Journal: Nucleic Acids Research

Article Title: Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9

doi: 10.1093/nar/gkae1069

Figure Lengend Snippet: esDNA templates (800 nM) improve mutational KI in HSPCs, T-cells and HUVECs but not iPSCs. HDR in CD34 + cells significantly increased with esDNA compared to end-modified ssDNA in the ( A ) HBB locus in the absence of AZD-7648 and ( B ) in the presence of AZD-7648. ( C ) The viability of CD34 + cells was not significantly different between edited CD34 + cells and unedited controls. HDR in CD34 + cells was also increased in the ( D ) CCR5 locus and ( E ) CFTR locus. ( F ) In T-cells, esDNA increased HDR in the CFTR locus. ( G ) In HUVECs, esDNA increased HDR in the CFTR locus from 4 ± 3% obtained with end-modified ssDNA to 12 ± 5% but the difference was not significant. Mutational KI with esDNA was significantly lower in iPSCs in the CFTR locus than unmodified ssDNA both in the ( H ) absence (not significant) and ( I ) presence of AZD-7648. 3 or more biological replicates were tested for each cell type. Groups in A–B and D–G were compared using a paired T-test. Statistical comparisons for panels (C), (H) and (I) were made using one-way ANOVA followed by Tukey’s test. ** and * represent P < 0.01 and P < 0.05, respectively, for all panels.

Article Snippet: G-CSF Mobilized Human Peripheral Blood CD34 + Cells were purchased from StemCell Technologies (Catalog: 70060.1) and the Hematopoietic Cell Procurement and Resource Development Center at The Fred Hutchison Cancer Center.

Techniques: Modification

Journal: Nucleic Acids Research

Article Title: Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9

doi: 10.1093/nar/gkae1069

Figure Lengend Snippet: Knocking out TREX1 improved mutational KI without chemically modifying ssDNA. ( A ) Western blot shows TREX1 expression in ABCs and HUVECs but not iPSCs. ABCs with TREX1 KO show reduced TREX1 expression. ( B ) HDR using unmodified ssDNA templates (800 nM) was improved when TREX1 was knocked out in non-CF ABCs. Knocking out TREX1 did not improve HDR by esDNA templates ( n = 4 biological replicates). ( C ) Mutational KI with ssDNA using combinations of chemical modifications in ABCs. Both esDNA OM (40 ± 21%) and esDNA PS (34 ± 16%) showed improved mutational KI relative to unmodified (3 ± 3%), end-modified (11 ± 6%) and Alt-R HDR modified ssDNA (23 ± 9%). All other combinations did not improve on esDNA OM (40 ± 21%) and esDNA PS (34 (± 16%) ( n = 4–6 biological replicates). ( D ) Modifying the 3′ half of the template alone with PS improved mutational KI compared to end modification ( n = 3 biological replicates). Including PS chemical modifications throughout the template improved editing slightly but the improvement was not statistically significant compared to templates in which only the 3′ half was modified. ( E ) Templates with PS modified internal bases spread throughout the template resulted in more consistent HDR compared to template with PS modified bases in just the last 20 bases ( n = 4 biological replicates). ( F ) Mutational KI in the HBB locus was significantly higher in ABCs using esDNA-6-PS templates (53 ± 14%) compared with Alt-R HDR templates (13 ± 12%) ( n = 4 biological replicates). G ) Mutational KI in the HBB locus was also significantly higher in CD34 + cells using esDNA-6-PS templates (66 ± 2%) compared with Alt-R HDR templates (17 ± 1%) ( n = 4 biological replicates). ( H ) Modification of every 10th base with PS groups also improved mutational KI compared to unmodified or end-modified ssDNA. Mutational KI was reduced when every fourth base was modified. Statistical comparison was made using one-way ANOVA. Multiple comparisons were performed using Tukey’s test. ***, ** and * represent P < 0.005, P < 0.01, P < and P < 0.05 respectively in all panels. All experiments used 800 nM template.

Article Snippet: G-CSF Mobilized Human Peripheral Blood CD34 + Cells were purchased from StemCell Technologies (Catalog: 70060.1) and the Hematopoietic Cell Procurement and Resource Development Center at The Fred Hutchison Cancer Center.

Techniques: Western Blot, Expressing, Modification, Comparison

Journal: Nucleic Acids Research

Article Title: Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9

doi: 10.1093/nar/gkae1069

Figure Lengend Snippet: Editing using esDNA-6-PS elicits less p21 activation in CD34 + cells. ( A ) In CD34 + cells, mutational KI by esDNA-6-PS was less than the mutational KI achieved using AAV templates ( n = 3–4 biological replicates). ( B ) There was no significant difference in the viability of edited CD34 + cells when measured using the trypan blue assay on day 2 after editing ( n = 3 biological replicates). Statistical comparison was made using ANOVA followed by Tukey’s multiple comparisons test. ( C ) CD34 + cells edited using AAV templates showed a greater increase in p21 expression when measured by ddPCR. Treatment using AAV alone also resulted in increased p21 expression. Editing with esDNA-6-PS resulted in less p21 activation ( n = 3 biological replicates with 2 technical replicates). Statistical comparison was made using one-way ANOVA. Multiple comparisons were performed using Tukey’s test. ** and * represent P < 0.01 and P < 0.05, respectively in all panels.

Article Snippet: G-CSF Mobilized Human Peripheral Blood CD34 + Cells were purchased from StemCell Technologies (Catalog: 70060.1) and the Hematopoietic Cell Procurement and Resource Development Center at The Fred Hutchison Cancer Center.

Techniques: Activation Assay, Comparison, Expressing