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. 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.

A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division

“…To confirm our findings, we conducted a validation screen targeting the top 25 25 lncRNA candidates identified in the initial screens for mitotic progression and cytokinesis (Supplementary Table 3). Depletion of each lncRNA was performed in two biological (and in total eight technical) replicates.…”

“…2a). Although loc100289019-depleted cells 5 also displayed an elevated mitotic index, levels of the lncRNA did not change upon RNAi 25 . For the cytokinesis category, we observed an increase in the number of cells with cytokinetic bridges after linc00840 depletion and a decrease after loc729970 depletion, but neither led to multinucleation ( Supplementary Fig.…”

“…1a). First, for defects in mitotic progression, we determined the percentage of mitotic cells (also called mitotic index), 25 because an increase in the mitotic index implies a delay or block in mitotic progression.…”

“…Systematic screens in human cells have identified new protein-coding genes 25 involved in cell survival, cell cycle progression and chromosome segregation [17][18][19][20] .…”

Long-lasting Analgesia via Targetedin vivoEpigenetic Repression of Nav1.7

“…Although other technologies, such as RNAi have been utilized to target Na V 1.7, a recent study has shown that the off-target effects of RNAi, as compared to CRISPRi, are far stronger and more pervasive than generally appreciated 76,77 . In addition, as an exogenous system, CRISPR and ZFPs (unlike RNAi) do not compete with endogenous machinery such as microRNA or RISC complex function.…”