基因组编辑对酿酒酵母DNA的损伤作用及修复响应  

DNA lesions and repair response caused by genome editing in Saccharomyces cerevisiae

作　　者：张首 王震[2] 蔺玉萍 戎倩倩 王丽贤 齐显尼[2] 刘浩[1] 王钦宏[2] Shou Zhang;Zhen Wang;Yuping Lin;Qianqian Rong;Lixian Wang;Xianni Qi;Hao Liu;Qinhong Wang(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;CAS Key Laboratory of Systems Microbial Biotechnology,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;University of Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所,中国科学院系统微生物工程重点实验室,天津300308 [3]中国科学院大学,北京100049

出　　处：《微生物学报》2020年第7期1384-1400,共17页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31700077);天津市自然科学基金(16JCYBJC43100)。

摘　　要：【目的】为了研究基因组编辑工具CRISPR/Cas9和CRISPR/Cpf1所产生的DNA双链断裂(DNA doublestrandbreak,DSB)对酿酒酵母DNA的损伤作用及修复响应情况,对比化学物质甲基磺酸甲酯(methyl methanesulfonate,MMS)对酿酒酵母基因组DNA的损伤和修复,阐明编辑细胞在细胞水平和转录水平上的变化。【方法】起始细胞分为两种情况,包括未进行细胞周期同步化和被α-因子同步化细胞周期至G0/G1期。检测CRISPR/Cas9和CRISPR/Cpf1处理后编辑细胞的生长情况。利用流式细胞术检测编辑细胞的细胞周期延滞的情况。利用荧光定量PCR检测编辑细胞和MMS处理细胞后DNA损伤响应关键基因转录表达水平的变化情况。【结果】起始细胞无论是未同步化还是同步化,其生长均受到基因组编辑抑制,细胞存活率降低,细胞周期被滞留在G2/M期,而MMS处理导致细胞周期S期的滞留。此外,随编辑时间的延长,突变率增加,细胞存活率降低。CRISPR/Cpf1编辑细胞的突变率和存活率均低于CRISPR/Cas9,由此可见,CRISPR/Cpf1对细胞的损伤强度高于CRISPR/Cas9。两种编辑均诱导酵母DNA损伤响应关键基因RNR3及HUG1转录水平显著上调,并且CRISPR/Cpf1介导的上调幅度大于CRISPR/Cas9,但两者均低于MMS的处理。【结论】本研究解析了CRISPR/Cas9和CRISPR/Cpf1介导的基因组编辑在细胞水平和转录水平上对DNA损伤作用及修复响应,初步揭示了酿酒酵母应对不同类型的DSB损伤时响应程度的差异,为提高基因组编辑工具的编辑能力和评估基因编辑安全性提供了重要依据。[Objective]To investigate DNA lesions and repair response caused by the DNA double strand break(DSB)generated by the genome editing tools including CRISPR/Cas9 and CRISPR/Cpf1 in Saccharomyces cerevisiae,we used the damage and repair of S.cerevisiae genomic DNA caused by a chemical substance methyl methane sulfonate(MMS)as a comparison and elucidated the changes of edited cells at the cellular and transcriptional levels.[Methods]Initial cells were divided into two situations,including unsynchronized cell cycle and synchronized cell cycle to G0/G1 phase byα-factor.We measured the growth profiles of CRISPR/Cas9-and CRISPR/Cpf1-mediated edited cells.We employed flow cytometry to detect the arrested cell cycle of edited cells.We used Real-time PCR to quantify the transcriptional expression changes of key genes involved in DNA damage response in edited cells and MMS-treated cells.[Results]Growth of initial cells,which were either unsynchronized or synchronized cell cycle byα-factor,were inhibited by genome editing.Cell viabilities of edited cells decreased,and the cell cycles were arrested at the G2/M phase.Furthermore,along with the prolongation of editing time,mutation efficiency of edited cells increased while cell viabilities decreased.The mutation efficiency and viabilities of CRISPR/Cpf1 edited cells were lower than those of CRISPR/Cas9,and thus the damage induced by CRISPR/Cpf1 was stronger than that of CRISPR/Cas9.Both these two editing tools induced significantly up-regulated transcriptional expressions of RNR3 and HUG1,which are key genes involved in DNA damage response in yeast.Additionally,the extent of CRISPR/Cpf1-mediated up-regulation was higher than that of CRISPR/Cas9,but both were lower than MMS treatment.[Conclusion]This study analyzed DNA lesions and repair response caused by CRISPR/Cas9-and CRISPR/Cpf1-mediated genome editing at the cellular and transcriptional levels,and preliminarily revealed the divergent extents of S.cerevisiae in response to different DSBs,thus providing an important guidan

关 键 词：酿酒酵母 CRISPR/Cas9 CRISPR/Cpf1 甲基磺酸甲酯 细胞周期 DNA损伤响应 

分 类 号：Q78[生物学—分子生物学]