An all-in-one UniSam vector system for efficient gene activation

AbstractWe have generated a drug-free, all-in-one dCAS9-SAM vector that can activate endogenous gene expression with the potential to modify cell fate. We demonstrate that this strategy can be used in a number of cell lines and avoids exceptionally high levels of gene expression that are observed in standard transgenic approaches. Compared to the multi-plasmid system, this all-in-one vector activates gene expression to a comparable level but the reduced overall DNA content results in significantly higher viability of transfected cells. This allowed us to use the RUNX1C-GFP human embryonic stem cell reporter cell line to monitor gene activation in individual cells and to show that activation could occur at all stages of the cell cycle.</jats:p

A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and in vivo tracking of therapeutic cells

We describe the production of a human induced pluripotent stem cell (iPSC) line, SFCi55-ZsGr, that has been engineered to express the fluorescent reporter gene, ZsGreen, in a constitutive manner. The CAG-driven ZsGreen expression cassette was inserted into the AAVS1 locus and a high level of expression was observed in undifferentiated iPSCs and in cell lineages derived from all three germ layers including haematopoietic cells, hepatocytes and neurons. We demonstrate efficient production of terminally differentiated macrophages from the SFCi55-ZsGreen iPSC line and show that they are indistinguishable from those generated from their parental SFCi55 iPSC line in terms of gene expression, cell surface marker expression and phagocytic activity. The high level of ZsGreen expression had no effect on the ability of macrophages to be activated to an M(LPS + IFNγ), M(IL10) or M(IL4) phenotype nor on their plasticity, assessed by their ability to switch from one phenotype to another. Thus, targeting of the AAVS1 locus in iPSCs allows for the production of fully functional, fluorescently tagged human macrophages that can be used for in vivo tracking in disease models. The strategy also provides a platform for the introduction of factors that are predicted to modulate and/or stabilize macrophage function. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’

Single cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs

Haematopoietic stem and progenitor cells (HSPCs) develop through distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) is able to recapitulate some of these processes, however, it has proven difficult to generate functional haematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we exploited single cell RNA sequencing (scRNAseq) in combination with single cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors as well as erythroid, megakaryocyte and leukocyte-committed progenitors and we identified CD44, CD326, ICAM2/CD9 and CD18 as markers of these progenitors, respectively. Using an artificial neural network (ANN), that we trained on a scRNAseq derived from human fetal liver, we were able to identify a wide range of hPSCs-derived HPSC phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded and was completely missing in the dataset that had been generated using cells selected on the basis of CD43expression. By comparing the single cell transcriptome of in vitro-generated HSC-like cells with those generated within the fetal liver we identified transcription factors and molecular pathways that can be exploited in the future to improve the in vitro production of HSCs

SplitAx:A novel method to assess the function of engineered nucleases

Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity. The assay involves splitting the N-terminal (amino acid 1-158) coding region of GFP and an out-of-frame of C-terminal region with a nuclease binding site sequence. Following exposure to the test nuclease, cutting and repair by error prone non-homologous end joining (NHEJ) restores the reading frame resulting in the production of a full length fluorescent GFP protein. Fluorescence can also be restored by complementation between the N-terminal and C-terminal coding sequences in trans. We demonstrate successful use of the SplitAx assay to assess the function of zinc finger nucleases, CRISPR hCAS9 and TALENS. We also test the activity of multiple gRNAs in CRISPR/hCas9/D10A systems. The zinc finger nucleases and guide RNAs that showed functional activity in the SplitAx assay were then used successfully to target the endogenous AAVS1, SOX6 and Cfms loci. This simple method can be applied to other unrelated proteins such as ZsGreen1 and provides a test system that does not require complex optimization

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

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 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

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