Optimizing Genome Engineering with Targetable Nucleases

描述(申请人提供)：在过去的10年里，随着靶向核酸酶的发展，在基因组DNA中进行非常具体的改变的能力已经显著提高，这种核酸酶可以被设计成基本上切割任何所需的序列。锌指核酸酶(ZFN)、TALENS和CRISPR/Cas核酸酶造成的双链断裂通过非同源末端连接(NHEJ)刺激局部突变的形成，并通过与设计的供体DNA的同源重组(HR)插入所需的序列变化。这些事件的频率可能非常高，在最好的情况下超过50%，但频率范围很大，包括一些完全故障。这种差异的部分原因是核酸酶设计的问题，特别是ZFN的问题。然而，TALEN识别代码通常是相当健壮的；CRISPR试剂的设计只是确保引导RNA和DNA靶标之间的碱基配对的问题。目标确定过程的一个很少考虑的参数是目标本身的可及性。染色质结构限制了其他DNA结合蛋白的访问，包括转录因子，因此它可能也对靶向核酸酶产生影响。这种影响在大多数靶向实验中可能并不明显，因为它们发生在较长的一段时间内，通常是在生长细胞中，因此染色质结构的动态可能掩盖了局部的时间限制。本研究的目的是利用酿酒酵母研究染色质结构对核酸酶裂解的特异性影响，这为此类研究提供了独特的优势。对于每个核酸酶平台，序列将被定位在两个酵母基因CLN2和HO启动子的强定位核小体和核小体缺失区域。酵母异染色质中沉默交配型基因座HML和HMR的靶标将被作为靶标，并与常染色质MAT基因座中的相同序列进行比较。影响染色质中DNA可及性的细胞过程将使用细胞周期控制和基因突变来测试它们的影响。这些包括DNA复制、转录、染色质重塑和沉默。这项研究的结果将在指导目标选择方面发挥重要作用，在这些情况下，基因组修饰的精确定位是必不可少的，对细胞活动的控制是有限的，在人类基因治疗和牲畜和农作物工程的应用中也是如此。

DESCRIPTION (provided by applicant): 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.