Academic journal article Genetics

RNA-Guided Nucleases: A New Era for Engineering the Genomes of Model and Nonmodel Organisms

Academic journal article Genetics

RNA-Guided Nucleases: A New Era for Engineering the Genomes of Model and Nonmodel Organisms

Article excerpt

In 1946, H. J. Muller was awarded the Nobel Prize in Med- icine for his discovery with Drosophila melanogaster, some 20 years earlier, that exposure to X-rays caused mutation (Muller 1927). This led to the identification of a large num- ber of Drosophila mutants and chromosome rearrangements, many of which are still frequendy used in fly labs. The dis- covery of an effective method to induce mutations made it feasible to undertake genetic studies of basic biological pro- cesses. Beadle and Tatum used X-ray mutagenesis of Neuros- pora crassa to identify metabolic mutations and prove the "oone gene-one enzyme" hypothesis (Beadle and Tatum 1941). Subsequently, Auerbach and Robson discovered the mutagenic properties of mustard gas (Auerbach and Robson 1946), and others found additional mutagenic chemicals (Beale 1993).

Though the methods of X-ray and chemical mutagenesis have been invaluable, their effects cannot be directed to specific genes, or to genes that control specific phenotypes. Genetic screening schemes or selections must be employed to find the mutants of interest. For instance, Beadle and Tatum screened for cultures that would grow on rich medium but not on minimal medium. This served to identify mutants that were deficient in basic metabolic processes, but was very labor intensive.

Since the advent of DNA sequencing, biologists have sought ways to produce mutations in chosen sequences. Yeast researchers were the first to achieve this with the discovery that exogenous DNA could integrate into a chromosome by homologous recombination (Hinnen et al. 1978). This quickly led to the realization that, by appropriate design of the donor DNA, predetermined changes could be introduced into the chromosomal DNA sequence (Scherer and Davis 1979). An especially significant advance for the future of gene targeting was the discovery that homologous recombination between the donor and the chromosomal target sequence could be gready stimulated by cutting the donor molecule with a re- striction enzyme (Orr-Weaver et aL 1981). The use of linear- ized donor DNA became part of the standard protocol for gene targeting in the mouse (Mansour et aL 1988), and much later, in Drosophila (Rong and Golic 2000; Rong et al. 2002; Gong and Golic 2003).

In spite of these successes, gene targeting generally remained a lengthy and involved process in organisms other than yeast. However, there were indications that homologous recombina- tion could be made much more efficient if a double-strand break were introduced at the target locus rather than in the donor DNA segment. For instance, mating type switching in yeast is initiated when the homing (HO) endonuclease makes a double- strand cut at the MAT locus (Strathem et al. 1982; Kostriken et aL 1983). The gene conversion that follows, in which homol- ogous sequences from HML or HMR are copied into MAT, is extremely efficient (Hicks and Herskowitz 1976). Breaks in- duced by the I-Scel homing endonuclease also induce high rates of homogolous recombination in yeast (Plessis et al. 1992).

In Drosophila, double-strand breaks produced by P transposon excision or I-Scel expression can be repaired by gene conversion at levels far higher than have been achieved by standard gene targeting procedures (Gloor et al. 1991; Johnson-Schlitz and Engels 1993; Rong and Golic 2003). In addition, such breaks can be repaired by copying information from engineered transgenes (Nassif et al. 1994), or from oligonucleotides (Banga and Boyd 1992) or plasmids (Keeler et al. 1996) injected into embryos. In mammalian cells, also, homologous recom- bination between introduced donor DNA and resident chromosomal DNA is greatly stimulated by breaks in the chromosomal sequence (Rouet et al. 1994; Choulika et al. 1995; Donoho et al. 1998).

These results suggested that if such breaks could be produced at desired locations, they might form the basis of an efficient gene targeting system. However, the problem was how to target the break to a specific location. …

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