Fig. 8: Cas9/gRNA complex activity is highly dependent on genomic context.

a A given gRNA can be expected to have different activity in different host organisms in a sequence specific manner. Cas9 dependent factors that are independent of host context present an orthogonal axis of activity. b Therefore, current algorithms trained on gRNA sequence features can perform well within the same context but will not accurately predict other species. However, the impact of context on gRNA activity can be reduced through c increasing the specificity of Cas9 PAM binding to reduce potential interactions at non-target sites (data from Moreb et al. 2020, each boxplot represents n > = 209 gRNA) and d increasing the expression of Cas9 and/or gRNA (data from Kim et al. 2019 and Kim et al. 2020b on left and right, respectively. Each boxplot represents n > = 169 gRNA). e Understanding the PAM proximal impact of context on gRNA activity also allows targeted gRNA libraries to specifically evaluate context effects (see Supplementary discussion on evaluating context).