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btxpress electroporation buffer  (Harvard Bioscience)

 
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    Harvard Bioscience btxpress electroporation buffer
    The method for delivery of DNA repair factors (RFs) for global overexpression in K562 differentially affects gene editing outcomes. (A) Experimental overview of K562 cell transduction and <t>electroporation</t> for delivery of DNA RFs and editing reagents. DNA RFs delivered as plasmid were co-electroporated with editing reagents, while delivery of DNA RFs as LVs occurred 24 h prior to electroporation. (B,C) Cas9 nuclease was delivered either as plasmid (1 μg) or RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA); donor template was delivered either as ssODN (3 μM) or AAV6 (MOI 2e4). DNA RFs were delivered as a plasmid. HDR levels were measured by qPCR. n = 2 biological replicates for CtIP DE + PALB2 KR + i53 experiments, n = 6 biological replicates for RAD52 + dn53BP1 experiments. Error bars, mean ± SD. (D) K562 cells were transduced with LVs expressing the indicated DNA RFs. Western Blot was performed on day 10 post-transduction. Vector copy number (VCN) was determined by droplet digital PCR (ddPCR). (E) K562 cells were transduced with DNA RF LVs and electroporated with editing reagents. n = 2 biological replicates. Data are normalized to “No RFs” conditions for each set of experiments. Error bars, mean ± SD.
    Btxpress Electroporation Buffer, supplied by Harvard Bioscience, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/btxpress+electroporation+buffer/btxpress+electroporation+buffer/pmc08525354-244-23-26
    Average 90 stars, based on 1 article reviews
    btxpress electroporation buffer - by Bioz Stars, 2026-09
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    1) Product Images from "Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells"

    Article Title: Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

    Journal: Frontiers in Genome Editing

    doi: 10.3389/fgeed.2020.601541

    The method for delivery of DNA repair factors (RFs) for global overexpression in K562 differentially affects gene editing outcomes. (A) Experimental overview of K562 cell transduction and electroporation for delivery of DNA RFs and editing reagents. DNA RFs delivered as plasmid were co-electroporated with editing reagents, while delivery of DNA RFs as LVs occurred 24 h prior to electroporation. (B,C) Cas9 nuclease was delivered either as plasmid (1 μg) or RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA); donor template was delivered either as ssODN (3 μM) or AAV6 (MOI 2e4). DNA RFs were delivered as a plasmid. HDR levels were measured by qPCR. n = 2 biological replicates for CtIP DE + PALB2 KR + i53 experiments, n = 6 biological replicates for RAD52 + dn53BP1 experiments. Error bars, mean ± SD. (D) K562 cells were transduced with LVs expressing the indicated DNA RFs. Western Blot was performed on day 10 post-transduction. Vector copy number (VCN) was determined by droplet digital PCR (ddPCR). (E) K562 cells were transduced with DNA RF LVs and electroporated with editing reagents. n = 2 biological replicates. Data are normalized to “No RFs” conditions for each set of experiments. Error bars, mean ± SD.
    Figure Legend Snippet: The method for delivery of DNA repair factors (RFs) for global overexpression in K562 differentially affects gene editing outcomes. (A) Experimental overview of K562 cell transduction and electroporation for delivery of DNA RFs and editing reagents. DNA RFs delivered as plasmid were co-electroporated with editing reagents, while delivery of DNA RFs as LVs occurred 24 h prior to electroporation. (B,C) Cas9 nuclease was delivered either as plasmid (1 μg) or RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA); donor template was delivered either as ssODN (3 μM) or AAV6 (MOI 2e4). DNA RFs were delivered as a plasmid. HDR levels were measured by qPCR. n = 2 biological replicates for CtIP DE + PALB2 KR + i53 experiments, n = 6 biological replicates for RAD52 + dn53BP1 experiments. Error bars, mean ± SD. (D) K562 cells were transduced with LVs expressing the indicated DNA RFs. Western Blot was performed on day 10 post-transduction. Vector copy number (VCN) was determined by droplet digital PCR (ddPCR). (E) K562 cells were transduced with DNA RF LVs and electroporated with editing reagents. n = 2 biological replicates. Data are normalized to “No RFs” conditions for each set of experiments. Error bars, mean ± SD.

    Techniques Used: Over Expression, Transduction, Electroporation, Plasmid Preparation, Expressing, Western Blot, Digital PCR

    (A) Experimental overview of CD34 + HSPC transduction and electroporation of DNA RFs and editing reagents. DNA RFs were co-expressed with the gene editing reagents. Cas9 nuclease was delivered either as RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA) or IVT Cas9 mRNA (5 μg) + IVT sgRNA (5 μg); donor template was delivered either as AAV6 (MOI 2e4) or ssODN (3 μg). HDR and NHEJ levels were measured by HTS four days post-electroporation. HDR/NHEJ ratio was calculated. (B) DNA repair factors delivered as LV 24 h prior to electroporation of editing reagents. n = 4 biological replicates for all CtIPDE+PALB2KR+i53 LV experiments (Cas9 RNP or Cas9mRNA); n = 8–12 for RAD52+dn53BP1 LV experiments using Cas9 RNP, or n = 4 for RAD52+dn53BP1 LV experiments using Cas9 mRNA. Error bars, mean ± SD. Differences are not significant if not specified, based on Wilcoxon rank sum test. (C) DNA RFs were co-delivered as IVT mRNA with editing reagents. n = 2–6 biological replicates for all conditions. Data are normalized to “no RFs” conditions for each set of experiments. Error bars, mean ± SD. Differences are not significant if not specified, based on Wilcoxon rank sum test.
    Figure Legend Snippet: (A) Experimental overview of CD34 + HSPC transduction and electroporation of DNA RFs and editing reagents. DNA RFs were co-expressed with the gene editing reagents. Cas9 nuclease was delivered either as RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA) or IVT Cas9 mRNA (5 μg) + IVT sgRNA (5 μg); donor template was delivered either as AAV6 (MOI 2e4) or ssODN (3 μg). HDR and NHEJ levels were measured by HTS four days post-electroporation. HDR/NHEJ ratio was calculated. (B) DNA repair factors delivered as LV 24 h prior to electroporation of editing reagents. n = 4 biological replicates for all CtIPDE+PALB2KR+i53 LV experiments (Cas9 RNP or Cas9mRNA); n = 8–12 for RAD52+dn53BP1 LV experiments using Cas9 RNP, or n = 4 for RAD52+dn53BP1 LV experiments using Cas9 mRNA. Error bars, mean ± SD. Differences are not significant if not specified, based on Wilcoxon rank sum test. (C) DNA RFs were co-delivered as IVT mRNA with editing reagents. n = 2–6 biological replicates for all conditions. Data are normalized to “no RFs” conditions for each set of experiments. Error bars, mean ± SD. Differences are not significant if not specified, based on Wilcoxon rank sum test.

    Techniques Used: Transduction, Electroporation

    CtIP DE +PALB2 KR + i53 overexpression in G1 phase of the cell cycle does not activate HDR. (A–D) Gene editing levels in sorted populations. Hoescht stain was used to discriminate G0/G1 and S/G2/M phases. Cells were pre-transduced with the indicated DNA RFs or GFP LVs, sorted into cell cycle phases and then electroporated with Cas9 RNP and ssODN or transduced with an AAV6 donor template immediately after electroporation. HDR and NHEJ levels were measured by HTS. (A) HDR, (B) NHEJ, (C) HDR/NHEJ, (D) MMEJ/indels, in unsorted, G0/G1-sorted, or S/G2/M-sorted populations. n = 2 biological replicates. Error bars, mean ± SD.
    Figure Legend Snippet: CtIP DE +PALB2 KR + i53 overexpression in G1 phase of the cell cycle does not activate HDR. (A–D) Gene editing levels in sorted populations. Hoescht stain was used to discriminate G0/G1 and S/G2/M phases. Cells were pre-transduced with the indicated DNA RFs or GFP LVs, sorted into cell cycle phases and then electroporated with Cas9 RNP and ssODN or transduced with an AAV6 donor template immediately after electroporation. HDR and NHEJ levels were measured by HTS. (A) HDR, (B) NHEJ, (C) HDR/NHEJ, (D) MMEJ/indels, in unsorted, G0/G1-sorted, or S/G2/M-sorted populations. n = 2 biological replicates. Error bars, mean ± SD.

    Techniques Used: Over Expression, Staining, Transduction, Electroporation

    Preliminary assessment of Cas9 variants to modulate local gene editing outcomes in a K562 BFP reporter cell line. (A) Experimental overview of electroporation of Cas9 variants as plasmid into a K562 BFP reporter cell line. K562 BFP cells were electroporated with 1 μg Cas9 variant plasmid, 1 μg of sgRNA plasmid targeting the BFP gene, and a ssODN donor (3 μM). Cells were cultured for 4 days post-electroporation prior to analysis by flow cytometry. (B–G) Proportion of GFP + , BFP + , or NF cells and GFP/NF ratio of cells edited with Cas9-hCtIP variants, n = 2–6 biological replicates (B,C) , and Cas9-UL12 variants, n = 3–8 biological replicates (D,E) , and Cas9-dn53BP1, n = 2–6 biological replicates (F,G) . Error bars, mean ± SD. Differences are not significant if not specified, * p < 0.05, based on Wilcoxon rank sum test.
    Figure Legend Snippet: Preliminary assessment of Cas9 variants to modulate local gene editing outcomes in a K562 BFP reporter cell line. (A) Experimental overview of electroporation of Cas9 variants as plasmid into a K562 BFP reporter cell line. K562 BFP cells were electroporated with 1 μg Cas9 variant plasmid, 1 μg of sgRNA plasmid targeting the BFP gene, and a ssODN donor (3 μM). Cells were cultured for 4 days post-electroporation prior to analysis by flow cytometry. (B–G) Proportion of GFP + , BFP + , or NF cells and GFP/NF ratio of cells edited with Cas9-hCtIP variants, n = 2–6 biological replicates (B,C) , and Cas9-UL12 variants, n = 3–8 biological replicates (D,E) , and Cas9-dn53BP1, n = 2–6 biological replicates (F,G) . Error bars, mean ± SD. Differences are not significant if not specified, * p < 0.05, based on Wilcoxon rank sum test.

    Techniques Used: Electroporation, Plasmid Preparation, Variant Assay, Cell Culture, Flow Cytometry

    Experimental outline of Cas9 variant gene editing in HSPCs. (A) Experimental overview of electroporation of Cas9 variants delivered as IVT mRNA to CD34 + HSPCs. Cas9 variants were electroporated at equimolar amounts (3 pmol) with 120 pmol of IVT sgRNA targeting the SCD mutation at the HBB locus, and either a ssODN (3 μM) or AAV6 donor (MOI 2e4). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. (B) Viability of CD34 + HSPCs edited with Cas9 variants, and an ssODN or AAV6 donor 24 h post-electroporation. n = 2–6 biological replicates. Center line represents mean. Differences are not significant if not specified, based on Wilcoxon rank sum test.
    Figure Legend Snippet: Experimental outline of Cas9 variant gene editing in HSPCs. (A) Experimental overview of electroporation of Cas9 variants delivered as IVT mRNA to CD34 + HSPCs. Cas9 variants were electroporated at equimolar amounts (3 pmol) with 120 pmol of IVT sgRNA targeting the SCD mutation at the HBB locus, and either a ssODN (3 μM) or AAV6 donor (MOI 2e4). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. (B) Viability of CD34 + HSPCs edited with Cas9 variants, and an ssODN or AAV6 donor 24 h post-electroporation. n = 2–6 biological replicates. Center line represents mean. Differences are not significant if not specified, based on Wilcoxon rank sum test.

    Techniques Used: Variant Assay, Electroporation, Mutagenesis

    Cas9 variant gene editing of the β-globin locus with an AAV6 donor reveals distinctive DNA repair outcomes in HSPCs. CD34 + HSPCs edited with Cas9 variants (3 pmol Cas9 mRNA + 120 pmol IVT sgRNA) and an AAV6 donor (MOI 2e4). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. Cas9 and Cas9-hGem were used as controls. (A–C) Gene editing outcomes with Cas9-hCtIP variants. (A) HDR and NHEJ. n = 4 biological replicates). Error bars, mean ± SD. (B) HDR/NHEJ. n = 4 biological replicates Center line represents mean (C) MMEJ/indels. n = 2 biological replicates. Center line represents mean. (D–F) Gene editing outcomes with Cas9-UL12 variants. (D) HDR and NHEJ. n = 2–6 biological replicates. Error bars, mean ± SD. (E) HDR/NHEJ. n = 2–6 biological replicates Center line represents mean (F) MMEJ/indels. n = 2–4 biological replicates. Center line represents mean. (G–I) Gene editing outcomes with Cas9-dn53BP1 variants. (G) HDR and NHEJ. n = 2–4 biological replicates. Error bars, mean ± SD. (H) HDR/NHEJ, n = 2–4 biological replicates. Center line represents mean (I) MMEJ/indels. n = 2–4 biological replicates. Center line represents mean. Differences are not significant if not specified, * p < 0.05, based on Wilcoxon rank sum test.
    Figure Legend Snippet: Cas9 variant gene editing of the β-globin locus with an AAV6 donor reveals distinctive DNA repair outcomes in HSPCs. CD34 + HSPCs edited with Cas9 variants (3 pmol Cas9 mRNA + 120 pmol IVT sgRNA) and an AAV6 donor (MOI 2e4). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. Cas9 and Cas9-hGem were used as controls. (A–C) Gene editing outcomes with Cas9-hCtIP variants. (A) HDR and NHEJ. n = 4 biological replicates). Error bars, mean ± SD. (B) HDR/NHEJ. n = 4 biological replicates Center line represents mean (C) MMEJ/indels. n = 2 biological replicates. Center line represents mean. (D–F) Gene editing outcomes with Cas9-UL12 variants. (D) HDR and NHEJ. n = 2–6 biological replicates. Error bars, mean ± SD. (E) HDR/NHEJ. n = 2–6 biological replicates Center line represents mean (F) MMEJ/indels. n = 2–4 biological replicates. Center line represents mean. (G–I) Gene editing outcomes with Cas9-dn53BP1 variants. (G) HDR and NHEJ. n = 2–4 biological replicates. Error bars, mean ± SD. (H) HDR/NHEJ, n = 2–4 biological replicates. Center line represents mean (I) MMEJ/indels. n = 2–4 biological replicates. Center line represents mean. Differences are not significant if not specified, * p < 0.05, based on Wilcoxon rank sum test.

    Techniques Used: Variant Assay, Electroporation

    Cas9 variant gene editing of the HBB locus with a ssODN donor. CD34 + HSPCs edited with Cas9 variants (3 pmol Cas9 mRNA + 120 pmol IVT sgRNA) and ssODN donor (3 μM). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. Cas9 and Cas9-hGem were used as controls. (A) HDR and NHEJ. (B) HDR/NHEJ n = 2 biological replicates. Error bars, mean ± SD. (C) MMEJ/indels. n = 2 biological replicates. Center line represents mean.
    Figure Legend Snippet: Cas9 variant gene editing of the HBB locus with a ssODN donor. CD34 + HSPCs edited with Cas9 variants (3 pmol Cas9 mRNA + 120 pmol IVT sgRNA) and ssODN donor (3 μM). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. Cas9 and Cas9-hGem were used as controls. (A) HDR and NHEJ. (B) HDR/NHEJ n = 2 biological replicates. Error bars, mean ± SD. (C) MMEJ/indels. n = 2 biological replicates. Center line represents mean.

    Techniques Used: Variant Assay, Electroporation

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    Article Snippet: 3-methyladenine (Sigma, M9281), freshly prepared for each experiment, was dissolved in phosphate-buffered saline (PBS; 8 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na2HPO4, 0.24 g/l KH2PO4) at 50 C at a stock concentration of 100 mM. .. N-acetyl-L-cysteine (NAC; Sigma, A7250) was dissolved in complete DMEM at a stock concentration of 500 mM and pH-balanced with sodium bicarbonate (Sigma, S5761). (2R, 4R)-APDC (P8765), bafilomycin A1 (B1793) and MitoTEMPO (SML0737) were from Sigma, AZD8055 from Synkinase (SYN-1166), Torin 1 from Cayman chemicals (10997) and rapamycin from Adipogen (AG-CN2-0025). siRNA transfection Mouse iBMM and RAW264.7 cells were transfected with siRNA as previously described.42 Briefly, cells were resuspended in 100 ml BTXpress electroporation buffer (BTX Harvard Apparatus, 45–0803) with 400 nM Atg5 or nontargeting (scrambled) ON-TARGETplus siRNA (SMARTpool, GE Healthcare Dharmacon) and electroporated using an Amaxa Nucleofector device (Lonza, Victoria, Australia). ..

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    btxpress electroporation buffer  (Harvard Bioscience)
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    Harvard Bioscience btxpress electroporation buffer
    The method for delivery of DNA repair factors (RFs) for global overexpression in K562 differentially affects gene editing outcomes. (A) Experimental overview of K562 cell transduction and <t>electroporation</t> for delivery of DNA RFs and editing reagents. DNA RFs delivered as plasmid were co-electroporated with editing reagents, while delivery of DNA RFs as LVs occurred 24 h prior to electroporation. (B,C) Cas9 nuclease was delivered either as plasmid (1 μg) or RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA); donor template was delivered either as ssODN (3 μM) or AAV6 (MOI 2e4). DNA RFs were delivered as a plasmid. HDR levels were measured by qPCR. n = 2 biological replicates for CtIP DE + PALB2 KR + i53 experiments, n = 6 biological replicates for RAD52 + dn53BP1 experiments. Error bars, mean ± SD. (D) K562 cells were transduced with LVs expressing the indicated DNA RFs. Western Blot was performed on day 10 post-transduction. Vector copy number (VCN) was determined by droplet digital PCR (ddPCR). (E) K562 cells were transduced with DNA RF LVs and electroporated with editing reagents. n = 2 biological replicates. Data are normalized to “No RFs” conditions for each set of experiments. Error bars, mean ± SD.
    Btxpress Electroporation Buffer, supplied by Harvard Bioscience, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/btxpress+electroporation+buffer/btxpress+electroporation+buffer/pmc08525354-244-23-26
    Average 90 stars, based on 1 article reviews
    btxpress electroporation buffer - by Bioz Stars, 2026-09
    90/100 stars
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    The method for delivery of DNA repair factors (RFs) for global overexpression in K562 differentially affects gene editing outcomes. (A) Experimental overview of K562 cell transduction and electroporation for delivery of DNA RFs and editing reagents. DNA RFs delivered as plasmid were co-electroporated with editing reagents, while delivery of DNA RFs as LVs occurred 24 h prior to electroporation. (B,C) Cas9 nuclease was delivered either as plasmid (1 μg) or RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA); donor template was delivered either as ssODN (3 μM) or AAV6 (MOI 2e4). DNA RFs were delivered as a plasmid. HDR levels were measured by qPCR. n = 2 biological replicates for CtIP DE + PALB2 KR + i53 experiments, n = 6 biological replicates for RAD52 + dn53BP1 experiments. Error bars, mean ± SD. (D) K562 cells were transduced with LVs expressing the indicated DNA RFs. Western Blot was performed on day 10 post-transduction. Vector copy number (VCN) was determined by droplet digital PCR (ddPCR). (E) K562 cells were transduced with DNA RF LVs and electroporated with editing reagents. n = 2 biological replicates. Data are normalized to “No RFs” conditions for each set of experiments. Error bars, mean ± SD.

    Journal: Frontiers in Genome Editing

    Article Title: Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

    doi: 10.3389/fgeed.2020.601541

    Figure Lengend Snippet: The method for delivery of DNA repair factors (RFs) for global overexpression in K562 differentially affects gene editing outcomes. (A) Experimental overview of K562 cell transduction and electroporation for delivery of DNA RFs and editing reagents. DNA RFs delivered as plasmid were co-electroporated with editing reagents, while delivery of DNA RFs as LVs occurred 24 h prior to electroporation. (B,C) Cas9 nuclease was delivered either as plasmid (1 μg) or RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA); donor template was delivered either as ssODN (3 μM) or AAV6 (MOI 2e4). DNA RFs were delivered as a plasmid. HDR levels were measured by qPCR. n = 2 biological replicates for CtIP DE + PALB2 KR + i53 experiments, n = 6 biological replicates for RAD52 + dn53BP1 experiments. Error bars, mean ± SD. (D) K562 cells were transduced with LVs expressing the indicated DNA RFs. Western Blot was performed on day 10 post-transduction. Vector copy number (VCN) was determined by droplet digital PCR (ddPCR). (E) K562 cells were transduced with DNA RF LVs and electroporated with editing reagents. n = 2 biological replicates. Data are normalized to “No RFs” conditions for each set of experiments. Error bars, mean ± SD.

    Article Snippet: For electroporation, 2 × 10 5 cells per condition were pelleted at 90×g for 15 min at RT, resuspended in 100 μl of BTXpress Electroporation buffer (Harvard Bioscience, Inc; Holliston, MA), combined with pre-aliquoted ssODN (where applicable), Cas9 mRNA (3 pmol) and IVT sgRNA (120 pmol), and pulsed once at 250 V for 5 ms in the BTX ECM 830 Square Wave Electroporator (Harvard Apparatus; Holliston, MA).

    Techniques: Over Expression, Transduction, Electroporation, Plasmid Preparation, Expressing, Western Blot, Digital PCR

    (A) Experimental overview of CD34 + HSPC transduction and electroporation of DNA RFs and editing reagents. DNA RFs were co-expressed with the gene editing reagents. Cas9 nuclease was delivered either as RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA) or IVT Cas9 mRNA (5 μg) + IVT sgRNA (5 μg); donor template was delivered either as AAV6 (MOI 2e4) or ssODN (3 μg). HDR and NHEJ levels were measured by HTS four days post-electroporation. HDR/NHEJ ratio was calculated. (B) DNA repair factors delivered as LV 24 h prior to electroporation of editing reagents. n = 4 biological replicates for all CtIPDE+PALB2KR+i53 LV experiments (Cas9 RNP or Cas9mRNA); n = 8–12 for RAD52+dn53BP1 LV experiments using Cas9 RNP, or n = 4 for RAD52+dn53BP1 LV experiments using Cas9 mRNA. Error bars, mean ± SD. Differences are not significant if not specified, based on Wilcoxon rank sum test. (C) DNA RFs were co-delivered as IVT mRNA with editing reagents. n = 2–6 biological replicates for all conditions. Data are normalized to “no RFs” conditions for each set of experiments. Error bars, mean ± SD. Differences are not significant if not specified, based on Wilcoxon rank sum test.

    Journal: Frontiers in Genome Editing

    Article Title: Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

    doi: 10.3389/fgeed.2020.601541

    Figure Lengend Snippet: (A) Experimental overview of CD34 + HSPC transduction and electroporation of DNA RFs and editing reagents. DNA RFs were co-expressed with the gene editing reagents. Cas9 nuclease was delivered either as RNP (100 pmol Cas9 protein + 4.5 μg of IVT sgRNA) or IVT Cas9 mRNA (5 μg) + IVT sgRNA (5 μg); donor template was delivered either as AAV6 (MOI 2e4) or ssODN (3 μg). HDR and NHEJ levels were measured by HTS four days post-electroporation. HDR/NHEJ ratio was calculated. (B) DNA repair factors delivered as LV 24 h prior to electroporation of editing reagents. n = 4 biological replicates for all CtIPDE+PALB2KR+i53 LV experiments (Cas9 RNP or Cas9mRNA); n = 8–12 for RAD52+dn53BP1 LV experiments using Cas9 RNP, or n = 4 for RAD52+dn53BP1 LV experiments using Cas9 mRNA. Error bars, mean ± SD. Differences are not significant if not specified, based on Wilcoxon rank sum test. (C) DNA RFs were co-delivered as IVT mRNA with editing reagents. n = 2–6 biological replicates for all conditions. Data are normalized to “no RFs” conditions for each set of experiments. Error bars, mean ± SD. Differences are not significant if not specified, based on Wilcoxon rank sum test.

    Article Snippet: For electroporation, 2 × 10 5 cells per condition were pelleted at 90×g for 15 min at RT, resuspended in 100 μl of BTXpress Electroporation buffer (Harvard Bioscience, Inc; Holliston, MA), combined with pre-aliquoted ssODN (where applicable), Cas9 mRNA (3 pmol) and IVT sgRNA (120 pmol), and pulsed once at 250 V for 5 ms in the BTX ECM 830 Square Wave Electroporator (Harvard Apparatus; Holliston, MA).

    Techniques: Transduction, Electroporation

    CtIP DE +PALB2 KR + i53 overexpression in G1 phase of the cell cycle does not activate HDR. (A–D) Gene editing levels in sorted populations. Hoescht stain was used to discriminate G0/G1 and S/G2/M phases. Cells were pre-transduced with the indicated DNA RFs or GFP LVs, sorted into cell cycle phases and then electroporated with Cas9 RNP and ssODN or transduced with an AAV6 donor template immediately after electroporation. HDR and NHEJ levels were measured by HTS. (A) HDR, (B) NHEJ, (C) HDR/NHEJ, (D) MMEJ/indels, in unsorted, G0/G1-sorted, or S/G2/M-sorted populations. n = 2 biological replicates. Error bars, mean ± SD.

    Journal: Frontiers in Genome Editing

    Article Title: Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

    doi: 10.3389/fgeed.2020.601541

    Figure Lengend Snippet: CtIP DE +PALB2 KR + i53 overexpression in G1 phase of the cell cycle does not activate HDR. (A–D) Gene editing levels in sorted populations. Hoescht stain was used to discriminate G0/G1 and S/G2/M phases. Cells were pre-transduced with the indicated DNA RFs or GFP LVs, sorted into cell cycle phases and then electroporated with Cas9 RNP and ssODN or transduced with an AAV6 donor template immediately after electroporation. HDR and NHEJ levels were measured by HTS. (A) HDR, (B) NHEJ, (C) HDR/NHEJ, (D) MMEJ/indels, in unsorted, G0/G1-sorted, or S/G2/M-sorted populations. n = 2 biological replicates. Error bars, mean ± SD.

    Article Snippet: For electroporation, 2 × 10 5 cells per condition were pelleted at 90×g for 15 min at RT, resuspended in 100 μl of BTXpress Electroporation buffer (Harvard Bioscience, Inc; Holliston, MA), combined with pre-aliquoted ssODN (where applicable), Cas9 mRNA (3 pmol) and IVT sgRNA (120 pmol), and pulsed once at 250 V for 5 ms in the BTX ECM 830 Square Wave Electroporator (Harvard Apparatus; Holliston, MA).

    Techniques: Over Expression, Staining, Transduction, Electroporation

    Preliminary assessment of Cas9 variants to modulate local gene editing outcomes in a K562 BFP reporter cell line. (A) Experimental overview of electroporation of Cas9 variants as plasmid into a K562 BFP reporter cell line. K562 BFP cells were electroporated with 1 μg Cas9 variant plasmid, 1 μg of sgRNA plasmid targeting the BFP gene, and a ssODN donor (3 μM). Cells were cultured for 4 days post-electroporation prior to analysis by flow cytometry. (B–G) Proportion of GFP + , BFP + , or NF cells and GFP/NF ratio of cells edited with Cas9-hCtIP variants, n = 2–6 biological replicates (B,C) , and Cas9-UL12 variants, n = 3–8 biological replicates (D,E) , and Cas9-dn53BP1, n = 2–6 biological replicates (F,G) . Error bars, mean ± SD. Differences are not significant if not specified, * p < 0.05, based on Wilcoxon rank sum test.

    Journal: Frontiers in Genome Editing

    Article Title: Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

    doi: 10.3389/fgeed.2020.601541

    Figure Lengend Snippet: Preliminary assessment of Cas9 variants to modulate local gene editing outcomes in a K562 BFP reporter cell line. (A) Experimental overview of electroporation of Cas9 variants as plasmid into a K562 BFP reporter cell line. K562 BFP cells were electroporated with 1 μg Cas9 variant plasmid, 1 μg of sgRNA plasmid targeting the BFP gene, and a ssODN donor (3 μM). Cells were cultured for 4 days post-electroporation prior to analysis by flow cytometry. (B–G) Proportion of GFP + , BFP + , or NF cells and GFP/NF ratio of cells edited with Cas9-hCtIP variants, n = 2–6 biological replicates (B,C) , and Cas9-UL12 variants, n = 3–8 biological replicates (D,E) , and Cas9-dn53BP1, n = 2–6 biological replicates (F,G) . Error bars, mean ± SD. Differences are not significant if not specified, * p < 0.05, based on Wilcoxon rank sum test.

    Article Snippet: For electroporation, 2 × 10 5 cells per condition were pelleted at 90×g for 15 min at RT, resuspended in 100 μl of BTXpress Electroporation buffer (Harvard Bioscience, Inc; Holliston, MA), combined with pre-aliquoted ssODN (where applicable), Cas9 mRNA (3 pmol) and IVT sgRNA (120 pmol), and pulsed once at 250 V for 5 ms in the BTX ECM 830 Square Wave Electroporator (Harvard Apparatus; Holliston, MA).

    Techniques: Electroporation, Plasmid Preparation, Variant Assay, Cell Culture, Flow Cytometry

    Experimental outline of Cas9 variant gene editing in HSPCs. (A) Experimental overview of electroporation of Cas9 variants delivered as IVT mRNA to CD34 + HSPCs. Cas9 variants were electroporated at equimolar amounts (3 pmol) with 120 pmol of IVT sgRNA targeting the SCD mutation at the HBB locus, and either a ssODN (3 μM) or AAV6 donor (MOI 2e4). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. (B) Viability of CD34 + HSPCs edited with Cas9 variants, and an ssODN or AAV6 donor 24 h post-electroporation. n = 2–6 biological replicates. Center line represents mean. Differences are not significant if not specified, based on Wilcoxon rank sum test.

    Journal: Frontiers in Genome Editing

    Article Title: Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

    doi: 10.3389/fgeed.2020.601541

    Figure Lengend Snippet: Experimental outline of Cas9 variant gene editing in HSPCs. (A) Experimental overview of electroporation of Cas9 variants delivered as IVT mRNA to CD34 + HSPCs. Cas9 variants were electroporated at equimolar amounts (3 pmol) with 120 pmol of IVT sgRNA targeting the SCD mutation at the HBB locus, and either a ssODN (3 μM) or AAV6 donor (MOI 2e4). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. (B) Viability of CD34 + HSPCs edited with Cas9 variants, and an ssODN or AAV6 donor 24 h post-electroporation. n = 2–6 biological replicates. Center line represents mean. Differences are not significant if not specified, based on Wilcoxon rank sum test.

    Article Snippet: For electroporation, 2 × 10 5 cells per condition were pelleted at 90×g for 15 min at RT, resuspended in 100 μl of BTXpress Electroporation buffer (Harvard Bioscience, Inc; Holliston, MA), combined with pre-aliquoted ssODN (where applicable), Cas9 mRNA (3 pmol) and IVT sgRNA (120 pmol), and pulsed once at 250 V for 5 ms in the BTX ECM 830 Square Wave Electroporator (Harvard Apparatus; Holliston, MA).

    Techniques: Variant Assay, Electroporation, Mutagenesis

    Cas9 variant gene editing of the β-globin locus with an AAV6 donor reveals distinctive DNA repair outcomes in HSPCs. CD34 + HSPCs edited with Cas9 variants (3 pmol Cas9 mRNA + 120 pmol IVT sgRNA) and an AAV6 donor (MOI 2e4). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. Cas9 and Cas9-hGem were used as controls. (A–C) Gene editing outcomes with Cas9-hCtIP variants. (A) HDR and NHEJ. n = 4 biological replicates). Error bars, mean ± SD. (B) HDR/NHEJ. n = 4 biological replicates Center line represents mean (C) MMEJ/indels. n = 2 biological replicates. Center line represents mean. (D–F) Gene editing outcomes with Cas9-UL12 variants. (D) HDR and NHEJ. n = 2–6 biological replicates. Error bars, mean ± SD. (E) HDR/NHEJ. n = 2–6 biological replicates Center line represents mean (F) MMEJ/indels. n = 2–4 biological replicates. Center line represents mean. (G–I) Gene editing outcomes with Cas9-dn53BP1 variants. (G) HDR and NHEJ. n = 2–4 biological replicates. Error bars, mean ± SD. (H) HDR/NHEJ, n = 2–4 biological replicates. Center line represents mean (I) MMEJ/indels. n = 2–4 biological replicates. Center line represents mean. Differences are not significant if not specified, * p < 0.05, based on Wilcoxon rank sum test.

    Journal: Frontiers in Genome Editing

    Article Title: Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

    doi: 10.3389/fgeed.2020.601541

    Figure Lengend Snippet: Cas9 variant gene editing of the β-globin locus with an AAV6 donor reveals distinctive DNA repair outcomes in HSPCs. CD34 + HSPCs edited with Cas9 variants (3 pmol Cas9 mRNA + 120 pmol IVT sgRNA) and an AAV6 donor (MOI 2e4). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. Cas9 and Cas9-hGem were used as controls. (A–C) Gene editing outcomes with Cas9-hCtIP variants. (A) HDR and NHEJ. n = 4 biological replicates). Error bars, mean ± SD. (B) HDR/NHEJ. n = 4 biological replicates Center line represents mean (C) MMEJ/indels. n = 2 biological replicates. Center line represents mean. (D–F) Gene editing outcomes with Cas9-UL12 variants. (D) HDR and NHEJ. n = 2–6 biological replicates. Error bars, mean ± SD. (E) HDR/NHEJ. n = 2–6 biological replicates Center line represents mean (F) MMEJ/indels. n = 2–4 biological replicates. Center line represents mean. (G–I) Gene editing outcomes with Cas9-dn53BP1 variants. (G) HDR and NHEJ. n = 2–4 biological replicates. Error bars, mean ± SD. (H) HDR/NHEJ, n = 2–4 biological replicates. Center line represents mean (I) MMEJ/indels. n = 2–4 biological replicates. Center line represents mean. Differences are not significant if not specified, * p < 0.05, based on Wilcoxon rank sum test.

    Article Snippet: For electroporation, 2 × 10 5 cells per condition were pelleted at 90×g for 15 min at RT, resuspended in 100 μl of BTXpress Electroporation buffer (Harvard Bioscience, Inc; Holliston, MA), combined with pre-aliquoted ssODN (where applicable), Cas9 mRNA (3 pmol) and IVT sgRNA (120 pmol), and pulsed once at 250 V for 5 ms in the BTX ECM 830 Square Wave Electroporator (Harvard Apparatus; Holliston, MA).

    Techniques: Variant Assay, Electroporation

    Cas9 variant gene editing of the HBB locus with a ssODN donor. CD34 + HSPCs edited with Cas9 variants (3 pmol Cas9 mRNA + 120 pmol IVT sgRNA) and ssODN donor (3 μM). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. Cas9 and Cas9-hGem were used as controls. (A) HDR and NHEJ. (B) HDR/NHEJ n = 2 biological replicates. Error bars, mean ± SD. (C) MMEJ/indels. n = 2 biological replicates. Center line represents mean.

    Journal: Frontiers in Genome Editing

    Article Title: Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells

    doi: 10.3389/fgeed.2020.601541

    Figure Lengend Snippet: Cas9 variant gene editing of the HBB locus with a ssODN donor. CD34 + HSPCs edited with Cas9 variants (3 pmol Cas9 mRNA + 120 pmol IVT sgRNA) and ssODN donor (3 μM). Editing outcomes were measured by MiSeq HTS 4 days post-electroporation. Cas9 and Cas9-hGem were used as controls. (A) HDR and NHEJ. (B) HDR/NHEJ n = 2 biological replicates. Error bars, mean ± SD. (C) MMEJ/indels. n = 2 biological replicates. Center line represents mean.

    Article Snippet: For electroporation, 2 × 10 5 cells per condition were pelleted at 90×g for 15 min at RT, resuspended in 100 μl of BTXpress Electroporation buffer (Harvard Bioscience, Inc; Holliston, MA), combined with pre-aliquoted ssODN (where applicable), Cas9 mRNA (3 pmol) and IVT sgRNA (120 pmol), and pulsed once at 250 V for 5 ms in the BTX ECM 830 Square Wave Electroporator (Harvard Apparatus; Holliston, MA).

    Techniques: Variant Assay, Electroporation