FIG 2.

The restriction of VACV by CHO cells can be abolished by knocking out chSAMD9L. (A to D) CHO cells were transduced with lentiviral vectors expressing Cas9 and a guide sequence targeting either chSAMD9 (A and B) or chSAMD9L (C and D). For each gene, two independent KOs with different guide sequences (1 [A and C] and 2 [B and D]) were performed, and the KO cell pools were infected with the panel of vK1⁻C7⁻-derived VACVs at a multiplicity of infection (MOI) of 1 PFU/cell. Viral titers at 0 and 24 h postinfection (hpi) were measured by plaque assay in Vero cells. (E) Several KO cell clones were isolated from the cell pools. Infection studies were performed on the cell clones, and the results were similar to those shown in panels A to D. Representative data with the ΔSAMD9-1D clone and the ΔSAMD9L-2F clone are shown. (F) The genotypes of the cell clones were determined by sequencing. The guide sequence is underlined, and the protospacer-adjacent motif (PAM) sequence is in bold italics. The starting and ending positions of the guide in the ORF and the encoded amino acid sequence are also shown. Shown below the target are the genomic sequences from cell clones. Red lines, deletions; ^, insertion. The numbers after the + and − denote the number of indels, and the number before the “x” denotes the number of times the sequence was detected from a total of 10 to 20 cloned PCR products.