Figure 4: Extending zinc-finger targets to increase CCR2 discrimination.

(a) The CCR5 target is shifted 6 nt 3′ relative to the target in Fig. 3 (bold and underlined). Mismatches with the CCR2 sequence are shown as red letters. Each ZFN monomer is increased from four to six fingers. (b) 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. 7. 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 CCR5 sequence but against the CCR2 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 CCR5 and CCR2 measured. Indel frequencies recovered at either target from cells that did not express a nuclease (GFP) are shown below each table. The ratio of CCR5 to CCR2 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.