Adenine base editing in an adult mouse model of tyrosinaemia

Abstract

In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH⁺ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.

Figure 1. ABE rescues liver disease phenotype in a mouse model of tyrosinemia.

(A) Fah^mut/mut mice harbor a G>A mutation (red) at the last nucleotide of exon 8, causing exon skipping. Exon sequences are in upper case. G>A mutation is at position 9 of the sgRNA target. (B) Hydrodynamic injection of ABE and sgRNA plasmids. (C) ABE6.3+sgRNA rescues body weight loss. Withdrawal of NTBC water is defined as Day 0. Error bars indicate s.d. (n=5 mice). In the ABE6.3+sgRNA group, the final body weight is significantly different from the lowest weight (P=0.02, one-tailed student t test). (D) ABE-treated mice show regions of Fah+ hepatocytes (n=3 mice, 32 days off NTBC). Scale bars = 75 μm.

Figure 2. ABE partially corrects the Fah mutation in mouse liver.

(A) RT-PCR in a representative ABE mouse (4 liver lobes) using primers spanning exon 5 and 9. Wildtype Fah (+/+) amplicon is 405bp and mutant Fah (lacking exon 8) is 305bp. Gapdh, control gene. Images of the uncropped gels are provided in Supplementary Fig. 10. (B) Representative Sanger sequencing of the 405bp RT-PCR bands. (C) Deep sequencing of Fah genomic region in liver DNA. Error bars are s.d. (n=6 liver samples from two mice). (D) Long-term survival of two ABE-treated mice (a and b) without NTBC.

Figure 3. Optimizing the coding sequence of ABE6.3 and adding N-terminal NLS sequences improves base editing.

(a) Schematic representation of codon-optimized ABE 6.3. (b) Frequency of A-to-G editing in HEK293T cells 5 days after ABE and sgRNA transfection. Graphs show mean values. Error bars indicate s.d. (n = 3 biologically independent samples). P values determined by one-tailed student t tests.

Figure 4. RA6.3 shows a higher editing efficiency compared to ABE6.3 and Cas9-mediated HDR at two genomic sites in HEK293T cells.

(a,b) Frequency of A-to-G editing at different positions at two genomic sites (sequences as indicated). The “A”s within the editing window are in red. (c,d) Frequency of A-to-G conversion at a targeted A (in red) mediated by ABE6.3, RA6.3 or HDR at two genomic sites (sequences as indicated). Cas9 indicates the group transfected with Cas9 plasmid alone. All error bars indicate s.d. (n = 3 biologically independent samples). P values determined by one-tailed student t tests.

Figure 5. RA6.3 increases editing efficiency in vivo compared to ABE6.3.

(a) Fah^mut/mut mice were injected with indicated plasmid combinations. To measure initial A->G conversion rate, mice were kept on NTBC to prevent expansion of corrected cells. (b) Quantification of Fah-positive hepatocytes by IHC at day 7. Error bars indicate s.d. (n=4 mice per group). *P = 0.0143 by one-tailed Mann-Whitney test. (c) Representative Fah IHC. Scale bars are 25um.