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Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor

Professor Rodrigo Ledesma Amaro

Reader in Synthetic Biology Department of Bioengineering - Faculty of Engineering at Imperial College London


Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

Yaokang Wu, Yang Li, Yanfeng Liu, Xiang Xiu, Jiaheng Liu, Linpei Zhang, Jianghua Li, Guocheng Du, Xueqin Lv, Jian Chen, Rodrigo Ledesma-Amaro, Long Liu, Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor, Nucleic Acids Research, Volume 52, Issue 8, 8 May 2024, Pages 4739–4755,

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