lentiviral vector pls mp  (Addgene inc)

Journal: bioRxiv

Article Title: Single-cell CRISPR activation screens in primary B cells discover gene regulatory mechanisms for hundreds of autoimmune risk loci

doi: 10.64898/2026.03.01.708923

Figure Lengend Snippet: (A) Lentiviral massively parallel reporter assay (lentiMPRA) to quantify variant-specific effects on transcriptional activity. (B) LentiMPRA transcription efficiencies (z-score) for reference (REF) and alternate (ALT) alleles with significant variant-specific effects ( p <0.05; paired sample t-test; n = 6). ( C ) LentiMPRA transcriptional efficiency (RNA/DNA, norm) for REF and ALT alleles at SCANDAL regions, with cis -regulatory target gene annotated below. p denotes paired sample t-test (n = 6, ±SD). ( D ) LentiMPRA transcriptional efficiency (RNA/DNA, norm) for REF (C) or ALT (T) alleles at rs1432296. ( E ) Genome snapshot of CRISPR-SAM targeting and Micro-Capture-C (MCC) at rs1432296, including SCANDAL effect on target gene REL as curved line. ( F ) REL expression after CRISPR-SAM perturbation for cells containing rs1432296-targeting or non-targeting control sgRNAs. Circular points reflect single cell expression values, while diamonds indicate mean expression values for all cells containing individual sgRNAs where size corresponds to percentage of cells in which the gene was detected. p denotes Benjamini-Hochberg-adjusted SCEPTRE p -value. ( G ) CRISPR-Cas9 Prime-Editing (PE7) in GC B cells to test risk variant-specific effects in situ via quantile-based sorting of target expression and amplicon sequencing. ( H ) rs1432296 ALT (T) allele frequency and REL transcript mean fluorescence intensity (MFI) in prime-edited GC B cells (n=5). Spearman’s rank correlation (ρ) and p -value are shown. ( I ) REL ChIP-seq signal at SCANDAL risk loci and their SCANDAL target promoters. CREs are centred on open chromatin peak or TSS. ( J ) Genome snapshot of REL-bound rs74405933 SCANDAL risk locus, with motifbreakR prediction for A allele effect on REL motif. SCANDAL effect on target gene REL as curved line. ( K ) CD83 expression after CRISPR-SAM perturbation for cells containing rs74405933-targeting or non-targeting control sgRNAs, with formatting as detailed in (F). ( L ) LentiMPRA transcriptional efficiency (RNA/DNA, norm) for REF (G) or ALT (A) alleles at rs74405933. ( M ) rs1432296 ALT (A) allele frequency and CD83 surface expression (MFI) in prime-edited GC B cells (n=5). Spearman’s rank correlation (ρ) and p -value are shown. ( N ) Number of SCANDAL loci bound by REL, denoting enrichment of REL-bound loci within each trait as compared to all SCANDAL loci with gene targets for this trait. ( O ) Binding of SCANDAL target transcription factors (TF) to other SCANDAL loci with targets, showing if the bound locus has the same trait as the locus controlling the TF expression. ( P ) Proposed model of genetic pleiotropy underlying pan-disease autoimmune risk. While cis -regulatory effects on TF expression may be mediated through a GWAS risk variant associated with Trait A, the downstream targets of that TF include many non-coding risk loci (or their regulatory target genes) with genetic associations for different traits.

Article Snippet: To clone the MPRA library into lentiviral vector pLS-mP (a gift from Nadav Ahituv; Addgene #81225), 125 ng of the oligonucleotide pool DNA was amplified in 4 × 50 μL reactions with 0.5 μM MPRAlib_PCR1_FW and MPRAlib_PCR1_RV primers and following conditions: 98°C for 1 min, (98°C for 10 s, 60°C for 15 s, 72°C for 20 s) × 5 cycles, 72°C for 5 min. 150 ng of purified PCR product split into 4 × 50 μL reactions and amplified with 0.5 μM MPRAlib_PCR2_FW and MPRAlib_BC_PCR2_RV primers under following conditions: 98°C for 2 min, (98°C for 15 s, 60°C for 20 s, 72°C for 30 s) × 4 cycles, 72°C for 5 min. At this step, a unique 13 bp molecular barcode (from primer MPRA_PCR2_FW) was added to each CRE.

Techniques: Reporter Assay, Variant Assay, Activity Assay, CRISPR, Capture-C, Expressing, Control, Single Cell, In Situ, Amplification, Sequencing, Fluorescence, ChIP-sequencing, Binding Assay