CRISPR adenine and cytosine base editors with reduced RNA off-target activities [ABE]

CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications. Overexpression of adenine base editors in HEK293T, GFP-based cell sorting and RNA-seq for evaluating the degree of off-target editing from ABEs.

CRISPR引导的DNA碱基编辑器（CRISPR-guided DNA base editors）能够高效介导靶向单核苷酸改变的精准引入。胞嘧啶碱基编辑器（Cytosine base editor, CBE）与腺嘌呤碱基编辑器（Adenine base editor, ABE）分别是将胞苷脱氨酶、腺苷脱氨酶融合至CRISPR-Cas切口酶（CRISPR-Cas nickases）的融合蛋白，可分别高效诱导DNA发生C→T与A→G的碱基替换。我们此前的研究证实，当前广泛使用的携带大鼠APOBEC1胞苷脱氨酶的CBE BE3，以及优化后的ABEmax编辑器，均可在人类细胞中以高效率诱导出数以万计的不依赖向导RNA（guide RNA）的全转录组范围（transcriptome-wide）RNA碱基编辑事件。此外，我们证实了构建选择性抑制非靶向RNA编辑（SElective Curbing of Unwanted RNA Editing, SECURE）-BE3变体的可行性，这类变体可在保留高效且更精准的靶向DNA编辑活性的同时，大幅降低不必要的RNA编辑活性。本研究报道了SECURE-ABE变体的结构引导工程化改造策略：这类变体不仅可在保持相当靶向DNA编辑活性的前提下降低脱靶RNA编辑水平，同时也是目前已报道的最小尺寸的酿脓链球菌Cas9（Streptococcus pyogenes Cas9, SpCas9）碱基编辑器。此外，我们测试了除APOBEC1之外的其他胞苷脱氨酶构建的CBE，发现人类APOBEC3A（hA3A）胞苷脱氨酶构建的CBE可高效诱导大规模全转录组范围的RNA碱基编辑。与之相反，此前报道的“增强型”A3A（eA3A）胞苷脱氨酶CBE，或是人类活化诱导胞苷脱氨酶（human activation-induced cytidine deaminase, hAID）构建的CBE，其诱导的RNA编辑水平则大幅降低，甚至接近背景基线。综上，本研究报道了具备广泛应用价值的SECURE-ABE与SECURE-CBE碱基编辑器，并进一步证实了在研究与治疗应用中，降低DNA碱基编辑器的RNA编辑活性的重要性。本研究通过在HEK293T细胞中过表达腺嘌呤碱基编辑器，结合基于GFP的细胞分选与RNA测序（RNA-seq）技术，评估ABE的脱靶编辑程度。