CRISPR/Cas9-mediated deletion of miR-146a enhances antiviral response in HIV-1 infected cells

Abstract

The human immunodeficiency virus type 1 (HIV-1) causes persistent infection in human and induces miR-146a expression in infected cells. miR-146a represses the innate immune response by inhibiting the expression of TRAF6 and IRAK1 genes, thus negatively controls the NF-κB-related cytokines and interferon stimulated genes. Here we reported that lentiviral CRISPR/Cas9 system was highly efficient in introducing mutations in the precursor miR-146a genomic sequences, resulting in a loss of miR-146a expression and function. miR-146a ablation led to increasing cytokines production in LPS-stimulated A549 cells. Moreover, miR-146a knockout in HIV-1 infected MT2 cells markedly increased the expression of cytokines and HIV-1 restriction factors and reversed T cell exhaustion markers expression, thus influencing HIV-1 replication. Our study indicates that lentiviral CRISPR/Cas9-mediated gene editing is an effective approach to abrogate miR-146a expression, which consequently inhibits HIV-1 replication as well as proviral reactivation by enhancing the expression of cytokines and HIV-1 restriction factors.

CRISPR/Cas9-Based Antiviral Strategy: Current Status and the Potential Challenge

“…Suppression of an HIV entry by disruption of its co-receptors, CCR5 and CXCR4 is one of the most typical examples for this host-targeting antiviral approach [18,19,20]. So far, fourteen studies described inhibition of HIV infection by disrupting essential host dependency factors required for an HIV infection (Table 1) [13,14,15,16,17,18,19,20,21,22,23,24,25,35]. These CRISPR/Cas9-targeted HIV host dependency factors include HIV co-receptors, CCR5 [13,14,15,17,18,20,22,23,24,25] and CXCR4 [16,17,19,20,21,25], and microRNA-146 [35].…”

“…So far, fourteen studies described inhibition of HIV infection by disrupting essential host dependency factors required for an HIV infection (Table 1) [13,14,15,16,17,18,19,20,21,22,23,24,25,35]. These CRISPR/Cas9-targeted HIV host dependency factors include HIV co-receptors, CCR5 [13,14,15,17,18,20,22,23,24,25] and CXCR4 [16,17,19,20,21,25], and microRNA-146 [35]. For CRISPR/Cas9 delivery, most studies used a lipofectamine-based transfection [13,14,15,24,25,35] while some used a lentiviral [16,18,20,21,22,25,35], adenoviral transduction [18,21], and even electroporation [17,20,25].…”

“…These CRISPR/Cas9-targeted HIV host dependency factors include HIV co-receptors, CCR5 [13,14,15,17,18,20,22,23,24,25] and CXCR4 [16,17,19,20,21,25], and microRNA-146 [35]. For CRISPR/Cas9 delivery, most studies used a lipofectamine-based transfection [13,14,15,24,25,35] while some used a lentiviral [16,18,20,21,22,25,35], adenoviral transduction [18,21], and even electroporation [17,20,25]. In particular, three studies reported a CRISPR/Cas9-mediated simultaneous disruption of both CCR5 and CXCR4 [17,20,25].…”

“…In particular, one study demonstrated the in vivo antiviral activity of the CRISPR/Cas9 system targeting CCR5 by using NOD/Prkdc-scid/IL-2Rγnull mice [23]. Expression of a GFP reporter virus [16,17,21] or p24 viral protein [16,18,19,20,21,25], and viral RNA load [16,19,23,35] were all reduced by ablation of HIV co-receptors by CRISPR/Cas9. The elimination of microRNA-146α by CRISPR/Cas9 led to a marked increase in the expression levels of cytokines and HIV-1 restriction factors [35].…”

“…Expression of a GFP reporter virus [16,17,21] or p24 viral protein [16,18,19,20,21,25], and viral RNA load [16,19,23,35] were all reduced by ablation of HIV co-receptors by CRISPR/Cas9. The elimination of microRNA-146α by CRISPR/Cas9 led to a marked increase in the expression levels of cytokines and HIV-1 restriction factors [35]. Although one study pointed out off-target activity in a high-homology host gene such as CCR2 [15], most of the studies reported no significant off-target activity [14,16,18,19,20,21,22,24,25,35].…”