Enhancers are cis-regulatory genomic regions that can modulate gene expression in a cell type-specific and time-controlled manner to regulate cellular behavior. ChIPseq, 4C-seq, RNA-seq and reporter assays have allowed the genome-wide identification of potential enhancers based on the correlation amongst specific chromatin marks, topological chromosome conformation, transcription factor biding sites and gene expression. However, strategies to validate their function are still limited to slow individual assays. Recently, it has been demonstrated that the CRISPR/Cas9 genome editing technology can be used to delete large genomic regions at high frequency by co-transfection of two guide RNAs (gRNAs) targeting collinear genomic sites and subsequent NHEJ-mediated repair. Nevertheless, this system is still limited to individual tests. Here we aimed to generate a novel lentiviral dual-gRNA expression system that enables large scale functional enhancer screening. Embryonic development involves a plethora of processes that need to be tightly coupled in order to ensure proper generation of all the specialized tissues in an organism from a single cell. Cellular identity and specialization is achieved and maintained through expression of specific sets of genes, therefore demonstrating differential usage of the same genetic information by different cell types. To modulate the transcriptional output, additional DNA sequences at distant genomic locations can recruit cell-specific transcriptional regulators and interact with the basal promoter by DNA looping. These sequences, also known as cis-regulatory modules (CRMs) or enhancers, consist of relatively small DNA elements that can act in a distance- and orientation-independent manner. The major objective of this proposal was to develop a novel system for large scale functional assessment of enhancers, to generate an unprecedent map of the regulatory regions essential for the maintenance of pluripotent cell identity. These aims are of crucial importance for the complete understanding of the biology underlying the maintenance of cell identity and allows us take a step further into the achievement of specific cell types for replacemente cell and tissue therapies in regenerative medicine. By analyzing published ChipSeq data, we have identified key regulatory elements potentially driving the exit from the self-renewing state. After selecting the top 2000 elements based on grouped Chipseq scores, we have classified these elements into mouse Embryonic Stem Cell (mESC)-specific, Differentiated-specific and common. We have developed an automatic pipeline capable of identifying flanking unique gRNA pairs, for each of such regulatory elements, and generated a complex oligonucleotide pool library. We have developed and validated a vector capable of expressing specific gRNA pairs, and optimized it for pooled cloning. We have determined the maximum efficacy of NHEJ-mediated repair by developing innovative cell surface marker surrogate assays. We have identified bona fide regulatory gene networks driving the exit from pluripotency upon LIF deprivation, and validated them in dual gRNA approachs. Finally, we have generated a dual gRNA library into appropriate vectors, whis we will sequence to verify its composition before embarking into genome-wide screening. Overall, our work is the first functional genetic screen on enhancers in the exit from pluripotency.
