A long-term goal in the field of restrictionmodification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.Since their discovery nearly 25 years ago (1), type II restriction enzymes have played a crucial role in the development of the recombinant DNA technology and the field of molecular biology. The type II restriction endonucleases and modification methylases are relatively simple bacterial enzymes that recognize specific sequences in duplex DNA. While the former cleave DNA, the latter methylate adenine or cytosine residues within the recognition site so as to protect the host genome against cleavage. So far, over 2500 restriction-modification (R-M) enzymes have been identified, and these are found in widely diverse organisms (2). These enzymes fall into numerous "isoschizomer" (identically cleaving) groups with about 200 sequence specificities. Discovery of new enzymes involves tedious and time-consuming effort that requires extensive screening of bacteria and other microorganisms (3). Even when one finds a new enzyme, more often than not, it falls into the already discovered isoschizomer groups. Furthermore, most naturally occurring restriction enzymes recognize sequences that are 4-6 bp long. Although these enzymes are very useful in manipulating recombinant DNA, they are not suitable for producing large DNA segments. For example, restriction enzymes that recognize DNA sequences 6 bp long result in cuts as often as every 4096 bases. In many instances, it is preferable to have fewer but longer DNA strands, especially during genome mapping. Rare cutters that recognize 8-bp-long sequences cut human DNA (which contains about 3 billion bp) every 65,536 bases on average. So far, only a few restriction endonucleases with recognition sequences longer than 6 bp (rare cutters) have been identified (New England Biolabs catalog). R-M systems appear to have a single biological function-namely, to protect cells from infection by foreign DNA that would otherwise destroy them. The phage genomes are usually small. It stands to reason, then, that bacteria select for R-M systems with small recognition sites (4-6 bp) because these sites occur more frequently in the phages. Therefore, aThe publication costs of this article were defrayed in part by page charge ...

We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified Saccharomyces cerevisiae genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by "endoreduplication intercross. " Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy.

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...

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871 bp designer eukaryotic chromosome, synIII, which is based on the 316,617 bp native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, tRNAs, transposons and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in “a mater” derivatives resulting from loss of the MATα allele on synIII. The total synthesis of synIII represents the first complete design and synthesis of a eukaryotic chromosome, establishing S. cerevisiae as the basis for designer eukaryotic genome biology.

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This study concerns chimeric restriction enzymes that are hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain from the natural restriction enzyme FOK:I. Because of the flexibility of DNA recognition by zinc fingers, these enzymes are potential tools for cleaving DNA at arbitrarily selected sequences. Efficient double-strand cleavage by the chimeric nucleases requires two binding sites in close proximity. When cuts were mapped on the DNA strands, it was found that they occur in pairs separated by approximately 4 bp with a 5' overhang, as for native FOK:I. Furthermore, amino acid changes in the dimer interface of the cleavage domain abolished activity. These results reflect a requirement for dimerization of the cleavage domain. The dependence of cleavage efficiency on the distance between two inverted binding sites was determined and both upper and lower limits were defined. Two different zinc finger combinations binding to non-identical sites also supported specific cleavage. Molecular modeling was employed to gain insight into the precise location of the cut sites. These results define requirements for effective targets of chimeric nucleases and will guide the design of novel specificities for directed DNA cleavage in vitro and in vivo.

The DNA double helix is not a regular, featureless barberpole molecule. Different base sequences have their own special signature, in the way that they influence groove width, helical twist, bending, and mechanical rigidity or resistance to bending. These special features probably help other molecules such as repressors to read and recognize one base sequence in preference to another. Single crystal x-ray structure analysis is beginning to show us the various structures possible in the B-DNA family. The DNA decamer C-C-A-A-G-A-T-T-G-G appears to be a better model for mixed-sequence B-DNA than was the earlier C-G-C-G-A-A-T-T-C-G-C-G, which is more akin to regions of poly(dA).poly(dT). The G.A mismatch base pairs at the center of the decamer are in the anti-anti conformation about their bonds from base to sugar, in agreement with nuclear magnetic resonance evidence on this and other sequences, and in contrast to the anti-syn geometry reported for G.A pairs in C-G-C-G-A-A-T-T-A-G-C-G. The ordered spine of hydration seen earlier in the narrow-grooved dodecamer has its counterpart, in this wide-grooved decamer, in two strings of water molecules lining the walls of the minor groove, bridging from purine N3 or pyrimidine O2, to the following sugar O4'. The same strings of hydration are present in the phosphorothioate analog of G-C-G-C-G-C. Unlike the spine, which is broken up by the intrusion of amine groups at guanines, these water strings are found in general, mixed-sequence DNA because they can pass by unimpeded to either side of a guanine N2 amine. The spine and strings are perceived as two extremes of a general pattern of hydration of the minor groove, which probably is the dominant factor in making B-DNA the preferred form at high hydration.

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.