Genetic analysis of zinc-finger nuclease-induced gene targeting in Drosophila

Abstract

Using zinc-finger nucleases (ZFNs) to cleave the chromosomal target, we have achieved high frequencies of gene targeting in the Drosophila germline. Both local mutagenesis through nonhomologous end joining (NHEJ) and gene replacement via homologous recombination (HR) are stimulated by target cleavage. In this study we investigated the mechanisms that underlie these processes, using materials for the rosy (ry) locus. The frequency of HR dropped significantly in flies homozygous for mutations in spnA (Rad51) or okr (Rad54), two components of the invasion-mediated synthesis-dependent strand annealing (SDSA) pathway. When single-strand annealing (SSA) was also blocked by the use of a circular donor DNA, HR was completely abolished. This indicates that the majority of HR proceeds via SDSA, with a minority mediated by SSA. In flies deficient in lig4 (DNA ligase IV), a component of the major NHEJ pathway, the proportion of HR products rose significantly. This indicates that most NHEJ products are produced in a lig4-dependent process. When both spnA and lig4 were mutated and a circular donor was provided, the frequency of ry mutations was still high and no HR products were recovered. The local mutations produced in these circumstances must have arisen through an alternative, lig4-independent end-joining mechanism. These results show what repair pathways operate on double-strand breaks in this gene targeting system. They also demonstrate that the outcome can be biased toward gene replacement by disabling the major NHEJ pathway and toward simple mutagenesis by interfering with the major HR process.

Figure 1.—

Molecular mechanisms of gene targeting after a ZFN-induced DSB in the target. The target locus is shown on the left with thin lines illustrating the DNA strands. The donor strands are shown as thick lines on the right, flanked by recognition sites for FLP (FRT, open triangles) and I-SceI (IRS, vertical bars): asterisks indicate the mutant sequence in the donor. ZFN action cleaves the target, which can be repaired directly by NHEJ (left); the star indicates mutations that may arise by inaccurate joining. Target ends can also be processed by 5′ → 3′ exonuclease activity. The donor is excised as a circle by FLP-mediated recombination between the two FRTs. If I-SceI is also present, it makes the donor linear in an ends-out configuration relative to the target. Invasion of the excised donor by one 3′ end of the resected target (center) is followed by priming of DNA synthesis (dashed line). Arrows from both circular and linear donors are intended to indicate that either configuration can serve as a substrate for invasion and synthesis. Withdrawal of the extended strand, annealing with the other resected target end, additional DNA synthesis, and ligation complete the SDSA process, resulting in donor sequences copied into the target. The SSA mechanism is illustrated on the right. Both donor and target ends are resected to reveal complementary single-stranded sequences that anneal. Removal of redundant sequences, possibly some DNA synthesis, and ligation restore the integrity of the target with inclusion of donor sequences.

Figure 2.—

Schematic illustration of genetic procedures for gene targeting at ry. (A) Locations of genes and transgenes. Open bars represent D. melanogaster chromosomes: X, left; 2, middle; and 3, right. Shaded circles represent centromeres. The locations of endogenous genes are as follows: lig4, X, 12B2; okr, 2L, 23C4; mei-W68, 2R, 56D9; ry, 3R, 87D9; and spnA, 3R, 99D3. The transgenes shown below chromosomes 2 and 3 are known to lie on those chromosomes, but their exact locations have not been mapped. {ryAB2} and {ryAB3} are pairs of ZFNs. The mutant ry donor is {ry^M}. (B) Illustration of the cross to produce flies with the gene targeting materials in an spnA^−/− background. The Y chromosome is shown simply as Y. + indicates the wild-type ry gene. Typically crosses were done in both directions with each set of components coming from males or females. CyO and TM6 are balancers for chromosomes 2 and 3, respectively. Flies with the desired genotype and their siblings were heat-shocked as larvae and then identified as adults on the basis of the absence of markers on the balancers. New ry mutants were revealed by crossing those adults to a known ry deletion mutant.

Figure 3.—

Histograms showing data from spnA experiments. The three tiers show the percentage of heat-shocked parents that yielded at least one ry mutant offspring (% Yielders, top), the percentage of all offspring that were new ry mutants (% ry, middle), and the percentage of analyzed mutants that were products of homologous recombination between target and donor (% HR, bottom). Data are presented separately for male and female parents and for linear and circular donor configurations. Genotypes of the parents are indicated along the x-axis; the numbers correspond to entries in Table 2, and the spnA genotype is shown explicitly. Results of comparisons to the corresponding wild type are indicated: *0.05 > P > 0.005; **0.005 > P > 0.001; ***P < 0.001.

Figure 4.—

Histograms showing data from lig4 experiments. Data are presented as in Figure 3. Both the lig4 and spnA genotypes are shown explicitly at the bottom. Combined lig4 and spnA mutations were analyzed only in males, and results for spnA^−/− only are included for comparison.

Figure 5.—

Illustration of the relationship between the D. melanogaster gish gene (top) and the insert found in one NHEJ product (bottom). Positions in the gene are numbered from the transcription start. Corresponding sequences in the insert begin at position 50 and extend to position 1574, except that intron 1 (positions 325–1145) is cleanly missing. In addition, there are 7 bp on the upstream end and 9 bp on the downstream end of the insert that do not match either the gish gene or the ry target.