NEW YORK (GenomeWeb) – Researchers interested in untangling the functional roles of regulatory elements have a new screening tool at their disposal: a CRISPR-Cas9-based epigenomic editing scheme.
In a study appearing online today in Nature Biotechnology, Duke University researchers introduced a strategy known as "CRISPR-Cas9-based epigenomic regulatory element screening," or CERES, designed for profiling regulatory elements in a high-throughput manner in a native chromosome context. By using CERES in screens for gain- or loss-of-regulatory element function in a handful of human cell lines, for example, they narrowed in on new and known expression regulators at the beta-globin and HER2 loci.
"Ultimately, we envision that these methods will provide information on how altered regulation of gene expression contributes to disease, drug response, and regeneration," co-corresponding authors Charles Gersbach, Timothy Reddy, and Gregory Crawford, and their co-authors wrote. "We also expect that by illuminating the function of the non-coding genome, these technologies will provide a new class of drug targets for diverse indications."
Though past genome-wide efforts have uncovered a slew of apparent regulatory elements, it is often difficult to verify the functional effects of the elements without altering native chromosomal interactions, the team explained. On the other hand, attempting to track regulatory element activity through CRISPR-Cas9-based DNA editing requires intensive targeting of potential transcription factors, allowing loss-of-function but not gain-of-function experiments.
To get around such problems, Gersbach, Reddy, Crawford, and colleagues focused on a strategy that fuses nuclease-deactivated Cas9 to repressor- or activator-coding domains to directly alter the expression activity associated with suspected regulatory sequences.
In particular, they used carefully designed guide RNAs in combination with Cas9 constructs with KRAB or p300 domains that either recruit histone methylation or acetylation marks, leading to the deactivation or activation of regulatory sites in the screen. Those constructs were targeted to sites along chromosomes with high susceptibility to DNase I enzyme cutting — a feature associated with chromatin accessibility.
"Because screening with epigenome editing perturbs regulatory element activity directly rather than via DNA mutation, the approach offers several advantages over genome editing screens," the authors wrote, noting that "use of the [KRAB and p300-fused Cas9 constructs] in parallel screens around the same loci uniquely facilitates the identification of elements that are necessary and sufficient, respectively, for target gene expression."
After validating their approach by characterizing beta-globin regulatory elements in a reporter gene-containing human myelogenous leukemia cell line, the researchers set out to characterize regulatory elements for the oncogene HER2 with epigenetic editing.
Using almost 300 control guide RNAs — and more than 12,200 guide RNAs targeting sites identified by DNase-seq in a breast cancer cell line with HER2 amplifications — the team screened an epidermoid carcinoma cell line with the repressive Cas9 construct, plucking out cells and sequencing guide RNAs in cells with the highest and lowest levels of HER2 gene expression. With gain-of-function CERES screening in multiple cell lines, the group fleshed out HER2-related regulatory features further.
"Although this study used chromatin-accessibility DNase-seq maps to define the regulatory element landscape," the authors noted, "the [guide RNA] library could be designed on the basis of any input, including locations from chromatin immunoprecipitation followed by sequencing (ChIP-seq), global run-on sequencing (GRO-seq), or single nucleotide polymorphisms identified in genome-wide association studies or analyses of expression quantitative trait loci."