Preassembled zinc-finger arrays for rapid construction of ZFNs

To the Editor:

Since the publication of our Correspondence¹ and the reply of Joung et al.², we improved zinc-finger nuclease (ZFN) modular assembly. ZFNs are artificial restriction enzymes³ composed of tailor-made zinc-finger DNA-binding arrays and the FokI nuclease domain, which can induce site-specific mutations⁴ and large chromosomal deletions⁵ in higher eukaryotic cells and organisms.

To reduce the number of ZFNs that need to be synthesized to identify a functional enzyme, we previously compared zinc fingers with equivalent DNA-binding specificity and chose ones that are often found in functional ZFNs⁴. Based on that analysis, we recommended 37 zinc fingers for use in genome editing⁴. Here we tested 33 of these fingers, which collectively recognize 39 of 64 three–base-pair subsites (15 GNN subsites and 24 non-GNN subsites, where G is guanine and N is any base; Supplementary Table 1). We prepared a combinatorial library of two-finger modules (Supplementary Methods) consisting of 1,089 (33 × 33 zinc fingers) two-finger arrays, each linked to the FokI nuclease domain. This library allowed us to construct three-finger or four-finger ZFNs in a single subcloning step (Supplementary Fig. 1). Previously, assembling hundreds of ZFNs to target a single gene had taken at least several weeks because up to four repetitive subcloning steps were required to make four-finger ZFNs. With our preassembled two-finger library, preparing several ZFNs to target a gene of interest took only a few days.

We assembled and tested ZFNs to target a human gene and a mouse gene (Fig. 1, Supplementary Fig. 2 and Supplementary Table 2). Of six ZFNs, each targeting different sites at the CDKN2A locus, two ZFNs induced site-specific mutations in human cells. Of seven ZFNs targeting the Gt(ROSA)26Sor (Rosa26) gene, one had genome-editing activity in mouse cells. Thus, the success rate of our improved modular-assembly method was 23% (3/13 ZFNs), in good agreement with the success rate of 24% previously measured by the number of target sites at the human CCR5 locus⁴. In this study, we tested only a single ZFN pair at each target site. Our semi-assembled zinc-finger arrays allow rapid construction of ZFNs in a few days using conventional recombinant DNA technology, and fewer ZFNs need to be synthesized to obtain a functional enzyme.

Figure 1: Targeted genome modifications in human and mouse cells using modularly assembled ZFNs.

Insertion-deletions induced by ZFNs designed for CDKN2A (CDKN-1 to CDKN-6) and for Rosa26 (Rosa-1 to Rosa-7) were detected by digesting PCR amplicons of the respective genes with T7 endonuclease I, which cleaves heteroduplexes but not homoduplexes. Arrow indicates uncut bands, and bracket marks cut bands. Asterisks denote functional ZFNs.