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Precision genome editing using cytosine and adenine base editors in mammalian cells

Abstract

Genome editing has transformed the life sciences and has exciting prospects for use in treating genetic diseases. Our laboratory developed base editing to enable precise and efficient genome editing while minimizing undesired byproducts and toxicity associated with double-stranded DNA breaks. Adenine and cytosine base editors mediate targeted A•T-to-G•C or C•G-to-T•A base pair changes, respectively, which can theoretically address most human disease-associated single-nucleotide polymorphisms. Current base editors can achieve high editing efficiencies—for example, approaching 100% in cultured mammalian cells or 70% in adult mouse neurons in vivo. Since their initial description, a large set of base editor variants have been developed with different on-target and off-target editing characteristics. Here, we describe a protocol for using base editing in cultured mammalian cells. We provide guidelines for choosing target sites, appropriate base editor variants and delivery strategies to best suit a desired application. We further describe standard base-editing experiments in HEK293T cells, along with computational analysis of base-editing outcomes using CRISPResso2. Beginning with target DNA site selection, base-editing experiments in mammalian cells can typically be completed within 1–3 weeks and require only standard molecular biology techniques and readily available plasmid constructs.

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Fig. 1: Overview of base editing.
Fig. 2: Key properties of base editors.
Fig. 3: Example base-editing target selection.
Fig. 4: Typical workflow and timeline of a base-editing experiment.
Fig. 5: Base editor classes and usage.
Fig. 6: Cas proteins compatible with base editing.
Fig. 7: Common base editor architectures and their key properties.
Fig. 8: Anticipated results for a typical base-editing transfection experiment in HEK293T cells with FACS sorting.

Data availability

The data that support the test findings in this study are available from the corresponding author upon reasonable request. The protein expression vector shown in Supplementary Data 1 has been deposited to Addgene. A subset of the data used to generate Fig. 8 can be found in Supplementary Data 2 and 3. Raw HTS files have been deposited to the NCBI Sequence Read Archive (PRJNA655949).

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Acknowledgements

We thank the Pattern team at the Broad Institute for data visualization assistance and preparation of the associated website; K. Zhao, S.Miller, B. Mok and T. Blum for helpful discussions; A. Vieira for assistance editing the manuscript; and all Liu laboratory members and alumni who contributed to the development of these methods. This work was supported by US NIH U01 AI142756, RM1 HG009490, R35 GM118062 and HHMI. G.A.N. was supported by a Helen Hay Whiteney post-doctoral fellowship.

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Authors

Contributions

T.P.H., G.A.N. and D.R.L. developed the protocol. T.P.H. and G.A.N. designed and performed the test experiments and computational analyses. D.R.L. designed and supervised the test experiments. T.P.H. and D.R.L. drafted the manuscript, and all authors contributed to editing the manuscript.

Corresponding author

Correspondence to David R. Liu.

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Competing interests

The authors declare competing financial interests. D.R.L. is a consultant and co-founder of Prime Medicine, Beam Therapeutics, Pairwise Plants and Editas Medicine, companies that use genome editing. The authors are co-inventors on patent applications on base editing.

Additional information

Peer review information Nature Protocols thanks Sangsu Bae, Zhanjun Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Komor, A. C. et al. Nature 533, 420–424 (2016): https://doi.org/10.1038/nature17946

Gaudelli, N. M. et al. Nature 551, 464–471 (2017): https://doi.org/10.1038/nature24644

Arbab, M. et al. Cell 182, 463–480.e430 (2020): https://doi.org/10.1016/j.cell.2020.05.037

Anzalone, A. V. et al. Nat. Biotechnol. 38, 824–844 (2020): https://doi.org/10.1038/s41587-020-0561-9

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Tables 1–6

Additional information on base editor components and a calculator for determining HTS library concentrations.

Supplementary Data 1

A plasmid map annotated with example primer sequences (Table 2) for cloning a base editor into a protein expression vector

Supplementary Data 2

Sequencing files associated with the data shown in Fig. 8, d and e. The raw fastq files are provided, along with a CRISPRessoBatch parameter file for each amplicon in the pre-processing folder. Successfully analyzed files can be found in the post-processing folder.

Supplementary Data 3

Sequencing files associated with the data shown in Fig. 8, f and g. The raw fastq files are provided, along with a CRISPRessoBatch parameter file for each amplicon. Fastq files are named by the delivery method used.

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Huang, T.P., Newby, G.A. & Liu, D.R. Precision genome editing using cytosine and adenine base editors in mammalian cells. Nat Protoc 16, 1089–1128 (2021). https://doi.org/10.1038/s41596-020-00450-9

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