A CRISPR-Cas9 Technique for Gene Editing in T Cells

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

1. 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://chopchop.cbu.uib.no), and the gRNA design portal from the Broad Institute (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design).

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 C.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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