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. 2017 Oct 24;6:e29702. doi: 10.7554/eLife.29702

Figure 2. CD95 and CD95L derived si/shRNAs kill cells in the absence of the targeted sites in CD95 or CD95L.

(A) Schematic of the genomic locations and sequences of the gRNAs used to excise the siL3 (Δ64bp) and shL3 (Δ41bp) target sites from CD95L. PAM site is underlined. Green indicates a gRNA targeting the sense strand. Blue indicates a gRNA targeting the antisense strand. (B) Schematic showing the genomic locations and sequences of the gRNAs used to excise the shR6 (Δ227bp) target site. Mix, pool of three 293T clones with the homozygous shL3 deletion. (C) PCR with flanking (top panels) and internal (bottom panels) primers used to confirm the Δ41 deletion in the shL3 site in one of the three homozygous deletion 293T clones generated. Cells transfected with Cas9 only (Cas9) are wild-type. (D) Quantitative PCR for endogenous CD95L with a primer downstream of the Δ41 shL3 deletion and another primer internal to the deleted region. nd, not detectable. Each bar represents mean ±SD of three replicates. (E) PCR with flanking (top row) and internal (bottom row) primers used to confirm the presence of the shL3 Δ41 (top panel), siL3 Δ64 (middle panel), and shR6 Δ227 (bottom panel) deletions in HeyA8 clones. Mix, HeyA8 cells after transfection with Cas9 and gRNAs but before single cell cloning. (F) Quantitative PCR for CD95 in HeyA8 cells transfected with Cas9 plasmid (Cas9) alone, or the HeyA8 ΔshR6 clone #11. RNA was extracted 5 days after infection with pLKO-shScr, pLKO-shR6, pLKO-shR2, or pLKO-shR6’ (targeting the 3'UTR). Each bar represents mean ±SD of three replicates. (G) Percent cell confluence over time of 293T cells (left) and a pool of three 293T clones with a homozygous deletion of the shL3 target site (right) infected with pTIP-shScr or pTIP-shL3 and treatment with or without Dox. Data are representative of two independent experiments. Each data point represents mean ±SE of six replicates. (H) Left: Percent confluence over time of HeyA8 cells infected with pLKO-shScr, pLKO-shR6, or pLKO-shL3. Center: Percent confluence over time of a HeyA8 clone with a homozygous deletion of the shR6 target site infected with either pLKO-shScr or pLKO-shR6. Right: Percent confluence over time of a pool of three HeyA8 clones with a homozygous deletion of the shL3 site infected with either pLKO-shScr or pLKO-shL3. Data are representative of two independent experiments. Each data point represents mean ±SE of three replicates. (I) Percent confluence over time of a pool of three HeyA8 clones harboring a homozygous deletion of the siRNA siL3 target site after transfection with different concentrations of siScr or siL3. Data are representative of three independent experiments. Each data point represents mean ±SE of three replicates.

Figure 2.

Figure 2—figure supplement 1. Knockout of CD95 in HeyA8 cells.

Figure 2—figure supplement 1.

(A) PCR showing a Δ227 shR6 deletion and insertions in HeyA8 clones #1 and #2. (B) Schematic of the Δ227 deletion in allele #1 and partial insertion of a pSC-B plasmid fragment in allele #2 in HeyA8 clone #2 based on Sanger sequencing of isolated bands from PCR shown in A. Note, cl#1 and #2 have the expected Δ227 shR6 deletion in one allele and an insertion in the other. cl#11 has a homozygous Δ227 shR6 deletion. The deleted region is shown in green containing the shR6 target site in red. pSC-B vector sequences are shown in blue letters, and the insertion is shown in orange. (C) Western blot for CD95 and β-actin in Cas9-control transfected HeyA8 cells and HeyA8 shR6 k.o. clones #1, #2, and #11. Shown is one of two repeats of this analysis. (D) Surface staining for CD95 in parental HeyA8 cells and HeyA8 shR6 knockout clones #1, #2, and #11. Shown is one of two repeats of this analysis. (E) Images showing apoptosis induction with LzCD95L treatment (4.5 hr) in parental HeyA8 cells but not in clone #2.