Research ArticleGENOME EDITING

Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity

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Science Advances  30 Aug 2017:
Vol. 3, no. 8, eaao4774
DOI: 10.1126/sciadv.aao4774
  • Fig. 1 Effects of knocking out UNG on base editing product purity.

    (A) Architecture of BE3. (B) Protospacers and PAM (blue) sequences of the genomic loci tested, with the target C’s analyzed in (A) shown in red. (C) HAP1 (UNG+) and HAP1 UNG cells were treated with BE3, as described in Materials and Methods. The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown. See fig. S1 for C-to-T editing efficiencies, which generally varied between 15 and 45%. (D) Frequency of indel formation following treatment with BE3 in HAP1 or HAP1 UNG cells. Values and error bars reflect the means and SD of three independent biological replicates performed on different days. ns (not significant), P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by two-tailed Student’s t test.

  • Fig. 2 Effects of multi-C base editing on product purity.

    (A) Architectures of BE3, CDA1-BE3, and AID-BE3. (B) Representative high-throughput sequencing data of BE3-, CDA1-BE3–, and AID-BE3–treated human HEK293T cells. The sequence of the protospacer is shown at the top, with the PAM in blue and the target C’s in red, with subscripted numbers indicating their position within the protospacer. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. The relative percentage of target C’s that are cleanly edited to T rather than to non-T bases are much higher for cells treated with AID-BE3, which edits three C’s at this locus, than for cells treated with BE3, which edits only one C. (C) HEK293T cells were treated with BE3, CDA1-BE3, and AID-BE3, as described in Materials and Methods. The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown. (D) Protospacers and PAM (blue) sequences of genomic loci studied, with the target C’s analyzed in (B) shown in red. (E) Frequency of indel formation (see Materials and Methods) following the treatment in (A). Values and error bars reflect the means and SD of three independent biological replicates performed on different days. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by two-tailed Student’s t test.

  • Fig. 3 Effects of changing the architecture of BE3 on C-to-T editing efficiencies and product purities.

    (A) Architectures of BE3, SSB-BE3, N-UGI-BE3, and BE3-2xUGI. (B) Protospacers and PAM (blue) sequences of genomic loci studied, with the target C’s in (C) shown in purple and red, and the target C’s in (B) shown in red. (C) HEK293T cells were treated with BE3, SSB-BE3, N-UGI-BE3, and BE3-2xUGI, as described in Materials and Methods. The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown for BE3, N-UGI-BE3, and BE3-2xUGI. (D) C-to-T base editing efficiencies. Values and error bars reflect the means and SD of three independent biological replicates performed on different days. ns, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by two-tailed Student’s t test.

  • Fig. 4 Effects of linker length variation in BE3 on C-to-T editing efficiencies and product purities.

    (A) Architecture of BE3, BE3C, BE3D, and BE3E. (B) Protospacers and PAM (blue) sequences of genomic loci studied, with the target C’s in (C) shown in purple and red, and target C’s in (D) shown in red. (C) HEK293T cells were treated with BE3, BE3C, BE3D, or BE3E, as described in Materials and Methods. C-to-T base editing efficiencies are shown. (D) The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown for BE3, BE3C, BE3D, and BE3E. Values and error bars reflect the means and SD of three independent biological replicates performed on different days. ns, P ≥ 0.05; *P < 0.05; **P < 0.01, by two-tailed Student’s t test.

  • Fig. 5 BE4 increases base editing efficiency and product purities compared to BE3.

    (A) Architectures of BE3, BE4, and Target-AID. (B) Protospacers and PAM (blue) sequences of genomic loci studied, with the target C’s in (C) shown in purple and red, and the target C’s in (D) shown in red. (C) HEK293T cells were treated with BE3, BE4, or Target-AID, as described in Materials and Methods. C-to-T base editing efficiencies are shown. (D) The product distribution among edited DNA sequencing reads (reads in which the target C is mutated) is shown for BE3 and BE4. Values and error bars reflect the means and SD of three independent biological replicates performed on different days. ns, P ≥ 0.05; *P < 0.05; **P < 0.01, by two-tailed Student’s t test.

  • Fig. 6 Fusion with Gam from bacteriophage Mu reduces indel frequencies.

    (A) Architectures of BE3-Gam and BE4-Gam. (B) HEK293T cells were treated with BE3, BE3-Gam, BE4, BE4-Gam, SaBE3, SaBE3-Gam, SaBE4, or SaBE4-Gam, as described in Materials and Methods. C-to-T base editing efficiencies are shown. (C) Frequency of indel formation (see Materials and Methods) following the treatment in (B). (D) Product distribution among edited DNA sequencing reads (reads in which the target C is mutated). (E) Protospacers and PAM (blue) sequences of genomic loci studied, with the target Cs in (B) shown in purple and red, and the target Cs in (D) shown in red. Values and error bars of BE3-Gam, SaBE3-Gam, BE4-Gam, and SaBE4-Gam reflect the means and SD of three independent biological replicates performed on different days. Values and error bars of BE3, SaBE3, BE4, and SaBE4 reflect the means and SD of six independent biological replicates performed on different days by two different researchers. ns, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by two-tailed Student’s t test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/eaao4774/DC1

    fig. S1. Base editing efficiencies in UNG knockout cells.

    fig. S2. CDA1-BE3 and AID-BE3 edit C’s following target G’s more efficiently than BE3.

    fig. S3. Uneven editing in sites with multiple editable C’s results in lower product purity.

    fig. S4. Base editing of multiple C’s results in higher base editing product purity.

    fig. S5. Base editing of multiple C’s results in higher base editing product purity at the HEK3 and RNF2 loci.

    fig. S6. BE4 induces lower indel frequencies than BE3, and Target-AID exhibits similar product purities as CDA1-BE3.

    fig. S7. SaBE4 exhibits increased base editing yields and product purities compared to SaBE3.

    table S1. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the EMX1 locus.

    table S2. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the FANCF locus.

    table S3. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the HEK2 locus.

    table S4. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the HEK3 locus.

    table S5. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the HEK4 locus.

    table S6. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the RNF2 locus.

    note S1. Python script to detect linkage disequilibrium in base editing outcomes at target sites with multiple target cytidines.

    Supplementary Sequences. Amino acid sequences of CDA1-BE3, AID-BE3, BE3-Gam, SaBE3-Gam BE4, BE4-Gam, SaBE4, and SaBE4-Gam.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Base editing efficiencies in UNG knockout cells.
    • fig. S2. CDA1-BE3 and AID-BE3 edit C’s following target G’s more efficiently than BE3.
    • fig. S3. Uneven editing in sites with multiple editable C’s results in lower product purity.
    • fig. S4. Base editing of multiple C’s results in higher base editing product purity.
    • fig. S5. Base editing of multiple C’s results in higher base editing product purity at the HEK3 and RNF2 loci.
    • fig. S6. BE4 induces lower indel frequencies than BE3, and Target-AID exhibits similar product purities as CDA1-BE3.
    • fig. S7. SaBE4 exhibits increased base editing yields and product purities compared to SaBE3.
    • table S1. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the EMX1 locus.
    • table S2. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the FANCF locus.
    • table S3. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the HEK2 locus.
    • table S4. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the HEK3 locus.
    • table S5. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the HEK4 locus.
    • table S6. Base editing outcomes from treatment with BE3, CDA1-BE3, AID-BE3, or APOBEC3G-BE3 at the RNF2 locus.
    • note S1. Python script to detect linkage disequilibrium in base editing outcomes at target sites with multiple target cytidines.
    • Supplementary Sequences. Amino acid sequences of CDA1-BE3, AID-BE3, BE3-Gam, SaBE3-Gam BE4, BE4-Gam, SaBE4, and SaBE4-Gam.

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