PMC

The generation of Hoxd13 and Tyr mutant mouse models by SaBE3 and SpABE. a Fused digits and white hair phenotypes in founder mice. Left: Mutant mouse shows whole-body white hair and fused digits (purple arrowhead). Right: all the four mice displayed whole-body white hair and fused digits. b Representative alignments of modified sequences from newborn pups after microinjection of SpABE/SaBE3 mRNA and sgRNAs targeting Hoxd13 and Tyr simultaneously. The PAM sequences and substitutions are highlighted in blue and red, respectively; N/N indicates positive colonies out of the total sequenced. The corresponding targeted codons are shown. c Summary of phenotypes. Three mice displayed normal hair and fused digits; nine mice displayed black-and-white hair and fused digits; four mice displayed whole-body white hair and fused digits; nine mice displayed normal hair and normal digits; one mouse displayed black-and-white hair and normal digits; no mice displayed whole-body white hair and normal digits. d Representative alignments of modified sequences from newborn pups harboring indel. The PAM sequences and substitutions are highlighted in blue and red, respectively; N/N indicates positive colonies out of the total sequenced. The corresponding targeted codons are shown. e Summary of the genotyping of Hoxd13 and Tyr mutations of newborns by SaBE3 and SpABE-mediated base editing

Whole-genome analysis of Hoxd13 (H029) mutant and wild-type (Wt) mice. a Summary of genome sequencing analysis. A Hoxd13 mutant mouse (H029) and a wild-type mouse (Wt) were sequenced separately using Illumina HiSeq X Ten. A total of 4,404,011 and 3,185,078 SNPs were identified for H029 and Wt, respectively. After filtering out dbSNP (naturally occurring variants in the SNP database), 45,946 SNPs were obtained over the H029 genome. The unwanted base substitutions (A→T/C SNPs, T→A/G SNPs) were excluded next. Then the sequences at the remaining SNP sites were compared with all on-/off-target sequences (20 bp). b Summary of on-/off-target site information. A total of 1248 sites, including 1 on-target site and 1, 1, 16, 117, and 1112 off-target sites with 1, 2, 3, 4, and 5 mismatch/es, respectively, were analyzed. c Summary of the whole-genome sequencing. d Summary of off-target analysis. After comparing the sequences at the remaining SNP sites with the 1248 on-/off-target sequence (20 bp), the A-to-G substitution was only detected within the on-target sequencing in H029. e Confirmation of the on-target mutation by the analysis of whole-genome sequencing. Red arrow indicates the A-to-G substitution within 20 bp on-target sequence in H029

ABE-mediated efficient A-to-G conversion at Ar and Hoxd13 loci in mouse embryos. a GFP-to-BFP conversion as a reporter for ABE-mediated base editing. b Analysis of base editing by FACS (left) and Sanger sequencing (right). Scramble: Scrambled sgRNA as negative control; sgRNA: sgRNA targeting GFP; PC: BFP expression plasmid only as positive control. Data were analyzed by Student’s t-test (***p < 0.001) and shown as mean ± s.e.m. (n = 3 from three independent experiments). c ABE-mediated base editing in vivo. ABE mRNA and sgRNA were co-injected into one-cell embryos, and the editing efficiency examined at blastocyst stage. d Efficiency of A-to-G substitution in mouse embryos. TA clones of PCR amplicons from the target regions in Ar (for sgAr-1, sgAr-15) and Hoxd13 (sgHoxd13) were analyzed by Sanger sequencing. Each dot indicates one individual mouse. At least 10 TA clones were analyzed for each sample. e The editing frequencies of individual A-to-G conversion of samples described in c were analyzed. A₃, A₇, C₅, and T₁₁ indicate edited positions of the protospacer for sgAr-1; A₆, A₈, and A₉ indicate edited positions of the protospacer for sgHoxd13. f Representative alignments of modified sequences from embryos after microinjection of ABE mRNA and sgRNAs into one-cell embryos. The PAM sequences and substitutions are highlighted in red and blue, respectively; the target codons are underlined; N/N represents positive colonies out of the total sequenced

The generation of Ar and Hoxd13 mutant mouse models by ABE. a The editing efficiencies were detected by Sanger sequencing of TA clones of mouse pups for sgAr-1, sgAr-15, and Hoxd13, respectively. Each dot indicates one individual mouse. At least ten TA clones were analyzed for each sample. Blue dots represent male mice and red dots represent female mice. Asterisks represent the mice with defects. b The editing efficiencies of different positions from individual samples described in a were analyzed. Left: A₃, A₇, and C₅ indicate the edited positions of the protospacer for sgAr-1; A₆, A₈, and A₉ indicate edited positions of the protospacer for sgHoxd13. Blue dots represent male mice and red dots represent female mice. Right: Editing frequencies in As of each mice with defects for sgHoxd13 and sgAr-1 are displayed in heatmap. Blue rectangles represent male mice and red rectangles represent female. Editing frequencies increase with the increase of color shades. c Representative alignments of modified sequences from pups after microinjection of ABE mRNA and sgRNAs into one-cell embryos. The PAM sequences and substitutions are highlighted in red and blue, respectively; the target codons are underlined; N/N represents positive colonies out of the total sequenced. d Characterization of the targeted modifications by deep sequencing. The frequencies were calculated from three (n = 3) replicates. e Sex reversal in founder mouse. Left: a 5-week-old mouse with female genitalia (green arrowhead) and nipples (red arrowheads); middle: Wt male with normal male genital (blue arrowhead); right: founder A021 with internal genitalia of male (orange arrowhead) and smaller testes and founder A063 with smaller testes. f Syndactyly phenotypes of founder mouse. Digits of mutant mouse are fused (purple arrowhead)