A Precise Genome Editing Method Reveals Insights into the Activity of Eukaryotic Promoters
A Precise Genome Editing Method Reveals Insights into the Activity of Eukaryotic Promoters
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DOI：
10.1016/j.celrep.2016.12.014
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发表时间：
2017-01-03
期刊：
影响因子：
8.8
通讯作者：
Acar, Murat
中科院分区：
文献类型：
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作者：
Elison, Gregory L.;Song, Ruijie;Acar, Murat
尽管几乎所有模式生物都有全基因组序列，但仅根据相应的启动子序列对基因表达水平进行准确预测仍然是一个挑战。由于拷贝数的波动及其破坏性的性质，涉及选择标记的基于质粒的途径和方法并不理想。在这里，我们提出了一种使用CRISPR/Cas9复合体的基因组编辑方法，并阐明了对活酵母细胞中典型启动子活性的见解。该方法包括将新的切割位点引入特定的基因组位置，然后以无疤痕的方式将编辑后的序列整合到相同位置。用这种方法编辑GAL1和GAL80启动子序列，我们发现启动子元件的相对定位对于在单个细胞中设定启动子活性水平至关重要。该方法可以推广到其他生物，以破译各种基因网络中的基因-表型关系。
Despite the availability of whole-genome sequences for almost all model organisms, making faithful predictions of gene expression levels based solely on the corresponding promoter sequences remains a challenge. Plasmid-based approaches and methods involving selection markers are not ideal due to copy-number fluctuations and their disruptive nature. Here, we present a genome editing method using the CRISPR/Cas9 complex and elucidate insights into the activity of canonical promoters in live yeast cells. The method involves the introduction of a novel cut site into a specific genomic location, followed by the integration of an edited sequence into the same location in a scarless manner. Using this method to edit the GAL1 and GAL80 promoter sequences, we found that the relative positioning of promoter elements was critically important for setting promoter activity levels in single cells. The method can be extended to other organisms to decode genotype-phenotype relationships in various gene networks.