Editing the VACV Genome using CRISPR-Cas9

The pCMV-Cas9 vector expressing the Streptococcus pyogenes Cas9 protein was purchased from Sigma-Aldrich. The nuclear localization signal (NLS) sequence (PKKKRKV) fused to the C-terminal extremity of Cas9 was removed by PCR-directed mutagenesis of pCMV-Cas9 using primers described in Table 1.
The pU6-gRNA plasmids encoding guide RNAs targeting the E9L gene (Table 2) were obtained from Sigma-Aldrich. To avoid off-target cleavage in the VACV genome, gRNA sequences were submitted to the Cas-OFFinder algorithm [15 (link)]. pCMV-Cas9ΔNLS and pU6-gRNA were amplified in DH5 α E. coli.
The VACV (Copenhagen strain) E9L gene was cloned into the pGEM-T vector (Promega, Charbonnières, France) with primers described in Table 1. The various mutations introduced into the E9L gene were generated by PCR-based site-directed mutagenesis (Table 1). pGEM-T-E9L constructs were amplified in JM109 cells (Promega).