RT Journal Article
SR Electronic
T1 Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 234930
DO 10.1101/234930
A1 Lovorka Stojic
A1 Aaron Lun
A1 Jasmin Mangei
A1 Patrice Mascalchi
A1 Valentina Quarantotti
A1 Alexis R Barr
A1 Chris Bakal
A1 John C Marioni
A1 Fanni Gergely
A1 Duncan T Odom
YR 2017
UL http://biorxiv.org/content/early/2017/12/15/234930.abstract
AB 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.