Identifying Novel Enhancer Elements with CRISPR-Based Screens

Abstract

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 advances in scanning noncoding regions of the genome for enhancer activity using CRISPR-based tools.

Figure 1.

CRISPR-based functional screening for enhancer elements. (a) Designed gRNAs targeting potential enhancer regions are synthesized on a microarray and cloned into viral constructs, which are used to construct lentivirus libraries. Cells are infected with the lentiviral library at a low MOI to confer stable expression of a single construct per cell. (b) Sequence disruption: Putative enhancer regions are either targeted by an individual or paired gRNA construct. Individual gRNAs induce double stranded breaks, which can be repaired by nonhomologous end joining (NHEJ) resulting in short insertions and deletions. Paired gRNAs can result in a drop-out of the intervening sequence, resulting in targeted, large deletions. (c) Epigenetic perturbation: Inactive CRISPR-Cas9 (dCas9) can be fused with effectors for epigenetic modification. LSD1 and KRAB have been used to repress transcription while VP64 and p300 have been used to activate transcription. Effectors directly or indirectly act on histones by introducing histone marks, such as H3K4/H3K9 methylation or H3K27 acetylation. (d) Effects of enhancer perturbation have been measured in at least three ways: Flow sorting based on expression of fluorescently tagged endogenous genes, cellular proliferation advantage/disadvantage controlled by genes of interest, and single cell RNA-seq. Sci-RNA-seq figure adapted from ref with permission from AAAS. Functional enhancers are detected by the identification of the enrichment/depletion of gRNAs or by gene expression changes.