<i>CCR5</i> gene disruption by RNA-guided Cas9 endonuclease in transduced TZM.bl cells.

Abstract

<p>A. Schematic diagrams of lentiviral transfer vectors containing Cas9 endonuclease or sgRNAs CR1, CR2, Cr3 and GF1. HA: HA epitope tag; NLS: nuclear localization signal; PGK: promoter sequence derived from phosphoglycerate kinase-1; U6: promoter sequence derived from polymerase III U6. B. Cell surface expression of CCR5 on TZM.bl cells co-transduced with lentiviral vectors expressing Cas9-HA-NLS and sgRNAs CR1, CR2, CR3 or GF1. Transduced (open curves) and mock-transduced (shaded curves) cells were stained with anti-CCR5 antibody followed by FACS analysis. C. FSC and SSC analysis mock-transduced TZM.bl cells and TZM.bl cells transduced with lentiviral vectors expressing Cas9-HA-NLS fusion protein and sgRNAs CR1, CR2, CR3 or GF1, respectively. Percentages of gated (live) cells are shown at the lower and left corner of the figures. D. <i>CCR5</i> gene disruption analysis by T7EI assay using prime pairs CR1/F990-CR1/R1750 and CR2/3F593-CR2/3R1254 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115987#pone.0115987.s003" target="_blank">S1 Table</a>). E. <i>CCR5</i> gene disruption analysis by T7EI assay using prime pair CR1/2/3F2559-CR1/2/3R3893 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115987#pone.0115987.s003" target="_blank">S1 Table</a>).</p