The ability to make very specific changes in genomic DNA has advanced dramatically in the past 10 years with the development of targetable nucleases that can be designed to cleave essentially any desired sequence. The double-strand breaks made by zinc-finger nucleases (ZFNs), TALENs, and CRISPR/Cas nucleases stimulate the formation of local mutations, by nonhomologous end joining (NHEJ), and the insertion of desired sequence changes, by homologous recombination (HR) with a designed donor DNA. The frequency of these events can be very high, over 50% in the best cases, but the range of frequencies is large, including some complete failures. Some of this variability is due to problems with nuclease design, particularly for ZFNs. The TALEN recognition code, however, is generally quite robust;and design of the CRISPR reagents is simply a matter of ensuring base pairing between a guide RNA and the DNA target. A parameter of the targeting process that is rarely considered is the accessibility of the target itself. Chromatin structure restricts access o other DNA-binding proteins, including transcription factors, so it likely has an influence on the targetable nucleases as well. Such an influence may not be evident in most targeting experiments because they take place over an extended period of time, often in growing cells, so the dynamics of chromatin structure may mask local, temporal restrictions. This proposal is to investigate the specific effects of chromatin structure on nuclease cleavage using the yeast Saccharomyces cerevisiae, which offers unique advantages for such a study. With each of the nuclease platforms, sequences will be targeted within strongly positioned nucleosomes and in nucleosome-depleted regions in the promoters of two yeast genes, CLN2 and HO. Targets in yeast heterochromatin at the silent mating type loci, HML and HMR, will be targeted and compared to identical sequences in the euchromatic MAT locus. Cellular processes that influence DNA accessibility in chromatin will be tested for their effects using cell cycle control and genetic mutations. These include DNA replication, transcription, chromatin remodeling, and silencing. The results of this study will be important in guiding target selection in cases in whih the precise location of genomic modification is essential and control over cellular activities is limited, as will be true in applications to human gene therapy and the engineering of livestock and crops.
Targetable nucleases have produced a revolution in our ability to modify genomic DNA sequences, but not all of these reagents are effective when designed for new targets. The proposed study aims to determine what governs access of the nucleases to their intended targets. This has important implications for applications of the nucleases to making experimental models of human genetic diseases, and ultimately to gene therapy for the treatment of these conditions.
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