Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases

Abstract

We report very high gene targeting frequencies in Drosophila by direct embryo injection of mRNAs encoding specific zinc-finger nucleases (ZFNs). Both local mutagenesis via nonhomologous end joining (NHEJ) and targeted gene replacement via homologous recombination (HR) have been achieved in up to 10% of all targets at a given locus. In embryos that are wild type for DNA repair, the products are dominated by NHEJ mutations. In recipients deficient in the NHEJ component, DNA ligase IV, the majority of products arise by HR with a coinjected donor DNA, with no loss of overall efficiency in target modification. We describe the application of the ZFN injection procedure to mutagenesis by NHEJ of 2 new genes in Drosophila melanogaster: coil and pask. Pairs of novel ZFNs designed for targets within those genes led to the production of null mutations at each locus. The injection procedure is much more rapid than earlier approaches and makes possible the generation and recovery of targeted gene alterations at essentially any locus within 2 fly generations.

Fig. 1.

Repair outcomes after a targeted, ZFN-induced DSB. ZFNs make a DSB in the chromosomal target, leaving a 4-base 5′ overhang. The break can be repaired by NHEJ, leading to localized mutations (star). Alternatively, a marked donor DNA can be used as a template to repair the break by HR, leading to the incorporation of specific mutant sequences (black box). Shading in the HR product indicates that sequences for some distance on either side of the break may be incorporated from the donor.

Fig. 2.

ZFN targets in the coil and pask genes. Both strands are shown in the top lines for each target, and the triplets to which ZFs were designed are in bold, red type. Points of expected cleavage on each strand are indicated with arrowheads. The start codon for the long form of coilin is underlined. Mutations recovered from the offspring of injected parents are shown in the context of the top strand. Dashes indicate deleted residues. Substitutions and insertions are shown in bold, blue type. The first coil mutant was recovered twice independently. The 4-bp insertion in the bottom pask mutant is a fill-in and blunt join of the 4-base overhang created by ZFN cleavage.

Fig. 3.

Scheme for isolation of ZFN-induced mutations in the coil gene. mRNAs for the designed ZFNs were injected into wild-type embryos. When adults eclosed they were crossed to parents carrying a deficiency (Df) that includes the coil gene on the second chromosome and a balancer with a dominant marker (CyO). Candidate chromosomes (*) carried over the balancer were isolated individually by crossing again to the same strain. From the second cross, and in some cases from the first cross, individual alleles carried over the deficiency were subjected to molecular analysis.

Fig. 4.

Cells of the ejaculatory duct stained with antibodies against Drosophila coilin (green) and Drosophila Lsm11 (red). DNA is stained with DAPI (blue). (A) In flies heterozygous for coil¹⁹⁹ and the balancer CyO, a single coilin-positive Cajal body and a single Lsm11-positive histone locus body are detectable in each nucleus. (B) In the homozygous coil¹⁹⁹ fly, coilin is absent and only the histone locus body is detectable.