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Creating functional chromosome fusions in yeast with CRISPR–Cas9
Nature Protocols ( IF 14.8 ) Pub Date : 2019-07-12 , DOI: 10.1038/s41596-019-0192-0
Yangyang Shao 1, 2 , Ning Lu 1, 2 , Xiaoli Xue 1 , Zhongjun Qin 1
Affiliation  

CRISPR–Cas9-facilitated functional chromosome fusion allows the generation of a series of yeast strains with progressively reduced chromosome numbers that are valuable resources for the study of fundamental concepts in chromosome biology, including replication, recombination and segregation. We created a new yeast strain with a single chromosome by using the protocol for chromosome fusion described herein. To ensure the accuracy of chromosome fusions in yeast, the long redundant repetitive sequences near linear chromosomal ends are deleted, and the fusion orders are correspondingly determined. Possible influence on gene expression is minimized to retain gene functionality. This protocol provides experimentally derived guidelines for the generation of functional chromosome fusions in yeast, especially for the deletion of repetitive sequences, the determination of the fusion order and cleavage sites, and primary evaluation of the functionality of chromosome fusions. Beginning with design, one round of typical chromosome fusion and functional verifications can be accomplished within 18 d.



中文翻译:

使用 CRISPR-Cas9 在酵母中创建功能性染色体融合

CRISPR-Cas9 促进的功能性染色体融合允许产生一系列染色体数量逐渐减少的酵母菌株,这些菌株是研究染色体生物学基本概念(包括复制、重组和分离)的宝贵资源。我们通过使用本文所述的染色体融合方案创建了具有单条染色体的新酵母菌株。为保证酵母染色体融合的准确性,删除线性染色体末端附近的长冗余重复序列,并相应确定融合顺序。对基因表达的可能影响被最小化以保留基因功能。该协议为在酵母中产生功能性染色体融合提供了实验得出的指导方针,特别是对于重复序列的删除,确定融合顺序和切割位点,以及对染色体融合功能的初步评估。从设计开始,18d内即可完成一轮典型的染色体融合和功能验证。

更新日期:2019-11-18
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