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1 VOLUME: 1 ARTICLE NUMBER: 0066 In the format provided by the authors and unedited. Engineering CRISPR-Cpf1 crrnas and mrnas to maximize genome editing efficiency Bin Li 1, Weiyu Zhao 1, Xiao Luo 1, Xinfu Zhang 1, Chenglong Li 2, Chunxi Zeng 1 and Yizhou Dong 1,3,4,5,6,7* 1 Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA. 2 Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida 32610, USA. 3 Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio 43210, USA. 4 The Center for Clinical and Translational Science, The Ohio State University, Columbus, Ohio 43210, USA. 5 James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA. 6 Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University, Columbus, Ohio 43210, USA. 7 Department of Radiation Oncology, The Ohio State University, Columbus, Ohio 43210, USA. * dong.525@osu.edu. NATURE BIOMEDICAL ENGINEERING DOI: /s

2 Supplementary Figure 1 Schematic diagram of chemical modifications applied to CRISPR-Cpf1 crrnas. Modified positions (phosphate backbone and sugar modifications) of nucleotides were marked with the dotted circle. NATURE BIOMEDICAL ENGINEERING DOI: /s

3 Supplementary Figure 2 Gel analysis of Cpf1 mrnas. mrnas were treated with glyoxal to disrupt secondary structure and run on an agarose gel. NATURE BIOMEDICAL ENGINEERING DOI: /s

4 Supplementary Figure 3 AsCpf1: maximizing gene editing efficiency through combination of chemically modified crrna and AsCpf1 mrna. a, AsCpf1-mediated gene cutting efficiency for the human DNMT1 site 3 in HEK293T, Hep3B and U87 cells. b, AsCpf1-mediated gene cutting efficiency for the human AAVS1 in HEK293T, Hep3B and U87 cells. c, AsCpf1-mediated gene cutting efficiency for the human FANCF site 2 in HEK293T cells. Indel percentage at each locus was determined using the T7E1 assay by quantification of the uncut (red arrow) and cut DNA bands (green arrow), and expressed as the mean ± s.d. from three biological replicates (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed t-test). NATURE BIOMEDICAL ENGINEERING DOI: /s

5 Supplementary Figure 4 LbCpf1-mediated gene cutting efficiency in human cells. Indel percentage at the DNMT1 site 3 was determined using the T7E1 assay by quantification of the uncut (red arrow) and cut DNA bands (green arrow), and expressed as the mean ± s.d. from three biological replicates (**, P < 0.01; two-tailed t-test). denotes an empty lane. ND, not detectable. NATURE BIOMEDICAL ENGINEERING DOI: /s

6 Supplementary Figure 5 Off-target effects of CRISPR-Cpf1 system characterized by T7E1 cleavage assays. The anticipated size of fragments were shown at the bottom of gels. * denotes non-specific bands. NATURE BIOMEDICAL ENGINEERING DOI: /s

7 Supplementary Figure 6 Gene cutting efficiency of crrnas with interspersed modifications. a, Schematic illustration of crrnas with interspersed modifications. Unmodified nucleotides are shown in black. 2'-F modifications are shown in red. 2'-O-methyl modifications are shown in blue. 2'-O-methyl combined with phosphorothioate modifications are shown in light blue. b, Gene cutting efficiency of crrnas with interspersed modifications in the present of ψ-modified AsCpf1 mrna. Gene cutting efficiency was determined by the T7E1 cleavage assay, and normalized to that of the treatment with cr3 5F and ψ-modified AsCpf1 mrna. ND, Not detectable. Data were expressed as the mean ± s.d. from three biological replicates. NATURE BIOMEDICAL ENGINEERING DOI: /s

8 Supplementary Figure 7 Indel size distribution induced by CRISPR-Cpf1 system. Plot of indel size distribution of all reads for crwt plus AsCpf1 plasmid (a), cr3 5F plus AsCpf1 mrna (ψ) (b) and cr3 5F plus LbCpf1 mrna (ψ) (c) from biological replicates 2 and 3 of each treatment group. NATURE BIOMEDICAL ENGINEERING DOI: /s

9 Supplementary Figure 8 Indel position distribution induced by CRISPR-Cpf1 system. Plot of indel position distribution of all reads for crwt plus AsCpf1 plasmid (a), cr3 5F plus AsCpf1 mrna (ψ) (b) and cr3 5F plus LbCpf1 mrna (ψ) (c) from biological replicates 2 and 3 of each treatment group. NATURE BIOMEDICAL ENGINEERING DOI: /s

10 Supplementary Table 1 Engineered crrnas and their mass spectrometry data. No. AscrRNA Modification pattern (5'- to -3') M.W. Chemically modified crrnas targeting DNMT1 site 3 Calcd. Found WT crwt UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr42ps UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr5'&'3f2ps UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr5'20m UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr5'10m UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr5'5m UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr5'20f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr5'10f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr5'5f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC crs3m3ps UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC crs3f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC crs2f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC crs1f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr3'10m UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr3'5m UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr3'10f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr3'5f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr3'5f4ps UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr3'5u UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr3'5l UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cri21f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cri16m5f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cri16m10ps5f UAAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC Stem-engineered crrnas targeting DNMT1 site 3 23 crsplit-l UAAUUUCUACUC crsplit-r UUGUAGAUCUGAUGGUCCAUGUCUGUUACUC crdel2 UAAUU-CUACUCUUGUAG-UCUGAUGGUCCAUGUCUGUUACUC crdel4 UAAUUUCU--UCUU--AGAUCUGAUGGUCCAUGUCUGUUACUC crdel8 UAAUUU----UCUU----AUCUGAUGGUCCAUGUCUGUUACUC crins4 UAAUUUCUACACUCUUGUGUAGAUCUGAUGGUCCAUGUCUGUUACUC crins4' UAAUUUCUACUCUCUUGUGUAGAUCUGAUGGUCCAUGUCUGUUACUC crins6' UAAUUUCUACUGCUCUUGCUGUAGAUCUGAUGGUCCAUGUCUGUUACUC crins8 UAAUUUCUACACACUCUUGUGUGUAGAUCUGAUGGUCCAUGUCUGUUACUC crins12 UAAUUUCUACACACACUCUUGUGUGUGUAGAUCUGAUGGUCCAUGUCUGUUAC UC Loop-engineered crrnas targeting DNMT1 site 3 32 FncrRNA UAAUUUCUACUGUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC Lb3crRNA UAAUUUCUACGUUCUGUAGAUCUGAUGGUCCAUGUCUGUUACUC BpcrRNA UAAUUUCUACGUAAUGUAGAUCUGAUGGUCCAUGUCUGUUACUC NATURE BIOMEDICAL ENGINEERING DOI: /s

11 35 Pb/Pe/LicrRNA UAAUUUCUACUUUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC SscrRNA UAAUUUCUACACGCGGUAGAUCUGAUGGUCCAUGUCUGUUACUC Lb2/Pc/PmcrRNA UAAUUUCUACUAUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC CMtcrRNA UAAUUUCUACUCUUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC EecrRNA UAAUUUCUACU-UUGUAGAUCUGAUGGUCCAUGUCUGUUACUC MbcrRNA UAAUUUCUACUGUUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC PdcrRNA UAAUUUCUACUUCGGUAGAUCUGAUGGUCCAUGUCUGUUACUC LbcrRNA AAUUUCUACUAAGUGUAGAUCUGAUGGUCCAUGUCUGUUACUC Chemically modified crrnas targeting AAVS1 WT crwt UAAUUUCUACUCUUGUAGAUCUUACGAUGGAGCCAGAGAGGAU cr3'5f UAAUUUCUACUCUUGUAGAUCUUACGAUGGAGCCAGAGAGGAU Chemically modified crrnas targeting FANCF site 2 WT crwt UAAUUUCUACUCUUGUAGAUGUCGGCAUGGCCCCAUUCGCACG cr3'5f UAAUUUCUACUCUUGUAGAUGUCGGCAUGGCCCCAUUCGCACG No. LbcrRNA Modification pattern (5'- to -3') M.W. Chemically modified crrnas targeting DNMT1 site 3 Calcd. Found WT crwt AAUUUCUACUAAGUGUAGAUCUGAUGGUCCAUGUCUGUUACUC cr3'5f AAUUUCUACUAAGUGUAGAUCUGAUGGUCCAUGUCUGUUACUC Unmodified nucleotides are shown in black. Full-length PS modifications are shown in pink. 2'-O-methyl modifications are shown in blue. 2'-F modifications are shown in red. 2'-O-methyl combined with PS modifications are shown in light blue. 2'-F combined with PS modifications are shown in green. Unlock nucleotides are shown in purple. Locked nucleotides are shown in yellow. The dashes in the stem-engineered crrnas denote deleted nucleotides, and the orange letters denote inserted nucleotides. Red letters in the loop-engineered crrnas denote nucleotides difference between AscrRNA and crrnas from other 15 Cpf1 family orthologues. NATURE BIOMEDICAL ENGINEERING DOI: /s

12 Supplementary Table 2 A list of genomic locus and primers used for T7E1 assay. Locus Genome Location Protospacer(5'-3') Mismatch (bp) DNMT1 On target Homo sapiens NC_ TTTCCTGATGGTCCATGTCTGTTACTC 0 - DNMT1 OT1 Homo sapiens NC_ TTTCCTGCTGGTCCATGTCTAATACTC 3 - DNMT1 OT2 Homo sapiens NC_ TTTTCTGATGGTCCATACCTGTTACAC 3 + DNMT1 OT3 Homo sapiens NC_ TTTTCTTATTGTACATGTCTGTAACTC 4 - DNMT1 OT4 Homo sapiens NC_ TTTCCTGATGGTCCACACCTGTTACAC 4 + AAVS1 On target Homo sapiens NC_ TTTGCTTACGATGGAGCCAGAGAGGAT 0 - FANCF On target Homo sapiens NC_ TTTGGTCGGCATGGCCCCATTCGCACG 0 - Strand (Continued) Locus Forward primer Reverse primer Amplicon (bp) Predicted fragment (bp) DNMT1 On target CTGGGACTCAGGCGGGTCAC CCTCAGCCAGAAGTCCCGTGC DNMT1 OT1 AGGAAAGCCATGCCAGAGACTCA CACCGCCACTCTGTTTCCAAG DNMT1 OT2 GTTGGGACATGAAGGTCAAGTGTG TTTGTCTCCTGTTGCCTTCAGGCC DNMT1 OT3 GAGGCATAGCAAGGTCATGCCTTT TGCTTCCCTTGGTGGAGCTG DNMT1 OT4 TTTCCATGTAGGCCCATGCCC CCAGGTTACCAGCAACAGATCTC AAVS1 On target GGGCTGGCTACTGGCCTTAT ATGGCATCTTCCAGGGGTCC FANCF On target AGCTCCGCCTGGGTCTTCAT GCGGAGACGTTCATGACTGG PAM is shown in red. Mismatched bases are shown in green. NATURE BIOMEDICAL ENGINEERING DOI: /s

13 Supplementary Table 3 A list of primers used for the first round of PCR for targeted deep sequencing. Locus Forward primer * Reverse primer* DNMT1 On target TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCCTC ACTCCTGCTCGGTGAA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGTCA CTCTGGGGAACACGCC DNMT1 OT1 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACCTTT TGGGCGTGGAGAAGGG GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGAGG GGGTCAGCATGAAAGG DNMT1 OT2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTCCCC CACCCCCTAGGAAAGT GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCTTT CTGGTGGAGTGTCCCC DNMT1 OT3 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGAAGG TATAGGAGAGGTTTTGGGCT GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGACGA CCTTAGATGGAGTGTTGTGT DNMT1 OT4 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGCCAG TGGAAGGAGGGAGTGT GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCCCA GGAAGTTGCTTCTCCC * Red letters indicate the Illumina overhang adapter sequences. NATURE BIOMEDICAL ENGINEERING DOI: /s

14 Supplementary Table 4 AsCpf1: A list of index sequences used for the second round of PCR of targeted deep sequencing. Index No. Sample name Index 1 Sequence Index 2 Sequence 1 AscrWT/AsCpf1 plasmid_1_ot1 N701 TAAGGCGA N517 GCGTAAGA 2 AscrWT/AsCpf1 plasmid_2_ot1 N701 TAAGGCGA N502 CTCTCTAT 3 AscrWT/AsCpf1 plasmid_3_ot1 N701 TAAGGCGA N503 TATCCTCT 4 AscrWT/AsCpf1 plasmid_1_ot2 N701 TAAGGCGA N504 AGAGTAGA 5 AscrWT/AsCpf1 plasmid_2_ot2 N701 TAAGGCGA N505 GTAAGGAG 6 AscrWT/AsCpf1 plasmid_3_ot2 N701 TAAGGCGA N506 ACTGCATA 7 AscrWT/AsCpf1 plasmid_1_ot3 N701 TAAGGCGA N507 AAGGAGTA 8 AscrWT/AsCpf1 plasmid_2_ot3 N701 TAAGGCGA N508 CTAAGCCT 9 AscrWT/AsCpf1 plasmid_3_ot3 N702 CGTACTAG N517 GCGTAAGA 10 AscrWT/AsCpf1 plasmid_1_ot4 N702 CGTACTAG N502 CTCTCTAT 11 AscrWT/AsCpf1 plasmid_2_ot4 N702 CGTACTAG N503 TATCCTCT 12 AscrWT/AsCpf1 plasmid_3_ot4 N702 CGTACTAG N504 AGAGTAGA 13 Ascr3'5F/AsCpf1 ψ mrna_1_ot1 N702 CGTACTAG N505 GTAAGGAG 14 Ascr3'5F/AsCpf1 ψ mrna_2_ot1 N702 CGTACTAG N506 ACTGCATA 15 Ascr3'5F/AsCpf1 ψ mrna_3_ot1 N702 CGTACTAG N507 AAGGAGTA 16 Ascr3'5F/AsCpf1 ψ mrna_1_ot2 N702 CGTACTAG N508 CTAAGCCT 17 Ascr3'5F/AsCpf1 ψ mrna_2_ot2 N703 AGGCAGAA N517 GCGTAAGA 18 Ascr3'5F/AsCpf1 ψ mrna_3_ot2 N703 AGGCAGAA N502 CTCTCTAT 19 Ascr3'5F/AsCpf1 ψ mrna_1_ot3 N703 AGGCAGAA N503 TATCCTCT 20 Ascr3'5F/AsCpf1 mrna_2_ot3 N703 AGGCAGAA N504 AGAGTAGA 21 Ascr3'5F/AsCpf1 mrna_3_ot3 N703 AGGCAGAA N505 GTAAGGAG 22 Ascr3'5F/AsCpf1 ψ mrna_1_ot4 N703 AGGCAGAA N506 ACTGCATA 23 Ascr3'5F/AsCpf1 ψ mrna_2_ot4 N703 AGGCAGAA N507 AAGGAGTA 24 Ascr3'5F/AsCpf1 ψ mrna_3_ot4 N703 AGGCAGAA N508 CTAAGCCT 25 AscrWT/AsCpf1 plasmid_1_on target N704 TCCTGAGC N517 GCGTAAGA 26 AscrWT/AsCpf1 plasmid_2_on target N704 TCCTGAGC N502 CTCTCTAT 27 AscrWT/AsCpf1 plasmid_3_on target N704 TCCTGAGC N503 TATCCTCT 28 Ascr3'5F/AsCpf1 ψ mrna_1_on target N704 TCCTGAGC N504 AGAGTAGA 29 Ascr3'5F/AsCpf1 ψ mrna_2_on target N704 TCCTGAGC N505 GTAAGGAG 30 Ascr3'5F/AsCpf1 ψ mrna_3_on target N704 TCCTGAGC N506 ACTGCATA NATURE BIOMEDICAL ENGINEERING DOI: /s

15 Supplementary Table 5 LbCpf1: A list of index sequences used for the aecond round of PCR of targeted deep sequencing. Index No. Sample name Index 1 Sequence Index 2 Sequence 1 LbcrWT/LbCpf1 plasmid_1_ot1 N701 TAAGGCGA N517 GCGTAAGA 2 LbcrWT/LbCpf1 plasmid_2_ot1 N701 TAAGGCGA N502 CTCTCTAT 3 LbcrWT/LbCpf1 plasmid_3_ot1 N701 TAAGGCGA N503 TATCCTCT 4 LbcrWT/LbCpf1 plasmid_1_ot2 N701 TAAGGCGA N504 AGAGTAGA 5 LbcrWT/LbCpf1 plasmid_2_ot2 N701 TAAGGCGA N505 GTAAGGAG 6 LbcrWT/LbCpf1 plasmid_3_ot2 N701 TAAGGCGA N506 ACTGCATA 7 LbcrWT/LbCpf1 plasmid_1_ot3 N701 TAAGGCGA N507 AAGGAGTA 8 LbcrWT/LbCpf1 plasmid_2_ot3 N701 TAAGGCGA N508 CTAAGCCT 9 LbcrWT/LbCpf1 plasmid_3_ot3 N702 CGTACTAG N517 GCGTAAGA 10 LbcrWT/LbCpf1 plasmid_1_ot4 N702 CGTACTAG N502 CTCTCTAT 11 LbcrWT/LbCpf1 plasmid_2_ot4 N702 CGTACTAG N503 TATCCTCT 12 LbcrWT/LbCpf1 plasmid_3_ot4 N702 CGTACTAG N504 AGAGTAGA 13 Lbcr3'5F/LbCpf1 ψ mrna_1_ot1 N702 CGTACTAG N505 GTAAGGAG 14 Lbcr3'5F/LbCpf1 ψ mrna_2_ot1 N702 CGTACTAG N506 ACTGCATA 15 Lbcr3'5F/LbCpf1 ψ mrna_3_ot1 N702 CGTACTAG N507 AAGGAGTA 16 Lbcr3'5F/LbCpf1 ψ mrna_1_ot2 N702 CGTACTAG N508 CTAAGCCT 17 Lbcr3'5F/LbCpf1 ψ mrna_2_ot2 N703 AGGCAGAA N517 GCGTAAGA 18 Lbcr3'5F/LbCpf1 ψ mrna_3_ot2 N703 AGGCAGAA N502 CTCTCTAT 19 Lbcr3'5F/LbCpf1 ψ mrna_1_ot3 N703 AGGCAGAA N503 TATCCTCT 20 Lbcr3'5F/LbCpf1 ψ mrna_2_ot3 N703 AGGCAGAA N504 AGAGTAGA 21 Lbcr3'5F/LbCpf1 ψ mrna_3_ot3 N703 AGGCAGAA N505 GTAAGGAG 22 Lbcr3'5F/LbCpf1 ψ mrna_1_ot4 N703 AGGCAGAA N506 ACTGCATA 23 Lbcr3'5F/LbCpf1 ψ mrna_2_ot4 N703 AGGCAGAA N507 AAGGAGTA 24 Lbcr3'5F/LbCpf1 ψ mrna_3_ot4 N703 AGGCAGAA N508 CTAAGCCT 25 LbcrWT/LbCpf1 plasmid_1_on target N704 TCCTGAGC N517 GCGTAAGA 26 LbcrWT/LbCpf1 plasmid_2_on target N704 TCCTGAGC N502 CTCTCTAT 27 LbcrWT/LbCpf1 plasmid_3_on target N704 TCCTGAGC N503 TATCCTCT 28 Lbcr3'5F/LbCpf1 ψ mrna_1_on target N704 TCCTGAGC N504 AGAGTAGA 29 Lbcr3'5F/LbCpf1 ψ mrna_2_on target N704 TCCTGAGC N505 GTAAGGAG 30 Lbcr3'5F/LbCpf1 ψ mrna_3_on target N704 TCCTGAGC N506 ACTGCATA 31 Untreated_1_OT1 N705 GGACTCCT N517 GCGTAAGA 32 Untreated_2_OT1 N705 GGACTCCT N502 CTCTCTAT 33 Untreated_3_OT1 N705 GGACTCCT N503 TATCCTCT 34 Untreated_1_On target N705 GGACTCCT N504 AGAGTAGA 35 Untreated_2_On target N705 GGACTCCT N505 GTAAGGAG 36 Untreated_3_On target N705 GGACTCCT N506 ACTGCATA NATURE BIOMEDICAL ENGINEERING DOI: /s

16 Supplementary Table 6 Top ten high-frequent on-target mutagenesis from biological replicates 2 and 3 induced by CRISPR-Cpf1 system Indel pattern On-target mutagenesis induced by the combination of AscrWT and AsCpf1 plasmid_2 Read (%) WT CAGTACGTTAATGTTTCCTGATGGTCCATGTCTGTTACTCGCCTGTCAAGTGGCGTGACACCGGG 71.4 D6 CAGTACGTTAATGTTTCCTGATGGTCCATGT------CTCGCCTGTCAAGTGGCGTGACACCGGG 2.5 D6 CAGTACGTTAATGTTTCCTGATGGTCCA------TTACTCGCCTGTCAAGTGGCGTGACACCGGG 1.4 D7 CAGTACGTTAATGTTTCCTGATGGTCCA TACTCGCCTGTCAAGTGGCGTGACACCGGG 1.3 D5 CAGTACGTTAATGTTTCCTGATGGTCCATG-----TACTCGCCTGTCAAGTGGCGTGACACCGGG 1.3 D8 CAGTACGTTAATGTTTCCTGATGGTCCATG TCGCCTGTCAAGTGGCGTGACACCGGG 0.9 D6 CAGTACGTTAATGTTTCCTGATGGTCCATG------ACTCGCCTGTCAAGTGGCGTGACACCGGG 0.8 D4 CAGTACGTTAATGTTTCCTGATGGTCCA----TGTTACTCGCCTGTCAAGTGGCGTGACACCGGG 0.7 D16 CAGTACGTTAATGTTTCCTGATGGT CCTGTCAAGTGGCGTGACACCGGG 0.5 D8 CAGTACGTTAATGTTTCCTGATGGTCCAT CTCGCCTGTCAAGTGGCGTGACACCGGG 0.5 D4 CAGTACGTTAATGTTTCCTGATGGTCCA----TGTAACTCGCCTGTCAAGTGGCGTGACACCGGG 0.5 Indel pattern On-target mutagenesis induced by the combination of AscrWT and AsCpf1 plasmid_3 Read (%) WT CAGTACGTTAATGTTTCCTGATGGTCCATGTCTGTTACTCGCCTGTCAAGTGGCGTGACACCGGG 77.0 D6 CAGTACGTTAATGTTTCCTGATGGTCCATGT------CTCGCCTGTCAAGTGGCGTGACACCGGG 1.8 D6 CAGTACGTTAATGTTTCCTGATGGTCCA------TTACTCGCCTGTCAAGTGGCGTGACACCGGG 0.9 D7 CAGTACGTTAATGTTTCCTGATGGTCCA TACTCGCCTGTCAAGTGGCGTGACACCGGG 0.9 D5 CAGTACGTTAATGTTTCCTGATGGTCCATG-----TACTCGCCTGTCAAGTGGCGTGACACCGGG 0.8 D8 CAGTACGTTAATGTTTCCTGATGGTCCAT CTCGCCTGTCAAGTGGCGTGACACCGGG 0.8 D8 CAGTACGTTAATGTTTCCTGATGGTCCATG TCGCCTGTCAAGTGGCGTGACACCGGG 0.7 D16 CAGTACGTTAATGTTTCCTGATGGT CCTGTCAAGTGGCGTGACACCGGG 0.6 D4 CAGTACGTTAATGTTTCCTGATGGTCCA----TGTTACTCGCCTGTCAAGTGGCGTGACACCGGG 0.5 D7 CAGTACGTTAATGTTTCCTGATGGTCCATGTCT GCCTGTCAAGTGGCGTGACACCGGG 0.4 D4 CAGTACGTTAATGTTTCCTGATGGTCCA----TGTAACTCGCCTGTCAAGTGGCGTGACACCGGG 0.4 Indel pattern On-target mutagenesis induced by the combination of Ascr3'5F and AsCpf1 mrna (ψ)_2 Read (%) WT CAGTACGTTAATGTTTCCTGATGGTCCATGTCTGTTACTCGCCTGTCAAGTGGCGTGACACCGGG 28.0 D6 CAGTACGTTAATGTTTCCTGATGGTCCATGT------CTCGCCTGTCAAGTGGCGTGACACCGGG 5.3 D16 CAGTACGTTAATGTTTCCTGATGGT CCTGTCAAGTGGCGTGACACCGGG 4.6 D7 CAGTACGTTAATGTTTCCTGATGGTCCA TACTCGCCTGTCAAGTGGCGTGACACCGGG 3.1 D15 CAGTACGTTAATGTTTCCTGATGGTCCA TGTCAAGTGGCGTGACACCGGG 2.1 D8 CAGTACGTTAATGTTTCCTGATGGTCCAT CTCGCCTGTCAAGTGGCGTGACACCGGG 2.0 D8 CAGTACGTTAATGTTTCCTGATGGTCCATG TCGCCTGTCAAGTGGCGTGACACCGGG 2.0 D26 CAGTACGTTAATGTT CCTGTCAAGTGGCGTGACACCGGG 1.9 D6 CAGTACGTTAATGTTTCCTGATGGTCCA------TTACTCGCCTGTCAAGTGGCGTGACACCGGG 1.4 D5 CAGTACGTTAATGTTTCCTGATGGTCCATG-----TACTCGCCTGTCAAGTGGCGTGACACCGGG 1.4 D6 CAGTACGTTAATGTTTCCTGATGGTCCATG------ACTCGCCTGTCAAGTGGCGTGACACCGGG 1.2 Indel pattern On-target mutagenesis induced by the combination of Ascr3'5F and AsCpf1 mrna (ψ)_3 Read (%) WT CAGTACGTTAATGTTTCCTGATGGTCCATGTCTGTTACTCGCCTGTCAAGTGGCGTGACACCGGG 33.3 NATURE BIOMEDICAL ENGINEERING DOI: /s

17 D6 CAGTACGTTAATGTTTCCTGATGGTCCATGT------CTCGCCTGTCAAGTGGCGTGACACCGGG 7.9 D7 CAGTACGTTAATGTTTCCTGATGGTCCA TACTCGCCTGTCAAGTGGCGTGACACCGGG 4.0 D16 CAGTACGTTAATGTTTCCTGATGGT CCTGTCAAGTGGCGTGACACCGGG 3.3 D8 CAGTACGTTAATGTTTCCTGATGGTCCAT CTCGCCTGTCAAGTGGCGTGACACCGGG 3.2 D5 CAGTACGTTAATGTTTCCTGATGGTCCATG-----TACTCGCCTGTCAAGTGGCGTGACACCGGG 2.9 D8 CAGTACGTTAATGTTTCCTGATGGTCCATG TCGCCTGTCAAGTGGCGTGACACCGGG 2.1 D6 CAGTACGTTAATGTTTCCTGATGGTCCATG------ACTCGCCTGTCAAGTGGCGTGACACCGGG 1.8 D6 CAGTACGTTAATGTTTCCTGATGGTCCA------TTACTCGCCTGTCAAGTGGCGTGACACCGGG 1.6 D15 CAGTACGTTAATGTTTCCTGATGGTCCA TGTCAAGTGGCGTGACACCGGG 1.4 D4 CAGTACGTTAATGTTTCCTGAGGGTCCA----TGTAACTCGCCTGTCAAGTGGCGTGACACCGGG 1.1 Indel pattern On-target mutagenesis induced by the combination of Lbcr3'5F and LbCpf1 mrna (ψ)_2 Read (%) WT CAGTACGTTAATGTTTCCTGATGGTCCATGTCTGTTACTCGCCTGTCAAGTGGCGTGACACCGGG 44.3 D TGTCAAGTGGCGTGACACCGGG 7.5 D34 CAGTA CGCCTGTCAAGTGGCGTGACACCGGG 4.7 D15 CAGTACGTTAATGTTTCCTGATGGTCCA TGTCAAGTGGCGTGACACCGGG 3.8 D22 CAGTACGTTAATGTTTCCTGATGGTCCA TGGCGTGACACCGGG 2.8 D27 CAGTACGTTAATGTTTCCTGATGGTCCA TGACACCGGG 2.8 D16 CAGTACGTTAATGTTTCCTGATGGT CCTGTCAAGTGGCGTGACACCGGG 2.8 D TTACTCGCCTGTCAAGTGGCGTGACACCGGG 2.8 D35 CAGTACGTTAATGTTTCCTGATGGT CCGGG 1.9 D23 CAGTACGTTAATGTTTCCTGATGGTACAT GCGTGACACCGGG 1.9 D37 CAGTACGTTAAT GTGGCGTGACACCGGG 1.9 Indel pattern On-target mutagenesis induced by the combination of Lbcr3'5F and LbCpf1 mrna (ψ)_3 Read (%) WT CAGTACGTTAATGTTTCCTGATGGTCCATGTCTGTTACTCGCCTGTCAAGTGGCGTGACACCGGG 51.7 D15 CAGTACGTTAATGTTTCCTGATGGTCCA TGTCAAGTGGCGTGACACCGGG 10.1 D16 CAGTACGTTAATGTTTCCTGATGGT CCTGTCAAGTGGCGTGACACCGGG 4.9 D21 CAGTACGTTAATGTTTCCTGATG GTCAAGTGGCGTGACACCGGG 1.2 D20 CAGTACGTTAATGTTTCCTGATGGTCCAT GTGGCGTGACACCGGG 1.1 D GCCTGTCAAGTGGCGTGACACCGGG 1.1 D38 CAGTACGTT AAGTGGCGTGACACCGGG 1.0 D CTGTCAAGTGGCGTGACACCGGG 0.9 D CTGTCAAGTGGCGTGACACCGGG 0.9 D40 CAGTACGTTAATGTTTCCT ACCGGG 0.9 D55 CAGT ACCGGG 0.8 Indel-pattern numbers refer to the size of the deletions. The protospacer-adjacent motif is highlighted in red. Deletions are marked as dashes (-). Read (%) refers to the read ratio of each mutated site. WT, wild-type target sequence of the DNMT1 locus. NATURE BIOMEDICAL ENGINEERING DOI: /s

18 Supplementary Note 1. Amplicons used for targeted deep sequencing analysis. PAM is shown in red. Protospacer is underlined. DNMT1 on-target (245 bp) >NC_ Homo sapiens chromosome 19, GRCh38.p7 Primary Assembly TCCCTCACTCCTGCTCGGTGAATTTGGCTCAGCAGGCACCTGCCTCAGCTGCTCACTTGAGCCTCTGGGTCTAGAAC CCTCTGGGGACCGTTTGAGGAGTGTTCAGTCTCCGTGAACGTTCCCTTAGCACTCTGCCACTTATTGGGTCAGCTGT TAACATCAGTACGTTAATGTTTCCTGATGGTCCATGTCTGTTACTCGCCTGTCAAGTGGCGTGACACCGGGCGTGTT CCCCAGAGTGACTT DNMT1 off-target 1 (OT1, 243 bp) >NC_ Homo sapiens chromosome 7, GRCh38.p7 Primary Assembly ACCTTTTGGGCGTGGAGAAGGGGTGTAGGGAGTGTGCATGTGTGCATGCACACATGTGTGTGTTTGTGAGCATGGGC ACGTGCCTGTGAACGTGTGCTTTCCTGCTGGTCCATGTCTAATACTCTTCTTGTTGTGAGAAGACCATAGCGGCCGT GGCAACAGCAGGGACTGACATTGTTGAGAAACATGGAAGGTGTGTGAATTGTCCATTTCACACTGCTCCTTTCATGC TGACCCCCTCCC DNMT1 off-target 2 (OT2, 230 bp) >NC_ Homo sapiens chromosome 16, GRCh38.p7 Primary Assembly CTCCCCCACCCCCTAGGAAAGTCAGGTGATGGTTCAGCAAGTATCACATCGCCTCTGTAAAGGTGATAAACTGGCTG CCAGGGCCAGGGAGAGGCCATTTTCTGATGGTCCATACCTGTTACACTAAAGTGTTAATTGAATGCAGATGCCAGGG AGGAGCAACTTCCAGGGCATGTGCATCAAGAGACAAAACAGTGGAATATGTCCTGGGGACACTCCACCAGAAAGGG DNMT1 off-target 3 (OT3, 226 bp) >NC_ Homo sapiens chromosome 11, GRCh38.p7 Primary Assembly TGAAGGTATAGGAGAGGTTTTGGGCTTAAGGTTTATGTTTGTTTTTAATAAGAGAGATAGGAAATTCAATTCAAATT CTTATATATTCAGATGTTTTTGTCCGAAATTTTCCTAATTTTCTTATTGTACATGTCTGTAACTCTCACGTGTCAAG AAGGGGACTGATCACGATAAAGCACAATGGTAACTCTGTTTGAAATACACAACACTCCATCTAAGGTCGTCT DNMT1 off-target 4 (OT4, 236 bp) >NC_ Homo sapiens chromosome X, GRCh38.p7 Primary Assembly TGCCAGTGGAAGGAGGGAGTGTTACAGGTAGTTAAGCAGGCATGAGCTGGGCTGGAGAGGGCTGTCCTCCACCCACT AGGAATGTCAGGTGATGGCTCAGCAATTATCACATTGACTCTCTAAAAGTGACAAATTGGCAGCCAGTGCCAGGGAG AAGCCATTTCCTGATGGTCCACACCTGTTACACTAAAGGGTTAATTGAATGCAGATGCCAGGGAGAAGCAACTTCCT GGGCA NATURE BIOMEDICAL ENGINEERING DOI: /s

19 Supplementary Note 2. Sequencing data analysis. CRISPResso is utilized to analyze the deep sequencing data. 22 nt (T rather than U) from 3' of the PAM (5 -TTTN-3 ) is designated as crrna sequences. The amplicon containing the PAM was used for alignment. To quantify mutation events and avoid false positives, the window size is set as 10 (10 nt before and after each side of the predicted cleavage site). Minimum average reading quality (phred33 scale) and minimum single bp quality (phred33 scale) is greater than 30 and 20 (recommended values), respectively. Default values are used for other parameters. Deletion and insertion are considered as indels. NATURE BIOMEDICAL ENGINEERING DOI: /s