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.
[1]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;
[2]Univ Chinese Acad Sci, Coll Life Sci, Beijing, Peoples R China;
[3]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;
[4]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;
[5]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;
[6]Shanghai Tech Univ, Sch Life Sci & Technol, Shanghai, Peoples R China;
[7]Chinese Acad Agr Sci, Agr Genome Inst Shenzhen, Ctr Anim Genom, Shenzhen, Peoples R China;
[8]Fudan Univ, Shanghai Acad Sci & Technol, Shanghai Jiao Tong Univ, Shanghai, Peoples R China
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