Large-scale GMP-compliant CRISPR-Cas9–mediated deletion of the glucocorticoid receptor in multivirus-specific T cells

Basar R, Daher M, Uprety N, Gokdemir E, Alsuliman A, Ensley E, Ozcan G, Mendt M, Hernandez Sanabria M, Kerbauy LN, Nunez Cortes AK, Li L, Banerjee PP, Muniz-Feliciano L, Acharya S, Fowlkes NW, Lu J, Li S, Mielke S, Kaplan M, Nandivada V, Bdaiwi M, Kontoyiannis AD, Li Y, Liu E, Ang S, Marin D, Brunetti L, Gundry MC, Turk R, Schubert MS, Rettig GR, McNeill MS, Kurgan G, Behlke MA, Champlin R, Shpall EJ, Rezvani K.
Source: Blood
Publication Date: (2020)
Issue: 4(14): 3357-3367
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Species: human
Tissue Origin: kidney
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® 96-well Systems
4D-Nucleofector® LV-Unit

For the large-scale CRISPR-Cas9 protocol, we used the 4D Nucleofector from Lonza. The program that was adopted was EO-115 and the buffer used was P3 buffer for primary cells. As in the small-scale CRISPR-Cas9 protocol, we first prepared the crRNA plus tracrRNA duplex for each crRNA by incubating them at 95°C for 5 minutes in a thermocycler at equimolar concentrations. Cas9 protein (IDT) and gRNA (crRNA plus tracrRNA combination) were then incubated at room temperature for 15 minutes. To electroporate 5 x 10^6 cells, we used 2.2 µM Cas9 and 2.4 µM gRNA. For the larger cell doses, reagents were then scaled up by dividing the cell dose of interest by 5 x 10^6 and then multiplying this number by the final amount of RNP complex used to electroporate 5 x 10^6 cells.

Off-target identification: The GUIDE-seq method was used for unbiased discovery of off-target editing events.8 In this study, HEK293 cells that constitutively express the S.p. Cas9 nuclease (“HEK293-Cas9” cells) were used as the source of Cas9. Alt-R gRNA complexes were formed by combining Alt-R tracrRNA and Alt-R crRNA XT at a 1:1 molar ratio. gRNA complexes were delivered by nucleofection using the Amaxa Nucleofector 96-well Shuttle System (Lonza). For each nucleofection, 3.5 x 10^5 HEK293-Cas9 cells were washed with 13 phosphate-buffered saline, resuspended in 20 µL of solution SF (Lonza), and combined with 10 µM gRNA together with 0.5 µM GUIDE-seq double-stranded DNA donor fragment. This mixture was transferred into 1 well of a Nucleocuvette plate (Lonza) and electroporated using protocol 96-DS-150.


Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)–grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.