Identifying Novel Regulators of LIN28B Through a Genome-Wide CRISPR/Cas9 Screen

LIN28 is a family of RNA-binding proteins that are well-conserved across species. It is well-known to regulate developmental timing by inhibiting the biogenesis of the let-7 microRNAs. Numerous studies have shown that LIN28 is dysregulated in a wide spectrum of cancer types, especially pediatric cancers such as hepatoblastoma and Wilms' tumor. Our laboratory previously showed that reactivation of LIN28B, one of the two LIN28 homologs, is sufficient to drive liver cancer, and that LIN28B deletion is detrimental to tumor development. LIN28B exerts its oncogenic function by inhibiting the maturation of let-7 precursors, as well as directly binding to and enhancing translation of growth-promoting mRNA targets, such as members of the IGF2BP family. While our work and others established that LIN28B functions as an oncogene, the identity of factors that regulate LIN28B expression during normal development and cancer remains elusive. As LIN28B is a driver of oncogenesis in various cancers, understanding its regulation in the process of oncogenesis will help uncover novel therapeutic targets. Here, we show an original approach for identifying regulators of the human LIN28B gene utilizing the CRISPR/Cas9 genome engineering system. Traditional transgenic approaches to study gene function often fail to capture transcriptional regulation at distal promoter and enhancer sequences. Using the CRISPR/Cas9 system, we knocked a GFP reporter sequence into the endogenous locus of LIN28B in human cancer cell lines, engineering a fusion LIN28B-GFP protein. This approach is unique in that GFP expression will be altered not only by changes in regulation at the coding sequence, mRNA and protein levels, but also changes at distal regulatory sequences. To identify unknown regulators of LIN28B, we will perform a genome-wide CRISPR/Cas9-mediated knockout screen in human cells expressing the fusion LIN28B-GFP protein. Using a genome-scale library with 76,441 sgRNAs, we will knock out 19,114 genes individually and assess their effects on LIN28B levels by measuring GFP expression. sgRNAs that are enriched in the high GFP-expressing population suggest genes that normally function as inhibitors of LIN28B. On the other hand, sgRNAs that are depleted suggest activators of LIN28B. Through this screen, we hope to gain further insight into how LIN28B is regulated in normal development and cancer. Furthermore, identifying regulators of LIN28B can provide novel avenues for developing cancer therapeutics.