No abstract

Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.Procedures and reagents that allow the directed alteration of genes in situ constitute a powerful toolbox for experimental genetics and potentially for agricultural and therapeutic applications. In many organisms, however, and particularly in higher eukaryotes, the efficiency of recombination between an introduced DNA and the homologous chromosomal target is discouragingly low. For example, such events typically occur in mammalian cells at a frequency of only about 1 for each 10 6 cells treated (3, 31). We are interested in developing procedures that would substantially improve the frequency of gene targeting.A major impediment to efficient gene replacement is the status of the chromosomal target. Increasing the number of target sequences has little or no effect on targeting efficiency (54, 60). In contrast, making an intentional double-strand break (DSB) in the target DNA increases the yield of specific homologous recombination events up to 1,000-fold or more (10,11,14,44,46). It is believed that exonucleases act at broken ends to generate single-stranded tails that are recombinagenic in any of several pathways. In particular, the singlestrand annealing mechanism (33), by which homologous recombination involving exogenous DNA usually occurs in higher eukaryotes (53), cannot proceed unless both the donor and target have ends (5, 48).Whatever the mechanism of recombination, it is clear that the frequency of targeted recombination can be substantially improved by i...

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.coilin ͉ DNA ligase IV ͉ DNA repair ͉ PAS kinase ͉ targeted mutagenesis

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.

SignificanceThe efficiency of genome editing with CRISPR-Cas9 can vary widely at different targets and in different cells. Some of this variability may be due to the inherent quality of different guide RNAs, but it may also depend on the cellular context of the genomic target DNA. In this report, we demonstrate that targets bound by nucleosomes are cut much less efficiently than targets from which nucleosomes are absent or have been depleted. This information can inform target selection, particularly in cases where cells are quiescent or nucleosome mobility is limited.