Plasmid-Based Donor Templates for Nonviral CRISPR/Cas9-Mediated Gene Knock-In in Human T Cells.
Curr Protoc
; 2(9): e538, 2022 Sep.
Article
em En
| MEDLINE
| ID: mdl-36130036
ABSTRACT
Effective and precise gene editing of T lymphocytes is critical for advancing the understanding of T cell biology and the development of next-generation cellular therapies. Although methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a simple and efficient method for nonviral CRISPR/Cas9-based gene knock-in utilizing plasmid-based donor DNA templates. © 2022 Wiley Periodicals LLC. Basic Protocol 1 Purification of human CD4+ or CD8+ T cells from blood Basic Protocol 2 Activation of purified CD4+ or CD8+ T cells using TransAct CD3/CD28 agonist-conjugated nanomatrix Basic Protocol 3 Preparation of Cas9/sgRNA RNPs Basic Protocol 4 Transfection of CAS9-RNP and knock-in template into human T cells Support Protocol 1 Purity check following magnetic T cell isolation Support Protocol 2 Dextramer staining of TCR-edited T cells Support Protocol 3 Functional characterization of TCR knock-in T cells Support Protocol 4 Detection of knock-in reporter activity in CRISPR/CAS9-edited T cells.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Linfócitos T CD8-Positivos
/
Sistemas CRISPR-Cas
Tipo de estudo:
Guideline
Limite:
Humans
Idioma:
En
Revista:
Curr Protoc
Ano de publicação:
2022
Tipo de documento:
Article