Fig 2: Identification of LNPs for efficient TTR gene editing.(A) Comparison of mouse serum TTR protein levels after treatment by different LNP formulations. (B) Analysis of mouse liver TTR gene editing efficacy induced by different LNP formulations. (C and D) Effects of different sgRNA: SpCas9-HF1 mRNA mass ratios on mouse serum TTR protein levels and mouse liver TTR gene editing efficacy induced by LNP-01, respectively. (E and F) Comparison of off-target edits and their frequencies induced by different ratios of sgTTR-1: SpCas9-HF1 mRNA. In (A) and (B), the dosage administered to TTR-humanized mice was fixed at 0.3 mpk. The mass ratio between sgTTR-1 and SpCas9-HF1 mRNA was 1:4. In (C) and (D), the dosage administered to TTR-humanized mice was fixed at 1.0 mpk. In (E) and (F), the total mass of sgTTR-1 and SpCas9-HF1 mRNA cotransfected with 106 HepG2 cells was 1.0 μg. NS represents not significant. Error bars indicate means ± SD (n ≥ 3; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig 3: Characterization of SpCas9 variants.(A) Conformations of SpCas9 residues that could form potential interactions with guide RNA or target DNA, based on SpCas9-sgRNA-target DNA complex structure (PDB_ID: 5FW3). (B) Mutations in SpCas9-HF1, SpCas9-HF4, and six SpCas9 variants designed in this study. (C) T7E1 assays showing that all novel SpCas9 variants can edit the TTR gene in HEK293T cells. (D) Comparison of TTR protein reduction in serum of TTR-humanized mice induced by WT SpCas9, SpCas9-HF1, SpCas9-HF4, or our SpCas9 variants. NS represents not significant. Error bars indicate means ± SD (n ≥ 4 independent experiments; **P < 0.01; ****P < 0.0001).

Fig 4: Analysis of chromosomal translocations induced by SpCas9 proteins.(A) Summary of chromosomal translocation counts and rates associated with TTR gene editing in HepG2 cells. (B) Comparison of chromosomal translocation sites and counts induced by WT SpCas9 and SpCas9-Mut5. (C) Distribution of detected chromosomal translocations. Translocations between the target site and off-target site(s) are highlighted by curved lines in the center. The number of reads for each translocation corresponds to line darkness. Darker lines indicate more translocation events.

Fig 5: Comparison of the gene editing activity and fidelity of ABE8e assisted by WT SpCas9 or SpCas9-Mut5.(A to C) Comparison of the editing activity and fidelity of ABE8e and ABE8e-mut5 at the TTR, VEGFA, and EMX1 genes, respectively. (D to F) Comparison of the editing activity and fidelity of ABE8e and ABE8e-mut5 at HEK293T sites 1 to 3. Off-target sites were reported in the literatures, and the mismatched nucleotides are highlighted in red. ND represents not detectable. NS represents not significant. Error bars indicate means ± SD (n ≥ 3 independent experiments; **P < 0.01; ***P < 0.001; ****P < 0.0001).