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.

Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

“…By contrast, the alternative pathway of homology-directed repair (HDR) can be utilized for the introduction of precise genetic modifications such as codon replacements or reporter insertions by recombination with exogenous targeting vectors, serving as repair template 5 . We reasoned that the efficiency of HDR and thus the construction of precise genetic modifications could be boosted by the transient inhibition of NHEJ key molecules, similar to what has been observed for Drosophila embryos with a genetic DNA Ligase IV deficiency 6 .…”

Sources and Structures of Mitotic Crossovers That Arise When BLM Helicase Is Absent inDrosophila

“…Knocking out NHEJ had no effect on crossover frequency ( Figure 1C), despite previous studies in Drosophila that have revealed roles for both NHEJ and HR in repairing DSBs in the male germline (Preston et al 2006;Bozas et al 2009;Beumer et al 2013). These experiments involved enzymatic induction of DSBs, probably throughout the cell cycle.…”

“…We did not observe any decrease in the number of progeny produced by Blm males when they were treated with HU (data not shown), suggesting that cell death was not pervasive, but modest elevations in cell death frequency might still go undetected due to rapid proliferation in the germline. Knocking out NHEJ had no effect on crossover frequency ( Figure 1C), despite previous studies in Drosophila that have revealed roles for both NHEJ and HR in repairing DSBs in the male germline (Preston et al 2006;Bozas et al 2009;Beumer et al 2013). These experiments involved enzymatic induction of DSBs, probably throughout the cell cycle.…”

The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

“…Whereas this effect could be further enhanced by siRNA-mediated knockdown of the second Ku70 allele (Fattah et al, 2008), knockdown of DNAPKcs did not have an effect on AAV-mediated gene targeting (Vasileva et al, 2006). On the other hand, although it has been shown that Rad51 and Rad54 are essential for both ZFN and AAV-mediated gene targeting (Vasileva et al, 2006;Bozas et al, 2009), human cells stably over-expressing RAD51 revealed only a two-fold increase in conventional, plasmid-based gene targeting (Yanez and Porter, 1999). On the whole, it remains open to discussion whether a genetic intervention to shift the balance in AAV or ZFN-mediated gene targeting from NHEJ to HR is meaningful in a therapeutic context.…”