BIG-TREE: Base-Edited Isogenic hPSC Line Generation Using a Transient Reporter for Editing Enrichment.
Stem Cell Reports
; 14(2): 184-191, 2020 02 11.
Article
em En
| MEDLINE
| ID: mdl-32004495
ABSTRACT
Current CRISPR-targeted single-nucleotide modifications and subsequent isogenic cell line generation in human pluripotent stem cells (hPSCs) require the introduction of deleterious double-stranded DNA breaks followed by inefficient homology-directed repair (HDR). Here, we utilize Cas9 deaminase base-editing technologies to co-target genomic loci and an episomal reporter to enable single-nucleotide genomic changes in hPSCs without HDR. Together, this method entitled base-edited isogenic hPSC line generation using a transient reporter for editing enrichment (BIG-TREE) allows for single-nucleotide editing efficiencies of >80% across multiple hPSC lines. In addition, we show that BIG-TREE allows for efficient generation of loss-of-function hPSC lines via introduction of premature stop codons. Finally, we use BIG-TREE to achieve efficient multiplex editing of hPSCs at several independent loci. This easily adoptable method will allow for the precise and efficient base editing of hPSCs for use in developmental biology, disease modeling, drug screening, and cell-based therapies.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Genes Reporter
/
Células-Tronco Pluripotentes
/
Edição de Genes
Limite:
Humans
Idioma:
En
Revista:
Stem Cell Reports
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos