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Development of multiplexed orthogonal base editor (MOBE) systems

Nature Biotechnology (2024)Cite this article

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Abstract

Base editors (BEs) enable efficient, programmable installation of point mutations while avoiding the use of double-strand breaks. Simultaneous application of two or more different BEs, such as an adenine BE (which converts A·T base pairs to G·C) and a cytosine BE (which converts C·G base pairs to T·A), is not feasible because guide RNA crosstalk results in non-orthogonal editing, with all BEs modifying all target loci. Here we engineer both adenine BEs and cytosine BEs that can be orthogonally multiplexed by using RNA aptamer–coat protein systems to recruit the DNA-modifying enzymes directly to the guide RNAs. We generate four multiplexed orthogonal BE systems that enable rates of precise co-occurring edits of up to 7.1% in the same DNA strand without enrichment or selection strategies. The addition of a fluorescent enrichment strategy increases co-occurring edit rates up to 24.8% in human cells. These systems are compatible with expanded protospacer adjacent motif and high-fidelity Cas9 variants, function well in multiple cell types, have equivalent or reduced off-target propensities compared with their parental systems and can model disease-relevant point mutation combinations.

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Fig. 1: A schematic overview of multiplexed orthogonal base editing.
Fig. 2: Engineering of an ABE aptamer system.
Fig. 3: Engineering of a CBE aptamer system.
Fig. 4: Characterization of optimized CBE and ABE aptamer systems.
Fig. 5: On-target and crosstalk editing efficiencies of MOBE systems and parental BEs when multiplexing CBE and ABE.
Fig. 6: On-target and crosstalk editing efficiencies of MOBEs when performing multiplexed editing followed by FACS enrichment for episomal edits.

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Data availability

NGS data are available on the National Center for Biotechnology Information Sequencing Read Archive database under project number PRJNA836633 ref. 89. Plasmids from this study will be available at Addgene (plasmids 219934–219949; https://www.addgene.org/Alexis_Komor/). Homo sapiens Hg38 was used as the reference genome for MOBEnto quantification of co-occurring edits.

Code availability

Source code for MOBEnto (quantification of haplotypes from multiplexed genome editing using next-generation sequencing data) is available at https://github.com/Komorlab/MOBEnto (ref. 90).

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Acknowledgements

We are grateful to all members of the Komor laboratory for helpful discussions. We are thankful to G. Debelouchina for use of the Neon transfection system. We would also like to thank A. Nelson, N. Chi, C. Sawyer and A. Goren. This research was supported by the University of California, San Diego and the National Institutes of Health (NIH) through grant no. 1R35GM138317 (to A.C.K) and the Research Corporation for Science Advancement through award number 28385 (to A.C.K). Q.T.C. was supported by the Molecular Biophysics Training grant, NIH grant T32 GM008326. B.L.R. was supported by the Chemistry–Biology Interface Training Program, NIH grant T32 GM112584.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA

    Quinn T. Cowan, Sifeng Gu, Brodie L. Ranzau & Alexis C. Komor

  2. Department of Medicine, Division of Pulmonary, Critical Care, Sleep Medicine, and Physiology, University of California San Diego, La Jolla, CA, USA

    Wanjun Gu & Tatum S. Simonson

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Contributions

Q.T.C. contributed to conceptualization of the research project, experimental design, data curation, data analysis and writing of the manuscript. S.G. contributed to data curation and data analysis. W.G. contributed to data analysis. B.L.R. contributed to experimental design. T.S.S. contributed to data analysis and supervision of the work. A.C.K. contributed to conceptualization of the research project, experimental design, data analysis, supervision of the work, writing of the manuscript and acquisition of the funding. All authors contributed to editing of the manuscript.

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Correspondence to Alexis C. Komor.

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Competing interests

A.C.K. is a member of the SAB of Pairwise Plants, is an equity holder for Pairwise Plants and Beam Therapeutics, and receives royalties from Pairwise Plants, Beam Therapeutics and Editas Medicine via patents licensed from Harvard University. A.C.K.’s interests have been reviewed and approved by the University of California, San Diego, in accordance with its conflict of interest policies. A.C.K. and Q.T.C. are listed as inventors in a Regents of the University of California patent application (PCT/US23/66812) on the development and application of multiplexed orthogonal base editing systems in living cells. All other authors declare no competing financial interests.

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Nature Biotechnology thanks Nozomu Yachie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 On-target and crosstalk editing efficiencies of MOBEs and parental BEs when targeted to two protospacers at distinct chromosomes.

(a–f) Editing efficiencies by the MOBE1-4 systems and the evoBE4/ABE8e and evoBE4/ABE8.20 combinations at the HIRA.0/HEK3.0 (A and D), HEK2.0/RNF2.0 (B and E), and HIRA.3/RNF2.0 (C and F) loci. HEK293T cells were treated as previously described in Fig. 5. (a–c) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target (y-axis, filled circles), C·G edited to T·A at the ABE target (y-axis, circles with X inset), C·G edited to T·A at the CBE target (x-axis, filled circles), and A·T edited to G·C at the CBE target (x-axis, circles with X inset). Dot plots and error bars represent the average and SEM for n = 3 biological replicates. (d–f) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target and C·G edited to T·A at the CBE target (on-target efficiencies only). Bar graphs and error bars represent the average and SEM for n = 3 biological replicates (each replicate is marked individually). Negative control (NC) samples are also shown, in which cells were treated identically but transfected with a non-targeting gRNA. (g) Orthogonality scores for the MOBE1-4 systems and the evoBE4/ABE8e and evoBE4/ABE8.20 combinations at the three protospacer combinations from (A) through (C). Plotted are the log2 of the ‘CBE orthogonality scores’, which we define for a given protospacer combination as the percent of total DNA sequencing reads with C·G edited to T·A at the CBE target divided by the percent of total DNA sequencing reads with C·G edited to T·A at the ABE target. The ‘ABE orthogonality scores’ are the A·T to G·C editing efficiency equivalent. Dot plots and error bars represent the average and propagation of uncertainty of the SEM for n = 3 biological replicates.

Extended Data Fig. 2 On-target and crosstalk editing efficiencies of MOBEs and parental BEs when targeted to two protospacers within the same locus.

(a–f) Editing efficiencies by the MOBE1-4 systems and the evoBE4/ABE8e and evoBE4/ABE8.20 combinations at the HEK3 (A and D), EMX1 (B and E), and RNF2 (C and F) loci. Cells were treated as previously described in Fig. 5. (a–c) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target (y-axis, solid circles), C·G edited to T·A at the ABE target (y-axis, circles with X inset), C·G edited to T·A at the CBE target (x-axis, solid circles), and A·T edited to G·C at the CBE target (x-axis, circles with X inset). Dot plots and error bars represent the average and SEM for n = 3 biological replicates. (d–f) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target and C·G edited to T·A at the CBE target (on-target efficiencies only). Bar graphs and error bars represent the average and SEM for n = 3 biological replicates at EMX1 and HEK3 or n = 4 biological replicates at RNF2 (each replicate is marked individually). Negative control (NC) samples are also shown, in which cells were treated identically but transfected with a non-targeting gRNA. (g) Orthogonality scores for the MOBE1-4 systems and the evoBE4/ABE8e and evoBE4/ABE8.20 combinations at the three protospacer combinations from (A) through (C). Plotted are the log2 of the ‘CBE orthogonality scores’, which we define for a given protospacer combination as the percent of total DNA sequencing reads with C·G edited to T·A at the CBE target divided by the percent of total DNA sequencing reads with C·G edited to T·A at the ABE target. The ‘ABE orthogonality scores’ are the A·T to G·C editing efficiency equivalent. Dot plots and error bars represent the average and propagation of uncertainty of the SEM for n = 3 biological replicates at EMX1 and HEK3 or n = 4 biological replicates at RNF2.

Extended Data Fig. 3 Quantification of indel rates for parental and MOBE systems.

(a, b) Indel introduction rates by the MOBE1-4 systems and the evoBE4/ABE8e and evoBE4/ABE8.20 combinations at the HIRA.0/HEK3.0, HEK2.0/RNF2.0, HIRA.3/RNF2.0 (A) distinct loci protospacer combinations and the HEK3, EMX1, and RNF2 (B) single-amplicon protospacer combinations. Cells were treated as previously described in Fig. 5. Plotted are the percent of total DNA sequencing reads with indels at each target site. Bar graphs and error bars represent the average and SEM for n = 3 biological replicates (for most sites) or n = 4 biological replicates at RNF2 (each replicate is marked individually). Negative control (NC) samples are also shown, in which cells were treated identically but transfected with a non-targeting gRNA.

Extended Data Fig. 4 Quantification of co-occurring orthogonal edits.

Cells were treated as previously described in Fig. 6. Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target and C·G edited to T·A at the CBE target, with no crosstalk edits when all multiplexing systems are targeted to two protospacers within the HEK3 (A), EMX1 (B), or RNF2 (C) locus. Bar graphs and error bars represent the average and SEM for n = 3 biological replicates at EMX1 and HEK3 or n = 4 biological replicates at RNF2 (each replicate is marked individually).

Extended Data Fig. 5 On-target and crosstalk editing efficiencies of MOBEs when targeted to two protospacers at distinct chromosomes followed by FACS enrichment for episomal edits.

(a–f) Editing efficiencies by the MOBE1-4 systems at the HIRA.0/HEK3.0 (A and D), HEK2.0/RNF2.0 (B and E), and HIRA.3/RNF2.0 (C and F) loci. HEK293T cells were treated as previously described in Fig. 6. (a-c) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target (y-axis, solid circles), C·G edited to T·A at the ABE target (y-axis, circles with X inset), C·G edited to T·A at the CBE target (x-axis, solid circles), and A·T edited to G·C at the CBE target (x-axis, circles with X inset). Dot plots and error bars represent the average and SEM for n = 3 biological replicates. (d-f) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target and C·G edited to T·A at the CBE target (on-target efficiencies only). Bar graphs and error bars represent the average and propagation of uncertainty of the SEM for n = 3 biological replicates (each replicate is marked individually). Negative control (NC) samples are also shown, in which cells were not transfected with any plasmid.

Extended Data Fig. 6 On-target and crosstalk editing efficiencies of MOBEs when targeted to two protospacers within the same locus followed by FACS enrichment for episomal edits.

(a-f) Editing efficiencies by the MOBE1-4 systems at the HEK3 (A and D), EMX1 (B and E), and RNF2 (C and F) loci. Cells were treated as previously described in Fig. 6. (a–c) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target (y-axis, solid circles), C·G edited to T·A at the ABE target (y-axis, circles with X inset), C·G edited to T·A at the CBE target (x-axis, solid circles), and A·T edited to G·C at the CBE target (x-axis, circles with X inset). Dot plots and error bars represent the average and SEM for n = 3 biological replicates at EMX1 and HEK3 or n = 4 biological replicates at RNF2. (d-f) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target and C·G edited to T·A at the CBE target (on-target efficiencies only). Bar graphs and error bars represent the average and SEM for n = 3 biological replicates at EMX1 and HEK3 or n = 4 biological replicates at RNF2 (each replicate is marked individually). Negative control (NC) samples are also shown, in which cells were not transfected with any plasmid.

Extended Data Fig. 7 MOBEs can be used in HeLa cells.

(a-c) Editing efficiencies by the MOBE2 and MOBE3 systems at the RNF2 (A), HEK3 (B), and HIRA.0/HEK3.0 (C) protospacer combinations. HeLa cells were transfected with plasmids encoding nCas9-NG-P2A-mCherry, tandem CP-deaminase fusions, both gRNA aptamers targeting endogenous loci, and the 2x-dead-GFP reporter. After 72 hours, the population of GFP+/mCherry+ ‘enriched’ cells was collected by FACS. Genomic loci of interest were then amplified and analyzed by NGS. Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target (y-axis, solid circles), C·G edited to T·A at the ABE target (y-axis, circles with X inset), C·G edited to T·A at the CBE target (x-axis, solid circles), and A·T edited to G·C at the CBE target (x-axis, circles with X inset). (d) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target and C·G edited to T·A at the CBE target, with no crosstalk edits when all multiplexing systems are targeted to two protospacers within the HEK3 or RNF2 loci. (e) Plotted are the percent of total DNA sequencing reads with indels at each target site. (f) Orthogonality scores for the MOBE systems at the two protospacer combinations from (A) through (C). Plotted are the log2 of the ‘CBE orthogonality scores’, which we define for a given protospacer combination as the percent of total DNA sequencing reads with C·G edited to T·A at the CBE target divided by the percent of total DNA sequencing reads with C·G edited to T·A at the ABE target. The ‘ABE orthogonality scores’ are the A·T to G·C editing efficiency equivalent. Dot plots and error bars represent the average and standard deviation for n = 3 biological replicates. Negative control (NC) samples are also shown, in which cells were treated identically but transfected with a non-targeting gRNA.

Extended Data Fig. 8 MOBEs can be used in SH-SY5Y cells.

(a-b) Editing efficiencies by the MOBE1 and MOBE3 systems at the HEK3 (A), and RNF2 (B) single-amplicon protospacer combinations. SH-SY5Y cells were electroporated with plasmids encoding nCas9-NG-P2A-mCherry, tandem CP-deaminase fusions, and both gRNA aptamers. After 72 hours, the population of transfected (mCherry+) cells was collected by FACS. Genomic loci of interest were then amplified and analyzed by NGS. Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target (y-axis, solid circles), C·G edited to T·A at the ABE target (y-axis, circles with X inset), C·G edited to T·A at the CBE target (x-axis, solid circles), and A·T edited to G·C at the CBE target (x-axis, circles with X inset). (c) Plotted are the percent of total DNA sequencing reads with A·T edited to G·C at the ABE target and C·G edited to T·A at the CBE target, with no crosstalk edits when all multiplexing systems are targeted to two protospacers within the HEK3 or RNF2 loci. (d) Plotted are the percent of total DNA sequencing reads with indels at each target site. (e) Orthogonality scores for the MOBE systems at the two protospacer combinations from (A) and (B). Plotted are the log2 of the ‘CBE orthogonality scores’, which we define for a given protospacer combination as the percent of total DNA sequencing reads with C·G edited to T·A at the CBE target divided by the percent of total DNA sequencing reads with C·G edited to T·A at the ABE target. The ‘ABE orthogonality scores’ are the A·T to G·C editing efficiency equivalent. Dot plots and error bars represent the average and standard deviation for n = 3 biological replicates. Negative control (NC) samples are also shown, in which cells were treated identically but transfected with a non-targeting gRNA.

Extended Data Fig. 9 MOBEs enable the installation of orthogonal point mutations known to cause Kallmann syndrome (OLI368).

(a) Schematic of the DNA and protein sequences of the OLI368 combination, FGFR1 c.2075A>G and DUSP6 c.545C>T, with MOBE protospacer and PAMs indicated. (b) Optimization of gRNA pairs with MOBE1 and MOBE3 to avoid bystander adenine editing in HEK293T cells. (c) Bulk editing efficiencies of the FGFR1 A7 protospacer combined with DUSP6 C7 and C8 protospacers with inset (D) to display crosstalk in HEK293T cells. (e) Editing efficiencies of MOBE3 with the optimal A7-C8 protospacer combination after enrichment with the all-in-one fluorescent reporter plasmid in HEK293T cells. (f) Editing efficiencies of MOBE3 (A7-C8 protospacers) sorted for nCas9-P2A-mCherry expression after electroporation in SH-SY5Y cells. (g) Orthogonality scores for the A7-C8 protospacer combination in all tested cell types. (h) Indel rates of MOBE3 with A7-C8 protospacer combination in all tested cell types. All values represent the mean and SEM of n = 3 or 4 independent biological replicates (shown individually).

Extended Data Fig. 10 MOBEs enable the installation of orthogonal point mutations known to cause isolated anencephaly (OLI736).

(a) Schematic of the DNA and protein sequences of the OLI736 combination, CELSR2 c.3800A > G and INVS c.725C > T, with MOBE protospacer and PAMs indicated. (b) On-target editing efficiencies of gRNA pair optimization with MOBE1 and MOBE3 in HEK293T cells. (c) Editing efficiencies of CELSR2 A6 protospacer combined with INVS C3 and C5 protospacers with inset (D) to display crosstalk in HEK293T cells. (e) Editing efficiencies of MOBE3 with the optimal A6-C5 protospacer combination sorted for nCas9-P2A-mCherry expression after electroporation in SH-SY5Y cells. (f) Orthogonality scores for the A6-C5 protospacer combination in both cell types. (g) Indel rates of MOBE3 with A6-C5 protospacer combination in both cell types. All values represent the mean and SEM of n = 3 independent biological replicates (shown individually).

Supplementary information

Supplementary Information

Supplementary Figs. 1–16 and Note 1.

Reporting Summary

Supplementary Data 1

Sequences of constructs, aptamers, protospacers and primers used.

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Cowan, Q.T., Gu, S., Gu, W. et al. Development of multiplexed orthogonal base editor (MOBE) systems. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02240-0

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