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. 2017 Sep 21;9:89–99. doi: 10.1016/j.omtn.2017.08.016

Figure 4.

Figure 4

In Vivo Gene Editing following FACS Analysis

(A–E) Following subretinal injection of LV/Cas9-sgRNA1 (n = 13), LV/Cas9-sgRNA2 (n = 5), LV/Cas9-sgRNA3 (n = 5), or LV/Cas9-sgRNA-Irr (n = 10) in C57BL/6J mice, animals were euthanized at day 35 PI, and the retinas were dissected. (A) FACS analysis of the resultant RPE cells. The RPE cells from all mice in each group were pooled before FACS analysis. Representative results from the FACS analysis (LV/Cas9-sgRNA3). No cells were excluded because of size (forward-scattered light [FSC]) or granularity (side-scattered light [SSC]). (B) Illustration of eGFP versus SSC FACS analysis (LV/Cas9-sgRNA3), including thresholds for eGFP+ and eGFP− cells. Because of these criteria, cells were marked as eGFP+ or eGFP−. (C) Numbers of FACS-mediated isolation of eGFP+ RPE cells from the four groups resulted in LV/Cas9-sgRNA-Irr 10,285 cells (n = 10), LV/Cas9-Cas9-sgRNA1 11,554 cells (n = 10), LV/Cas9-sgRNA2 1,936 cells (n = 5), and LV/Cas9-sgRNA3 10,842 cells (n = 5). (D) Sequencing chromatograms of PCR amplicons following administration of LV/Cas9-sgRNA1, LV/Cas9-sgRNA2, or LV/Cas9-sgRNA3. Chromatograms were assessed for indels by TIDE in comparison to LV/Cas9-sgRNA-Irr. (E) Sequencing analysis of TOPO-cloned PCR amplicons obtained from FACS-isolated eGFP+ RPE cells. PCR amplicons following delivery of LV/Cas9-sgRNA1, LV/Cas9-sgRNA2, or LV/Cas9-sgRNA3 were TOPO cloned into the pCR-Blunt-II-TOPO vector. Purified DNA samples were amplified by means of PCR and Vegfa exon 3-specific amplicons sequenced. The resultant sequences were designated with the number of sequences (1× by default), altered length, and overall indel formation frequency. The wild-type is shown in red; insertions and deletions are indicated in bold and cyan. Top: schematic overview of the three target sites in Vegfa exon 3. Blue arrows shown on top of the sequence represent the different Cas9-sgRNAs. PAM sequences are underlined and indicated beneath the sequence. PAM, protospacer-adjacent motif; FSC, forward-scattered light; SSC, side-scattered light.