Supplementary Figure 6: Testing “PAM-out” target configurations by fusing FokI to Cas6.
From: Harnessing type I CRISPR–Cas systems for genome engineering in human cells
EcoCascade-FokI editing efficiency as a function of FokI-Cas6 linker length and interspacer distance after nucleofection with a paired gRNA expression plasmid and a polycistronic plasmid encoding all 5 proteins separate by 2A peptides. For FokI-Cas6 samples, each data point represents the average of three independent nucleofections at a unique “PAM-out” target site in HEK293 cells, n=4 unique target sites per interspacer distance. Positive control nucleofections with FokI-EcoCascade containing a 30-aa FokI-Cas8 linker and targeting “PAM-in” site 7 from Fig. 2a is shown in grey. No editing was observed for any FokI-Cas6 fusion constructs with the “PAM-out” configuration.
