Custom-designed molecular scissors for site-specific manipulation of the plant and mammalian genomes

Abstract

Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

Fig. 1

Selection of ZFN target sites within the nucleotide sequences of mouse tyrosinase (mTYR) gene. The nucleotide sequence of the mTYR exon 1 is shown. The best targets are inverted sequences of the form (NNC)_{3 or 4}…(GNN)_{3 or 4} separated by 5 or 6 bp. The ZFN designs for the chosen targets that have been constructed and characterized for their DNA binding and cleavage properties are shaded. Other potential target sites for ZFN designs are boxed. The site of point mutation within the tyrosinase gene responsible for transition from pigmented (black) to nonpigmented (albino) mice is shown in bold.

Fig. 2

Assembly of 3-finger ZFPs by using PCR. (a) The genes for the ZFPs are first assembled using the overlapping BBOs and SDOs (60-mers) in a Klenow reaction, which is then amplified by PCR using the outside forward and reverse primers, which are flanked by unique restriction sites (NdeI and SpeI sites, respectively) to facilitate cloning. BBO1, BBO2, and BBO3 correspond to the consensus backbone oligonucleotides whereas SDO1, SDO2, and SDO3 correspond to specificity-determining oligonucleotides for ZF1, ZF2, and ZF3, respectively. (b) Scheme for assembling the 3-finger ZFPs via the oligonucleotide assembly strategy using the consensus framework residues and the chosen contact amino acid residues at positions −1, +1, +2, +3, +4, +5, and +6 of the α-helix, which confer specificity to each of the ZFs. The indicated top strand (bold) and bottom strand oligonucleotides overlap and will be assembled using PCR. The bottom strand oligonucleotides are depicted as having NNN, which code for the contact residues that confer specificity to each ZF.

Fig. 3

Converting ZFPs into ZFNs. The NdeI/SpeI-cut ZFPs are ligated into the pET15b:N, the plasmid containing the FokI cleavage domain to form pET15b:ZFN.

Fig. 4

Cell-free ZFN cleavage assays using the IVTT system. (a) Western blot profile of the fusion proteins made using the in vitro transcription-translation (IVTT) system. This yields sufficient fusion protein for rapid characterization of the cleavage specificity of the custom-designed ZFNs. (b) Nucleotide sequences of the ZFN target sites (TS) for mTYR, the gene encoded in the plasmid substrates (pUC18: TS) for use in the cleavage reactions. (c) Schematic representation of the plasmid substrates (pUC18: TS) encoding the ZFN target site of the mTYR gene at the multiple cloning site of pUC18. Four unique restriction enzyme sites, namely AatII, SspI, and XmnI, within the plasmid substrates are indicated. The expected sizes of the fragments upon cleavage by ZFNs, followed by AatII, SspI, or XmnI, respectively, are shown. (d) Agarose gel profile of engineered ZFN cleavage of mTYR plasmid substrates. The plasmid substrate was digested by the ZFNs, followed by one of the restriction enzymes, namely AatII, SspI, or XmnI. The particular restriction enzyme used in the reactions after the corresponding ZFN digestion is indicated on top of each lane. Plasmid substrates digested with the control IVTT product (which contained no ZFN plasmids), followed by one of the enzymes, AatII, SspI, or XmnI, respectively, for each are also shown. The 1-kb ladder marker is included in the gel profile.

Fig. 5

Structure of pIRES: ZFNs. The pair of ZFNs that bind to the mTYR site, are cloned into a single pIRES plasmid at two different multiple cloning sites (MCS) downstream of the CMV promoter for use in the co-transfection of mouse melanocytes along with the exogenous donor DNA during ZFN-mediated gene targeting experiments.