Supplementary Information Targeting fidelity of adenine and cytosine base editors in mouse embryos

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1 Supplementary Information ing fidelity of adenine and cytosine base s in mouse embryos Lee et al.

2 a P = 1.012e-14 b Frequency (%) 100% 80% 60% 40% 20% 0% CB AB On-target Bystander Proximal Indels Frequency of bystander (%) P = CB AB c d Frequency of proximal off-target (%) P = e-06 Frequency of indels (%) P = e-07 0 CB AB 0 CB AB e dited alleles HF2- B2 B3 CB SaB3 VQR- B3 AB B4 AB a AB b AB c Bystander # of alleles # of mutations Proximal off-target # of alleles # of mutations NA

3 Supplementary Fig. 1 Statistical analysis of base editing frequencies. a Frequency of alleles carrying different categories of editing outcomes for cytosine base editing (CB) and adenine base editing (AB). Note that a single allele can carry more than one mutation. The total number of mutant alleles obtained with CB and AB was set as 100% (CB, 222; AB, 214). The applied Chi-Squared test is significant (P = 1.012e-14) and the power analysis results in a 83% probability that the test correctly rejects the null hypothesis, using a small effect size and a significance level of b-d Statistical significance in different categories of off-target mutations. diting frequencies (Table 1) of each base were used the values for comparison of CB and AB. Median, middle bar inside the box; IQR, 50% of the data; whiskers, minimum and maximum. Chi- Squared test was applied and each P-value was Bonferroni corrected for multiple testing. Bystander editing (b) shows similar frequency for CB and AB (P = ). In contrast, frequencies of proximal off-targets (P = e-06) (c), and indels (P = e-07) (d) are highly significant between CB and AB. e Number of edited alleles carrying bystander and proximal off-target mutations as well as all mutations on edited alleles for each base. Some alleles can carry more than one mutation.

4 a This study Mouse VQR-B3 Csn2 enhancer C -10 AGAAATGAACAGAGTTCAAAGAAGGCAGGAAAGAGACAAT 17x T T T T------T A----T T T----A T T------T b This study Mouse B4 Csn2 enhancer -50 8x 6x 3x TCTCCTT CAGCCCTTCCTTGTTCACACCCTTTGGGTGACT TT T T TT-----T T TT TT TT AT A C T c (sgrna-1) GGTGATGGGAGTCCCTGCGGCCAGCTTTCAGGCAGAGGTTCCTGCC TT T T A T C Mouse HF2-B2 (sgrna-2) C CTTGTTATTGTGGGAACAAGAAATTCGAGAACTAACTGGGGATGAG T x A AAA A G G A G AAA G A TA T A T AAT AA T A AA-----A A-----A T--A A A T T T T T-A--T G T T-G

5 d Dmd ACAGCAATTAAAAGCCAGTTAAAAATTTGTAAGG 18x T x T x TT x A TA GT AT A Mouse B3 GTGGCACCATCTGGACCTCAGTTCCCCTTCAAAGGGGTGG T x G x A T----T T--T T----A T T A T T e Mouse SaB3 10x 5x CATCTGGACCTCAGTTCCCCTTCAAAGGGGT T T----T TT T TA T T T----TT T T----T-T T----A T T TTT _

6 Supplementary Fig. 2 ing six loci in the mouse genome with CBs. a-e Alignment of sequences from founder mice or edited embryos injected with VQR-B3 (a), B4 (b), HF2-B2 (c), B3 (d), or SaB3 (e), and corresponding sgrnas. The editing window is shown in grey overlay. The sgrna sequences are underlined. The target nucleotides for base editing are shown in bold black. The PAM site and nucleotide substituted by cytosine base editing are shown in purple and green, respectively. Opposite-strand edits are labelled in blue and inaccurate mutations are in red. Deletions are shown as underlines.

7 a This study Mouse AB a Wap Csn2 enhancer H GAACAGAGCTCTGGCTCCTAAGACACAGGGCCTTCTGGGAAACTCAAGCAGCC 3x G G-G G GG---G GAGAGTTGCTCTTTCTAATTCTATGAAGAATTGAGTTGGAATTTTGATGGGAT G b Mouse AB b CAATGCACCTATCGGCCATAACAGAGACTCTTACATGGTTCCTTTCATACCGC 10x GG x G x GG-G x G-G x G-GG x G--G G-G--G G--G-G c Ar (sgrna-1) Ar-1 34x 19x 19x TTATCTAGCCTCAATGAGCTTGG --G---G G G G-G-G A-G G-T-G Mouse AB c Ar (sgrna-15) Ar-15 ATGATCTCTGCCATCATTTCAGG 34x ---G Hoxd13 Hoxd13 GTTTCAGAATCGAAGGGTGAAGG 40x -----G x G x -----G-G GG Supplementary Fig. 3 ing three genomic loci using AB. a-c Alignment of sequences from founder mice or embryos derived from edited zygotes that had been injected with AB and corresponding sgrnas in our study (a), the (b), the Ar and Hoxd13 genes (c). The editing window is shown in grey overlay. The sgrna sequences are underlined. The target nucleotides for base editing are shown in bold black. The PAM site and nucleotide substituted by adenine base editing are shown in purple and green, respectively. Inaccurate mutations are in red.