Journal: Nucleic Acids Research

Article Title: Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

doi: 10.1093/nar/gky437

Figure Lengend Snippet: No overlap in DEGs between the different LOF methods upon depletion of SLC25A25-AS1 . ( A ) Expression levels of SLC25A25-AS1 after RNAi, LNA and CRISPRi-mediated depletion. qPCR revealed only a 25% reduction in SLC25A25-AS1 transcription after siRNA-mediated knockdown relative to negative control siRNA from Dharmacon (Control Dharm), and no significant difference relative to negative control siRNA from Ambion (Control Ambion). LNA-mediated knockdown of SLC25A25-AS1 was performed using LNA oligonucleotide sequence 2 (LNA 2), and showed 90% reduction. CRISPRi-mediated repression of SLC25A25-AS1 using two guide RNAs targeting the TSS of SLC25A25-AS1 relative to the negative (non-targeting) guide RNA 2 yielded 70–90% knockdown in clonal cells. Only one guide RNA (guide 9) was efficient in depleting SLC25A25-AS1 in non-clonal cells. Statistical significance by two-tailed Student's t -test: ** P < 0.01, *** P < 0.001 and **** P < 0.0001. For all graphs, expression levels of SLC25A25-AS1 were measured by qPCR using primers spanning exons 1–4, and normalized to the geometric mean of GAPDH and RPS18 . Error bars, s.e.m. ( n = 4 biological replicates). ( B ) Volcano plots of transcriptional differences induced by RNAi, LNA and CRISPRi-mediated depletion of SLC25A25-AS1 . After siRNA-mediated depletion of SLC25A25-AS1 , seven genes were differentially expressed compared to the negative control siRNAs. LNA-mediated depletion with LNA 2 identified 370 DEGs compared to negative control oligonucleotides. CRISPRi-mediated depletion of SLC25A25-AS1 using guide RNA 1 and 9 revealed only two DEGs compared to negative guide RNA 2. In non-clonal cells, only four DEGs were identified using guide RNA 9 compared to the negative guides. The red horizontal line represents the significance threshold corresponding to an FDR of 5%. Red vertical lines are log 2 -fold change thresholds of ±0.5. The red dot corresponds to the SLC25A25-AS1 itself. ( C ) Venn diagram showing no overlap between the sets of DEGs identified after using LNA and CRISPRi to deplete SLC25A25-AS1 . The only gene in common between LNA and CRISPRi-mediated depletion is SLC25A25-AS1 itself. The total number of genes used for this analysis was 18224. ( D ) qPCR confirmation of the downregulation of two DEGs ( SEPTIN2 and GM130 ) identified in B after LNA-mediated depletion of SLC25A25-AS1 with LNA 2. Expression levels were normalized to the geometric mean of GAPDH and RPS18 . Error bars, s.e.m. ( n = 3 biological replicates). Statistical significance by two-tailed Student's t -test: **** P < 0.0001. ( E ) Quantification results from time-lapse microscopy of mitotic progression of HeLa cells incubated with Sir-Hoechst after LNA and CRISPRi-mediated (clonal and non-clonal) depletion of SLC25A25-AS1 . Mitotic duration was measured from nuclear envelope breakdown (NEBD) to anaphase onset. Bars show mean±s.d. ( n = 2 independent biological replicates). Statistical significance by Mann–Whitney test: **** P < 0.0001. ( F ) Left panel : Expression analysis of SLC25A25-AS1 by qPCR using two additional LNAs targeting SLC25A25-AS1 (LNA 1 and LNA 3). Expression levels were normalized to the geometric mean of GAPDH and RPS18 . Error bars, s.e.m. ( n = 3 biological replicates). Statistical significance by two-tailed Student's t -test: **** P < 0.0001. Right panel : Quantification results from time-lapse microscopy using HeLa Kyoto cells after depletion of SLC25A25-AS1 using three different LNAs. Bars show mean±s.d. ( n = 2 biological replicates). Statistical significance by Mann–Whitney test: **** P < 0.0001. The list of DEGs identified after RNAi, LNA and CRISPRi-mediated depletion of SLC25A25-AS1 are shown in .

Article Snippet: The MIT CRISPR ( http://crispr.mit.edu ) and the gUIDEbookTM gRNA design (Desktop Genetics Ltd) web tools were used to design the gRNA sequence.

Techniques: Expressing, Knockdown, Negative Control, Control, Sequencing, Two Tailed Test, Time-lapse Microscopy, Incubation, MANN-WHITNEY