Fig. 2: Screening of sgRNA for CRISPR-edited KO of GM-CSF and anti-CD19 CART-aIL6/IL1RA with GM-CSF/TCR KO in xenograft study. | Cell Discovery

Fig. 2: Screening of sgRNA for CRISPR-edited KO of GM-CSF and anti-CD19 CART-aIL6/IL1RA with GM-CSF/TCR KO in xenograft study.

From: CRISPR-edited CART with GM-CSF knockout and auto secretion of IL6 and IL1 blockers in patients with hematologic malignancy

Fig. 2

a sgRNA candidates targeting the first exon of GM-CSF were electroporated with Cas9 protein in normal donor T cells and subsequently tested for cytokine expression via intracellular staining. b, c Normal donor T cells were engineered with CRISPR/Cas9 RNP for TCR KO (b) or GM-CSF KO (c). The edited T cells were then collected for PCR amplification of a region spanning the PAM (NGG) site targeted by sgRNA. Amplified PCR products were Sanger sequenced. PCR products amplified from edited T cells were also ligated to a pUC-19 vector and subsequently transformed into competent cells. Single colonies were picked up for preparation of plasmid DNA, which were then Sanger sequenced to reveal the genetic alteration after CRISPR editing. d Anti-CD19 CART co-expressing IL1RA and anti-IL6 scFv fused to GST/His tag were cocultured with Nalm6-GFP cells and the culture supernatant was collected for WB analysis with antibodies against human IL1RA and His tag (Lane 1). Non-transduced T cells were included as control (Lane 2). e, f Normal donor T cells were engineered with CRISPR/Cas9 RNP for GM-CSF/TCR KO and transduced with lentivector encoding 2nd generation 41BBζ CAR and anti-IL6 scFv and IL1RA: e the KO efficiency of TCR and GM-CSF; f CAR frequency and CD4/CD8 subpopulations in edited CD3– T cells. g Anti-CD19 CART cells with GM-CSF KO or WT were stimulated with Nalm6-GFP cells and analyzed by intracellular staining. The representative result of two independent experiments was shown. h Cytotoxicity of anti-CD19 CART-aIL6/IL1RA with GM-CSF/TCR KO or only TCR KO. Statistical analysis by Student’s t test showed no significance (ns). i Antitumor efficacy of anti-CD19 CART-aIL6/IL1RA with GM-CSF/TCR KO in xenograft model. Statistical analysis by Student’s t test was conducted for body weight (BW), GFP+ Nalm6 cell number, and CD45+CD3– T cell number, with Log-Rank test for survival curves. * Indicates statistical significance with P < 0.05 and ns indicates no significance.

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