Figure 1
From: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection
Guide RNA adjacent to the coding sequence D366 shows efficient disruption of the PSIP1 gene. (a) Schematic representation of LEDGF/p75 protein with indication of the epitope sites of respective antibodies used in Western analysis. Below the human PSIP1 locus on chromosome 9 is depicted showing the different exons as light grey boxes. IBD is underlined in green. (b) Schematic of representing the location of the different gRNA that were used (red lines), gRNA1 close to D366 and two additional supporting gRNAs (gRNA_A, gRNA_B). D366 is shown in yellow. The expected PCR fragment sizes are indicated as well as the predicted deletions for the different gRNA combinations. Below the targeted gDNA sequence is shown. D366 is boxed in green, the PAM site is shown in red and the landing site of gRNA1 is shown in blue. (c) Agarose gel analysis showing truncated amplicons generated by DNA cleavage guided by a pair of gRNAs. Genomic DNA was extracted from polyclonal cell populations and PCR amplified using Fwd and Rv primers indicated in panel (b). The WT amplicon is indicated by the large arrow head. The lower migrating bands (small arrow head) indicate segmental deletion. (d) Western blot analysis showing LEDGF protein in a polyclonal HEK293T population transfected with the indicated gRNA pairs. Wild-type 293T cells (WT) are shown as control. (e) Immunocytochemical staining of endogenous LEDGF showing nuclear localization in WT and CRISPRed polyclonal HEK239T cells. Phalloidin-stained F-actin in white is shown as a counterstain. The respective antibodies used are indicated above. Scale Bar: 10 μm.
