Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells.
Nucleic Acids Res
; 52(1): 141-153, 2024 Jan 11.
Article
em En
| MEDLINE
| ID: mdl-37985205
ABSTRACT
Genetic modification of specific genes is emerging as a useful tool to enhance the functions of antitumor T cells in adoptive immunotherapy. Current advances in CRISPR/Cas9 technology enable gene knockout during in vitro preparation of infused T-cell products through transient transfection of a Cas9-guide RNA (gRNA) ribonucleoprotein complex. However, selecting optimal gRNAs remains a major challenge for efficient gene ablation. Although multiple in silico tools to predict the targeting efficiency have been developed, their performance has not been validated in cultured human T cells. Here, we explored a strategy to select optimal gRNAs using our pooled data on CRISPR/Cas9-mediated gene knockout in human T cells. The currently available prediction tools alone were insufficient to accurately predict the indel percentage in T cells. We used data on the epigenetic profiles of cultured T cells obtained from transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Combining the epigenetic information with sequence-based prediction tools significantly improved the gene-editing efficiency. We further demonstrate that epigenetically closed regions can be targeted by designing two gRNAs in adjacent regions. Finally, we demonstrate that the gene-editing efficiency of unstimulated T cells can be enhanced through pretreatment with IL-7. These findings enable more efficient gene editing in human T cells.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Linfócitos T
/
Técnicas de Inativação de Genes
/
Sistemas CRISPR-Cas
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Japão