grna design web tools  (Benchling Inc)

Journal: Biogerontology

Article Title: Telomere shortening induces aging-associated phenotypes in hiPSC-derived neurons and astrocytes

doi: 10.1007/s10522-023-10076-5

Figure Lengend Snippet: TERT deficient isogenic clone generation and characterisation. a Targeting strategy to generate TERT low hiPSC lines with guide RNA and CRISPR/Cas9. b Diagram of the two TERT low clones obtained, TERT low C1 and TERT low C2. c Telomerase activity of TERT low clones via TRAP assay visualisation and quantification show TERT low clones have reduced telomerase activity. Relative telomerase activity is calculated from the following formula [(TRAP sample—negative control)/Internal control (IC) of sample]/[(WT sample—negative control)/Internal control (IC) of WT sample)]*100. Data is shown as mean ± SD, n = 3, **P < 0.01; ANOVA with Tukey’s multiple comparisons test. d Average telomere length of WT and TERT low C1 and low C2 using the Absolute Human Telomere Length Quantification qPCR Assay Kit showing shorter telomeres in TERT low clones. Data is shown as mean ± SD, n = 3, **** P < 0.0001; ANOVA with Tukey’s multiple comparisons test. e Immunostaining of telomeres in WT and TERT low hiPSCs. TERT low clones show a decrease in telomeres compared to WT iPSCs. Scale bar = 20 μm. f Immunostaining of pluripotency markers NANOG and OCT4. Both WT and TERT low hiPSC lines display pluripotent markers. Scale bar = 50 μm

Article Snippet: Guide RNAs (gRNA) were designed using the gRNA design web tools by Feng Zhang’s lab: crispr.mit.edu and the benchling software, https://www.benchling.com .

Techniques: CRISPR, Clone Assay, Activity Assay, TRAP Assay, Negative Control, Control, Immunostaining

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