Optimized base editors enable efficient editing in cells, organoids and mice (2024)

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Accession codes Primary accessions References Acknowledgements Author information Authors and Affiliations Contributions Corresponding author Ethics declarations Competing interests Integrated supplementary information Supplementary Figure 1 Optimized editing constructs show equivalent generation of viral particles and transduction of target cells Supplementary Figure 2 Codon usage across different Cas9 variants Supplementary Figure 3 RA increases target base editing in transfection assays and improves the ratio of desired to non-desired target editing Supplementary Figure 4 Optimizing the coding sequence of high-fidelity and PAM variant Cas9 enzymes improves protein expression Supplementary Figure 5 N-terminal Nuclear Localization Signal (NLS) sequences increase the efficiency and range of base editing Supplementary Figure 6 Expression of optimized base editors in stable NIH/3T3 and DLD1 cells Supplementary Figure 7 FNLS increases target base editing, the ratio of desired vs non-desired editing Supplementary Figure 8 FNLS increases editing and optimized BE4Gam reduces indel frequency in human cells Supplementary Figure 9 Optimized BE4Gam reduces non-desired base editing compared to FNLS Supplementary Figure 10 Editing at off target sites Supplementary Figure 11 2X increases the range of editing in human and mouse cells Supplementary Figure 12 Base editing induced mutational activation of CTNNB1 enables outgrowth following Tankyrase and MEK inhibition Supplementary Figure 13 Multiplexed editing in organoids, and dox-inducible base editing in mESCs Supplementary Figure 14 xFNLS and xF2X show increased editing in human cell lines Supplementary Figure 15 Lentiviral vectors generated in this manuscript Supplementary information Supplementary Text and Figures Life Sciences Reporting Summary (PDF 133 kb) Supplementary Tables Rights and permissions About this article Cite this article

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References

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Acknowledgements

This work was supported by a project grant from the NIH/NCI (CA195787-01), a U54 grant from the NIH/NCI (U54OD020355), a project grant from the Starr Cancer Consortium (I10-0095), a Research Scholar Award from the American Cancer Society (RSG-17-202-01), and a Stand Up to Cancer Colorectal Cancer Dream Team Translational Research Grant (SU2C-AACR-DT22-17). Stand Up to Cancer is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, a scientific partner of SU2C. M.P.Z. is supported in part by National Cancer Institute (NCI) grant NIH T32 CA203702. E.M.S. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD–PhD Program and an F31 Award from the NCI/NIH under grant number 1 F31 CA224800-01. E.R.K. is supported by an F31 NRSA predoctoral fellowship from the NCI/NIH under award number F31CA192835. F.J.S.-R. was supported by the MSKCC TROT program (5T32CA160001) and is supported as an HHMI Hanna Gray Fellow. S.W.L. is supported as the Geoffrey Beene Chair of Cancer Biology and as an Investigator of the Howard Hughes Medical Institute. D.F.T. is supported by the Helmholtz Association (VH-NG-1114) and by the German Research Foundation (DFG) project B05, SFB/TR 209 'Liver Cancer'. L.E.D. was supported by a K22 Career Development Award from the NCI/NIH (CA 181280-01). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We thank H. Varmus (Weill Cornell Medicine) for providing cells.

Author information

Author notes

  1. Maria Paz Zafra and Emma M Schatoff: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, New York, USA

    Maria Paz Zafra,Emma M Schatoff,Alyna Katti,Miguel Foronda,Amber Simon,Teng Han,Sukanya Goswami,Emma Montgomery&Lukas E Dow

  2. Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD–PhD program, New York, New York, USA

    Emma M Schatoff

  3. Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medicine, New York, New York, USA

    Alyna Katti,Teng Han,Jordana Thibado&Lukas E Dow

  4. Helmholtz-University Group 'Cell Plasticity and Epigenetic Remodeling', German Cancer Research Center (DKFZ) and Institute of Pathology, University Hospital, Heidelberg, Germany

    Marco Breinig,Anabel Y Schweitzer&Darjus F Tschaharganeh

  5. Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Edward R Kastenhuber,Francisco J Sánchez-Rivera&Scott W Lowe

  6. Gerstner Sloan Kettering Graduate School of Biomedical Sciences, New York, New York, USA

    Edward R Kastenhuber

  7. Cold Spring Harbor Laboratory, New York, New York, USA

    Junwei Shi&Christopher R Vakoc

  8. Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Junwei Shi

  9. Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Scott W Lowe

  10. Department of Biochemistry, Weill Cornell Medicine, New York, New York, USA

    Lukas E Dow

Authors

  1. Maria Paz Zafra

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  2. Emma M Schatoff

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  3. Alyna Katti

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  4. Miguel Foronda

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  5. Marco Breinig

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  6. Anabel Y Schweitzer

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  7. Amber Simon

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  8. Teng Han

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  9. Sukanya Goswami

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  10. Emma Montgomery

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  11. Jordana Thibado

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  12. Edward R Kastenhuber

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  13. Francisco J Sánchez-Rivera

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  14. Junwei Shi

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  15. Christopher R Vakoc

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  16. Scott W Lowe

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  17. Darjus F Tschaharganeh

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  18. Lukas E Dow

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Contributions

M.P.Z. and E.M.S. performed experiments, analyzed data, and wrote the paper. A.K., M.F., A.S., S.G., E.M., T.H., J.T., and F.J.S.-R. performed experiments and analyzed data. M.B. and A.Y.S. performed and analyzed in vivo experiments. D.F.T. designed and supervised in vivo experiments. E.R.K. performed computational analysis of MSKCC IMPACT data. J.S., S.W.L., and C.R.V. supplied critical previously unreported reagents. L.E.D. performed and supervised experiments, analyzed data, and wrote the paper.

Corresponding author

Correspondence to Lukas E Dow.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Optimized editing constructs show equivalent generation of viral particles and transduction of target cells

a. Concentration of viral particles (IU/ml) present in supernatants from all base editing lentiviral constructs. b. Number of genomic integrations of each lentiviral construct (prior to puro selection), as measured by a Taqman copy number assay to detect the puro resistance (Pac) gene. c. Number of live NIH/3T3 cells at day 3 of puro selection. All graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent experiments; statistics calculated using a two-way ANOVA with Tukey’s correction for multiple testing. No significant differences in either a or b; p>0.05.

Supplementary Figure 2 Codon usage across different Cas9 variants

a. Plots show frequency of codons across each of the 20 amino acids in different Cas9 variants. Green represents the most commonly used codon across all human genes. Red represents codons that are present in human genes less than 50% of the time that would be expected by chance. Grey represents codons that are neither the most frequent nor underrepresented. b. Percentage of favored, disfavored, and neutral codons across different Cas9 sequences.

Supplementary Figure 3 RA increases target base editing in transfection assays and improves the ratio of desired to non-desired target editing

Frequency (%) of C>T conversion and indel formation in co-transfected HEK293T cells with BE3 or RA, and FANCF.S1 (a) or CTNNB1.S45 (b) sgRNAs. Graphs show mean values. Error bars indicate s.e.m., n = 4 biologically independent experiments, asterisks (*) indicate a significant difference (p<0.05) between groups, using a two-way ANOVA with Sidak’s correction for multiple testing. c. Frequency (%) of unwanted target modifications (indels, C>A, C>G) in BE3 or RA expressing 3T3 cells generated with the PGK-Puro lentiviral vector. Graph shows mean values +/- s.e.m., n=3 biologically independent experiments. d. Relative increase in target base editing in RA-expressing lines, compared to BE3 cells. Error bars represent s.e.m., n = 12 different target cytosines among 5 different sgRNAs, includes values from day 2 and day 6; asterisks (*) indicate a significant difference (p<0.05) between groups, using a one-way ANOVA with Tukey’s correction for multiple testing.

Supplementary Figure 4 Optimizing the coding sequence of high-fidelity and PAM variant Cas9 enzymes improves protein expression

a. Giemsa stained NIH/3T3 cells following transduction with P2A-Puro lentiviruses, as indicated, and selection in puro for 6 days. Experiment was repeated 3 times with similar results. b. Flow cytometry plots showing fluorescence of GFP linked to original and optimized HF1, PAM variant, and BE3 enzymes. While most cells expressing optimized versions show much higher GFP fluorescence, a small fraction show low levels of GFP expression. This is likely due to integration-site specific effects on EF1-mediated transcription. c. Quantitation of mean GFP fluorescence intensity from original and optimized HF1, PAM variant, and BE3 enzymes. Error bars represent s.e.m., n = 3 biologically independent experiments.

Supplementary Figure 5 N-terminal Nuclear Localization Signal (NLS) sequences increase the efficiency and range of base editing

a. Schematic showing location of NLS sequences and linker size in each construct tested. To provide a fair comparison, each of the constructs shown carries the original (non-optimized) cDNA sequence. b. Frequency (%) of C>T conversion in co-transfected HEK293T cells with BE3, 2X, FNLS, FLAGlink, or BE4 CMV vectors and either FANCF.S1 or CTNNB1.S45 sgRNAs, as indicated. Graphs show mean values. Error bars represent s.e.m., n = 2–6 biologically independent experiments, as indicated; asterisks (*) indicate a significant difference (p<0.05) between groups, using a two-way ANOVA with Tukey’s correction for multiple testing. c. Frequency (%) of C>T conversion in the last edited cytosine relative to the first edited cytosine for each construct co-transfected with either FANCF.S1 or CTNNB1.S45 sgRNAs. Graphs show mean values. Error bars represent s.e.m., n=2-6 biologically independent experiments, as indicated; first number refers to FANCF.S1, the second to CTNNB1.S45. The BE3 condition for FANCF.S1 could not be calculated for more than one replicate as the other two showed zero editing at C11. Asterisks (*) indicate a significant difference (p<0.05) between groups, using a two-way ANOVA with Tukey’s correction for multiple testing.

Supplementary Figure 6 Expression of optimized base editors in stable NIH/3T3 and DLD1 cells

a. Immunoblot showing editor expression from PGK-Puro and P2A-Puro vectors in NIH/3T3 cells. b. Immunoblot showing editor expression from PGK-Puro and P2A-Puro vectors in DLD1 cells. c. Relative mRNA abundance of RA, 2X, and FNLS editors in NIH/3T3 stable cell lines. Graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent experiments; no significant differences (p<0.05) between any of the groups, using a one-way ANOVA with Tukey’s correction for multiple testing. d. Immunoblot showing expression of each optimized editor in NIH/3T3s, relative to Cas9. Each blot was repeated at least two times with similar results.

Supplementary Figure 7 FNLS increases target base editing, the ratio of desired vs non-desired editing

a. Frequency (%) of C>T conversion in NIH/3T3 cells transduced with RA- or FNLS-P2A-Puro lentiviral vectors 2 days following introduction of different sgRNAs, as indicated. Editing in BE3-PGK-Puro cells (from Figure 1e) is shown for comparison. Graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent experiments; asterisks (*) indicate a significant difference (p<0.05) between groups, using a two-way ANOVA with Tukey’s correction for multiple testing. b. Frequency (%) of unwanted target modifications (indels, C>A, C>G) in RA and FNLS expressing 3T3 cells generated with the P2A-Puro lentiviral vector. Graphs shows mean values +/- s.e.m.; n=3 biologically independent experiments. c. Relative change in base editing in FNLS-expressing lines, compared to RA cells. Graphs show mean values. Error bars represent s.e.m., n = 12 target cytosines across 5 different sgRNAs, includes day 2 and day 6; asterisks (*) indicate a significant difference (p<0.05) between groups, using an ANOVA with Tukey’s correction for multiple testing.

Supplementary Figure 8 FNLS increases editing and optimized BE4Gam reduces indel frequency in human cells

a. Frequency (%) of C>T conversion in H23 and DLD1 cells transduced with BE3-PGK-Puro, FNLS or BE4GamRA-P2A-Puro lentiviral vectors 6 days following introduction of sgRNAs targeting either FANCF.S1 or CTNNB1.S45. Graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent experiments (n=2 for BE4Gam in H23 cells); asterisks (*) indicate a significant difference (p<0.05) between groups, using an ANOVA with Tukey’s correction for multiple testing. In cases where cultures were not completely transduced with sgRNA (due to incomplete antibiotic selection), editing was normalized to the percentage of tdTomato positive cells, as measured by flow cytometry at the time of collection. b. Frequency (%) of indels in DLD1, PC9, and, H23 cells expressing either BE3, RA, FNLS, or BE4Gam and infected with sgRNAs targeting either FANCF.S1 or CTNNB1.S45. Graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent experiments (n=2 for BE4Gam in H23 cells), asterisks (*) indicate a significant difference (p<0.05) between groups, using an ANOVA with Tukey’s correction for multiple testing.

Supplementary Figure 9 Optimized BE4Gam reduces non-desired base editing compared to FNLS

Frequency (%) of unwanted target modifications (C>A, C>G) in DLD1, PC9, and H23 cells expressing either BE3, FNLS, of BE4Gam and infected with sgRNAs targeting either FANCF.S1 or CTNNB1.S45. Graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent experiments.

Supplementary Figure 10 Editing at off target sites

a. Frequency (%) of C>T conversion of any C in the editing window at two predicted off target sites for FANCF.S1 and CTNNB1.S45 in DLD1 cells expressing BE3, RA, or FNLS. Graph shows mean values. Error bars represent s.e.m., n = 3 biologically independent experiments. b. Sanger sequencing chromatograms showing detectable off target editing for the Apc.492 sgRNA (indicated by blue arrowheads) in NIH/3T3 cells. No editing was detected for either of two predicted off-target sites for Apc.1405, or the top predicted off-target site for Pik3ca.545. We were unable to amplify the Pik3ca_OT2 target region from genomic DNA. Bases highlighted green represent the target cytosine, while bases in black represent mismatches to the perfect sgRNA target site. Chromatograms are representative of three independent experiments, each with similar results.

Supplementary Figure 11 2X increases the range of editing in human and mouse cells

a. Frequency (%) of C>T conversion in NIH/3T3 cells transduced with RA- or FNLS-P2A-Puro lentiviral vectors 2 and 6 days following introduction of different sgRNAs, as indicated. Editing in BE3-PGK-Puro cells (from Figure 1e) is shown for comparison. Graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent experiments; asterisks (*) indicate a significant difference (p<0.05) between groups, using a two-way ANOVA with Tukey’s correction for multiple testing. b. Frequency (%) of unwanted target modifications (indels, C>A, C>G) in RA or 2X expressing NIH/3T3 cells at Day 6. Graph shows mean values. Error bars represent s.e.m., n=3 biologically independent experiments. c and d. Frequency (%) of target C>T conversion in DLD1 cells expressing either BE3, RA, or 2X, and infected with sgRNAs targeting FANCF.S1 (c) or CTNNB1.S45 (d). e. Frequency (%) of target C>T conversion in NIH/3T3 cells expressing either BE3, BE3RA, or 2X, and infected with an sgRNA targeting (mouse) Ctnnb1.S45. Graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent experiments; asterisks (*) indicate a significant difference (p<0.05) between groups, using a two-way ANOVA with Tukey’s correction for multiple testing.

Supplementary Figure 12 Base editing induced mutational activation of CTNNB1 enables outgrowth following Tankyrase and MEK inhibition

a. Schematic overview of the fluorescence-based competitive proliferation assay. Parental cells are shown in gray, transduced cells (tdTomato+) are in red, and cells bearing the target editing are highlighted in blue. Neutral competition keeps both tdTomato+ and tdTomato- cell proportions constant, whereas positive or negative selection causes the tdTomato+ population to increase or decrease, respectively. b. BE3, RA, 2X, and FNLS-expressing DLD1 cells were transduced with CTNNB1.S45 sgRNAs and treated with DMSO (left) or XAV939 1uM + Trametinib 10nM (right). Graph shows the number of tdTomato+ cells relative to the start of the assay. Bars represents measurements every 5 days (0, 5, 10, 15). Graphs show mean values. Error bars represent s.e.m., n = 3 biologically independent experiments; asterisks (*) indicate a significant difference (p<0.05) between groups, using an ANOVA with Tukey’s correction for multiple testing. c. Same as in b. but using FANCF.S1 (control) sgRNA. Note the neutral impact on relative proliferation in all the conditions, in contrast to CTNNB1.S45.

Supplementary Figure 13 Multiplexed editing in organoids, and dox-inducible base editing in mESCs

a. Images show FNLS/Apc.1405 and FNLS/Apc.1405/Pik3ca.545 transfected organoids, following selection by RSPO1 withdrawal and treatment with 25nM Trametinib for 5 days. b. Sanger sequencing chromatograms of the Pik3ca target locus, showing enrichment of the Pik3caE545K mutation following selection with Trametinib. Multiplexed editing and MEK inhibitor selection experiments were repeated on three independent occasions with similar results. c. Sanger sequencing chromatogram showing inducible base-editing in the presence of doxycycline (dox) in mouse ES cell lines transduced with either Apc.1405 or Pi3kca.545 sgRNAs. Base editing only occurs in cells expressing RA. Chromatograms representative of experiments repeated at least two times with similar results.

Supplementary Figure 14 xFNLS and xF2X show increased editing in human cell lines

a. Immunoblot showing expression levels of different base editor variants in PC9 cells. Sanger sequencing chromatograms showing editing 6 days following introduction of FANCF.S1 or CTNNB1.S45 sgRNAs (cytosines highlighted in green) in human PC9 (b) or DLD1 (c) cells expressing stably expressing FNLS, xBE3, xF2X, or xFNLS. xFNLS and xF2X enhance editing relative to xBE3 but are not as effective as FNLS containing the original Cas9 sequence. As expected, xF2X markedly increases editing at cytosine 10 of the CTNNB1 target site, as noted for 2X. Chromatograms represent a single experiment performed in parallel with both cell lines.

Supplementary Figure 15 Lentiviral vectors generated in this manuscript

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Optimized base editors enable efficient editing in cells, organoids and mice (1)

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Zafra, M., Schatoff, E., Katti, A. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat Biotechnol 36, 888–893 (2018). https://doi.org/10.1038/nbt.4194

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Optimized base editors enable efficient editing in cells, organoids and mice (2024)
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