Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.
Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA.
Nature. 2019 May;569(7756):433-437. doi: 10.1038/s41586-019-1161-z. Epub 2019 Apr 17.
CRISPR-Cas base-editor technology enables targeted nucleotide alterations, and is being increasingly used for research and potential therapeutic applications. The most widely used cytosine base editors (CBEs) induce deamination of DNA cytosines using the rat APOBEC1 enzyme, which is targeted by a linked Cas protein-guide RNA complex. Previous studies of the specificity of CBEs have identified off-target DNA edits in mammalian cells. Here we show that a CBE with rat APOBEC1 can cause extensive transcriptome-wide deamination of RNA cytosines in human cells, inducing tens of thousands of C-to-U edits with frequencies ranging from 0.07% to 100% in 38-58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, and 5' and 3' untranslated region mutations. We engineered two CBE variants bearing mutations in rat APOBEC1 that substantially decreased the number of RNA edits (by more than 390-fold and more than 3,800-fold) in human cells. These variants also showed more precise on-target DNA editing than the wild-type CBE and, for most guide RNAs tested, no substantial reduction in editing efficiency. Finally, we show that an adenine base editor can also induce transcriptome-wide RNA edits. These results have implications for the use of base editors in both research and clinical settings, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
CRISPR-Cas 碱基编辑技术可实现靶向核苷酸改变，并且越来越多地用于研究和潜在的治疗应用。最广泛使用的胞嘧啶碱基编辑器（CBEs）使用大鼠 APOBEC1 酶诱导 DNA 胞嘧啶脱氨，该酶被连接的 Cas 蛋白指导 RNA 复合物靶向。先前对 CBEs 特异性的研究已经在哺乳动物细胞中鉴定出了脱靶 DNA 编辑。在这里，我们表明大鼠 APOBEC1 的 CBE 可以导致人类细胞中广泛的转录组范围内的 RNA 胞嘧啶脱氨，在 38-58%的表达基因中诱导数万种 C 到 U 的编辑，频率范围从 0.07%到 100%。CBE 诱导的 RNA 编辑发生在蛋白质编码和非蛋白质编码序列中，并产生错义、无义、剪接位点和 5'和 3'非翻译区突变。我们设计了两种在大鼠 APOBEC1 中带有突变的 CBE 变体，这大大减少了人类细胞中的 RNA 编辑数量（减少了 390 多倍和 3800 多倍）。这些变体在靶 DNA 编辑上也比野生型 CBE 更精确，对于大多数测试的指导 RNA，编辑效率没有显著降低。最后，我们表明腺嘌呤碱基编辑器也可以诱导转录组范围内的 RNA 编辑。这些结果对研究和临床环境中碱基编辑器的使用具有影响，说明了设计具有降低 RNA 编辑活性的改良变体的可行性，并表明需要更全面地定义和表征碱基编辑平台中脱氨酶的 RNA 脱靶效应。
