严谨实验结果现真知——中国学者成功解开基因编辑脱靶疑团
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摘要
基因编辑技术在修复致病突变与种质改良方面都有重要的发展前景,被广泛使用于基础与应用研究中。该技术的真实脱靶率一直存在争议,严重阻碍了其发展。2019年3月,杨辉、高彩霞分别领导的两项研究终于揭示了脱靶的奥秘,其成果同时在《Science》发表。在小鼠与水稻中的研究一致发现:胞嘧啶编辑器会造成严重的点突变,而腺嘌呤编辑器与经典的CRISPR/Cas9技术没有明显的脱靶效应。文章综述了脱靶的疑团为何长期未解,最新研究是如何通过构建严谨的实验对照实现了精密的脱靶检测并得到严谨的数据与结论,同时初步讨论了在风险未能排除之前,人类必须谨慎开展人体临床试验。
Genome editing is a high promising biotech for correcting pathogenic mutations and trial improvement,and is widely used in basic and applicable researches. However, the true off-target rate of this technology has long been controversial, which has seriously hindered its development. In March 2019, two scientific reports led by Yang Hui and Gao Caixia, respectively, finally revealed the mystery of off-target, and both studies published in Science. Both of the mouse and rice studies consistently found that the cytosine editor causes severe point mutations, while the adenine editor and the classic CRISPR/Cas9 have no significant off-target effects. This article reviews why the off-target suspects has been long remain unsolved, andhow the latest researches achieve in precise off-target detection and obtain very rigorous data and conclusions,which base on constructing rigorous experimental controls. At the same time,it was discussed here that humans must be cautious in conducting human clinical trials before the risks are truly eliminated.
Genome editing is a high promising biotech for correcting pathogenic mutations and trial improvement,and is widely used in basic and applicable researches. However, the true off-target rate of this technology has long been controversial, which has seriously hindered its development. In March 2019, two scientific reports led by Yang Hui and Gao Caixia, respectively, finally revealed the mystery of off-target, and both studies published in Science. Both of the mouse and rice studies consistently found that the cytosine editor causes severe point mutations, while the adenine editor and the classic CRISPR/Cas9 have no significant off-target effects. This article reviews why the off-target suspects has been long remain unsolved, andhow the latest researches achieve in precise off-target detection and obtain very rigorous data and conclusions,which base on constructing rigorous experimental controls. At the same time,it was discussed here that humans must be cautious in conducting human clinical trials before the risks are truly eliminated.
引文
[1] ZUO E,SUN Y,WEI W, et al. Cytosine base editor generates substantial offtarget single nucleotide variants in mouse embryos[J]. Science, 2019. doi:10.1126/science.aav9973.
[2] JIN S, ZONG Y,GAO Q, et al. Cytosine,but not adenine, base editors induce genome-wide off-target mutations in rice[J]. Science, 2019. doi:10.1126/science.aaw7166(2019).
[3] HAAPANIEMI E, BOTLA S, PERSSON J, et al. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response[J]. Nature Medicine, 2018,24:927-930.
[4] KOMOR A C, KIM Y B, PACKER M S,et al. Programmable editing of a target base in genomic DNA without doublestranded DNA cleavage[J]. Nature,2016, 533(7603):420-424.
[5] GAUDELLI N M, KOMOR A C, REES H A, et al. Programmable baseediting of A·T to G·C in genomic DNA without DNA cleavage[J].Nature, 2017,551(7681):464-471.
[6] WANG H,YANG H,SHIVALILA C S, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J]. Cell, 2013, 153(4):910-918.
[7] ZONG Y,WANG Y,LI C,et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion[J]. Nat Biotechnol, 2017, 35:438-440.
[8] DRAKE J W, CHARLESWORTH B,CHARLESWORTH D, et al. Rates of spontaneous mutation[J]. Genetics,1998, 1484:1667-1686.
[9]何静,程唯珈,黄辛·给基因编辑做“体检”:让脱靶无处隐藏[N].中国科学报,2019-03-01(1).
[10] HARRIS R S, PETERSEN-MAHRT S K, NEUBERGER M S. RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators[J].Mol Cell, 2002, 10:1247-1253.
[11] RADANY E H, DORNFELD K J,SANDERSON R J, et al. Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNAglycosylase[J]. Mutat Res, 2000, 461:41-58.
[12] KOSICKI M,TOMBERGK, BRADLEY A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements[J]. Nature Biotechnology, 2018,36:765-771.
[2] JIN S, ZONG Y,GAO Q, et al. Cytosine,but not adenine, base editors induce genome-wide off-target mutations in rice[J]. Science, 2019. doi:10.1126/science.aaw7166(2019).
[3] HAAPANIEMI E, BOTLA S, PERSSON J, et al. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response[J]. Nature Medicine, 2018,24:927-930.
[4] KOMOR A C, KIM Y B, PACKER M S,et al. Programmable editing of a target base in genomic DNA without doublestranded DNA cleavage[J]. Nature,2016, 533(7603):420-424.
[5] GAUDELLI N M, KOMOR A C, REES H A, et al. Programmable baseediting of A·T to G·C in genomic DNA without DNA cleavage[J].Nature, 2017,551(7681):464-471.
[6] WANG H,YANG H,SHIVALILA C S, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J]. Cell, 2013, 153(4):910-918.
[7] ZONG Y,WANG Y,LI C,et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion[J]. Nat Biotechnol, 2017, 35:438-440.
[8] DRAKE J W, CHARLESWORTH B,CHARLESWORTH D, et al. Rates of spontaneous mutation[J]. Genetics,1998, 1484:1667-1686.
[9]何静,程唯珈,黄辛·给基因编辑做“体检”:让脱靶无处隐藏[N].中国科学报,2019-03-01(1).
[10] HARRIS R S, PETERSEN-MAHRT S K, NEUBERGER M S. RNA editing enzyme APOBEC1 and some of its homologs can act as DNA mutators[J].Mol Cell, 2002, 10:1247-1253.
[11] RADANY E H, DORNFELD K J,SANDERSON R J, et al. Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNAglycosylase[J]. Mutat Res, 2000, 461:41-58.
[12] KOSICKI M,TOMBERGK, BRADLEY A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements[J]. Nature Biotechnology, 2018,36:765-771.