To further substantiate the biological role of DHX29 in type I IFN signaling in both human and mouse primary cells, we performed DHX29 knockout (KO) experiments in human peripheral blood mononuclear cells (PBMCs), mouse bone marrow-derived dendritic cells (BMDCs) and peritoneal macrophages using a lentiviral-based Cas9-P2A-puromycin fusion CRISPR (clustered regularly interspaced short palindromic repeats). Of note, both human and murine DHX29-sgRNA/Cas9 lentiCRISPR could efficiently knock out DHX29 in puromycin-selected bulk PBMCs, BMDCs, and peritoneal macrophages, as determined by western blot and a surveyor assay (Fig 3A–3C and S4A–S4D Fig). DHX29 KO markedly reduced TBK1 and IRF3 phosphorylation and IFN-β production in response to HMW Poly(I:C) in lyo/vec form (Vec) and EMCV, but did not affect IRF3 phosphorylation and IFN-β production in response to LMW Poly(I:C) Vec, Poly(dA:dT) or VSV in human PBMCs (Fig 3A and S4E Fig). Similar results were observed with DHX29 KO by DHX29 sgRNA/Cas9 lentiCRISPR in murine BMDCs and peritoneal macrophages (Fig 3B and 3C and S4F and S4G Fig). Thus, these results suggest that DHX29 KO markedly reduced type I IFN signaling in response to EMCV, but not VSV in immune cells, in contrast to what was observed in human airway epithelial cells . It should be noted that all our KO experiments were performed on day 3–4 post transduction, since further long-term culture (after day 4) of DHX29 KO cells caused cell death (S4H Fig), which is consistent with previous observations [31–33, 44], showing that DHX29 is essentially required for protein translation, cell proliferation and survival. Collectively, our results gathered by deploying DHX29 shRNA KD and DHX29 KO approaches in human and murine primary immune cells, clearly suggest that DHX29 is required for type I IFN signaling in response to HMW Poly(I:C) treatment and EMCV infection.