Abstract
The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9 (SpCas9) and its variants. The detailed mechanism remains unknown. Here, based on in vitro cleavage assays and base editing analysis, we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1). We also show that these nicks are made on the target DNA strand. These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets. This system provides a novel tool for achieving trait stacking in plants.
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Acknowledgements
This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102), the National Transgenic Science and Technology Program (2018ZX0801002B), the National Natural Science Foundation of China (31788103 and 31971370), the Chinese Academy of Sciences (QYZDY-SSW-SMC030), and the National Key R&D Program of China (2018YFA0900600, 2016YFD0100102-11, and 2016YFD0100605).
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Fan, R., Chai, Z., Xing, S. et al. Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1) to nick the target DNA strand. Sci. China Life Sci. 63, 1619–1630 (2020). https://doi.org/10.1007/s11427-020-1722-0
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DOI: https://doi.org/10.1007/s11427-020-1722-0