Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators

doi: 10.1016/j.omtn.2022.11.016

Figure Lengend Snippet: RD114-pseudotyped retroviral vectors efficiently transduce primary human B cells (A) Experimental design. Human naive B cells were negatively selected from buffy coat and activated with human CD40L multimer in presence of IL-4 and IL-21 for 2 days. Simultaneously, retroviral packaging cell lines were plated for 20 h before transfection. Two days post transfection, retroviral supernatants were harvested and used for human B cell transduction. Transduced B cells were harvested on day 4 after transduction and analyzed by flow cytometry. (Created with BioRender.com ). (B) Representative FACS plots show gating strategy to identify mCherry + B cells at day 4 post transduction. (C) Representative FACS plots (top) and the graph (bottom) show the percentages of mCherry + B cells 4 days post transduction. Non-transduced cells were used to define mCherry + gate. HEK-293T was co-transfected with sgRNA plasmid, packaging plasmid (pHIT60), and envelope plasmid (GaLV WT); Platinum-A, 293Vec-GaLV, and 293Vec-RD114 were transfected with sgRNA plasmid only. Data were pooled from eight donors, shown as mean ± SEM. Statistical significance was calculated using one-way ANOVA test, only significant p values are shown: ∗∗p < 0.01; ∗∗∗∗p < 0.0001.

Article Snippet: Human naive B cells were activated either with (1) human CD40L multimer (1 μg/mL) (Miltenyi Biotec, Cat. #130-098-776) in presence of IL-4 (10 ng/mL) and IL-21 (10 ng/mL), or with (2) 30 Gy irradiated 40LB feeder cells (1.5 × 10 4 cells/well in 96-well plate) in presence of IL-2 (5 ng/mL) and IL-21 (10 ng/mL) for 2 days before retroviral transduction.

Techniques: Retroviral, Transfection, Transduction, Flow Cytometry, Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators

doi: 10.1016/j.omtn.2022.11.016

Figure Lengend Snippet: Establishing efficient transduction conditions for primary human B cells with RD114-and GaLV-pseudotyped retroviral vectors (A) Experimental design. Negatively selected human naive B cells were activated either with (i) human CD40L multimer in the presence of IL-4 and IL-21, or with (ii) 40LB feeder cells, in the presence of IL-2 and IL-21. 40LB feeder cells were irradiated (30 Gy) and plated 1 day prior to the B cell culture. (Created with BioRender.com ). (B) Representative FACS plots (top) show the percentage of mCherry + B cells at day 4 post transduction using different plasmid conditions as indicated. Picomole of sgRNA plasmid increased as opposed of packaging and envelope plasmids, respectively. The graphs (bottom) show frequencies of mCherry + B cells obtained from three donors for condition (i) and (ii). (C) The graph shows the percentage of mCherry + B cells activated with condition (i) compared with condition (ii) at day 4 post transduction with retrovirus produced by the 293Vec-RD114 cell line. Groups were compared using unpaired t test, only significant p values are shown: ∗p < 0.1; ∗∗p < 0.01; ∗∗∗p < 0.001. Data shown as mean ± SEM.

Article Snippet: Human naive B cells were activated either with (1) human CD40L multimer (1 μg/mL) (Miltenyi Biotec, Cat. #130-098-776) in presence of IL-4 (10 ng/mL) and IL-21 (10 ng/mL), or with (2) 30 Gy irradiated 40LB feeder cells (1.5 × 10 4 cells/well in 96-well plate) in presence of IL-2 (5 ng/mL) and IL-21 (10 ng/mL) for 2 days before retroviral transduction.

Techniques: Transduction, Retroviral, Irradiation, Cell Culture, Plasmid Preparation, Produced

Journal: Molecular Therapy. Nucleic Acids

Article Title: Efficient CRISPR-Cas9-mediated mutagenesis in primary human B cells for identifying plasma cell regulators

doi: 10.1016/j.omtn.2022.11.016

Figure Lengend Snippet: Efficient CRISPR-Cas9-mediated knockout of ß2M housekeeping gene in primary human B cells (A) ß2M -specific targeting sequence was inserted into BbsI restriction site of sgRNA plasmid (left). The scheme shows the targeted site of ß2M exon 1 and two primer sites for amplifying the flanking region of ß2M -targeted site by PCR (right). (B) The sequence of T2A-GFP was amplified from plasmid MSCV-Cas9-2A-GFP-sgRNA and Furin-GSG sequence was included in the forward primer. HindIII restriction sites were added in both forward and reverse primers. The amplified fragment was digested with HindIII enzyme and replaced for T2A-mCherry sequence in plasmid MSCV-Cas9-T2A-mCherry. The reconstructed plasmid carries sequences of Cas9-Furin-GSG-T2A-GFP. (C) Experimental design. Negatively selected human naive B cells were activated with CD40L multimer in presence of IL-4 and IL-21 for 2 days. In parallel, 293Vec-RD114 retroviral packaging cells were plated for 20 h until transfection. Two days post transfection, retroviral supernatant was harvested for transduction. Day 4 post transduction, a half of B cells was harvested for FACS analysis or cell sorting and the other half was remained in culture with IL-10 and IL-21 for another 4 days. (Created with BioRender.com ). (D) Representative FACS plots show co-transduced B cells before (top) and after (bottom) sorting at day 4 post transduction. (E) Agarose gel from T7 endonuclease I assay shows digested bands from sg ß2M -targeted compared with non-targeted sample. sg ß2M : sgRNA plasmid with ß2M -targeted sequence; (−): non-targeted. Red asterisks highlight the digested DNA bands. (F) Sanger sequencing signal traces (left) show noise peaks at ß2M -targeted site compared with non-targeted sample. The dashed line indicates cleavage site of Cas9. The graph (right) shows the percentage of Indel mutations at ß2M -targeted site compared with non-targeted sample. Alignment and the percentage of Indel mutations were performed by ICE analysis online program (Synthego). (G) Representative FACS plots show co-transduced B cells with both sgRNA (mCherry) and Cas9 (GFP) (left) and β2M − B cells (right) at day 4 and 8 post transduction. mCherry + GFP + B cells were gated based on non-transduced sample, β2M − B cells were gated based on non-targeted sample (transduced with sgRNA and Cas9 plasmids but sgRNA has no targeted sequence). The graph (right) shows the percentage of mCherry + GFP + and surface β2M − B cells in mCherry + GFP + cells at day 0, 4, and 8 post transduction. Statistical significance was calculated using the unpaired t test: ∗∗p < 0.01; ∗∗∗∗p < 0.0001. Data were pooled from three donors (F) and six donors (G), shown as mean ± SEM.

Article Snippet: Human naive B cells were activated either with (1) human CD40L multimer (1 μg/mL) (Miltenyi Biotec, Cat. #130-098-776) in presence of IL-4 (10 ng/mL) and IL-21 (10 ng/mL), or with (2) 30 Gy irradiated 40LB feeder cells (1.5 × 10 4 cells/well in 96-well plate) in presence of IL-2 (5 ng/mL) and IL-21 (10 ng/mL) for 2 days before retroviral transduction.

Techniques: CRISPR, Knock-Out, Sequencing, Plasmid Preparation, Amplification, Retroviral, Transfection, Transduction, FACS, Agarose Gel Electrophoresis, T7EI Assay