Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
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. However, a major concern is non-specific targeting, which involves depletion of transcripts other than those intended. Little work has been performed to characterize the off-target effects of these common LOF methods at the whole-transcriptome level. Here, we experimentally compared the non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible off-target effects in gene expression, with CRISPRi also exhibiting strong clonal effects. As an illustrative example, we evaluated the performance of each method for determining the role of an uncharacterized long noncoding RNA (lncRNA). Several LOF methods successfully depleted the candidate lncRNA but yielded different sets of differentially expressed genes as well as a different cellular phenotype upon depletion. Similar discrepancies between methods were observed with a protein-coding gene (Ch-TOG/CKAP5) and another lncRNA (MALAT1). We suggest that the differences between methods arise due to method-specific off-target effects and provide guidelines for mitigating such effects in functional studies. Our recommendations provide a framework with which off-target effects can be managed to improve functional characterization of genes of interest.
Date Issued
2018-07-06
Date Acceptance
2018-05-09
Citation
Nucleic Acids Research, 2018, 46 (12), pp.5950-5966
ISSN
0305-1048
Publisher
Oxford University Press
Start Page
5950
End Page
5966
Journal / Book Title
Nucleic Acids Research
Volume
46
Issue
12
Copyright Statement
© 2018 The Author(s). 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.
Subjects
05 Environmental Sciences
06 Biological Sciences
08 Information And Computing Sciences
Developmental Biology
Publication Status
Published
Date Publish Online
2018-06-01