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Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis

机译：DNA碱基编辑诱导脱靶RNA突变，并通过诱变消除

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摘要

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks(1-3), but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA(4-8), it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA(9-13). For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA(12), and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA(9,11). However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern(14-16). Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.
机译：最近开发的DNA碱基编辑方法可以直接在基因组DNA中生成所需的点突变，而不会产生任何双链断裂（1-3），但是脱靶编辑的问题限制了这些方法的应用。尽管先前的几项研究已经评估了基因组DNA的脱靶突变（4-8），但现在很清楚，常用DNA碱基编辑器不可或缺的脱氨酶通常与RNA结合（9-13）。例如，用于胞嘧啶碱基编辑器（CBE）的胞嘧啶脱氨酶APOBEC1既可靶向DNA也可靶向RNA（12），而用于腺嘌呤碱基编辑器（ABEs）的腺嘌呤脱氨酶TadA可以诱导位点特异性肌苷。在RNA上形成（9,11）但是，尚未评估由DNA碱基编辑器引起的任何潜在RNA突变。腺相关病毒是涉及DNA编辑的基因治疗中最常见的传递系统。这些病毒可以在体内维持长期的基因表达，因此由DNA碱基编辑者诱导的潜在RNA突变的程度备受关注（14-16）。在这里，我们定量评估了由CBE或ABE诱导的RNA单核苷酸变异（SNV）。胞嘧啶碱基编辑器BE3和腺嘌呤碱基编辑器ABE7.10都产生了数以万计的脱靶RNA SNV。随后，通过工程脱氨酶，我们发现三个CBE变体和一个ABE变体显示脱靶RNA SNV降低至基线，同时保持了有效的DNA靶向活性。这项研究揭示了脱氧核糖核酸编辑中脱靶效应的一个先前被忽略的方面，并且还表明可以通过工程脱氨作用消除这种效应。

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  • 来源
    《Nature》 |2019年第7764期|275-278|共4页
  • 作者

    Zhou Changyang; Sun Yidi; Yan Rui; Liu Yajing; Zuo Erwei; Gu Chan; Han Linxiao; Wei Yu; Hu Xinde; Zeng Rong; Li Yixue; Zhou Haibo; Guo Fan; Yang Hui;

  • 作者单位

    Chinese Acad Sci, Inst Neurosci,Key Lab Primate Neurobiol, State Key Lab Neurosci,CAS Ctr Excellence Brain S, Shanghai Inst Biol Sci,Shanghai Res Ctr Brain Sci, Shanghai, Peoples R China|Univ Chinese Acad Sci, Coll Life Sci, Beijing, Peoples R China;

    Univ Chinese Acad Sci, Coll Life Sci, Beijing, Peoples R China|Chinese Acad Sci, CAS Key Lab Syst Biol,CAS Ctr Excellence Mol Cell, Inst Biochem & Cell Biol, Shanghai Inst Biol Sci, Shanghai, Peoples R China|Chinese Acad Sci, Univ Chinese Acad Sci,Key Lab Computat Biol, Biomed Big Data Ctr,CAS MPG Partner Inst Computat, Shanghai Inst Nutr & Hlth,Shanghai Inst Biol Sci, Shanghai, Peoples R China;

    Sichuan Univ, Coll Life Sci,West China Univ Hosp 2, Ctr Translat Med,Minist Educ, Dept Obstet & Gynecol,Key Lab Birth Defects & Rel, Chengdu, Sichuan, Peoples R China;

    Univ Chinese Acad Sci, Coll Life Sci, Beijing, Peoples R China|Shanghai Tech Univ, Sch Life Sci & Technol, Shanghai, Peoples R China;

    Chinese Acad Sci, Inst Neurosci,Key Lab Primate Neurobiol, State Key Lab Neurosci,CAS Ctr Excellence Brain S, Shanghai Inst Biol Sci,Shanghai Res Ctr Brain Sci, Shanghai, Peoples R China|Chinese Acad Agr Sci, Agr Genome Inst Shenzhen, Ctr Anim Genom, Shenzhen, Peoples R China;

    Sichuan Univ, Coll Life Sci,West China Univ Hosp 2, Ctr Translat Med,Minist Educ, Dept Obstet & Gynecol,Key Lab Birth Defects & Rel, Chengdu, Sichuan, Peoples R China;

    Chinese Acad Sci, Inst Neurosci,Key Lab Primate Neurobiol, State Key Lab Neurosci,CAS Ctr Excellence Brain S, Shanghai Inst Biol Sci,Shanghai Res Ctr Brain Sci, Shanghai, Peoples R China;

    Chinese Acad Sci, Inst Neurosci,Key Lab Primate Neurobiol, State Key Lab Neurosci,CAS Ctr Excellence Brain S, Shanghai Inst Biol Sci,Shanghai Res Ctr Brain Sci, Shanghai, Peoples R China;

    Chinese Acad Sci, Inst Neurosci,Key Lab Primate Neurobiol, State Key Lab Neurosci,CAS Ctr Excellence Brain S, Shanghai Inst Biol Sci,Shanghai Res Ctr Brain Sci, Shanghai, Peoples R China|Univ Chinese Acad Sci, Coll Life Sci, Beijing, Peoples R China;

    Chinese Acad Sci, CAS Key Lab Syst Biol,CAS Ctr Excellence Mol Cell, Inst Biochem & Cell Biol, Shanghai Inst Biol Sci, Shanghai, Peoples R China|Shanghai Tech Univ, Sch Life Sci & Technol, Shanghai, Peoples R China;

    Sichuan Univ, Coll Life Sci,West China Univ Hosp 2, Ctr Translat Med,Minist Educ, Dept Obstet & Gynecol,Key Lab Birth Defects & Rel, Chengdu, Sichuan, Peoples R China|Shanghai Tech Univ, Sch Life Sci & Technol, Shanghai, Peoples R China|Fudan Univ, Shanghai Acad Sci & Technol, Shanghai Jiao Tong Univ, Shanghai, Peoples R China;

    Chinese Acad Sci, Inst Neurosci,Key Lab Primate Neurobiol, State Key Lab Neurosci,CAS Ctr Excellence Brain S, Shanghai Inst Biol Sci,Shanghai Res Ctr Brain Sci, Shanghai, Peoples R China;

    Sichuan Univ, Coll Life Sci,West China Univ Hosp 2, Ctr Translat Med,Minist Educ, Dept Obstet & Gynecol,Key Lab Birth Defects & Rel, Chengdu, Sichuan, Peoples R China;

    Chinese Acad Sci, Inst Neurosci,Key Lab Primate Neurobiol, State Key Lab Neurosci,CAS Ctr Excellence Brain S, Shanghai Inst Biol Sci,Shanghai Res Ctr Brain Sci, Shanghai, Peoples R China;

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