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: bioRxiv

Article Title: Synaptopodin enables directional mechanoadaptation of integrin-based adhesions

doi: 10.64898/2026.01.25.701551

Figure Lengend Snippet: (a, b) Low-magnification immunofluorescence images of Synpo ⁻/⁻ primary podocytes transfected with the synaptopodin long isoform (Synpo-L) using nanofountain probe electroporation (NFP-E) and stained for synaptopodin using an N-terminal-specific antibody (green) (a) and myosin IIA (MyoIIA; purple). (b) Mosaic expression allows direct comparison of transfected and non-transfected cells within the same field. (c, d) Higher magnification of a non-transfected Synpo ⁻/⁻ podocyte showing MyoIIA alone (c) and merged with synaptopodin staining (d) . In the absence of synaptopodin, MyoIIA displays diffuse, disorganized distribution with no discernible periodic structure. (e, f) Higher magnification of a neighboring transfected podocyte showing MyoIIA alone (e) and merged with synaptopodin staining (f) . Following synaptopodin expression, MyoIIA adopts a sarcomeric pattern, forming alternating bands with synaptopodin along stress fibers. This cell-autonomous reorganization demonstrates that synaptopodin is sufficient to organize the actomyosin cytoskeleton into the sarcomere-like structures (SLSs) observed in WT cells and suggests that synaptopodin acts as a structural template for contractile machinery assembly. The single-cell transfection approach provides an internal control, as transfected and non-transfected cells experience identical culture conditions and imaging parameters. Scale bars: 20 μm (a, b) ; 10 μm (c–f) .

Article Snippet: Cells were transferred to a 4 mm electroporation cuvette (Bio-Rad, 1652088) and electroporated using a Gene Pulser Xcell system (Bio-Rad) with the following parameters: 140 V, square wave pulse, 25 ms duration.

Techniques: Immunofluorescence, Transfection, Electroporation, Staining, Expressing, Comparison, Control, Imaging

Journal: bioRxiv

Article Title: ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

doi: 10.1101/2022.06.01.494308

Figure Lengend Snippet: a , Schematic overview of long-dsDNA-based endogenous gene tagging in human RPE1 cells. The long dsDNA donor is amplified by PCR using primers containing 90 bases of HAs. The guide RNA transcribed in vitro from PCR-assembled DNA is mixed with recombinant Cas12a or Cas9 proteins to form RNP complexes, which are electroporated with the dsDNA donor into RPE1 cells. The transgene is expected to be inserted via the HDR pathway into the target locus of the Cas-RNP using the dsDNA donor as a template. b , Representative images of cells with Cas12a-mediated endogenous mNG tagging of the indicated genes. Cells at 7-12 days after electroporation were fixed and analyzed. Scale bar: 10 µm. c , Representative images of cells with Cas9-mediated endogenous mNG tagging of the indicated genes. Cells at 12-17 days after electroporation were fixed and analyzed. Scale bar: 10 µm. d , Genomic PCR detecting the mNG insertion into the HNRNPA1 or TOMM20 locus with Cas12a-mediated knock-in. The primers were designed to amplify the 5’ junction of the mNG insertion for each gene. LHA: left HA, RHA: right HA. e , Western blotting confirming the fusion of mNG to HNRNPA1 via the Cas12a-mediated knock-in method. The knock-in cells were sorted by flow cytometry to collect mNG positive cells. HSP90 was used as a loading control. f , Flow cytometric analysis of Cas12a-mediated HNRNPA1-mNG and TOMM20-mNG knock-in cells. Cells at 8 days after electroporation were analyzed. Percentages of cells with mNG signal are shown in the plots. g , Quantification of percentages of mNG positive cells from ( f ). Data from three biological replicates are shown. >5,000 cells were analyzed for each sample of HNRNPA1 and TOMM20. Data are represented as mean ± S.D. h , Flow cytometric analysis of Cas9-mediated HNRNPA1-mNG knock-in cells. Cells at 5 days after electroporation were analyzed. Percentages of cells with mNG signal are shown in the plots. i , Quantification of percentages of mNG positive cells from ( h ). Two different concentrations of the dsDNA donor were analyzed. Data from three biological replicates are shown. 10,000 cells were analyzed for each sample. Data are represented as mean ± S.D.

Article Snippet: A.s. Cas12a Ultra (1 μM) from Integrated DNA Technologies (IDT) and crRNA (1 μM) were pre-incubated in resuspension buffer R (Thermo Fisher Scientific) at room temperature and mixed with cells (0.125 ×10 5 /μL), Cpf1 electroporation enhancer (1.8 μM, IDT), and the HDR donors (33 nM).

Techniques: Amplification, In Vitro, Recombinant, Electroporation, Knock-In, Western Blot, Flow Cytometry, Control

Journal: bioRxiv

Article Title: ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

doi: 10.1101/2022.06.01.494308

Figure Lengend Snippet: a , Schematic of endogenous gene tagging using long ssDNA donors in RPE1 cells. ssDNA donors are produced by either the T7 or the T7RE method. b , Representative images of cells with mNG tagging to the indicated genes using T7 or T7RE donors. The indicated Cas nuclease was used for each condition. For ssDNA donors, sense strands were used. Cells at 7-13 days after electroporation were fixed and analyzed. Scale bar: 10 µm. c , Flow cytometric analysis of Cas12a-mediated HNRNPA1-mNG knock-in cells, using dsDNA, T7, and T7RE donors. Cells at 9 days after electroporation were analyzed. Percentages of cells with mNG signal are shown in the plots. d , Quantification of percentages of mNG positive cells from ( c ). Data from three biological replicates are shown. Approximately 10,000 cells were analyzed for each sample. e , Flow cytometric quantification of mNG positive cells in Cas12a-mediated TOMM20-mNG knock-in cells, using the indicated donors. Cells at 12 days after electroporation were analyzed. Data from three biological replicates are shown. >5,000 cells were analyzed for each sample. f , Titration of the indicated donors for mNG tagging of HNRNPA1 using Cas12a. Cells at 11 days (dsDNA) or 10 days (T7 and T7RE) after electroporation were analyzed. For ssDNA donors, sense strands were used. Data from three biological replicates are shown. Approximately 10,000 cells were analyzed for each sample. Data are presented as mean ± S.D. P-value was calculated by the Tukey–Kramer test. ***P <0.001, n.s.: Not significant.

Article Snippet: A.s. Cas12a Ultra (1 μM) from Integrated DNA Technologies (IDT) and crRNA (1 μM) were pre-incubated in resuspension buffer R (Thermo Fisher Scientific) at room temperature and mixed with cells (0.125 ×10 5 /μL), Cpf1 electroporation enhancer (1.8 μM, IDT), and the HDR donors (33 nM).

Techniques: Produced, Electroporation, Knock-In, Titration

Journal: bioRxiv

Article Title: ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

doi: 10.1101/2022.06.01.494308

Figure Lengend Snippet: a , Schematic overview of analysis of knock-in outcomes. After electroporation of Cas12a-RNP and HDR donors for mNG tagging of HNRNPA1, cells were expanded for two to three weeks. mNG positive cells were then collected by FACS, and genomic DNA was isolated. Libraries for sequencing were prepared from the amplified target locus and subjected to long-read amplicon sequencing by PacBio. After analysis of sequencing outputs, including CCS generation, knock-knock categorized each read into a specific category of a knock-in outcome. b , Representative plots generated by knock-knock showing the distribution frequency of amplicon length. The range of read lengths corresponding to WT and indels, perfect HDR, truncated integrations, and duplication of homology arm(s) are indicated. c , Distribution of integration events across the donor types. For each category, the percentage within total integration events was calculated. Data from three biological replicates are shown. For each sample, 11559-44431 reads were categorized as the integration events from 43697-91850 total reads. d , Data from c , the frequencies of perfect HDR are highlighted. A two-tailed, unpaired Student’s t-test was used to obtain the P-value. *P < 0.05. s: sense strand, as: antisense strand.

Article Snippet: A.s. Cas12a Ultra (1 μM) from Integrated DNA Technologies (IDT) and crRNA (1 μM) were pre-incubated in resuspension buffer R (Thermo Fisher Scientific) at room temperature and mixed with cells (0.125 ×10 5 /μL), Cpf1 electroporation enhancer (1.8 μM, IDT), and the HDR donors (33 nM).

Techniques: Knock-In, Electroporation, Isolation, Sequencing, Amplification, Generated, Two Tailed Test

Journal: bioRxiv

Article Title: ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

doi: 10.1101/2022.06.01.494308

Figure Lengend Snippet: a , Schematic for evaluating homology-independent integration of mNG donors into Cas nuclease-induced DSBs. Since the Cas12a cleavage site is located inside the coding region of the TOMM20 gene, homology-independent integration of mNG into the cleavage site in the correct orientation and a correct reading frame leads to the expression of TOMM20-mNG proteins. b , Flow cytometric analysis of the homology-independent integration experiment. Sense strands were used for T7 and T7RE donors. Cells at 9 days after electroporation were analyzed. Data from three biological replicates are shown. >5,000 cells were analyzed for each sample. c , Representative images from the homology-independent integration experiment. Cells at 11 days after electroporation were fixed and analyzed. Scale bar: 10 µm. d , Schematic overview of the workflow for evaluating homology-independent integration of GalNAcT2-mNG cassettes into Cas nuclease-induced DSBs. Non-integrated cassettes are cleared from cells during long-term culture for more than 10 days. The cassettes inserted into the genome produce doxycycline (dox)-induced expression of a Golgi protein GalNAcT2-mNG. e , Flow cytometric analysis of the cassette integration experiment. Cells at 20 days after electroporation were treated with 1 µg/mL of doxycycline (dox) for 24h and analyzed. Data from three biological replicates are shown. Approximately 20,000 cells were analyzed for each sample. f , Representative images from the cassette integration experiment. Cells at 13 days after electroporation were treated with 1 µg/mL doxycycline (dox) for 24h and fixed for analysis. Arrowheads indicate cells with Golgi-like mNG signals. Scale bar: 100 µm (the left panel), 10 µm (the right panel). Data are presented as mean ± S.D. A two-tailed, unpaired Student’s t-test was used to obtain the P-value. n.s.: Not significant.

Article Snippet: A.s. Cas12a Ultra (1 μM) from Integrated DNA Technologies (IDT) and crRNA (1 μM) were pre-incubated in resuspension buffer R (Thermo Fisher Scientific) at room temperature and mixed with cells (0.125 ×10 5 /μL), Cpf1 electroporation enhancer (1.8 μM, IDT), and the HDR donors (33 nM).

Techniques: Expressing, Electroporation, Two Tailed Test

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Electroporation of alphavirus RNA translational reporters into fibroblastic and myeloid cells as a tool to study the innate immune system

doi: 10.1007/978-1-4939-3625-0_8

Figure Lengend Snippet: Electroporation Conditions for Various Cell Types

Article Snippet: Electroporation Components list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Bio-Rad Gene Pulser II with capacitance extender or Bio-Rad Gene Pulser Xcell Pre-warmed complete media 0.4 cm gap sterile electroporation cuvette 1.5 mL Eppendorf tubes or 96-well U-bottom tissue culture treated plate 2.4.

Techniques: Electroporation, Concentration Assay