Optimizing sgRNA position markedly improves the efficiency of CRISPR/dCas9-mediated transcriptional repression

CRISPR interference (CRISPRi) represents a newly developed tool for targeted gene repression. It has great application potential for studying gene function and mapping gene regulatory elements. However, the optimal parameters for efficient single guide RNA (sgRNA) design for CRISPRi are not fully defined. In this study, we systematically assessed how sgRNA position affects the efficiency of CRISPRi in human cells. We analyzed 155 sgRNAs targeting 41 genes and found that CRISPRi efficiency relies heavily on the precise recruitment of the effector complex to the target gene transcription start site (TSS). Importantly, we demonstrate that the FANTOM5/CAGE promoter atlas represents the most reliable source of TSS annotations for this purpose. We also show that the proximity to the FANTOM5/CAGE-defined TSS predicts sgRNA functionality on a genome-wide scale. Moreover, we found that once the correct TSS is identified, CRISPRi efficiency can be further improved by considering sgRNA sequence preferences. Lastly, we demonstrate that CRISPRi sgRNA functionality largely depends on the chromatin accessibility of a target site, with high efficiency focused in the regions of open chromatin. In summary, our work provides a framework for efficient CRISPRi assay design based on functionally defined TSSs and features of the target site chromatin.