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Nucleic Acids Res. 2016 May 19;44(9):e84. doi: 10.1093/nar/gkw063. Epub 2016 Feb 4.

Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference.

Author information

1
MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, UK sanjay.ghosh@dpag.ox.ac.uk.
2
MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, UK.
3
MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, UK jilong.liu@dpag.ox.ac.uk.

Abstract

Long non-coding RNAs (lncRNAs) have emerged as regulators of gene expression across metazoa. Interestingly, some lncRNAs function independently of their transcripts - the transcription of the lncRNA locus itself affects target genes. However, current methods of loss-of-function analysis are insufficient to address the role of lncRNA transcription from the transcript which has impeded analysis of their function. Using the minimal CRISPR interference (CRISPRi) system, we show that coexpression of the catalytically inactive Cas9 (dCas9) and guide RNAs targeting the endogenous roX locus in the Drosophila cells results in a robust and specific knockdown of roX1 and roX2 RNAs, thus eliminating the need for recruiting chromatin modifying proteins for effective gene silencing. Additionally, we find that the human and Drosophila codon optimized dCas9 genes are functional and show similar transcription repressive activity. Finally, we demonstrate that the minimal CRISPRi system suppresses roX transcription efficiently in vivo resulting in loss-of-function phenotype, thus validating the method for the first time in a multicelluar organism. Our analysis expands the genetic toolkit available for interrogating lncRNA function in situ and is adaptable for targeting multiple genes across model organisms.

PMID:
26850642
PMCID:
PMC4872081
DOI:
10.1093/nar/gkw063
[Indexed for MEDLINE]
Free PMC Article

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