Figure 2.

Y239H is important for KKH-SaCas9-SAV2’s editing specificity and activity.(A) KKH-SaCas9-SAV2 carrying a Y239R substitution and/or additional mutations were individually constructed and characterized using GFP disruption assays. OVCAR8-ADR cells harboring reporter constructs with two on-target sgRNAs and three off-target sgRNAs were infected with lentiviruses encoding the individual KKH-SaCas9 mutants. After 7- and 15- day post-infection, the editing efficiency of the KKH-SaCas9 variants was measured as the percentage of cells with depleted GFP fluorescence using flow cytometry. Mean and standard deviation obtained from at least two biological replicates are shown. (B) Molecular modelling of Y239H/R mutations in SaCas9 depicts their differential interactions with F418 of SaCas9 and the sgRNA backbone.