Skotheim, J. | bioRxiv Science

bioRxiv2018

Reversible inhibition of specific transcription factor-DNA interactions using CRISPR

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it has been difficult to assay the function of single transcription factor binding sites within larger transcription networks. Here, we developed such a method by using deactivated Cas9 to disrupt binding to specific sites on the genome. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting deactivated Cas9 to a specific Oct4 binding site in the Nanog promoter blocked Oct4 binding, reduced Nanog expression, and slowed division. Multiple guide RNAs allows simultaneous inhibition of multiple binding sites and conditionally-destabilized dCas9 allows rapid reversibility. The method is a novel high-throughput approach to systematically interrogate cis-regulatory function within complex regulatory networks.

molecular biology↗

bioRxiv2017

Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

genomics↗