improves loss-of-function genetic screening approaches

The 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 sgRNAs to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgenee outperforms conventional CRISPRko, CRISPRi, or 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. To investigate the benefitial effect when screening using CRISPRgenee we performed a dropout loss-of-function screen in TF-1 cells. Cells were transduced with a dual sgRNA library targeting 1137 genes involved in epigenetic regulation and cultivated for 14 days (~4 splits). To investigate if truncating a sgRNA to the length of 15 nt does not induce DNA cleavage we transduced ZIM3-Cas9 expressing cells with full length and truncated sgRNAs targeting either CD13 or CD33, measured depletion of CD13 and CD33 and harvested the cells after 14 days. The genomic DNA was isolated and the region where the sgRNA should cut was amplified via PCR while simultaneously adding illumina adapter sequences.The amplicon was sequenced and the amount of InDels calculated for the truncated and normal lengths sgRNA as well as for a non-targeting sgRNA control. To investigate the off-target effects when truncating an sgRNA to 15 nt we designed two sgRNAs targeting CD33 and performed RNA sequencing on cells co-expressing CRISPRgate and one of the sgRNAs either full length or truncated.