Jonathan K. Trautman

Stimulation of homologous recombination through targeted cleavage by chimeric nucleases

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.

Efficient Gene Targeting in Drosophila With Zinc-Finger Nucleases

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.

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

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.

Genetic Analysis of Zinc-finger Nuclease-induced Gene Targeting in Drosophila

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.

Donor DNA Utilization During Gene Targeting with Zinc-Finger Nucleases

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.

Comparing Zinc Finger Nucleases and Transcription Activator-Like Effector Nucleases for Gene Targeting in Drosophila

Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.

Gene targeting in Drosophila and Caenorhabditis elegans with zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) are promising new tools for enhancing the efficiency of gene targeting in many organisms. Because of the flexibility of zinc finger DNA recognition, ZFNs can be designed to bind many different genomic sequences. The double-strand breaks they create are repaired by cellular processes that generate new mutations at the cleavage site. In addition, the breaks can be repaired by homologous recombination with an exogenous donor DNA, allowing the experimenter to introduce designed sequence alterations. We describe the construction of ZFNs for novel targets and their application to targeted mutagenesis and targeted gene replacement in Drosophila melanogaster and Caenorhabditis elegans.

High-efficiency gene targeting in Drosophila with zinc finger nucleases.

We describe a method for making targeted double-strand breaks in Drosophila melanogaster using zinc finger nucleases (ZFNs). After design and construction of the appropriate coding sequences, synthetic mRNAs for the ZFNs are injected directly into fly embryos. Frequencies of target cleavage and mutagenesis in the range of 1-10% have been achieved at several different loci. A donor DNA carrying desired sequence changes can be incorporated in the injection mix and leads to targeted gene replacement, with particularly good efficiency when the recipient embryos are defective for nonhomologous end joining.

Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo

Significance The 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. Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.