Supplementary Figure 1: Design and purification of FokI-EcoCascade.
From: Harnessing type I CRISPR–Cas systems for genome engineering in human cells
a, X-ray crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target (PDB ID: 5H9F)(Nature 530, 499–503 (2016)). The PAM is highlighted in the rotated view on the left, and the FokI domain (grey) is shown fused to the N-terminus of Cas8 via a linker sequence (red). This attachment point positions the nuclease domain in close proximity to dsDNA within the interspacer, such that FokI dimerization can occur between two adjacent, oppositely oriented FokI-Cascade monomers. The dsDNA flanking the PAM has been artistically extended beyond the experimental structure, for clarity. b, SDS-PAGE analysis of six purified FokI-Cas8 fusion proteins containing various linker lengths (left of ladder), and three Cas8-less Cascade subcomplexes containing either the λ1, λ2, or both λ1 and λ2 gRNAs (right of ladder). The complex with paired gRNAs (λ1+λ2) was purified from an E. coli strain in which a single CRISPR array encoded three repeats and both spacer sequences. FokI-Cas8 linker sequences were as follows: 8, TGPGAAAR; 10, GGSGSSGGSG; 12, TGPGAAARAASG; 15, GGSGSSGGSGSSGGS; 16, SGSETPGTSESATPES; 30, SGSETPGTSESATPESGGSGSSGGSGSSGG. aa, amino acid. *, contaminating protein. Experiment was repeated > two independent times with similar results; the uncropped gel can be found in Source Data Fig. 1.
