VATIS UPDATE Article

A team of researchers from Duke University, the United States, have developed a high-throughput screening technique that uses CRISPR-Cas9 epigenome editing to identify regulatory elements in the genomes of human cells. The researchers created lentiviral libraries of guide RNAs to target likely regulatory elements across several megabases of DNA surrounding two loci of interest: β-globin and HER2.

They then generated cell lines with an integrated fluorescent protein to report on target gene activation. The authors transduced their cell lines with one of two versions of the Cas9 protein with deactivated nuclease activity, dubbed dCas9. The repressor form of dCas9 recruits proteins that methylate lysine 9 on histone H3, leading to heterochromatin formation and gene repression in target sequences.

The activator form of dCas9 binds to targeted DNA enhancers or promoters and facilitates the acetylation of lysine 27 on histone H3, which results in gene activation. Next, the researchers transduced their cell lines with the libraries of guide RNAs at low levels to ensure that a single guide RNA would be present in each cell. They then sorted the cells based on fluorescence, and sequenced the guide RNA present in cells with especially high and low target gene expression.