Journal: Communications Biology

Article Title: Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects

doi: 10.1038/s42003-023-04994-w

Figure Lengend Snippet: We used allelic exchange mutagenesis approaches to generate catalytically inactive dcas9 with D10A and H845A missense mutations ( A ). The p3015b sgRNA shuttle expression vector accepts a 20-bp targeting protospacer cassette sequence that, in GBS, produces a compatible sgRNA molecule that directs dCas9 to a complementary target sequence located adjacent to a NGG PAM site ( B ).

Article Snippet: We used Gibson assembly to insert a synthesized, double-stranded GBS-optimized sgRNA cassette (Genscript custom order) with an upstream xyl / tet promoter that constitutively expresses the sgRNA.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Sequencing

Journal: Nature metabolism

Article Title: Cell-surface SLC nucleoside transporters and purine levels modulate BRD4-dependent chromatin states

doi: 10.1038/s42255-021-00386-8

Figure Lengend Snippet: a Schematic overview of FACS-based SLC loss-of-function screen in REDS clones. b, The enrichment of sgRNA-targeted SLCs in the highest 0.5% RFP-expressing REDS cells versus the unsorted population. The color-coded dots represent the top 16 enriched SLCs from the screen according to - log10(p-value). c, Validation of individual hits by SLC knockout. REDS1 and REDS15 cells were infected with lentivirus-based sgRNA expression cassettes and the RFP intensity was measured 12 days after sgRNA transduction. The log2 fold change of RFP intensity was calculated relatively to the cells transduced with control sgRNA (sgRen). Mean ± s.d., n =3 biologically independent samples. The p-value was calculated by paired t-tests. d, RFP expression in REDS15 cells with the identified SLC hits single, dual or triple knockout (SLC TKO) after sgRNA transduction for 12 days. e, RFP expression in REDS15 cells with SLC knockout exogenously supplied with purine (adenosine and guanosine) or pyrimidine (cytidine and thymidine) nucleosides for 72 h. The log2 fold change of RFP intensity was calculated relatively to sgRen cells with DMSO treatment. Mean ± s.d., n =3 biologically independent samples. f, Western blot analysis of the overexpression level of N- or C-terminal HA-tagged wild-type (WT) or transporter activity defective mutant SLC28A3 or SLC29A4 in REDS15 cells with respective SLC knockout. g, RFP expression in REDS15 cells as indicated.

Article Snippet: Codon-optimized sgRNA-resistance SLC28A3 and SLC29A4 plasmids were synthesized by GeneScript ( Supplementary Table 4 ).

Techniques: Clone Assay, Expressing, Biomarker Discovery, Knock-Out, Infection, Transduction, Control, Triple Knockout, Western Blot, Over Expression, Activity Assay, Mutagenesis

Journal: Nature metabolism

Article Title: Cell-surface SLC nucleoside transporters and purine levels modulate BRD4-dependent chromatin states

doi: 10.1038/s42255-021-00386-8

Figure Lengend Snippet: a,b, Relative RFP intensity (a) and cell viability (b) of SLC28A3 or SLC29A4 overexpressing REDS15 cells with or without PPAT knockout 10 days after sgRNA transduction. Mean ± s.d., n=3 biologically independent samples. The p-value was calculated by unpaired t-tests. c,d, Relative RFP expression (c) and cell viability (d) of REDS15-sgPPAT cells with nucleosides supplement for 72 h. Mean ± s.d., n=3 biologically independent samples. e, Stable isotope labeled +1 adenine or +3 glycine tracing into nucleotides in REDS15 vehicle cells, with triple SLC knockout (SLC TKO, 12 days after sgRNA transduction) or with 10 μM 6-MP treatment after 24 h. Mean ± s.d., n=3 biologically independent samples. The p-value was calculated by unpaired t-tests. f,g, Relative RFP intensity (f) and cell viability (g) of REDS15 cells with PPAT, triple SLC knockout (SLC TKO, 12 days after sgRNA transduction) or the combined knockout. Mean ± s.d., n=3 biologically independent samples. The p-value was calculated by unpaired t-tests. Cell viability was measured by CellTiter Glo assay. h, RFP expression in control sgRen cells and cells with triple SLC knockout (SLC TKO, 12 days after sgRNA transduction) treated with DMSO or 1 uM (+)-JQ1 for 72 h. i, Dose-response matrix displaying the RFP intensity in REDS1 and REDS15 clones treated with indicated concentrations of (+)-JQ1 or 6-MP, or in combination for 72 h, relative to treatment with DMSO.

Article Snippet: Codon-optimized sgRNA-resistance SLC28A3 and SLC29A4 plasmids were synthesized by GeneScript ( Supplementary Table 4 ).

Techniques: Knock-Out, Transduction, Expressing, Labeling, Glo Assay, Control, Clone Assay

Journal: Nature metabolism

Article Title: Cell-surface SLC nucleoside transporters and purine levels modulate BRD4-dependent chromatin states

doi: 10.1038/s42255-021-00386-8

Figure Lengend Snippet: a-c, Relative RFP expression in REDS1 and REDS15 cells co-treated with 100 μM nucleotide metabolites and 10 μM SLC29A4 inhibitor TC-T 6000 for 72 h (a), 10 nM dBET6 for 24 h (b) and 1 μM (+)-JQ1 for 72 h (c). The log2 fold change was normalized with DMSO treated cells. d, Western blot analysis of BRD4, c-MYC and BRD4-related histone acetylation markers expression in REDS15 cells co-treated with 100 μM nucleotide metabolites and 10 μM 6-MP or 1 μM (+)-JQ1 for 72 h. ns, non-specific band.

Article Snippet: Codon-optimized sgRNA-resistance SLC28A3 and SLC29A4 plasmids were synthesized by GeneScript ( Supplementary Table 4 ).

Techniques: Expressing, Western Blot