Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast.
Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast.
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DOI：
10.1093/nar/gkv112
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发表时间：
2015-04-30
影响因子：
14.9
通讯作者：
Kouprina N
中科院分区：
文献类型：
--
作者：
Lee NC;Larionov V;Kouprina N
转化相关重组（TAR）方案允许从酵母S.酿酒酵母已经在十多年前被开发出来。然而，其广泛的应用受到酵母转化期间染色体区域捕获效率低（0.5-2%）的阻碍，这反过来需要耗时筛选数百个菌落。在这里，我们证明了用CRISPR-Cas9核酸酶预处理基因组DNA以在靶向基因组区域附近产生双链断裂，导致基因阳性菌落的比例急剧增加（高达32%）。由于只需要筛选十几个或更少的酵母转化体就可以获得具有所需染色体区域的克隆，因此不再需要丰富的酵母经验。TAR-CRISPR协议可能有助于创建一个人类基因库，每个基因库都由包含其天然调节元件的基因组拷贝代表，这将导致功能、结构和比较基因组学、诊断、基因替代、动物模型的显着进步人类疾病并具有基因治疗的潜力。
Transformation-associated recombination (TAR) protocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low efficiency (0.5–2%) of chromosomal region capture during yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene replacement, generation of animal models for human diseases and has a potential for gene therapy.
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影响因子：
48
作者：
Mali, Prashant;Esvelt, Kevin M.;Church, George M.
通讯作者：
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作者：
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通讯作者：
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