Report
Teaser, summary, work performed and final results
Periodic Reporting for period 1 - DUALgRENP (A DUAL gRNA system for functional assessment of ENhancers in Pluripotency)
Teaser
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...
Summary
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.
Work performed
1. Generation of a custom dual-gRNA library targeting the main mouse ES cell enhancers.
2. Validation of the efficiency of the dual-gRNA method using using surrogate reporter cell surface markers.
3. Evaluation of the efficiency of NHEJ-mediated repair of large genomic regions (10% for ranges 0.5Kb-20Kb)
4. Development of a screening method in mouse ES cells. In either Nanog-GFP or Rex1-GFP transgenic backgrounds, we have obtained LIF-independent self-renewing clones using positive controls and custom libraries.
5. Optimization of the detection of differential gRNA enrichment by quantitative PCR on genome-integrated viral DNA.
Overall, we have achieved most of the major aims exposed in the grant agreement. The generation of dual gRNA genome-wide enhancer libraries is a groundbreaking step that will pave the way for upcoming studies on non-coding regions of the genome, not only in the context of pluripotency, but virtually any other context in which gene regulatory networks play a fundamental role (embryonic development, directed differentiation, cancer, genetic disorders, etc.
The results have been disseminated in the prestigious annual meeting for the International Society for Stem Cell Research (ISSCR) 2016, in San Francisco. My abstract was selected for an invited talk in one of the concurrent sessions, to which around 200 people attended. I have also participated in the International Science Festival, organized by the University of Edinburgh.
Final results
Here we describe a novel method to perform systematic enhancer screens using CRISPR. To develop this innovative method, we have employed a multidisciplinary collaborative approach combining the timely topics of Stem Cell biology (Kaji lab), Bioinformatics (Tomlinson lab) and Genome editing (Yusa lab) with state of the art technology (CRISPR/Cas9 genome editing, 3C-qPCR, RNA-seq). While important NGS studies (ChIP-seq, 3C-seq) have described potential enhancers and chromatin interactions associated to the maintenance of mES cell identity, functional relevance of such potential regulatory elements has not yet been addressed. This project aimed to fill the gap between cellular function and NGS-chromatin mapping, by providing a layer of functionality to these descriptive approaches. In addition, this versatile technology can be applied to a variety of research areas such as developmental biology, cancer, disease correction or in vitro disease modelling, for which enhancer function has been shown relevant. We hope that this technology will enable the answer of many important questions in Biology, such as the unravelling of the functions of the non-coding genome. Our studies will focus on the maintenance of stem cell identity, which is a timely subject as the first clinical trials with induced-pluripotent stem cells in humans are just being started.