Donor DNA Utilization During Gene Targeting with Zinc-Finger Nucleases

Abstract

Gene targeting is the term commonly applied to experimental gene replacement by homologous recombination (HR). This process is substantially stimulated by a double-strand break (DSB) in the genomic target. Zinc-finger nucleases (ZFNs) are targetable cleavage reagents that provide an effective means of introducing such a break in conjunction with delivery of a homologous donor DNA. In this study we explored several parameters of donor DNA structure during ZFN-mediated gene targeting in Drosophila melanogaster embryos, as follows. 1) We confirmed that HR outcomes are enhanced relative to the alternative nonhomologous end joining (NHEJ) repair pathway in flies lacking DNA ligase IV. 2) The minimum amount of homology needed to support efficient HR in fly embryos is between 200 and 500 bp. 3) Conversion tracts are very broad in this system: donor sequences more than 3 kb from the ZFN-induced break are found in the HR products at approximately 50% of the frequency of a marker at the break. 4) Deletions carried by the donor DNA are readily incorporated at the target. 5) While linear double-stranded DNAs are not effective as donors, single-stranded oligonucleotides are. These observations should enable better experimental design for gene targeting in Drosophila and help guide similar efforts in other systems.

Keywords: Gene targeting; conversion tracts; homologous recombination; nonhomologous end joining; zinc-finger nucleases.

Figure 1

Illustration of ZFN cleavage and various repair possibilities. The types of donor DNAs used are shown, and the issues addressed in this study are numbered and listed at the bottom.

Figure 2

Influence of the length of homology between donor and target DNAs on the proportion of all new mutants that are the result of homologous recombination (%HR). Gray bars are data obtained for the wild-type (lig4⁺) host, and black bars are for the lig4 mutant host. Additional data are provided in Table 1.

Figure 3

Conversion tracts during homologous recombination. (A)The 4.2-kb donor showing polymorphic sites between donor and target that define restriction enzyme sites. The ZFN site is indicated by an arrow. (B) %Conversion is the proportion of HR products that acquired sequences from the 4.2-kb donor at each site. The ZFN cut site is set to 100% as all HR events must capture donor sequence there. Black boxes are results from experiments in which ZFN expression and donor excision were induced in larvae with a heat shock (HS). Open and gray boxes are results from experiments with injected embryos, either wild type (Inj wt) or lig4 mutants (Inj lig4). (C) As in panel B, but showing independent experiments with 4.2-kb and 7.5-kb donors. HS data from panel B are repeated for comparison.

Figure 4

Histogram of data for the 4.2-kb donor in injection (Inj) and HS experiments shown in Figure 3C. The 5 sites assayed in both cases are illustrated on the left as having donor (black) or target (gray) sequence(s); the ZFN site (arrow) is in the middle and is from donors in all cases, as only HR products were included. The proportions of all tracts represented by each type are indicated by the lengths of the bars in the histogram.

Figure 5

Deletion donors. The genomic ry locus is shown, with exons as rectangles and introns and intergenic sequences as lines. Protein coding sequences are gray. The ZFN target sequence is shown by an arrow. Donor homologies are shown as black rectangles, dashes indicate sequences deleted, and the substitution at the ZFN site is indicated by a vertical white line. Data for the deletion donors are provided in Table 2.

Figure 6

Single-stranded oligonucleotide donors. The ZFN-cut target is illustrated with longer lines. Half arrowheads indicate 3′ ends. Null mutations in the oligonucleotides are shown as a black rectangle (deletion-substitution) or an x (single-base deletion). Dots indicate single-base substitutions. Oligonucleotides A, B, and F have the same polarity; oligonucleotide R has reverse polarity. Data for oligonucleotide donors are provided in Table 3.