Crispr-Based Editing of Human Pluripotent Stem Cells for Disease Modeling.
Stem Cell Rev Rep
; 20(5): 1151-1161, 2024 Jul.
Article
em En
| MEDLINE
| ID: mdl-38564139
ABSTRACT
The CRISPR system, as an effective genome editing technology, has been extensively utilized for the construction of disease models in human pluripotent stem cells. Establishment of a gene mutant or knockout stem cell line typically relies on Cas nuclease-generated double-stranded DNA breaks and exogenous templates, which can produce uncontrollable editing byproducts and toxicity. The recently developed adenine base editors (ABE) have greatly facilitated related research by introducing A/T > G/C mutations in the coding regions or splitting sites (AG-GT) of genes, enabling mutant gene knock-in or knock-out without introducing DNA breaks. In this study, we edit the AG bases in exons anterior to achieve gene knockout via the ABE8e-SpRY, which recognizes most expanded protospacer adjacent motif to target the genome. Except for gene-knockout, ABE8e-SpRY can also efficiently establish disease-related A/T-to-G/C variation cell lines by targeting coding sequences. The method we generated is simple and time-saving, and it only takes two weeks to obtain the desired cell line. This protocol provides operating instructions step-by-step for constructing knockout and point mutation cell lines.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Células-Tronco Pluripotentes
/
Sistemas CRISPR-Cas
/
Edição de Genes
Limite:
Humans
Idioma:
En
Revista:
Stem Cell Rev Rep
/
Stem cell reviews and reports (Online)
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China
País de publicação:
Estados Unidos