Citation

  • Authors: Van Zeebroeck L. et al.
  • Year: 2021
  • Journal: Front Immunol 12 655122
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T

Method

gRNA Activity Testing in HEK Cells: Ten gRNAs targeting IL6RA were designed and tested in HEK293T cells for their in vitro effectiveness of creating indels as described before (35). Briefly, HEK293T cells were transfected using jetOptimus buffer (Polyplus, #117-07) with 300 ng Cas9 plasmid (pU6-(BbsI)_Cbh-Cas9-T2A-mCherry; Addgene plasmid #64324) and 150 ng OOF plasmid (pBS SK mCherryROSAegfp; Addgene plasmid #54322) according to manufacturer’s protocol and incubated at 37°C for 48 hours before flow cytometry read-out. gRNAs were considered working when at least 33% of the transfected cells were GFP+.

Abstract

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.

Pubmed