DNA base editor may induce a large number of off-target RNA mutations

Source: CC0 Public DomainDNA base editing methods can directly correct dot mutations in genomic DNA without producing any double-stranded fractures (DSBs, double-breaks), but the potential off-target effect often limits the application of these methods, and adeno-related viruses (AAVs) are the most commonly used delivery systems in DNA editing gene therapy, as these viruses can sustain gene expression in the body, so the potential induced dna base is highly inducedprevious studies have evaluated genomic DNA off-target mutations induced by the DNA base editor, and the deaminase necessary for the commonly used DNA base editor usually exhibits the binding activity of DNA, such as cytosine deaminase APO, used in the cytosine base editor (CBEs) BEC1 is able to target DNA and RNA, while adenonine deaminase TadA in adenine base editor (ABEs) induces site-specific myoside formation on RNA, but any potential RNA mutations induced by the DNA base editor have not been evaluated by the researchersTo assess the off-target effect struck by THE DNA base editor at RNA levels, the researchers calculated the number of off-target RNA SNVs produced each replication of each CBE and ABE processed cells, and explored the effective elimination of off-target RNA SNVs through engineered DNA base editor deaminaseIn the paper, the researchers transfected BE3 (APOBEC1-nCas9-UGI) (a CBE), ABE7.10 (TadA-TadA-nCas9) (an ABE) and GFP carrying or not carrying single-stranded RNA In heK293T cultured cells, after confirming the effective DNA editing efficiency of BE3 and ABE7.10 on HEK293T cells, the researchers sequenced the samples rna at an average depth of 125X and quantitatively evaluated RNA SNVs in each replicated cellto ensure efficient editing, each cell replicator processed by CBE or ABE assessed its target editing efficiency, and then the researchers compared the number of off-target RNA SNVs in two groups with the GFP control group, and found that the number of RNA SNVs in the cells processed by the DNA base editor was surprisingly highIn addition, the researchers found that the cell mutation bias estheofs treated by BE3 and ABE7.0 were the same as those of APOBEC1 and TadA, respectively, suggesting that the off-target effect may have been caused by over-expression of the DNA base editor, and that the researchers identified C and ABE-specific base sequences and genetic regions in these off-target RNA SNVsTo eliminate RNA off-target activity in the base editor, the researchers also analyzed the effects of the introduction point mutation on APOBEC1 and TadA, and found that three high-fidelity mutations: BE3W90Y-R126E, THE BE3 (HA3AR128A) and BE3 (hA3AY130F) reduce RNA off-target SNVs at the baseline level, and similarly, the ABE mutation ABE7.10F148A completely eliminates the off-target effectthis study, the researchers obtained high fidelity mutations in CBEs and ABEs by introducing point mutations in deaminase, and proposed a new method to improve the specificity of the base editor by using engineered operationsoriginal origin:Changyang Zhou, Yidi Sun, Rui Yan, et alOff-target RNA ennal induced by DNA base editing and its sylwed by mutagenesis, Nature (2019)DOI: 10.1038/s41586-019-1314-0original title:Nature: Chinese Academy of Sciences, Kawada Cooperation New Achievements! DNA base editor may induce a large number of off-target RNA mutations!