Cytosine and adenosine base editing in human pluripotent stem cells using transient reporters for editing enrichment.
Nat Protoc
; 16(7): 3596-3624, 2021 07.
Article
em En
| MEDLINE
| ID: mdl-34172975
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Adenosina
/
Genes Reporter
/
Citosina
/
Células-Tronco Pluripotentes Induzidas
/
Edição de Genes
Limite:
Humans
Idioma:
En
Revista:
Nat Protoc
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos