Fig. 1: Summary of known factors that influence Cas9/gRNA complex activity and the reported gRNA sequence features identified as most important.

a The gRNA sequence can at least partially predict on-target activity. Here, we provide a model of factors reported to influence Cas9/gRNA complex activity and whether they impact finding the target site, cleaving the target, or repair of the target. b The gRNA:target duplex is highlighted in yellow and the PAM site is highlighted in green. Features that reportedly positively impact gRNA activity, have been identified as inhibitory to activity, or were specifically identified as not significant are labeled in blue, red, and gray, respectively. Position dependent features are labeled in the relevant position while position independent features are shown in separately labeled boxes. c When comparing all datasets, there was no feature identified with a common impact on activity across all datasets and only one feature identified across 75% of the datasets. d The consistency of reported features across datasets of the same species. Of features identified as important in human datasets, no feature consistently had the same impact across human datasets. For other species, we identified five, four, and 6 features that were consistent across datasets within the E. coli, mouse, and zebrafish datasets. Y. lipolytica was excluded from this analysis as there is only one dataset.