한빛사논문
Mieun Lee 1 2, Yu Been Heo 1 2, Han Min Woo 1 2
1Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon, 16419, Republic of Korea.
2BioFoundry Research Center, Institute of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon, 16419, Republic of Korea.
CORRESPONDING AUTHOR : Han Min Woo
Abstract
Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4 to C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs.
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