Figure 3.

Testing role of the leader–repeat junction in G1 and G2 integrations. A, sequence alignment showing the conserved WT leader–repeat region from the three type II-A groups and the E. coli CRISPR type I-E system. B, FAM image of urea-PAGE showing G1-IC and G2-IC integrating into linear cognate target, as well as randomized linear targets containing the conserved leader–repeat junction at different positions. The G1-L and G2-L integration products are 96 nt (leader side, LS) and 116 nt (spacer side, SS), and the Rand114 and Rand75 targets produce leader-side integration products that are 114 and 75 nt, respectively. The results show that although G1 shows reasonable insertion into random DNA (∼60%), the ability of G2 to insert into a minimized DNA backbone is significantly reduced.