Functional validation of the ZsGreen1-Cfms-SplitAx reporter assay with Cfms gRNAs and hCAS9 or D10A nickase.

<p>Schematic diagram of the 5’ and 3’end of Zs Green1 separated by the <i>Cfms</i> binding site. The DNA sequence of the Cfms binding site is shown and the location of the gRNA_Cfms-8a, 8b and 9b are underlined (a). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with ZsGreen1-Cfms-SplitAx with hCAS9 (b), ZsGreen1-Cfms-SplitAx, hCAS9 with gRNA_Cfms-8a (c), ZsGreen1-Cfms-SplitAx, hCAS9 with gRNA_Cfms-8b (d), ZsGreen1-Cfms-SplitAx, hCAS9 with gRNA_Cfms-9b (e). Quantification of flow cytometry data for the ZsGreen1-Cfms- SplitAx and hCAS9 with the gRNAs_Cfms (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (f). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with ZsGreen1-Cfms-SplitAx only (g), ZsGreen1-Cfms-SplitAx with D10A nickase (h), ZsGreen1-Cfms-SplitAx, D10A nickase with gRNA_Cfms-8a and8b (i), ZsGreen1-Cfms-SplitAx, D10A nickase with gRNA_Cfms-8a and 8b (j). Graphical representation of flow cytometry data for the ZsGreen1-Cfms- SplitAx and D10A nickase with the gRNAs_Cfms (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (k).</p

Functional validation of the GFP-AAVS1 SplitAx reporter assay with zinc fingers and CRISPR/CAS9 system.

<p>Schematic of the GFP cDNA with the N-terminus and C-terminus separated by the <i>AAVS1</i> binding site. The DNA sequence of the <i>AAVS1</i> binding site is shown and the location of zinc finger left (ZF L), Zinc finger right (ZF R), <i>AAVS1</i> guide RNAs T1 and T2 underlined (a). The translated DNA sequence of the <i>AAVS1</i> binding site with stop codons (-) (b). The translated DNA after genome editing. In this case a 1 bp deletion removes the stop codons and allows in frame of translation of the C-terminal GFP resulting in fluorescence (c). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with GFP-AAVS1 SplitAx only (d), GFP-AAVS1 SplitAx with single AAVS1 Zinc Finger Left (Zn L) (e), GFP-AAVS1 SplitAx with AAVS1 single Zinc Finger Right (Zn R) (f), and GFP-AAVS1 SplitAx with both AAVS1 Zinc Finger Lefand/Zinc Finger Right (Zn L, Zn R) (g). Quantification of flow cytometry data for the GFP-AAVS1 SplitAx with the AAVS1 Zinc Fingers (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (h). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with GFP-AAVS1 SplitAx only (i) GFP-AAVS1 SplitAx and hCAS9 (j), GFP-AAVS1 SplitAx, hCAS9 CRISPR and gRNA_AAVS1-T1 (k), GFP-AAVS1 SplitAx, hCAS9 CRISPR and gRNA_AAVS1-T2 (l). Quantification of flow cytometry data for the GFP-AAVS1 SplitAx with the CRIPSR gRNA_AAVS1- T1 or T2 and hCAS9 (+), cells not transfected with a plasmid (-). Data shown as +STDev (n = 3) (m).</p

Schematic diagram illustrating the different mechanisms of how the SplitAX assay functions.

<p>The vector consisting of the pCAG promoter, the GFP cDNA (N-terminus 1-474bp), a genome editing binding site containing a stop codon which is out of frame with the GFP cDNA C-terminus (475-end). In the absence of exposure to a specific genome editing tool, the full length GFP protein is not expressed. Exposure of the GFP-SplitAx to a genome editing tool creates a double strand break. Repair by non-homologous end joining (NHEJ) mutates the binding site restoring the open reading frame (ORF) of GFP resulting in fluorescence. The second mechanism involves the repair of the double strand break by NHEJ resulting in an N-terminal ORF in frame with the C-terminal GFP. The C-terminal GFP can complement with the N-terminal GFP expressed from a different vector leading to restored fluorescent activity.</p

Functional validation of the ZsGreen1-AAVS1 SplitAx reporter assay with AAVS1 zinc fingers.

<p>Schematic of the ZsGreen1 cDNA with the N-terminus and C-terminus separated by the AAVS1 binding site. The DNA sequence of the AAVS1 binding site is shown and the location of zinc finger left (ZF L), Zinc finger right (ZF R) (a). Representative flow cytometry plots of 293FT cells 44–48 hours after transfection with ZsGreen1-AAVS1 SplitAx only (b), ZsGreen1-AAVS1 SplitAx with AAVS1 Zinc Finger Left (Zn L) (c), ZsGreen1-AAVS1 SplitAx with AAVS1 Zinc Finger Right (Zn R) (d), and ZsGreen1-AAVS1 SplitAx with AAVS1 Zinc Finger Left/Zinc Finger Right (Zn L, Zn R) (e). Graphical representation of flow cytometry data for the ZsGreen1-AAVS1 SplitAx with the AAVS1 Zinc Fingers (+), cells not transfected with a plasmid (-). Data shown as mean +/- SD (n = 3) (f).</p

Supplementary Figures and Tables from A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and <i>in vivo</i> tracking of therapeutic cells

Figure S1: Production of SFCi55-ZsG iPSC line; Figure S2 and S3: Flow cytometry analysis of mature SFCi55 and SFCi55-ZsG- derived macrophages macrophages; Figure S4: Representative Images of phagocytosis assay; Supplementary Table 1: Primer sequences used in study; Supplementary Table 2: Antibodies used in study

Supplementary Figures and Tables from A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and <i>in vivo</i> tracking of therapeutic cells

Figure S1: Production of SFCi55-ZsG iPSC line; Figure S2 and S3: Flow cytometry analysis of mature SFCi55 and SFCi55-ZsG- derived macrophages macrophages; Figure S4: Representative Images of phagocytosis assay; Supplementary Table 1: Primer sequences used in study; Supplementary Table 2: Antibodies used in study