利用CRISPR/Cas9对基因组中高度同源DNA片段编辑多样性的遗传学研究

doi:10.16288/j.yczz.17-019

遗传 ›› 2017, Vol. 39 ›› Issue (4): 313-325.doi: 10.16288/j.yczz.17-019

利用CRISPR/Cas9对基因组中高度同源DNA片段编辑多样性的遗传学研究

汪乐洋(),黄海燕(),吴强()

上海交通大学系统生物医学研究院比较生物医学研究中心,系统生物医学教育部重点实验室,上海 200240

收稿日期:2017-01-16 修回日期:2017-02-28 出版日期:2017-04-20 发布日期:2017-03-08
作者简介:汪乐洋,硕士研究生,专业方向：遗传学。E-mail: wangleyang321@163.com|黄海燕,博士,副研究员,研究方向：药物分子遗传学。E-mail: hy_huang@sjtu.edu.cn|吴强,博士,教授,研究方向：基因表达调控及神经发育。E-mail: qwu123@gmail.com
基金资助:
国家自然科学基金项目(31470820,81302861)

The diversity of DNA fragment editing by CRISPR/Cas9 in highly homologous or repetitive sequences

Wang Leyang(),Huang Haiyan(),Wu Qiang()

Center for Comparative Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2017-01-16 Revised:2017-02-28 Online:2017-04-20 Published:2017-03-08
Supported by:
the National Natural Science Foundation of China(31470820,81302861)

摘要/Abstract

摘要：

在基因组中,编码区存在许多高度相似的基因簇或基因群(多拷贝基因),非编码区也存在大量的重复序列。这些重复序列能通过改变染色体的三维结构调控基因的转录,对于生物体的遗传与进化起到了重要的作用。其高度同源的特征使得利用CRISPR/Cas9技术进行基因组编辑时面临更加复杂的状况。如果编辑的片段是二倍体或多倍体,还会产生各条染色单体上的编辑情况不相同的现象。为此本文选择了2个位于同一染色体相距11 kb的高度同源300 bp片段(L1和L2)进行CRISPR介导的DNA片段编辑。采用一对sgRNA(分别共同靶向两片段的上、下游位点)引导Cas9对HepG2细胞两个高度相似的DNA片段进行切割。片段编辑的细胞进一步单克隆化后,对获得的22个L1/L2编辑的CRISPR单克隆细胞株进行详细的基因型鉴定。结果发现除了这两个DNA片段本身被删除外,它们之间的大片段也存在被删除的现象,三个片段的各种反转组合也很频繁。该研究结果对于采用CRISPR/Cas9系统编辑多拷贝基因或重复序列,尤其是对二倍体或多倍体生物进行基因组编辑时具有重要的借鉴和参考价值。

关键词: CRISPR/Cas9, DNA片段编辑, 单克隆, 多拷贝基因, 重复调控序列

Abstract:

In complex genomes, there are a large number of duplicated genes in the coding regions and many more repetitive sequences in the non-coding regions. Repetitive sequences can exert great impacts on the heredity and evolution of the organisms, as well as their genome 3D architecture and transcriptional regulation. The high homology nature of repetitive sequences renders their editing by CRISPR/Cas9 very complex. At diploid or polyploid situations, such repetitive sequences could be edited differently on each chromosome or chromatid. To explore such possibilities, we had studied the editing of two highly homologous DNA fragments (L1 and L2), each about 300 bp in size and 11 kb apart on the same chromosome. We designed a pair of sgRNAs targeting the upstream and downstream of the two DNA fragments to guide the Cas9 cleavage of the two fragments in the human HepG2 cells. We further established single-cell CRISPR clones for DNA-fragment-edited cells. A total of 22 CRISPR cell clones were characterized for their DNA fragment editing patterns. In addition to the deletion of L1/L2 fragments, we had also identified the deletion of the large internal fragment between L1 and L2 fragments, and the various combinations of inversions and deletions of the three DNA fragments. Our results have demonstrated the potential issues with important implications for using CRISPR/Cas9 to edit duplicated genes or repetitive sequences in diploid or polyploid species or cell lines.

Key words: CRISPR/Cas9, DNA fragment editing, monoclone, duplicated genes, repetitive sequences

汪乐洋,黄海燕,吴强. 利用CRISPR/Cas9对基因组中高度同源DNA片段编辑多样性的遗传学研究[J]. 遗传, 2017, 39(4): 313-325.

Wang Leyang,Huang Haiyan,Wu Qiang. The diversity of DNA fragment editing by CRISPR/Cas9 in highly homologous or repetitive sequences[J]. Hereditas(Beijing), 2017, 39(4): 313-325.

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利用CRISPR/Cas9对基因组中高度同源DNA片段编辑多样性的遗传学研究

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