Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses

Barnard, Karen N.; Wasik, Brian R.; LaClair, Justin R.; Buchholz, David W.; Weichert, Wendy S.; Alford-Lawrence, Brynn K.; Aguilar, Hector C.; Parrish, Colin R.

Cell line mutations and characterization. Two methods for utilizing CRISPR-Cas9 were used. For A549 and MDCK cells, paired Cas9 plasmids (PX459; Addgene plasmid 62988) targeted adjacent sites in early exons of CasD1, as diagrammed in Fig. 4A . Plasmids were transfected using TransIT-X2 (Mirus Bio LLC) (8) . For HEK-293 cells, nickase Cas9 plasmids (PX462; Addgene plasmid 62987) were used instead. Transfected cells were selected with puromycin, and single cell clones were screened with PToV-P4 HE-Fc to identify nonstaining variants. Edited sequences were confirmed by PCR amplification of the targeted regions and sequencing the PCR product for each allele. Knockout cell lines were used to prepare overexpression cell lines by transfection of a pcDNA3.1(-) plasmid expressing the complete human CasD1 cDNA open reading frame synthesized by Bio Basic (Markham, Ontario, Canada). Transfected cells were selected with G418, and single-cell clones were screened by staining with PToV-P4 HE-Fc to identify 9-O-Ac positive cell lines. Editing of the SIAE gene followed a protocol for CRISPR-Cas9 similar to that described above, and the gene regions targeted are shown in Fig. 5A . After transfection and selection, cells were cloned, and single-cell clones were screened by direct PCR amplification of the target gene region and analysis of the PCR product size for the edited form of both alleles. Full sequencing of each allele and qPCR were performed to confirm deletion of the gene.