Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis.

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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Stojic L,Lun ATL,Mangei J,Mascalchi P,Quarantotti V,Barr AR,Bakal C,Marioni JC,Gergely F,Odom DT

doi

10.1093/nar/gky437

subject

Has Abstract

pub_date

2018-07-06 00:00:00

pages

5950-5966

issue

12

eissn

0305-1048

issn

1362-4962

pii

5026268

journal_volume

46

pub_type

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