Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases

Abstract

Despite the recent discoveries of and interest in numerous structural variations (SVs)--which include duplications and inversions--in the human and other higher eukaryotic genomes, little is known about the etiology and biology of these SVs, partly due to the lack of molecular tools with which to create individual SVs in cultured cells and model organisms. Here, we present a novel method of inducing duplications and inversions in a targeted manner without pre-manipulation of the genome. We found that zinc finger nucleases (ZFNs) designed to target two different sites in a human chromosome could introduce two concurrent double-strand breaks, whose repair via non-homologous end-joining (NHEJ) gives rise to targeted duplications and inversions of the genomic segments of up to a mega base pair (bp) in length between the two sites. Furthermore, we demonstrated that a ZFN pair could induce the inversion of a 140-kbp chromosomal segment that contains a portion of the blood coagulation factor VIII gene to mimic the inversion genotype that is associated with some cases of severe hemophilia A. This same ZFN pair could be used, in theory, to revert the inverted region to restore genomic integrity in these hemophilia A patients. We propose that ZFNs can be employed as molecular tools to study mechanisms of chromosomal rearrangements and to create SVs in a predetermined manner so as to study their biological roles. In addition, our method raises the possibility of correcting genetic defects caused by chromosomal rearrangements and holds new promise in gene and cell therapy.

Figure 1.

Various chromosomal rearrangements induced by ZFNs. (A) Schematic of ZFN target sites and chemokine receptor genes on the human chromosome 3 (http://genome.ucsc.edu). Note that ZFN-224 has two target sites, one at the CCR5 locus and the other at the CCR2 locus. Colored triangles indicate approximate positions of PCR primers. (B) PCR products validating various chromosomal rearrangements. (+) indicates a positive control (cells transfected with plasmids encoding two ZFN pairs) and (−) indicates a negative control (cells transfected with empty plasmid). (C) DNA sequences of PCR products. Each ZFN target site is shown in boldface letters. K230 and CCR5 target sites are shown in green and blue letters, respectively. Note the absence of the intact CCR5 (ZFN-224) site in these clones. Microhomologies are underlined and inserted bases are shown in italics. Dashes indicate deleted bases. (D) Schematic of ZFN target sites in wild-type cells and in various clones with chromosomal rearrangements. Two probes (red and orange bars) and the site recognized by the restriction enzyme, XbaI, used for Southern blot analysis are indicated. (E) Southern blot analysis of chromosomal rearrangements. Genomic DNA digested with XbaI and hybridized with probe 1 (upper) or with probe 2 (lower). A direct evidence of the 15-kbp inversion was provided by DNA sequencing (Supplemental Fig. 1).

Figure 2.

Duplications induced by ZFNs. (A) Schematic representation of ZFN-mediated duplications. Zigzag lines indicate ZFN target sites. Colored triangles indicate approximate positions of PCR primers. (B) PCR products corresponding to the 15-kbp duplications in cells treated with various ZFNs. (−) indicates a negative control (cells transfected with empty plasmid). (C) DNA sequences of breakpoint junctions of the duplications. Nucleotide sequences of CCR5 and CCR2 sites are shown in blue and red colors, respectively. Dup indicates various duplication junctions induced by ZFN-224. Duplication junction sequences induced by other ZFNs are shown in Supplemental Figure 3. DNA sequences of breakpoint junctions in two clones, DUP1A and DUP1B, are also shown. Nonconserved bases at the CCR2 and CCR5 loci are shown in lowercase letters. Symbols are as in Figure 1. (D) PCR products validating duplications in two clones. No other mutations or rearrangements were detected in these clones. (+) is a positive control (cells treated with ZFN-224) and (−) is a negative control (cells transfected with empty plasmid). (E) Schematic of CCR2 and CCR5 loci in the wild-type and duplication alleles and Southern blot analysis. Symbols are as in Figure 1.

Figure 3.

Inversions induced by ZFNs. (A) Schematic representation of ZFN-mediated inversions. Zigzag lines indicate ZFN target sites. PCR primers (triangles) used for the detection of two breakpoint junctions that result from genomic inversions are shown. (B) PCR products corresponding to inversion events in cells treated with various combinations of two ZFN pairs. (C) DNA sequences of breakpoint junctions of the inversion events. Nucleotide sequences of CCR5 and CCR2 sites are shown in blue and red, respectively. (Inv) Various inversion junctions induced by ZFN-224. Inversion junction sequences induced by other ZFNs are shown in Supplemental Figure 4. The two breakpoint junction sequences in the INV1 clone are also shown. Nonconserved bases at the CCR2 and CCR5 loci are shown in lowercase letters. Symbols are as in Figure 1. (D) PCR products validating inversions in the INV1 clone. (+) and (−) are as in Figure 2D. (E) Schematic of CCR2 and CCR5 loci in the wild-type and inversion alleles and Southern blot analysis. Symbols are as in Figure 1.

Figure 4.

Targeted inversion of the F8 gene using ZFNs. (A) Schematic representation of a chromosomal inversion that causes severe hemophilia A. NAHR between two homologous regions, one in intron 1 of the F8 gene (here named homolog 1) and the other located 140 kbp upstream (homolog 2), gives rise to an inversion found in severe hemophilia A. The two homologous regions are oriented in opposite directions. PCR primers (colored triangles) used to detect the inversion are shown. Z10 target sites are indicated by arrows. (B) Site-specific mutations at the Z10 target site in the F8 intron 1 revealed by the T7 endonuclease assay. (C) PCR products corresponding to wild-type and inversion genotypes. Genomic DNA isolated from a hemophilia A patient was used as a positive control for the inversion-specific PCR. (D) DNA sequences of breakpoint junctions of the inversion events. Z10 target sites are shown in red (homolog 1) or blue (homolog 2).