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.

Glioblastoma frequently exhibits therapy-associated subtype transitions to mesenchymal phenotypes with adverse prognosis. Here, we perform multi-omic profiling of 60 glioblastoma primary tumours and use orthogonal analysis of chromatin and RNA-derived gene regulatory networks to identify 38 subtype master regulators, whose cell population-specific activities we further map in published single-cell RNA sequencing data. These analyses identify the oligodendrocyte precursor marker and chromatin modifier SOX10 as a master regulator in RTK I-subtype tumours. In vitro functional studies demonstrate that SOX10 loss causes a subtype switch analogous to the proneural–mesenchymal transition observed in patients at the transcriptomic, epigenetic and phenotypic levels. SOX10 repression in an in vivo syngeneic graft glioblastoma mouse model results in increased tumour invasion, immune cell infiltration and significantly reduced survival, reminiscent of progressive human glioblastoma. These results identify SOX10 as a bona fide master regulator of the RTK I subtype, with both tumour cell-intrinsic and microenvironmental effects.

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.