A CRISPR-Cas9 Technique for Gene Editing in T Cells

Published: May 31, 2024

Abstract

Source: Agarwal, S., et al. Production of Human CRISPR-Engineered CAR-T Cells. J. Vis. Exp. (2021).

This video demonstrates an assay for performing gene editing in human T cells using the CRISPR-Cas9 technology. A mixture of primary CD4+ and CD8+ T cells is combined with a CRISPR-Cas9 ribonucleoprotein complex, targeting specific genes for knockout. Upon electroporation, the sgRNA guides Cas9 to the target DNA sequence, creating precise cuts. These cuts are then repaired by the cell's non-homologous end-joining mechanism, leading to gene knockout.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Designing of sgRNAs and gene disruption in primary human T cells Designing CRISPR sgRNA's NOTE: Several servers and programs facilitate the design of target-specific single guide RNAs (sgRNA). In this protocol, clustered regularly interspaced short palindromic repeats (CRISPR) sgRNAs were designed using CHOPCHOP (https…

Representative Results

Figure 1. Expansion of edited CAR-T cells and their population doublings. (A) Timeline of CRISPR editing and manufacturing in primary human CART cells. (B) Population Doublings in Mock and CRISPR-edited CD19 CAR-T cells measured using a Coulter Counter during the expansion of the CAR-T cells (n=3 healthy donors; KO=knockout) (C) …

開示

The authors have nothing to disclose.

Materials

4D-Nucleofactor Core Unit Lonza AAF-1002B
4D-Nucleofactor X-Unit Lonza AAF-1002X
Cas9-Electroporation enhancers IDT 1075915
CD4+ T cell isolation Kit StemCell technologies 15062
CD8+ T cell isolation Kit StemCell technologies 15063
Corning 0.45 micron vacuum filter/bottle Corning 430768
Corning T150 cell culture flask Millipore Sigma CLS430825
DNAeasy Blood and Tissue Kit Qiagen 69504
Glutamax supplement ThermoFisher 35050061
HEPES (1 M) ThermoFisher 15630080
huIL-15 PeproTech 200-15
huIL-7 PeproTech 200-07
Lipofectamine 2000 ThermoFisher 11668019
Opti-MEM ThermoFisher 31985062
P3 Primary cell 4D-nucleofactor X Kit L Lonza V4XP-3024
Penicilin-Streptomycin-Glutamine ThermoFisher 10378016
RPMI1640 ThermoFisher 12633012
sgRNA IDT
Spy Fi Cas9 Aldevron 9214

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記事を引用
A CRISPR-Cas9 Technique for Gene Editing in T Cells. J. Vis. Exp. (Pending Publication), e22242, doi: (2024).

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