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

Nat Protoc. 2021 Jul;16(7):3596-3624. doi: 10.1038/s41596-021-00552-y. Epub 2021 Jun 25.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine / metabolism*
  • Base Sequence
  • Clone Cells
  • Cryopreservation
  • Cytosine / metabolism*
  • Flow Cytometry
  • Gene Editing / methods*
  • Genes, Reporter*
  • Humans
  • Induced Pluripotent Stem Cells / metabolism*
  • Plasmids / genetics
  • RNA, Guide, CRISPR-Cas Systems
  • Reproducibility of Results
  • Single-Cell Analysis
  • Transfection

Substances

  • Cytosine
  • Adenosine