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Cytosine and adenosine base editing in human pluripotent stem cells using transient reporters for editing enrichment

Abstract

Deaminase fused-Cas9 base editing technologies have enabled precise single-nucleotide genomic editing without the need for the introduction of damaging double-stranded breaks and inefficient homology-directed repair. However, current methods to isolate base-edited cell populations are ineffective, especially when utilized with human pluripotent stem cells, a cell type resistant to genome modification. Here, we outline a series of methods that employ transient reporters of editing enrichment (TREE) to facilitate the highly efficient single-base editing of human cells at precise genomic loci. Briefly, these transient reporters of editing enrichment based methods employ a transient episomal fluorescent reporter that allows for the real-time, flow-cytometry-based enrichment of cells that have had single nucleotide changes at precise genomic locations. This protocol details how these approaches can enable the rapid (~3–4 weeks) and efficient (clonal editing efficiencies >80%) generation of biallelic or multiplexed edited isogenic hPSC lines using adenosine and cytosine base editors.

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Fig. 1: Schematic of TREE reporters and TREE-based enrichment strategies.
Fig. 2: Overview of protocol to employ TREE-based methods for the generation of isogenic hPSC lines.
Fig. 3: sgRNA design and cloning into targeting vector.
Fig. 4: Representative fluorescent microscopy analysis of hPSCs after transfection with TREE plasmids.
Fig. 5: Representative flow cytometry analysis of hPSCs after transfection with TREE plasmids.
Fig. 6: Representative analysis of editing in bulk sorted hPSCs.
Fig. 7: Representative data from generation of clonal isogenic hPSCs.
Fig. 8: Phenotypic characterization of clonal isogenic hPSCs.

Data availability

No new data were generated or analyzed with this protocol. All presented data were previously published26,31,32.

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Acknowledgements

Funding for this work was provided by the National Institutes of Health (R01GM121698 to D.A.B, R21AG056706 to D.A.B, R01GM106081 to X.W.) and the Arizona Biomedical Research Commission (ADHS16-162401 to D.A.B). N.B. was supported by a fellowship from the International Foundation for Ethical Research. We would like to thank the ASU Biodesign Flow Cytometry core for assistance with flow-cytometry-related experiments.

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All authors contributed equally to this work. S.J.T, K.S.B., N.B. and D.A.B. wrote the paper. K.S.B. generated figures. S.J.T. and N.B. designed sample data. X.W. and D.A.B. oversaw the writing and layout of the manuscript. All authors commented on the manuscript.

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Correspondence to Xiao Wang or David A. Brafman.

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S.J.T., N.B., K.S.B., X.W. and D.A.B. are coinventors on a provisional patent application.

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

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Key references using this protocol

Standage-Beier, K. et al. Nucl. Acids Res. 47, e120 (2019): https://doi.org/10.1093/nar/gkz713

Brookhouser, N. et al. Stem Cell Reports. 14, 184–191 (2020): https://doi.org/10.1016/j.stemcr.2019.12.013

Brookhouser, N. et al. BMC Biol. 18, 193 (2020): https://doi.org/10.1186/s12915-020-00929-7

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Tekel, S.J., Brookhouser, N., Standage-Beier, K. et al. Cytosine and adenosine base editing in human pluripotent stem cells using transient reporters for editing enrichment. Nat Protoc 16, 3596–3624 (2021). https://doi.org/10.1038/s41596-021-00552-y

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