Optimization of S. aureus dCas9 and CRISPRi Elements for a Single Adeno-Associated Virus that Targets an Endogenous Gene

Figure 1. Analysis of SadCas9 (A) Effect of carboxyl-terminal truncations on nuclear targeting of SadCas9. All constructs had the EA linker and the myc-NLS at the extreme carboxyl-terminus (Table 1). The sequence begins at Arg1015 (“O”) and locations of β-structure are marked above the protein sequence (adapted from Nishimasu et al.8). Asterisks (∗∗) denote a significant difference (p < 0.01) between WT and Δ1,036 SadCas9. (B) Blot of extracts from ARPE-19 cell lines expressing WT or Δ1033 SadCas9 grown in the absence or presence of 5 μg Dox/mL. (C) ARPE-19 polyclonal cell line expressing WT SadCas9 (5 μg Dox/mL) displayed punctate labeling (green) within nuclei (blue).

Figure 2. Analysis of SadCas9 Constructs with the Transcriptional Repression KRAB Domain (A) The FLAG-tag was used to examine accessibility of the amino-terminal “domain” of SadCas9. Immunoreactive FLAG was barely detected with a single threonine residue between FLAG and SadCas9 (upper left panel) but was greatly enhanced with the 7-amino acid GS linker (lower left panel) and co-localized with imunoreactive SadCas9 (lower right panel). Therefore, the GS linker was used for the KRAB-SadCas9 (N-KRAB) construct. (B) Nuclear localization of N-KRAB in an ARPE-19 cell line that was treated with 5 μg Dox/mL. (C) Blot of immunoreactive SadCas9 proteins from the ARPE-19 cell lines. The N-KRAB construct migrated to a position between C-KRAB (lane 1) and SadCas9 at ~125 kDa (lane 3). The internal KRAB (I-KRAB) construct displayed significantly lower protein levels than the other constructs and was only detected by immunoprecipitation.

Figure 3. CRISPRi Activity Against the Secreted Protein Pcsk9 (A) Blot of soluble extracts from parental AML-12 mouse liver cells and cell lines expressing SadCas9, N-KRAB, or C-KRAB treated with 5 μg Dox/mL. The N-KRAB and C-KRAB constructs co-expressed U6-driven non-targeting (NT) or Pcsk9 gRNA. (B) Both N-KRAB and C-KRAB were localized exclusively in the nucleus. Results in (A) and (B) are representative of two independent experiments. (C) Levels of Pcsk9 from the supernatant of cells. Relative to parental cells, cells with SadCas9 decreased Pcsk9 levels 2-fold (∗∗p < 0.01). A comparison of NT versus Pcsk9 gRNA revealed a significant difference (∗p < 0.05) for N-KRAB, but not C-KRAB. The results (mean ± SD) are representative of three independent experiments.

Figure 4. Comparison of 7SK and U6 Promoters Driving Expression of gRNA AML-12 cell lines expressing N-KRAB and either 7SK or U6 promoters were treated with 2 μg Dox/mL for 3 days. (A) Blot of soluble protein from cell lines probed with antibodies against Sa Cas9 (top panel) or α-tubulin (bottom panel). The results are representative of two independent experiments. (B) Analysis of mRNA levels for N-KRAB relative to Gapdh [ΔCt = (Ct SadCas9) – (Ct Gapdh)]. (C) Levels of Pcsk9 mRNA relative to parental cells. The U6 promoter and Pcsk9 gRNA reduced levels of Pcsk9 mRNA 3.5-fold relative to gStop (∗∗p < 0.01), whereas there was no difference for the 7SK promoter. (D) Quantitation of Pcsk9 protein from the cell supernatants. A comparison between promoters with gStop gRNA was not significant, whereas promoters with Pcsk9 gRNA revealed that the U6 promoter decreased Pcsk9 levels 2.5-fold relative to the 7SK promoter (∗∗p < 0.01). The results (mean ± SD) presented in (B)–(D) are representative of three independent experiments.

Figure 5. Comparison of Promoters Driving Expression of KRAB-SadCas9 (N-KRAB) (A) Analysis of N-KRAB mRNA levels from the different promoters (see Table 2) relative to cells with CMV and U6:gStop. Levels of N-KRAB mRNA from the tested promoters were significantly lower than CMV (∗∗p < 0.01). (B) Pcsk9 mRNA levels and (C) Pcsk9 protein levels relative to parental AML-12 cells. The results (mean ± SD) are representative of three independent experiments. Asterisks denote significance at p < 0.05 (∗) or p < 0.01 (∗∗) relative to parental cells.

Figure 6. Analysis of a Single AAV CRISPRi that Targets Pcsk9 in Mice (A) Diagram of a representative AAV2 plasmid construct used to package AAV8.N-KRAB. The promoters included Alb-AAT or EF-1α (see Table 2) with either gStop or Pcsk9 gRNA. (B) Fold-increase in N-KRAB mRNA from different doses of rAAV.N-KRAB.Pcsk9 under control the Alb-AAT promoter relative to the EF-1α promoter at a dose of 2 × 1011 vg/mouse. The results are from one experiment and asterisks (∗∗) denote p < 0.01. Mice tail-injected with PBS or Alb-AAT viruses with either gStop or Pcsk9 gRNA at doses of 2 × 1011 vg/mouse were evaluated for (C) levels of Pcsk9 mRNA relative to control PBS mice and (D) concentration of Pcsk9 from sera. The results presented in (C) and (D) are representative of two independent experiments and asterisks denote p < 0.05 (∗) or p < 0.01 (∗∗). (E) Immunolabeling of liver sections from mice treated with either 2 × 1011 vg/mouse of AAV8.N-KRAB (Pcsk9; top panels) or PBS (bottom panels). Sections were labeled with rabbit anti-Sa Cas9 (left panels) and DAPI (middle panels). The arrows denote nuclear labeling of N-KRAB. The results are representative of three animals from each treatment.