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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