Journal: The CRISPR Journal

Article Title: Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies

doi: 10.1089/crispr.2022.0086

Figure Lengend Snippet: Study and optimization of electroporation conditions using CliniMACS Prodigy electroporator. (A) Small-scale pulse optimization using DsRed mRNA to determine the transfection efficiency (gray) and viability (white), n = 2. (B) Upscale BCL11A transfection using two of the most suitable electroporation settings in small-scale optimization led to moderate InDel efficiencies, n = 1. (C) Upscale comparison of different conditions to further optimize transfection efficiency. BCL11A editing is provided in InDel rate after Sanger sequencing of the PCR product (gray). Viability was determined by flow cytometry 2 days post-transfection (white), n = 2. Nonsignificant differences were observed for editing efficiencies in Mann–Whitney tests (p > 0.05). (D) Influence of different RNP concentrations (2250 pmol/mL, light gray; 4500 pmol/mL, dark gray; 6750 pmol/mL, black) with increasing cell concentrations (5 × 106 to 1.5 × 107 cells/mL) on the BCL11A editing rate, n = 1. (E) Same data as in (D), but displayed as editing rate in relation to RNP concentration per 106 cells, n = 1. (F) Effect of the electroporation volume in CliniMACS Prodigy EP-2 cuvette on the editing performance (gray) and viability (white), n = 2. Nonsignificant differences were observed for editing efficiencies and viabilities in Mann–Whitney tests (p > 0.05). (G) RNP stability controlling freshly prepared RNP (control) versus RNP recovered after a process run and storage time of 60 min, n = 1. (H) Effect of the RNP incubation time on the editing rate (light gray) and viability (white), n = 2. Nonsignificant differences were observed for editing efficiencies and viabilities in Mann–Whitney tests (p > 0.05). (I) Average editing rate for thawed HSPCs from different donors, n = 4. (J) Comparison of BCL11A transfection efficiency after cultivation in the CliniMACS Prodigy system versus a classical cell incubator, n = 1. HSPCs, hematopoietic stem and progenitor cells; PCR, polymerase chain reaction; RNP, ribonucleoprotein.

Article Snippet: Upscale HPSC electroporation The cuvette of the CliniMACS Prodigy EP-2 was manually filled with 600 μL of CD34+ cells in the CliniMACS Electroporation Buffer (Miltenyi Biotec) at a concentration of 5 × 106 to 1.5 × 107 cells/mL with either 30 μg/mL eGFP mRNA (Miltenyi Biotec) or 150–900 pmol ribonucleoprotein (RNP) complex per million cells.

Techniques: Electroporation, Transfection, Comparison, Sequencing, Flow Cytometry, MANN-WHITNEY, Concentration Assay, Control, Incubation, Polymerase Chain Reaction

Journal: The CRISPR Journal

Article Title: Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies

doi: 10.1089/crispr.2022.0086

Figure Lengend Snippet: Large-scale BCL11A editing of HSPCs using CliniMACS Prodigy system with electroporator compared with NTC, NTCe, and upscale controls using the CliniMACS Prodigy EP-2 cuvette. (A) BCL11A editing at the genomic level (n = 1 with technical replicates), cellular viability, and recovery at day 2 after electroporation (n = 1). (B) CFU assay of large-scale samples compared with upscale samples. Total colonies counted for 250 seeded HSPCs (left) and proportion of different colonies, n = 1 with technical replicates. (C) Erythroid differentiation staining on days 7, 14, and 21. Positive rate by flow cytometry for CD34, CD36, CD235a, CD71, CD233, and CD49d, mock electroporated cells (NTCe) (dark gray), upscale control electroporation (light gray), and cells electroporated by the CliniMACS Prodigy process (white), n = 1. (D) HbF levels of electroporated samples measured by flow cytometry on days 7, 14, and 21. (E) HbF/(HbF+HbA0) ratio as determined by HPLC analysis of normal control cells, HbF expressing control cells, and processed cells after additional erythroid differentiation: mock electroporated cells (NTCe), upscale control electroporated cells, and cells electroporated by the CliniMACS Prodigy process, n = 1. (F) HPLC chromatograms of HbF expressing control cells and electroporated samples after in vitro erythroid differentiation. BFU-E, burst-forming unit-erythroid; CFU, colony-forming unit; CFU-G, CFU-granulocyte; CFU-GEMM, CFU-granulocyte erythrocyte macrophage megakaryocyte; CFU-GM, CFU-granulocyte macrophage; CFU-M, CFU-macrophage; HbF, fetal hemoglobin; HPLC, high-performance liquid chromatography; NTC, nontransfected controls; NTCe, electroporated nontransfected controls.

Article Snippet: Upscale HPSC electroporation The cuvette of the CliniMACS Prodigy EP-2 was manually filled with 600 μL of CD34+ cells in the CliniMACS Electroporation Buffer (Miltenyi Biotec) at a concentration of 5 × 106 to 1.5 × 107 cells/mL with either 30 μg/mL eGFP mRNA (Miltenyi Biotec) or 150–900 pmol ribonucleoprotein (RNP) complex per million cells.

Techniques: Electroporation, Colony-forming Unit Assay, Staining, Flow Cytometry, Control, Expressing, In Vitro, High Performance Liquid Chromatography

Journal: Nature

Article Title: CRISPR Screen in Regulatory T Cells Reveals Modulators of Foxp3

doi: 10.1038/s41586-020-2246-4

Figure Lengend Snippet: a) Representative flow cytometry analysis of the YFP + Treg population (gated on CD4 + cells) from the spleen and lymph nodes of Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice. b) Statistical analysis of YFP MFI in CD4 + YFP + Tregs from the thymus (Thy), peripheral lymph nodes (pLN), and spleen (Spl) of Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice. c) Statistical analysis of CD4 + YFP + Treg frequencies in Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice, corresponding to panel b. d) Volcano plot for RNA sequencing of Usp22 RNP KO Tregs vs Rnf20 RNP KO murine Tregs. X-axis shows log2FoldChange (LFC). Y-axis shows the –log10 of the p-value as calculated by DESeq2. Genes downregulated in the Usp22 RNP KO compared to Rnf20 RNP KO are shown in red and genes upregulated are shown in blue defined by p-value <5e-3 and LFC > 0.8. Foxp3 (shown in green) trended down but did not reach significance. e) qPCR analysis of FOXP3 mRNA levels in human Tregs from 2 donors 8 days post-electroporation with Cas9 RNPs targeting NTC , FOXP3, USP22, RNF20 or both USP22 and RNF20. Normalized to the expression of β -ACTIN transcripts. Data are presented as mean ±SEM and are representative of at least two independent experiments. f) qPCR analysis of Foxp3 mRNA levels in mouse Tregs 4 and 8 days post-electroporation with Cas9 RNPs targeting NTC, Foxp3, Usp22, Rnf20 or both Usp22 and Rnf20. Normalized to the expression of β -actin transcripts. g) Western blot analysis of ubiquitinated histone 2A (H2AK119Ub; H2A-ub) and ubiquitinated histone 2B (H2BK120Ub; H2B-ub) from iTregs from Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice. Gapdh was used as a loading control. Source data can be found in . h) Schematic of Foxp3 locus depicting PCR products used for ChIP-qPCR data shown in panel i and panel j. i) ChIP-qPCR data analysis for H2AK119Ub (H2A-ub) where primers amplified across the transcriptional start site (TSS) and the CNS1 enhancer region of the Foxp3 locus. Data are normalized to the input and are presented as mean ±SD. j) ChIP-qPCR data analysis for H2BK120Ub (H2B-ub) for PCR across the transcriptional start site (TSS) and across the CNS1 enhancer region of the Foxp3 locus. Data are normalized to the input and are presented as mean ±SD. k) Heatmap of ChIP-seq read density for Foxp3, Usp22, and Rnf20 at sites bound by Foxp3 (using previously published Foxp3 ChIP data ), ranked by highest to lowest Foxp3 binding signal. The corresponding log2 fold change (log2fc) for either H2BK120Ub or H2AK119Ub upon Usp22 or Rnf20 deletion at these sites are plotted on the right, with each biological replicate shown as an individual column. l) Average ChIP-seq read density of H2BK120Ub at Treg super enhancers in control versus Usp22-deficient Tregs. m) Co-occurrence analysis showing the natural log of the ratio of the observed number of overlapping regions over the expected values for sites that either gain or lose H2BK120Ub in Usp22-deficient Tregs against publicly available histone modifications H3K4me, H3K4me3 and H3K27ac as well as enhancer classes, as described in the Methods. n) Analysis of reciprocal regulation of Foxp3 by deubiquitinase Usp22 and E3 ubiquitin ligase Rnf20. YFP MFI of Tregs sorted from Usp22 +/+ Foxp3 YFP-Cre WT or Usp22 fl/fl Foxp3 YFP-Cre KO mice and then electroporated with either NT control (NTC-RNP) or Rnf20 RNP, corresponding with where Foxp3 MFI from the same experiment is shown. All data are presented as mean ±SEM, unless otherwise stated. ns indicates no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The exact sample sizes (n), p-values, statistical tests and number of times the experiment was replicated can be found in the “Statistics and Reproducibility” section.

Article Snippet: Tregs were collected from their culture vessels and centrifuged for 5 min at 300g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μl buffer per 200,000 cells.

Techniques: Flow Cytometry, RNA Sequencing Assay, Electroporation, Expressing, Western Blot, Amplification, ChIP-sequencing, Binding Assay