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

Stefan J. Tekel, Nicholas Brookhouser, Kylie Standage-Beier, Xiao Wang, David A. Brafman

Research output: Contribution to journalReview articlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)3596-3624
Number of pages29
JournalNature protocols
Volume16
Issue number7
DOIs
StatePublished - Jul 2021

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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