Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis
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Authors
Mangei, Jasmin
Mascalchi, Patrice
Publication Date
2017Publisher
Cold Spring Harbor Laboratory
Type
Article
Metadata
Show full item recordCitation
Stojic, L., Lun, A., Mangei, J., Mascalchi, P., Quarantotti, V., Barr, A., Bakal, C., et al. (2017). Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis. https://doi.org/10.1101/234930
Abstract
<h4>ABSTRACT</h4> Loss-of-function (LOF) methods, such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing, provide unparalleled power for studying the biological function of genes of interest. When coupled with transcriptomic analyses, LOF methods allow researchers to dissect networks of transcriptional regulation. However, a major concern is nonspecific targeting, which involves depletion of transcripts other than those intended. The off-target effects of each of these common LOF methods have yet to be compared at the whole-transcriptome level. Here, we systematically and experimentally compared non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible offtarget effects in gene expression, with CRISPRi exhibiting clonal variation in the transcriptional profile. As an illustrative example, we evaluated the performance of each method for deciphering the role of a long noncoding RNA (lncRNA) with unknown function. Although all LOF methods reduced expression of the candidate lncRNA, each method yielded different sets of differentially expressed genes upon knockdown as well as a different cellular phenotype. Therefore, to definitively confirm the functional role of a transcriptional regulator, we recommend the simultaneous use of at least two different LOF methods and the inclusion of multiple, specifically designed negative controls.
Sponsorship
Cancer Research UK (CB4180)
Cancer Research UK (C14303/A17197)
Cancer Research UK (20412)
Wellcome Trust (202878/Z/16/Z)
European Research Council (615584)
Identifiers
External DOI: https://doi.org/10.1101/234930
This record's URL: https://www.repository.cam.ac.uk/handle/1810/280572
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