1. Molecular Therapy - Nucleic Acids
CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus 
HyunJun Kang, Petra Minder, Mi Ae Park, Walatta-Tseyon Mesquitta, Bruce E Torbett, Igor I Slukvin 
Molecular Therapy - Nucleic Acids 
Volume 4, (January 2015)
DOI: /mtna
Copyright © 2015 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Generation of CCR5-mut iPSC cell lines using single gRNA. (a) Schematic diagram of the target site of the gRNA1 in CCR5 gene. Protospacer-adjacent motif sequence is written in bold, and the target sequence of gRNA1 is underlined. (b) Detection of gRNA1/Cas9-promoted CCR5 gene alterations. Surveyor nuclease assay was used to detect mutations caused by gRNA1/Cas9 system. The exact sequence of the CCR5 mutation was determined by direct sequencing of multiple individual bacterial colonies transformed with T vector with subcloned PCR product from the indicated iPSC lines. (c) Editing efficiency of single gRNA/Cas9. (d) Flow cytometric analysis of CCR5-mut iPSCs established following single cell sorting. Plots depict isotype control (open) and specific antibody (gray) histograms. (e) Teratoma assay shows derivatives of all three germ layers. C, cartilage; ML, melanocytes, NE, rosettes of neural epithelium; PA, pancreatic acininar cells. (f) Karyotype of CCR5-mut iPSCs.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Generation of CCR5-mut iPSC cell lines using two gRNAs. (a) Schematic diagram of the CCR5 genomic target sites and genetic distances of the combinatorial gRNAs; combination 1 (Comb1) is gRNA1 and gRNA2, and Comb2 is gRNA1 and gRNA3. (b) Detection of large deletion caused by dual gRNAs/Cas9 cleavage. Genomic DNA-PCR analysis shows unaffected (1,265 bp), monoallelic (1,265 and 736 bp), and biallelic deletion (736 bp) of genomic alterations in clones isolated after transfection with Comb2-dual gRNAs/Cas9 plasmids. Representative eight clones out of 120 clones obtained from dual gRNAs targeting are shown. (c) Editing efficiency of dual gRNA/Cas9. (d) Flow cytometric analysis of CCR5-mut iPSCs established following single cell isolation. Plots depict isotype control (open) and specific antibody (gray) histograms. (e) Teratoma assay shows derivatives of all three germ layers. C, cartilage; ML, melanocytes; NE, rosettes of neural epithelium; RE, respiratory epithelium. (f) Karyotype of CCR5-mut iPSCs.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Surveyor nuclease assay for potential off-target mutation in selected CCR5-mut iPSC clones. Several genomic off-target sites were predicted by Cas-OFFinder, online software; the Surveyor nuclease assay shows no off-target genomic alterations during the gRNA/Cas9-mediated CCR5 editing. (a) Two predicted OTSs for gRNA1. (b) Four predicted OTSs for gRNA2. (c) One predicted OTSs for gRNA3. Con+: amplified DNA fragment mixture with only single base substitution provided by Surveyor Mutation Detection Kit (Transgenomics), Con-: amplified DNA product from parental GFP-iPSC cell line.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Hematopoietic differentiation and generation of macrophages from CCR5mut iPSCs. (a) Multipotent hematopoietic progenitors generated from parental and CCR5mut-hiPSCs. No significant differences were observed in the hematopoietic differentiation efficiency of parental and CCR5mut-hiPSCs. SC7, GFP-CCR5mut-SC7-; SC8, GFP-CCR5mut-SC8-; Comb1-SC8, GFP-CCR5mut Comb1-SC8-; Comb2-SC1, GFP-CCR5mut-Comb2-SC1-derived CD43+ hematopoietic progenitors. (b) Colony-forming potential of hematopoietic progenitors generated from parental and CCR5mut-hiPSCs. (c) Morphology of macrophages generated from parental and CCR5mut-hiPSCs. (d) Flow cytometric analysis of macrophage-specific markers and GFP expression in macrophages generated from parental and CCR5mut-hiPSCs. From the top row, parental-, GFP-CCR5mut-SC7-, GFP-CCR5mut-SC8-, GFP-CCR5mut-Comb1-SC8-, GFP-CCR5mut-Comb2-SC1-derived macrophages. Plots depict isotype control (open) and specific antibody (gray) histograms.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
HIV challenge of iPSC derived CCR5mut macrophages. Macrophages derived from the parental and CCR5mut iPSC-derived macrophages were challenged with (a) A R5-tropic R8-Bal (three independent experiments), (b) a R5-tropic SF162 (two independent experiments), (c) A X4-tropic LAI (two independent experiments), or (d) A X4-tropic NL4-3 (two independent experiments) HIV-1. On the designated days post-infection, supernatants were collected and analyzed for p24 antigen by an enzyme-linked immunosorbent assay.
Molecular Therapy - Nucleic Acids 2015 4, DOI: ( /mtna )
Copyright © 2015 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions