Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.
Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
Nat Biotechnol. 2019 Sep;37(9):1041-1048. doi: 10.1038/s41587-019-0236-6. Epub 2019 Sep 2.
Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE, human activation-induced cytidine deaminase-based CBE, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.
胞嘧啶或腺嘌呤碱基编辑器（CBE 或 ABE）可引入特定的 DNA C 到 T 或 A 到 G 改变。然而，我们最近证明，它们还可以诱导 RNA 碱基的全转录组向导 RNA 独立编辑，并创建了选择性抑制不必要的 RNA 编辑（SECURE）-BE3 变体，这些变体降低了不必要的 RNA 编辑活性。在这里，我们描述了经过结构指导的 SECURE-ABE 变体的工程改造，这些变体具有降低的脱靶 RNA 编辑活性和可比的靶 DNA 编辑活性，并且是迄今为止描述的最小的酿脓链球菌 Cas9 碱基编辑器之一。我们还测试了除 APOBEC1 以外的其他胞嘧啶脱氨酶的 CBE，并发现基于人类 APOBEC3A 的 CBE 会诱导大量的 RNA 碱基编辑，而增强的基于 APOBEC3A 的 CBE、基于人类激活诱导胞嘧啶脱氨酶的 CBE 和基于 Petromyzon marinus 胞嘧啶脱氨酶的 CBE Target-AID 则会导致更少的 RNA 编辑。最后，我们发现表现出 RNA 脱靶编辑活性的 CBE 和 ABE 也可以自我编辑其自身的转录本，从而导致碱基编辑器编码序列的异质性。
