Targeted, homology-driven gene insertion in stem cells by ZFN-loaded ‘all-in-one’ lentiviral vectors
- Aarhus University, Denmark
- Technical University of Denmark, Denmark
- Beijing Genomics Institute, China
Figures
Figure 1 with 1 supplement
Proof-of-efficacy studies and characterization of ZFN-loaded IDLVs carrying a transgene flanked by homology arms facilitating insertion by homologous recombination.
(A) Schematic illustration of the production, transduction and integration of IDLVs carrying ZFNs and transgene. Enrichment of ZFN-fused viral polypeptides and donor-carried viral RNAs at the cell …
Figure 1—figure supplement 1
Characterization of protein delivery by ZFN-loaded IDLVs.
(A– C) Immunostaining and confocal microscopy of producer cells and virions. Green, eGFP or HA-tagged ZFN; blue, nuclei; red, p24; scale bar represents 5 μm. (A) HEK293T cells co-transfected with …
Figure 2 with 1 supplement
Targeted gene integration mediated by IDLV-ZFN(AAVS1)/egfp in CD34+ cells.
(A) Schematic illustration of protocols used for IDLV-ZFN(AAVS1)/egfp transduction in CD34+ cells. (B) Flow cytometry analysis showing the percentage of eGFP+ cells after transduction using protocol …
Figure 2—figure supplement 1
Allelic disruption and gene insertion after lentiviral ZFN delivery in human CD34+ cells.
(A) Allelic disruption at the AAVS1 locus in CD34+ cells by LP-ZFN(AAVS1) transduction at a dose of 500 ng p24 per 105 cells.LP entry was facilitated by RetroNectin. (B) Flow cytometry analysis of …
Figure 3 with 1 supplement
Targeted gene integration in iPSCs by IDLV-ZFN(AAVS1)/puro and IDLV-ZFN(CCR5)/puro.
(A) Gene disruption mediated by transduction of LP-ZFN(AAVS1) and LP-ZFN(CCR5) in iPSCs. (B) Puromycin-resistant iPSC clones appearing after transduction with IDLV-ZFN(AAVS1)/puro. Clones were …
Figure 3—figure supplement 1
Deep sequencing analysis of cleavage frequencies at the on-target and top ranking off-target loci of AAVS1 (A) and CCR5 (B) in pools of iPSC clones.
Error bars indicate 95% Wilson score intervals. For this analysis, HiSeq reads were trimmed with Sickle, merged with FLASH, and mapped to the amplicons using bowtie2 (described in Material and …
Additional files
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Supplementary file 1
(A) Sanger sequencing of donor-genome junctions and the AAVS1 locus. The PCR products of 5’ junction and 3’ junction sites as well as the AAVS1 locus from 8 analyzed iPSC clones were Sanger sequenced. The black letters represent genomic DNA at the AAVS1 locus, whereas blue letters represent donor-derived sequences. WT indicates wild-type sequence; indel (+1bp) indicates 1-bp insertion; N.A., not available. (B) Sanger sequencing of PROGNOS-predicted off-target site 1, 2 and 3. The blue sequence indicates the predicted binding site of the right ZFN; the green sequence indicates predicted binding site of the left ZFN; letters highlighted in red indicate mismatches.
- https://doi.org/10.7554/eLife.12213.008
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Supplementary file 2
(A) Primers used for molecular cloning and PCR amplification of junction sites. (B) Primers used to amplify PROGNOS-predicted off-target sites.
- https://doi.org/10.7554/eLife.12213.009
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Supplementary file 3
Donor sequences.
Homology arms and transgene expression cassettes in pLV/AAVS1-donor-egfp-PGK, pLV/AAVS1-donor-fluc-PGK, pLV/AAVS1-donor-puro-PGK and pLV/CCR5-donor-puro-PGK are provided. For each transgene expression cassette, sequences are annotated as listed below the sequence and indicated with different colors.
- https://doi.org/10.7554/eLife.12213.010
