Fig. 6.

Knockout of p65 or IRF3 abrogates ISG activation upon ZIKV infection. a Schematic representation of the paired-KO strategy for NF-κB p65 or IRF3 knockout. The cleavage sites are pointed out by the scissors. Genomic DNA sequences before and after the paired-gRNA mediated cleavage and repair are shown in red. A representative Sanger sequencing peak map verifies precise re-joining of the double blunt ends. The positions of the designed primer sets for genomic PCR are shown as arrows. b Genomic DNA PCR results showing 5 colonies retrieved from p65 or IRF3 paired-KO in H9 hESCs. Clones #2, #4 and #5 are p65 KO (upper). Clones #3 and #5 are IRF3 KO (lower). c Western blot results further verify complete lack of target protein expression of clones #2, #5 of p65 KO, and #3, #5 of IRF3 KO. d Confocal images showing NPCs within FD organoids from WT, p65 KO or IRF3 KO are all Pax6⁺/Sox1⁺/Sox2⁺/Foxg1⁺/Nestin⁺/Nkx2.1⁻. Scale bar, 100 μm. e qRT-PCR analysis of ISG mRNA expression of WT, p65 KO and IRF3 KO FD organoids infected with ZIKV at MOI = 0.5 alone or with IFNAR blocking antibody (2 μg/ml, mouse IgG2a, pbi assay science), or control mouse IgG2a (2 μg/ml, mouse IgG2a, R&D) at 5 dpi as well as each mock groups. Data are presented as mean ± SEM. n = 3. Unpaired two-tailed Student’s t-test. **p < 0.01 compared to mock controls