Identifying Novel Enhancer Elements with CRISPR-Based Screens

Jason C. Klein, Wei Chen, Molly Gasperini, Jay Shendure
2018 ACS Chemical Biology  
Enhancers control the spatiotemporal expression of genes and are essential for encoding differentiation and development. Since their discovery more than three decades ago, researchers have largely studied enhancers removed from their genomic context. The recent adaptation of CRISPR/Cas9 to genome editing in higher organisms now allows researchers to perturb and test these elements in their genomic context, through both mutation and epigenetic modulation. In this Perspective, we discuss recent
more » ... vances in scanning noncoding regions of the genome for enhancer activity using CRISPR-based tools. Graphical Abstract * Corresponding Authors: shendure@u.washington.edu., jcklein@u.washington.edu. The authors declare no competing financial interest. KEYWORDS Enhancer: Classically defined as short sequences of DNA, which are able to increase expression of a gene independent of their relative position or orientation to the transcriptional start site CRISPR: Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is a bacterial defense system that recognizes and destroys foreign DNA. CRISPR has recently been modified for genome engineering in mammalian cells. Epigenetics: Usually biochemical modifications to DNA or relevant proteins that cause heritable changes in gene function without changing the nucleotide sequence. In the context of this review, we are mainly focusing on activating and repressing histone modifications. Massively Parallel Reporter Assay: A plasmid-based assay to measure the regulatory effects on gene expression of thousands of independent sequences at the same time Guide RNA: An RNA sequence that contains a scaffold for Cas-binding and a spacer sequence that targets DNA. In the context of this review, guide RNAs target Cas9 to specific genomic sites. Episomal: DNA that replicates independently of chromosomal DNA. While traditional MPRAs are episomal assays, CRISPR-based screens identify enhancer elements directly in their endogenous genomic loci. Nonhomologous end joining: A repair mechanism for double stranded breaks, which directly ligates broken DNA without a template. In the context of this review, the field relies on imperfect NHEJ to create insertions and deletions while repairing double-stranded breaks induced by Cas9. ChIP-seq: Chromatin immunoprecipitation followed by sequencing of cross-linked DNA is used to identify protein-DNA interactions. In the context of this review, ChIP-seq has been used to identify regions of DNA bound by proteins and modified-histones associated with enhancer activity, such as P300, H3K27ac, and H3K4me1. Rather than testing candidate enhancer elements for their positive activity when removed from context, a complementary approach is to perturb these same sequences in their Klein et al.
doi:10.1021/acschembio.7b00778 pmid:29300083 pmcid:PMC6218247 fatcat:74ydykbdnjaitkoclgaxemkaqm