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Figure S6. Analysis of liver tissue from hPCSK9-KI mice 3 weeks after BE3 treatment. 10-week-old hPCSK9-KI mice were injected with adenoviral vectors encoding BE3 alone or together with gMH. Representative micrographs show staining with hematoxylin and eosin (H&E) and antibodies against human PCSK9 (hPCSK9, brown) and LDL receptors (LDL-R, brown). Scale bars, 200 μm. CV, central vein of the liver. (PDF 1664 kb)

Figure S1. Liver-specific expression of human PCSK9 in the hPCSK9-KI mouse model. (a) Representative micrographs of liver tissues from a WT (left) and a hPCSK9-KI mouse (right) showing expression of human PCSK9 (hPCSK9, brown) only in the hPCSK9-KI mouse liver. (b) Representative micrographs of tissues from a hPCSK9-KI mouse showing no hPCSK9 expression in pancreatic islets, skeletal muscle, kidney, white adipose tissue (WAT), brown adipose tissue (BAT), and spleen. Tissues were incubated with antibodies against hPCSK9. Scale bars, 200 μm. (PDF 3790 kb)

Figure S5. Analysis of single nucleotide substitutions at the human PCSK9 locus. (a) Percentage of single base changes at the human PCSK9 target site in BE3-gMH-treated HEK293T cells. Cells were co-transfected with plasmids encoding BE3 and gMH and genomic DNA was analyzed by deep sequencing after 3 days. gMH targets codon W159 (TGG) within the human PCSK9 locus; the two targeted Gs are in positions 13 and 14 of the protospacer adjacent motif (G13 and G14, respectively). (b) Percentage of single base changes at the human PCSK9 (left) and mouse Pcsk9 (right) target sites in the liver from BE3-gMH-treated hPCSK9-KI mice; mice were 10 weeks old at the time of injection, and genomic DNA was analyzed by deep sequencing 3 weeks after treatment. (PDF 236 kb)

Figure S4. Cas9-gH treatment in hPCSK9-KI mice. (a) Surveyor mismatch cleavage assay on genomic DNA from liver tissue of hPCSK9-KI mice 3 weeks after injection with adenoviral vectors encoding Cas9 together with GFP (as control) or gH; mice were 10 weeks old at the time of injection. The gel image demonstrates cleaving efficacy of gH at the human PCSK9 locus. (b) Plasma concentrations of human PCSK9 protein after treatment with Cas9-gH or Cas9-GFP in hPCSK9-KI mice (normalized to pretreatment plasma concentrations; n = 6 per group). (c, d) Plasma concentrations of (c) mouse PCSK9 protein and (d) total cholesterol 3 weeks after treatment with Cas9-gH or Cas9-GFP in WT or hPCSK9-KI mice (normalized to pretreatment plasma concentrations; n = 6 per group). Data were analyzed with univariate linear regression; reported p values correspond to t tests for estimated regression coefficients (effects). *p

Table S7. List of primers used in this study. (PDF 215Â kb)

Table S3. Frequency of null alleles generated by BE3-gMH and Cas9-gMH treatment in hPCSK9-KI mice. (PDF 202Â kb)

Figure S2. CRISPR-Cas9 targeting strategy and editing efficiency. (a) Schematic of exon 1 of human PCSK9 (top) and mouse Pcsk9 (bottom) loci. Nucleotides are depicted as the distance from the ATG (orange box). The guide RNA gH has perfect complementarity to a sequence within exon 1 of human PSCK9 while gM has eight mismatches to the most similar sequence within that exon, and there is no NGG protospacer adjacent motif (PAM) in the proximity. The guide RNA gM has perfect complementarity to a sequence within exon 1 of mouse Pcsk9 while gH has six mismatches to the most similar sequence within that exon. Therefore, active cleavage is expected with gH only at human PCSK9 and with gM only at mouse Pcsk9 with no cross reactivity. (b) Surveyor mismatch cleavage assay shows gH cleavage activity in HEK293T cells. Cells were co-transfected with plasmids encoding Cas9 and gH and genomic DNA was analyzed 3 days later. The gel image demonstrates cleaving efficacy of gH at the human PCSK9 locus. (c) Surveyor mismatch cleavage assay on genomic DNA from liver tissue of hPCSK9-KI mice 3 weeks after injection with adenoviral vectors encoding Cas9 together with gH, gM, both gH and gM (gH/gM), or GFP; mice were 28 weeks old at the time of injection. The gel image demonstrates cleaving efficacy of gH at the human PCSK9 locus, gM at the mouse Pcsk9 locus, and gM/gH at both loci. (PDF 1479 kb)

Table S6. List of off-target sites for gMH. (PDF 439Â kb)

Table S4. Most frequent mutant alleles generated by BE3-gMH and Cas9-gMH treatment in hPCSK9-KI mice. (PDF 678Â kb)

Figure S3. Analysis of liver tissue from hPCSK9-KI mice 3 weeks after Cas9 treatment. Twenty-eight-week-old hPCSK9-KI mice were injected with adenoviral vectors encoding Cas9 together with gH, gM, both gH and gM (gH/gM), or GFP. Representative micrographs show staining with hematoxylin and eosin (H&E) and antibodies against human PCSK9 (hPCSK9, brown), mouse Pcks9 (mPCSK9, brown), and LDL receptors (LDL-R, brown). Scale bars, 200 μm. CV, central vein of the liver. (PDF 1391 kb)

Table S5. Frequency of targeted stop codons generated by BE3-gMH. (PDF 179Â kb)

Table S2. List of GUIDE-Seq-detected off-target sites for gH. (PDF 274Â kb)

Table S1. Lipoprotein profile of WT and hPCSK9-KI mice. Plasma concentrations (in nanomolar) of HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), VLDL-cholesterol (VLDL-C), and total cholesterol in WT and hPCSK9-KI mice at 10 and 28 weeks of age. HDL-C, LDL-C, VLDL-C, and total cholesterol concentrations are presented as group means ± SD; n = 4–29; data were analyzed with univariate linear regression, p values correspond to t tests for estimated regression coefficients (effects) for the comparison between hPCSK9 and WT mice at 10 weeks or 28 weeks of age. *p