Efficient gene targeting in Drosophila with zinc-finger nucleases - PubMed (original) (raw)
Efficient gene targeting in Drosophila with zinc-finger nucleases
Kelly Beumer et al. Genetics. 2006 Apr.
Abstract
This report describes high-frequency germline gene targeting at two genomic loci in Drosophila melanogaster, y and ry. In the best case, nearly all induced parents produced mutant progeny; 25% of their offspring were new mutants and most of these were targeted gene replacements resulting from homologous recombination (HR) with a marked donor DNA. The procedure that generates these high frequencies relies on cleavage of the target by designed zinc-finger nucleases (ZFNs) and production of a linear donor in situ. Increased induction of ZFN expression led to higher frequencies of gene targeting, demonstrating the beneficial effect of activating the target. In the absence of a homologous donor DNA, ZFN cleavage led to the recovery of new mutants at three loci-y, ry and bw-through nonhomologous end joining (NHEJ) after cleavage. Because zinc fingers can be directed to a broad range of DNA sequences and targeting is very efficient, this approach promises to allow genetic manipulation of many different genes, even in cases where the mutant phenotype cannot be predicted.
Figures
Figure 1.
The Drosophila ry gene showing the ZFN target and its modification in the ryM donor. The lower line shows the structure of the gene: exons are shown as boxes, with protein-coding sequences shaded and introns as lines. The sequence of the ZFN-targeted site is shown below with triplets bound by the zinc fingers in capital letters. Zinc fingers are illustrated as shaded ovals (f1, f2, f3), the _Fok_I cleavage domains attached to each set of fingers as lightly shaded ovals, and the expected cleavage sites on each strand with carats. The 4.16-kb segment of the ry gene that was included in the donor is shown above. It is flanked by recognition sites for I-_Sce_I and for FLP (FRTs). The specific modifications made in the donor are shown: the two in-frame stop codons are underlined, and the new _Xba_I site is boxed.
Figure 2.
Initial set-up and expected products of the gene-targeting experiments at ry. The target gene (ry+) is on chromosome 3, and one of these chromosomes carries transgenes for the ZFNs (ryA and ryB). (The locations of these transgenes are not known.) The transgenes for FLP and I-_Sce_I are on one second chromosome; the ryM donor is on the other. Angled arrows indicate heat-inducible promoters. Upon heat shock, the ZFNs make a DSB in the ry+ gene, as illustrated (only one is shown for simplicity). Expression of FLP will excise the donor as an extrachromosomal circle, and coexpression of I-_Sce_I will convert it to an ends-out linear molecule. The break at ry can be restored to wild type, or it can acquire a mutant sequence either by NHEJ or by HR with the donor.
Figure 3.
Gene targeting at ry. (Top) The percentage of heat-shocked flies that gave at least one ry mutant offspring is shown for both females (open bars) and males (solid bars). Above each bar in the histogram is the number of flies screened in each category. (Bottom) The number of ry mutants per parent, and the proportion that were the result of NHEJ and HR are given. Above each bar the total number of ry mutants recovered is shown for each case. Comparisons are made among independent experiments with ZFNs only (“No Donor,” data from Table 2) and those that included the ryM donor (“Linear Donor”) at several temperatures.
Figure 4.
Structure of the yM donor. The 6.7-kb segment of the y gene, with surrounding sequences, that was included in the donor is shown in the center of the diagram; introns and exons are labeled. Above this, the specific sequence targeted by ZFNs is shown, as in Figure 1. Below this is the sequence of this same region as modified in the yM donor. The two in-frame stop codons are underlined, and the new _Xho_I site is boxed. The locations of the two _Nde_I sites used in construction of the yM modification are also indicated.
Figure 5.
Gene targeting at y as a function of heat-shock temperature. Data are presented as in Figure 3, including the number of parents screened in each category (top) and the total number of new y mutants recovered (bottom).
Figure 6.
Mechanisms of homologous recombination that could explain gene targeting after ZFN cleavage, illustrated for the case of a linear donor. The target sequence is shown as thin lines, the donor as thick lines, with each line representing one DNA strand. After cleavage by the ZFNs, the target and donor ends are resected by a 5′-to-3′ exonuclease activity. In the SDSA mechanism (left), one of the resulting 3′ single-stranded tails invades homologous sequence in the donor and begins to copy (dashed line). After some synthesis, this end withdraws and pairs with the single-stranded tail from the other end at the original break, and any remaining gaps are filled by further DNA synthesis. In the SSA mechanism (right), resected ends from the target and linear donor anneal to each other by simple base pairing. Excess DNA is removed by nuclease action, and the junctions are completed by a combination of DNA synthesis and ligation. Because SDSA uses internal sequences of the donor as a template, it would work equally well with circular or integrated donor configurations, whereas SSA requires molecular ends on the donor as well as on the target.
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