Role of p38a MAPK in Monkeypox Virus Replication and Neuroinvasion: Implications for Therapeutic Targeting (Human CRISPR Deletion Library Screen)

To identify key phosphatase targets associated with MPXV resistance, we employed a human CRISPR deletion library (encompassing druggable genes, kinases, and phosphatases, constructed using the lentiGuide-Puro two-vector system; Addgene 101927). We first established a stable U87MG-Cas9 cell line by transducing parental U87MG cells with a Cas9-encoding lentiviral vector, then transduced these cells with a 2,333-gene gRNA library at a multiplicity of infection (MOI) of 0.3 (to minimize single-cell integration of multiple constructs).This was followed by selection with 5 ug mL-1 puromycin for 2-3 days to enrich successfully transduced cells.Cells were subsequently infected with MPXV at an MOI of 1, cultured in 5% FBS-supplemented DMEM for 5 days, and surviving MPXV-resistant cells were harvested. Genomic DNA was isolated from these cells; sgRNA sequences were PCR-amplified using barcode-containing primers, pooled equimolarly, and deep-sequenced on the Illumina Nove6000 platform. Reads were aligned to the reference gRNA library (mismatched reads filtered out), sgRNA abundance was quantified, and candidate resistance genes were scored via the MAGeCK RRA algorithm (v0.5.6). This work identified core genetic determinants of MPXV resistance, delivering critical insights to support the development of novel anti-MPXV therapeutic targets.