Increasing cloning possibilities using artificial zinc finger nucleases

Abstract

The ability to accurately digest and ligate DNA molecules of different origins is fundamental to modern recombinant DNA research. Only a handful of enzymes are capable of recognizing and cleaving novel and long DNA sequences, however. The slow evolution and engineering of new restriction enzymes calls for alternative strategies to design novel and unique restriction enzymes capable of binding and digesting specific long DNA sequences. Here we report on the use of zinc finger nucleases (ZFNs)-hybrid synthetic restriction enzymes that can be specifically designed to bind and cleave long DNA sequences-for the purpose of DNA recombination. We show that novel ZFNs can be designed for the digestion of specific sequences and can be expressed and used for cloning purposes. We also demonstrate the power of ZFNs in DNA cloning by custom-cloning a target DNA sequence and assembling dual-expression cassettes on a single target plasmid, a task that rarely can be achieved using type-II restriction enzymes. We demonstrate the flexibility of ZFN design and the ability to shuffle monomers of different ZFNs for the digestion of compatible recognition sites through ligation of compatible ends and their cleavage by heterodimer ZFNs. Of no less importance, we show that ZFNs can be designed to recognize and cleave existing DNA sequences for the custom-cloning of native target DNA molecules.

Fig. 1.

Structure and expression of ZFNs and their use for DNA cloning. (A) The structure of a typical 24-bp-long ZFN recognition site and its corresponding zinc finger protein, exemplified by ZFN10. DNA triplets (blue) and their binding zinc fingers (ovals) are numbered correspondingly. The unique amino acid sequences in each zinc finger are in purple. The FokI cleavage domain (F) is linked to the zinc finger protein's carboxyl terminus, and the predicted cleavage sites are indicated by arrowheads. (B) Scheme of the pSAT10-MCS plasmid. Sequences of the ZFN10 palindrome-like recognition sites are in blue. (C) Separation of total crude extract from induced (+IPTG) and noninduced (-IPTG) ZFN10 protein-expressing E. coli cells and of purified ZFN10 protein (P). The 34-kDa band corresponding to the ZFN10 protein is indicated. (D) Restriction analysis of the parental pSAT10-MCS and its progeny plasmid pSAT10-. (E) Sequence analysis of the ZFN10 ligation site in one of the pSAT10- plasmids. Sequences of the reconstructed ZFN10 palindrome-like recognition sites are in blue.

Fig. 2.

Assembly of dual-expression cassettes by ZFNs. (A) Schemes of pSAT10-YFP-CHS and pSAT11-DsRed2-P. Sequences of ZFN10 and ZFN11 recognition sites are in purple, and the predicted cleavage sites in each sequence are indicated by arrowheads. (B) Scheme of the pRCS11 acceptor plasmid. Sequences of ZFN10 and ZFN11 recognition sites in the plasmid's MCS are in blue. (C) Detection of YFP-CHS and DsRed2-P expression from pRCS11[YFP-CHS][ DsRed2-P]-bombarded plant cells in the presence (+N) or absence (-N) of the N protein of SYNV. Expression of YFP-CHS, DsRed-P, and chloroplast autofluorescence is shown in yellow, blue, and red, respectively. The images are projections of several confocal sections. (D) Sequence analysis of the ZFN10 and ZFN11 ligation sites in pRCS11[YFP-CHS][DsRed2-P]. ZFN10 and ZFN11 sequences derived from the acceptor plasmid pRCS11 are in blue, and those derived from the inserted expression cassettes are in purple.

Fig. 3.

Custom-cloning of a target DNA sequence. (A) Outline of the Arabidopsis FtsH2 genomic clone. Sequences of the ZFN-H2a and ZFN-H2b binding sites are shown in blue. (B) Sequence analysis of ZFN-H2a-ZFN-H2b/SmaI and KpnI/KpnI junctions in various pSL301-AtFtsH2 clones. Sequences derived from the SmaI site on acceptor plasmid pSL301 are in purple, and those derived from the inserted DNA are in blue. Filled-in nucleotides are underlined.

Fig. 4.

DNA cloning using compatible ZFNs. (A) Scheme of pSAT12.1-CHRD-RFP. The ZFN-H2a recognition-site sequence is in purple, and the predicted cleavage sites are indicated by arrowheads. (B) Scheme of the pRCS11 acceptor plasmid. The ZFN10 recognition site in the plasmid's MCS is in blue. (C) Sequence analysis of the ZFN10/ZFN-H2a junctions at the ligation sites in pRCS11[10/12.1-CHRD-RFP]. The ZFN10 recognition sequences derived from the acceptor plasmid pRCS11 are in blue, and the ZFN-H2a sequences derived from pSAT12.1-CHRD-RFP are in purple. (D) Restriction analysis of an ≈1-kb-long ApaLI-HindIII fragment from pRCS11[10/12.1-CHRD-RFP]. The fragment's restriction map, which includes a ZFN-H2a/ZFN10 ligation junction, is on the right.