Wenxia Yu1,2, Jianan Li1,2,3, Shisheng Huang1,2, Xiangyang Li1,2, Ping Li4, Guanglei Li1, Aibin Liang4, Tian Chi5,6, Xingxu Huang7,8. 1. School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. 2. University of Chinese Academy of Sciences, Beijing, 100049, China. 3. Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, 200031, China. 4. Department of Hematology, Tongji Hospital of Tongji University, Shanghai, 200092, China. 5. School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. chitian@shanghaitech.edu.cn. 6. Department Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA. chitian@shanghaitech.edu.cn. 7. School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. huangxx@shanghaitech.edu.cn. 8. CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, 200031, China. huangxx@shanghaitech.edu.cn.
Abstract
BACKGROUND: Site-specific C>T DNA base editing has been achieved by recruiting cytidine deaminases to the target C using catalytically impaired Cas proteins; the target C is typically located within 5-nt editing window specified by the guide RNAs. The prototypical cytidine base editor BE3, comprising rat APOBEC1 (rA1) fused to nCas9, can indiscriminately deaminate multiple C's within the editing window and also create substantial off-target edits on the transcriptome. A powerful countermeasure for the DNA off-target editing is to replace rA1 with APOBEC proteins which selectively edit C's in the context of specific motifs, as illustrated in eA3A-BE3 which targets TC. However, analogous editors selective for other motifs have not been described. In particular, it has been challenging to target a particular C in C-rich sequences. Here, we sought to confront this challenge and also to overcome the RNA off-target effects seen in BE3. RESULTS: By replacing rA1 with an optimized human A3G (oA3G), we developed oA3G-BE3, which selectively targets CC and CCC and is also free of global off-target effects on the transcriptome. Furthermore, we created oA3G-BE4max, an upgraded version of oA3G-BE3 with robust on-target editing. Finally, we showed that oA3G-BE4max has negligible Cas9-independent off-target effects at the genome. CONCLUSIONS: oA3G-BE4max can edit C(C)C with high efficiency and selectivity, which complements eA3A-editors to broaden the collective editing scope of motif selective editors, thus filling a void in the base editing tool box.
BACKGROUND: Site-specific C>T DNA base editing has been achieved by recruiting cytidine deaminases to the target C using catalytically impaired Cas proteins; the target C is typically located within 5-nt editing window specified by the guide RNAs. The prototypical cytidine base editor BE3, comprising ratAPOBEC1 (rA1) fused to nCas9, can indiscriminately deaminate multiple C's within the editing window and also create substantial off-target edits on the transcriptome. A powerful countermeasure for the DNA off-target editing is to replace rA1 with APOBEC proteins which selectively edit C's in the context of specific motifs, as illustrated in eA3A-BE3 which targets TC. However, analogous editors selective for other motifs have not been described. In particular, it has been challenging to target a particular C in C-rich sequences. Here, we sought to confront this challenge and also to overcome the RNA off-target effects seen in BE3. RESULTS: By replacing rA1 with an optimized humanA3G (oA3G), we developed oA3G-BE3, which selectively targets CC and CCC and is also free of global off-target effects on the transcriptome. Furthermore, we created oA3G-BE4max, an upgraded version of oA3G-BE3 with robust on-target editing. Finally, we showed that oA3G-BE4max has negligible Cas9-independent off-target effects at the genome. CONCLUSIONS: oA3G-BE4max can edit C(C)C with high efficiency and selectivity, which complements eA3A-editors to broaden the collective editing scope of motif selective editors, thus filling a void in the base editing tool box.
Authors: Reuben S Harris; Kate N Bishop; Ann M Sheehy; Heather M Craig; Svend K Petersen-Mahrt; Ian N Watt; Michael S Neuberger; Michael H Malim Journal: Cell Date: 2003-06-13 Impact factor: 41.582
Authors: Andrew V Anzalone; Peyton B Randolph; Jessie R Davis; Alexander A Sousa; Luke W Koblan; Jonathan M Levy; Peter J Chen; Christopher Wilson; Gregory A Newby; Aditya Raguram; David R Liu Journal: Nature Date: 2019-10-21 Impact factor: 69.504