Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype

Abstract

We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.

Figure 1

Hydrodynamic injection of CRISPR components rescues lethal phenotype of Fah-deficient mice. (a) Experimental design. Fah^mut/mut mice harbor a homozygous G→A point mutation at the last nucleotide of exon 8 (red), causing skipping of exon 8 during splicing. pX330 plasmids expressing Cas9 and a sgRNA targeting the Fah locus are delivered to the liver by hydrodynamic tail vein injection. A ssDNA oligo with the correct fragment of Fah sequence (i.e., the G allele) is co-injected to serve as a donor template to repair the ‘A’ mutation. Exon and intron sequences are in upper and lower cases, respectively. (b) Fah^mut/mut mice were injected with saline only, ssDNA oligo only, ssDNA oligo plus pX330 (unguided Cas9), or ssDNA oligo plus pX330 expressing Cas9 and one of the three Fah sgRNAs (FAH1, FAH2 and FAH3). Body weight was monitored over time and normalized to pre-injection weight. Arrow indicates withdrawal of NTBC water (defined as day 0, which is 3 d after injection). (c) Mice injected with FAH1 or FAH3 in b were put back on NTBC water for 7 d and then again withdrawn from NTBC for 28 d. (d) H&E staining of liver sections from wild-type (Fah^+/+) or Fah^mut/mut mice injected with unguided Cas9 or Cas9 with the FAH2 sgRNA and kept off NTBC water. The FAH2 sample is from a mouse 30 d after NTBC withdrawal. Scale bars, 100 µm for upper panels, 20 µm for lower panels. (e–g) Liver damage markers (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and bilirubin) were measured in peripheral blood from Fah^mut/mut mice injected with saline or ssDNA oligo only or unguided Cas9 (NTBC off) or FAH2 (NTBC off + FAH2, day 30). Fah^mut/mut mice on NTBC water (NTBC on) served as a control. * P < 0.01 (n = 3 mice) using one-way ANOVA. Error bars, mean ± s.e.m.

Figure 2

CRISPR-Cas9–mediated editing corrects Fah splicing mutation in the liver. (a) Fah immunohistochemistry (IHC) of Fah^mut/mut mice injected with unguided Cas9 or Cas9 plus the FAH2 sgRNA. Upper panel: FAH2 mice were off NTBC water for 30 d as in Figure 1d. There are 33.5% ± 3.3% Fah⁺ cells (n = 3 mice). Lower panel: mice were kept on NTBC water and euthanized at day 6 to estimate initial repair rate. Fah⁺ cell counts were 0.40 ± 0.12% for FAH2 and 0.01 ± 0.02% for unguided Cas9. P < 0.01 (n = 3 mice) using an unpaired t-test. Fah^+/+ mice are shown as a control. Scale bars, 100 µm. (b) RT-PCR in liver RNA from wild-type (Fah^+/+), Fah^mut/mut and Fah^mut/mut mice injected with FAH1, 2 or 3, using primers spanning exons 5–9 to amplify wild-type Fah (405 bp) and mutant Fah (305 bp, lacking exon 8). FAH-treated mice were harvested at the endpoints of NTBC withdrawal. (c) Representative sequence of the 405-bp bands in FAH2-treated mice. The corrected G nucleotide is highlighted in gray. (d) Quantitative RT-PCR measurement of wild-type Fah mRNA expression using primers spanning exons 8 and 9. Error bars, s.d. from three technical replicates for each individual mouse.