sgBE: a structure-guided design of sgRNA architecture specifies base editing window and enables simultaneous conversion
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sgBE: a structure-guided design of sgRNA architecture specifies base editing window and enables simultaneous conversion of cytosine and adenosine Yanhong Wang†, Lifang Zhou†, Rui Tao†, Nan Liu, Jie Long, Fengming Qin, Wenling Tang, Yang Yang, Qiang Chen and Shaohua Yao* * Correspondence: shaohuayao@ scu.edu.cn † Yanhong Wang, Lifang Zhou and Rui Tao contributed equally to this work. Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
Abstract We present a base editing system, in which base editors are attached to different sites of sgRNA scaffold (sgBE). Each independent sgBE has its own specific editing pattern for a given target site. Among tested sgBEs, sgBE-SL4, in which deaminase is attached to the last stem-loop of sgRNA, yields the highest editing efficiency in the window several nucleotides next to the one edited by BE3. sgBE enables the simultaneous editing of adenine and cytosine. Finally, in order to facilitate in vivo base editing, we extend our sgBE system to an AAV-compatible Cas9, SaCas9 (Staphylococcus aureus), and observe robust base editing.
Introduction The clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing tools have shown significant successes in basic biomedical research and also provided great promise in clinical translation [1–3]. Recently, the development of base editing technique has enabled the conversion of one target DNA base pairs to another (C/G➔T/A or A/T➔G/C) in an efficient and irreversible way without causing robust double-strand breaks (DSBs), which significantly reduced off-target effects [4, 5] and made it practicable to correct genetic lesions of point mutation in inherited diseases [3]. Basically, base editors consist of a catalytically impaired Cas9 protein tethered with cytidine or adenosine deaminases that are active on ssDNA substrates. Cas9 binds a genomic locus of interest through the guidance of single sgRNA to form a proteinRNA-DNA ternary “R-loop” complex, in which the nontarget strand of sgRNA (NTS) is partially detached from the complex [6]. The exposed NTS provided a feasible substrate for deaminases to action. The most frequent way to tether deaminases is fusing them, through various linkers, to the N-terminus of Cas9 protein [4, 7]. Base editors © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not
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