Figure 5: MR-B1H-produced zinc fingers provide high HBB activity with strong discrimination against HBD.

(a) The HBB target is shown, mismatches to the HBD sequence are shown as red letters. The sickle cell causing mutation that separates the left target here from the HBD sequence is shown as a green letter. (b) As in Fig. 4, for each target, two overlapping four-finger libraries are produced. The right monomer pools are shown here while the left monomer pools are shown in Supplementary Fig. 8. Pools of these libraries are colour coded to emphasize that the overlapping zinc fingers target the same sequences. Zinc fingers are selected from each pool by selection for the HBB sequence but against the HBD sequence. Targets are shown 3′ to 5′ to emphasize the overlap in the targets of the four-finger selections. From each of these selections, 10 of the selected ZFPs are shown. Candidates (Cand.) used to design the four- and six-finger monomers employed as nucleases are bold and underlined. All enriched amino acids for each of the four-finger selections are shown below as a sequence logo with the overlapping two fingers boxed in purple. (c) Candidate ZFN pairs were expressed in vivo and the percentage of indels at HBB and HBD measured. Indel frequencies recovered at either target from cells that did not express a nuclease (GFP) are shown below each table. The ratio of HBB to HBD indel frequency is shown below. A table of the four- and six-finger zinc-finger helices used in the nuclease studies, shown N term to C term, is provided.