CRISPRing the Human Genome for Functional Regulatory Elements

The sequence of DNA is a code that contains all the information that is required for life (as we know it). DNA is stored inside the nucleus of cells and its sequence is replicated during cell division to ensure that the genetic information is transmitted to the daughter cells. The information contained in DNA is copied into RNA by a process called transcription. RNA acts as a messenger (mRNA) to carry the information between the nucleus and the cytoplasm, where it is used as a template to produce proteins through a process called translation. Proteins are the main effectors of all biological functions in the cell. However, the information required to make proteins (called “coding DNA sequence”) comprises only a small portion (~2%) of the entire human genome sequence. For several decades, it was generally accepted that the remaining 98% of the genome sequence had no biological function and, because of that, it was dubbed “junk DNA”. The discovery of non-coding DNA sequences that control the expression of genes challenged this idea, and revealed that there is biological function beyond protein-coding sequences. These non-coding sequences are called “regulatory elements” and they are classified into four classes according to their function: promoters, enhancers, insulators and silencers. Among them, enhancers play a critical role in activating the expression of genes in response to intra- and extra-cellular stimuli – which is essential for the development of complex organisms. Previous studies suggest that the human genome might contain more than one million enhancers – a much higher number compared to the number of protein-coding genes (~22.000). However, not much is known about the biological function of most enhancers since only a handful of them were studied in detail to present date.
In recent years, the use of next-generation sequencing technologies (e.g. RNA-seq) revealed that the vast majority of the human genome is transcribed into non-coding RNA species. Initially, it was thought that these transcripts were “junk RNA” transcribed from “junk DNA”. This hypothesis was refuted by thousands of studies reporting that non-coding RNAs regulate a remarkably broad spectrum of cellular processes – including transcription and translation. Moreover, it was shown that the dysregulation of regulatory elements (and the non-coding RNAs transcribed from them) is associated with different human pathologies such as cancer. In this work, we describe a detailed protocol of Global Run-on sequencing (GRO-seq), which is a high-throughput sequencing technique that measures nascent RNA transcription. We applied GRO-seq to detect enhancer-associated RNAs (eRNAs), which are non-coding RNAs transcribed from active enhancers. Our experiments identified thousands of enhancers that are activated by critical transcription factors (e.g. p53 and ER) and might play a role in cancer development. In order to characterize their function, we used a recently developed technology called CRISPR-Cas9. This system is composed of a protein that can cleave DNA (Cas9) and a nucleic acid sequence that can guide Cas9 to the target site (CRISPR). CRISPR-Cas9 triggered a revolution in biology because it allows editing specific DNA sequences in a very fast and easy way. Importantly, Cas9 can be directed to virtually any sequence of the human genome, thus allowing to study the function of enhancers and other regulatory elements in a comprehensive manner. We pioneered the application of CRISPR-Cas9 to test the function of enhancers by mutating them and examining the resulting phenotype in a high-throughput manner (i.e. genetic screening). Our experiments led to the identification of several enhancers that regulate the expression of critical genes (e.g. CCND1) and poorly-characterized genes (e.g. CUEDC1) in human cells. In both cases, we showed that these enhancers are absolutely required for the growth of cancer cells, and our findings provide the basis for better diagnosis and therapies of human cancer.