Research Article: Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes

Date Published: February 16, 2018

Publisher: Public Library of Science

Author(s): Harshavardhan Janga, Marina Aznaourova, Fabian Boldt, Katrin Damm, Arnold Grünweller, Leon N. Schulte, Stefan Maas.


CRISPR/Cas9-based approaches have greatly facilitated targeted genomic deletions. Contrary to coding genes however, which can be functionally knocked out by frame-shift mutagenesis, non-coding RNA (ncRNA) gene knockouts have remained challenging. Here we present a universal ncRNA knockout approach guided by epigenetic hallmarks, which enables robust gene silencing even in provisionally annotated gene loci. We build on previous work reporting the presence of overlapping histone H3 lysine 4 tri-methylation (H3K4me3) and DNaseI hypersensitivity sites around the transcriptional start sites of most genes. We demonstrate that excision of this gene-proximal signature leads to loss of microRNA and lincRNA transcription and reveals ncRNA phenotypes. Exemplarily we demonstrate silencing of the constitutively transcribed MALAT1 lincRNA gene as well as of the inducible miR-146a and miR-155 genes in human monocytes. Our results validate a role of miR-146a and miR-155 in negative feedback control of the activity of inflammation master-regulator NFκB and suggest that cell-cycle control is a unique feature of miR-155. We suggest that our epigenetically guided CRISPR approach may improve existing ncRNA knockout strategies and contribute to the development of high-confidence ncRNA phenotyping applications.

Partial Text

Since the decryption of the human genome sequence thousands of non-coding RNA (ncRNA) genes have been discovered. Meanwhile ncRNAs have been implicated in transcriptional and post-transcriptional control of major cellular processes, ranging from cell-cycle progression to lineage commitment and immune-responses. While microRNAs exhibit a size range of 20–22 nt and regulate gene expression through translational repression and destabilization of target transcripts [1], long non-coding RNAs (lncRNAs) are less uniform in function. By definition, lncRNAs are > 200 nt in size and lack coding sequences (CDS). LncRNAs have been suggested to interact with proteins to function e.g. as decoys, scaffolds or guides [2] and can be found both the nucleus and the cytosol.

Our study introduces an improved method for the generation of ncRNA gene knockouts. While previous strategies have relied on individual determination of essential gene regions we demonstrate that excision of DNaseI/H3K4me3 signatures, which constitute universal TSS hallmarks, is sufficient to confer robust gene silencing.




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