In vitro gene silencing with dSaCas9-based repressors

While SaCas9 has been described as a nuclease for gene editing27, our goal in this study was to adapt SaCas9 for targeted gene silencing. We first sought to show that dSaCas9KRAB could effectively silence genes in vitro. As a model, we harvested primary mouse fibroblasts from a mouse strain that constitutively expresses a luciferase reporter from a CAG promoter. We stably expressed dSaCas9KRAB and gRNAs targeted to the CAG promoter by lentiviral transduction (Fig. 1a, Supplementary Fig 1a, Supplementary Table 1). Seven days after transduction, three of six promoter-targeting gRNAs significantly reduced luciferase expression compared to negative controls of untransduced cells and cells transduced with dSaCas9KRAB but no gRNA (Supplementary Fig 1b, c).

Fig. 1 Targeted gene silencing of endogenous Pcsk9 in vitro. a Deactivated S. aureus dCas9 was fused to a KRAB repressor motif and delivered by lentivirus for in vitro gRNA screening. The lentiviral vector also contained a puromycin resistance gene and a gRNA expression cassette. b A panel of eight S. aureus gRNAs were designed to target the accessible chromatin region of the mouse Pcsk9 promoter region in AML12 cells, a mouse hepatocyte cell line with high expression of Pcsk9. An ENCODE wild-type mouse liver DNase I hypersensitivity-sequencing track is included to highlight the accessible chromatin region around the Pcsk9 transcription start site38. c Single gRNAs were screened for silencing efficacy by qRT-PCR. (mean ± s.e.m., n = 2 biological replicates). P < 0.05 indicated by * compared with the non-transduced (NT) control (Student’s t-test) Full size image

For an endogenous gene target for in vivo studies, we selected Pcsk9, a regulator of LDL cholesterol levels targeted for repression in therapies for familial hypercholesterolemia. We screened several gRNAs for optimal repression of the mouse Pcsk9 gene, our target for in vivo transcriptional repression (Supplementary Table 2). Pcsk9 is highly expressed in the liver, and we designed gRNAs to target the DNase I hypersensitivity site surrounding the transcription start site in Pcsk9 in adult mouse liver tissue (Fig. 1b)38. We tested these gRNAs in the AML12 mouse hepatocyte cell line. When delivered stably with dSaCas9KRAB by lentiviral transduction, seven of eight gRNAs repressed Pcsk9 transcript expression >90% by qRT-PCR, compared to non-treated controls and controls without a gRNA (Fig. 1c). These results demonstrate the effectiveness of RNA-guided SaCas9-based repressors for silencing target gene transcription in vitro.

In vivo gene silencing in an adult wild-type mouse

For targeted gene repression in vivo, we generated two AAV vectors, one encoding dSaCas9KRAB (4.7 kb between inverted terminal repeats) and the other containing an expression cassette of the human U6 promoter driving a Pcsk9-targeting gRNA (4.2 kb between inverted terminal repeats including a stuffer sequence) (Fig. 2a). We selected Pcsk9-targeting gRNA 2 from our screen in AML12 cells for in vivo studies. We used two separate vectors to achieve high levels of dSaCas9KRAB expression by including the full-length CMV promoter and the bGH-derived polyadenylation signal. Separating dSaCas9KRAB and the gRNA on two vectors also maximized flexibility of experimental design and AAV production, including testing the effects of dSaCas9KRAB and the gRNA independently.

Fig. 2 Targeted gene silencing in adult wild-type mice with S. aureus dCas9KRAB. a A dual AAV vector system was designed to deliver dSaCas9KRAB and Pcsk9-targeting gRNA to adult-wild type mice via tail-vein injection. qRT-PCR for b dSaCas9KRAB and c Pcsk9 expression was performed on mRNA from livers harvested from treated mice at 6 weeks post-injection (mean ± s.e.m, n = 4 mice). P < 0.05 is indicated by *, determined by Student’s t-test compared to controls. Serial serum collections were assayed for secreted d Pcsk9 protein levels and e low-density lipoprotein cholesterol (mean ± s.e.m., n = 4 mice, * indicates P < 0.05 by mixed design ANOVA with Tukey’s post-hoc analysis) Full size image

We administered AAV to 6–8-week-old C57Bl/6 mice systemically by tail-vein injection using an AAV8 serotype to target hepatocytes in the liver that highly express Pcsk9. We tested two different doses of AAV expressing dSaCas9KRAB and Pcsk9-targeting gRNA at 2×1011 and 1×1012 viral genomes per vector per mouse (vg/v/m). Age-matched controls received a PBS sham injection or dSaCas9KRAB AAV injection without gRNA at 1×1012 vg/v/m. At 6 weeks post-treatment, we detected expression of dSaCas9KRAB in the liver via qRT-PCR (Fig. 2b). Compared to PBS sham and dSaCas9KRAB-only controls, we observed significant transcriptional silencing of the Pcsk9 gene in mouse livers treated with dSaCas9KRAB and Pcsk9-targeting gRNA AAVs at a dose of 2×1011 vg/v/m (Fig. 2c). We collected serum from treated mice longitudinally to track Pcsk9 protein levels over time. Delivery of dSaCas9KRAB and Pcsk9-targeting gRNA AAVs dramatically reduced Pcsk9 serum levels to 20% of levels in PBS-treated controls within 2 weeks of treatment (Fig. 2d). With the higher AAV dose of 1×1012 vg/v/m, Pcsk9 serum levels were reduced >90% over 4 weeks after treatment, but this magnitude of silencing was not sustained (Supplementary Fig. 2). In fact, at 6 weeks post-administration of the higher AAV dose, Pcsk9 transcript levels were indistinguishable from non-treated controls and serum protein levels displayed an increasing trend relative to earlier time points. In contrast, Pcsk9 silencing in mice receiving the lower dose of AAV was sustained throughout 6 weeks post-AAV delivery (Fig. 2d).

Concomitant with reduced Pcsk9 serum levels, we also observed significant reductions in serum levels of LDL and total cholesterol over 6 weeks post-treatment compared to PBS and dSaCas9KRAB controls without the gRNA vector (Fig. 2e, Supplementary Fig. 3a). Reductions in LDL cholesterol serum levels were corroborated by western blot showing increases in LDL receptor expression in liver tissue expressing dSaCas9KRAB and Pcsk9 gRNA (Supplementary Fig. 3b). Together these results demonstrate that AAV delivery and CRISPR-mediated transcriptional silencing of Pcsk9 in adult mice is sufficient to modulate downstream effects on cholesterol regulation.

Host responses to CRISPR-based repression and AAV vectors

We next investigated the genome-wide effects of CRISPR-mediated gene silencing on transcriptional regulation. We performed RNA-sequencing on mouse liver tissue at 6 weeks post-treatment. When comparing treatment with dSaCas9KRAB and Pcsk9 gRNA to dSaCas9KRAB without the gRNA vector, no gene expression changes achieved genome-wide significance (defined by a false discovery rate, FDR < 0.05) (Fig. 3a, b). However, when ranking genes by raw P-value, Pcsk9 repression was the seventh most significant transcriptional change genome-wide (Fig. 3a, b, Supplementary Data 1).

Fig. 3 Genome-wide analysis of gene expression in dSaCas9KRAB-treated mice. a–d RNA-sequencing and differential expression analysis was performed comparing liver tissue from mice treated with a, b AAVs expressing dSaCas9KRAB and Pcsk9-targeting gRNA vs. dSaCas9KRAB alone, c AAVs expressing dSaCas9KRAB with Pcsk9-targeting gRNA to PBS, or d AAV expressing dSaCas9KRAB vs. PBS. Red data points indicate FDR < 0.05 by differential-expression analysis with a negative binomial generalized linear model and Wald test for significance52 (n = 4, mice 6–8 weeks old at time of treatment). The data point representing the Pcsk9 transcript is highlighted in blue (FDR > 0.05). Extended gene lists found in Supplementary Data 1, 4, 5 Full size image

Previous studies performed with S. pyogenes dCas9KRAB have demonstrated that CRISPR-guided dCas9KRAB binding and gene regulation can be highly specific for the target gene locus in cell culture systems15. To explore potential off-target gene regulation effects with RNA-guided dSaCas9KRAB silencing in vivo, we identified the top 100 differentially regulated genes by p-value when comparing liver tissue from mice treated with dSaCas9KRAB and Pcsk9-targeting gRNA compared to dSaCas9KRAB alone (Supplementary Data 1). We also computationally predicted 159 potential off-target binding sites for the Pcsk9-targeting gRNA in the mouse genome39, selected based on the presence of a 6 bp PAM-proximal seed sequence and fewer than ten total mismatches to the cognate target sequence40 (Supplementary Data 2). Of the top 100 differentially expressed genes identified, only one gene, Sox1ot, contained a predicted off-target binding site for the Pcsk9-targeting gRNA. However, Sox1ot expression was upregulated 1.9-fold in dSaCas9KRAB and gRNA-treated mice, suggesting this gene regulation effect is not a direct result of dSaCas9KRAB transcriptional repression. To explore the possibility of distal effects of dSaCas9KRAB binding, we matched each of the top 100 differentially expressed genes to the closest computationally predicted off-target site of the Pcsk9-targeting gRNA (Supplementary Data 3). With the exceptions of Pcsk9 and Sox1ot, none of the top 100 genes contained a predicted off-target site within 100 kilobases of the gene body. These results suggest that the top gene regulation changes observed in this study are not a function of off-target gRNA-mediated binding of dSaCas9KRAB and may instead reflect downstream responses to AAV transduction and transgene expression or Pcsk9 silencing and cholesterol reductions in a complex, heterogeneous tissue.

We also compared gene expression by RNA-sequencing in liver tissue treated with dSaCas9KRAB and Pcsk9 gRNA to PBS-treated controls. Here, we observed significant enrichment of nine genes (FDR < 0.05), including a subset specific to immune cells (Btla, Cd8, Ccl5, Gzma, Irf7, and Pdcd1), suggesting immune cell infiltration (Fig. 3c, Supplementary Data 4). Similar immune gene enrichment has been observed in recent studies with SpCas9-based activators delivered to skeletal muscle41. Delivery of dSaCas9KRAB AAV alone without gRNA AAV was also sufficient to cause immune gene enrichment when compared to PBS controls (Fig. 3d, Supplementary Data 5).

To understand the contributions the dSaCas9KRAB protein and Pcsk9-targeted gRNA in generating a host response, we compared mice treated with dSaCas9KRAB AAV only, gRNA AAV only, and an equal mixture of two AAVs expressing dSaCas9KRAB and gRNA at 4×1011 vg/v/m. Treatment with dSaCas9KRAB and gRNA resulted in reduced Pcsk9 and LDL cholesterol serum in mice compared to controls within 2 weeks post-treatment (Supplementary Fig. 4). Immune cell-specific gene enrichment occurred primarily in response to dSaCas9KRAB expression, indicating that this effect is independent of gRNA expression or Pcsk9 repression (Supplementary Fig. 5, Supplementary Data 6−9). Widespread expression of genes related to immune response was accompanied by attenuation in Pcsk9 silencing at the transcriptional level (Supplementary Fig. 5b, Supplementary Data 6−9) and at the protein level in serum over 6 weeks post-treatment (Supplementary Fig. 4). These results highlight the importance of tuning AAV doses for dSaCas9KRAB and gRNA delivery in order to maximize silencing effect and mitigate immune response.

To further investigate consequences of AAV delivery of dSaCas9KRAB and the Pcsk9-targeted gRNA, we measured secretion of alanine transaminases (ALT). Elevated ALT secretion is a marker of liver toxicity and increases in ALT serum levels have been observed with AAV-mediated transgene expression targeted to the liver42. In this study, we detected increased ALT serum levels at 4 weeks after AAV administration, with up to a sixfold increase in mice treated with AAV-dSaCas9KRAB compared to either PBS injection or the gRNA AAV alone (Fig. 4a). Despite these increases, ALT levels were within physiological ranges for male C57Bl/6 mice for all conditions at 6 weeks post-treatment. Furthermore, we observed similar normal tissue morphology across livers isolated from PBS-treated controls, gRNA-only controls, and dSaCas9KRAB-treated mice at 6 weeks after treatment (Fig. 4b). Together, these studies show that the host response, indicated by enrichment in immune cell gene expression and concurrent increase in ALT levels, are primarily a result of dSaCas9KRAB expression and not simply the AAV capsid proteins.

Fig. 4 Transient effects of AAV-mediated dSaCas9KRAB gene silencing on host liver. a Secreted alanine transaminase as a measure of liver toxicity was assayed in the serum of treated mice over 6 weeks post-treatment (mean ± s.e.m., n = 4 mice at 6–8 weeks old when treated). P < 0.05 is indicated by * and calculated by mixed design ANOVA with Tukey’s post-hoc analysis compared to day 0 controls. Dotted line marks the upper limit of physiological levels of ALT in adult C57Bl/6 mice. b H&E staining was performed on sections from livers of mice harvested 6 weeks post-treatment (representative images shown, scale bars = 100 μm) Full size image

Long-term efficacy of in vivo transcriptional repression

We tracked the long-term efficacy of dSaCas9KRAB-based silencing in vivo to further assess its usefulness as a research or gene therapy tool. We treated mice with two doses of AAVs expressing dSaCas9KRAB and Pcsk9-targeted gRNA (2×1011 and 4×1011 vg/v/m) and collected serum over 24 weeks post-treatment. We measured ALT serum levels as an indication of how well this treatment was tolerated over time. The acute elevation of ALT levels we observed in our shorter-term studies dissipated past 6 weeks post-treatment and stabilized to levels within physiological ranges for the duration of the study, similar to what has been observed in clinical studies43 (Fig. 5a). This suggests that despite an initial toxicity response to AAV dSaCas9KRAB expression, long-term liver function is not acutely compromised.

Fig. 5 Long-term efficacy of targeted transcriptional silencing with dSaCas9KRAB. In mice treated with AAVs expressing dSaCas9KRAB and Pcsk9-targeting gRNA at 2×1011 and 4×1011 viral genomes/vector/mouse, serum was collected over 168 days post-treatment and analyzed for a alanine transanimase secretion, b Pcsk9 protein concentration, and c low-density lipoprotein-cholesterol concentration (mean ± s.e.m., n = 4 mice at 6–8 weeks old when treated). * and # mark P < 0.05 in 2e11 and 4e11 doses respectively, calculated by mixed design ANOVA with Tukey’s post-hoc analysis compared to PBS controls Full size image

Notably, we observed durable dSaCas9KRAB-mediated silencing of Pcsk9 expression, with both doses maintaining significantly reduced serum Pcsk9 levels up to 24 weeks post-treatment compared to PBS-treated controls (Fig. 5b). Similar to ALT levels, we observed the most acute effects early after AAV injection followed by an attenuation and then stabilization of repression. In mice treated with 4×1011 vg/v/m of dSaCas9KRAB and Pcsk9 gRNA AAVs, we measured up to 90% repression of Pcsk9 levels until 4 weeks after treatment, after which Pcsk9 was repressed to 27–41% of day 0 levels for the duration of the study. Reduction in LDL-cholesterol levels was sustained through 10 weeks post-treatment, suggesting potential compensatory effects for cholesterol regulation in response to Pcsk9 repression at later timepoints (Fig. 5c). Overall, these results demonstrate that RNA-guided dSaCas9KRAB repressors are capable of durable, long-term target gene silencing in vivo.