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

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 insuffic...

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Detalles Bibliográficos
Autores principales: Ghosh, Sanjay, Tibbit, Charlotte, Liu, Ji-Long
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
RNA
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872081/
https://www.ncbi.nlm.nih.gov/pubmed/26850642
http://dx.doi.org/10.1093/nar/gkw063
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author Ghosh, Sanjay
Tibbit, Charlotte
Liu, Ji-Long
author_facet Ghosh, Sanjay
Tibbit, Charlotte
Liu, Ji-Long
author_sort Ghosh, Sanjay
collection PubMed
description 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.
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spelling pubmed-48720812016-05-27 Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference Ghosh, Sanjay Tibbit, Charlotte Liu, Ji-Long Nucleic Acids Res RNA 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. Oxford University Press 2016-05-19 2016-02-04 /pmc/articles/PMC4872081/ /pubmed/26850642 http://dx.doi.org/10.1093/nar/gkw063 Text en © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle RNA
Ghosh, Sanjay
Tibbit, Charlotte
Liu, Ji-Long
Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference
title Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference
title_full Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference
title_fullStr Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference
title_full_unstemmed Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference
title_short Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference
title_sort effective knockdown of drosophila long non-coding rnas by crispr interference
topic RNA
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872081/
https://www.ncbi.nlm.nih.gov/pubmed/26850642
http://dx.doi.org/10.1093/nar/gkw063
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