Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs.

Retinoblastoma tumor suppressor proteins (Rb) are highly conserved metazoan transcriptional corepressors involved in regulating the expression of thousands of genes. The vertebrate lineage and the Drosophila genus independently experienced an Rb gene duplication event, leading to the expression of several Rb paralogs whose unique and redundant roles in gene regulation remain to be fully explored. Here, we used a novel CRISPRi system in Drosophila to identify the significance of paralogy in the Rb family. We engineered dCas9 fusions to the fly Rbf1 and Rbf2 paralogs and deployed them to gene promoters in vivo, studying them in their native chromatin context. By directly querying the in vivo response of dozens of genes to Rbf1 and Rbf2 targeting, using both transcriptional as well as sensitive developmental readouts, we find that Rb paralogs function as "soft repressors" and have highly context-specific activities. Our comparison of targeting endogenous genes to reporter genes in cell culture identified striking differences in activity, underlining the importance of using CRISPRi effectors in a physiologically relevant context to identify paralog-specific activities. Our study uncovers the complexity of Rb-mediated transcriptional regulation in a living organism, and serves as a stepping stone for future CRISPRi development in Drosophila.