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CRISPR-PCDup: a novel approach for simultaneous segmental chromosomal duplication in Saccharomyces cerevisiae.
AMB Express ( IF 3.7 ) Pub Date : 2020-02-03 , DOI: 10.1186/s13568-020-0957-4
Naim Hassan 1 , Yu Sasano 1 , Shunta Kimura 2 , Farhana Easmin 1 , Keisuke Ekino 1 , Hisataka Taguchi 1 , Satoshi Harashima 1
Affiliation  

In our previous study, a novel genome engineering technology, PCR-mediated chromosome duplication (PCDup), was developed in Saccharomyces cerevisiae that enabled the duplication of any desired chromosomal region, resulting in a segmental aneuploid. From one round of transformation, PCDup can duplicate a single chromosomal region efficiently. However, simultaneous duplication of multiple chromosomal regions is not possible using PCDup technology, which is a serious drawback. Sequential duplication is possible, but this approach requires significantly more time and effort. Because PCDup depends upon homologous recombination, we reasoned that it might be possible to simultaneously create duplications of multiple chromosomal regions if we could increase the frequency of these events. Double-strand breaks have been shown to increase the frequency of homologous recombination around the break point. Thus, we aimed to integrate the genome editing tool CRISPR/Cas9 system, which induces double-strand breaks, with our conventional PCDup. The new method, which we named CRISPR-PCDup increased the efficiency of a single duplication by up to 30 fold. CRISPR-PCDup enabled the simultaneous duplication of long chromosomal segments (160 kb and 200 kb regions). Moreover, we were also able to increase the length of the duplicated chromosome by up to at least 400 kb, whereas conventional PCDup can duplicate up to a maximum of 300 kb. Given the enhanced efficiency of chromosomal segmental duplication and the saving in both labor and time, we propose that CRISPR-PCDup will be an invaluable technology for generating novel yeast strains with desirable traits for specific industrial applications and for investigating genome function in segmental aneuploid.

中文翻译:

CRISPR-PCDup:一种在酿酒酵母中同时进行分段染色体复制的新方法。

在我们之前的研究中,在酿酒酵母中开发了一种新的基因组工程技术,即PCR介导的染色体复制(PCDup),该技术能够复制任何所需的染色体区域,从而产生节段非整倍体。从一轮转化中,PCDup可以有效地复制单个染色体区域。但是,使用PCDup技术无法同时复制多个染色体区域,这是一个严重的缺点。顺序复制是可能的,但是这种方法需要更多的时间和精力。因为PCDup依赖于同源重组,所以我们认为,如果我们可以增加这些事件的发生频率,则有可能同时创建多个染色体区域的重复。已经显示双链断裂增加了在断裂点附近的同源重组的频率。因此,我们旨在将基因组编辑工具CRISPR / Cas9系统与常规PCDup集成在一起,该系统可诱导双链断裂。我们将其命名为CRISPR-PCDup的新方法将单次复制的效率提高了30倍。CRISPR-PCDup能够同时复制长染色体片段(160 kb和200 kb区域)。此外,我们还能够将复制的染色体的长度增加至少400 kb,而传统的PCDup最多可以复制300 kb。鉴于提高了染色体节段复制的效率,并节省了人工和时间,
更新日期:2020-02-03
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