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.

中文翻译：

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由基于 dCas12a 的双功能碱基编辑器驱动的多重原位诱变

驱动遗传多样性的诱变对于理解和设计生物系统至关重要。然而，缺乏在多个基因组位点组合产生原位诱变的有效方法限制了复杂生物学功能的研究。在这里，我们设计并构建了 MultiduBE，一种基于 dCas12a 的多重双功能碱基编辑器，在一体化质粒中用于执行组合原位诱变。两个合成效应子 duBE-1a 和 duBE-2b 是通过合并胞嘧啶脱氨酶（来自 hAPOBEC3A 或 hAID*Δ）、腺嘌呤脱氨酶（来自 TadA9）和 crRNA 阵列处理（来自 dCas12a）的功能而创建的。此外，引入合成分离器 Sp4 可最大程度地减少 crRNA 阵列中的干扰，从而促进大肠杆菌和枯草芽孢杆菌中的多重原位诱变。在相应的 crRNA 阵列的指导下，MultiduBE 成功用于细胞生理重编程和代谢调节。在枯草芽孢杆菌中发现了一种赋予链霉素抗性的新突变，并将其整合到具有多种抗生素抗性的突变菌株中。此外，与单基因突变对照菌株相比，组合突变菌株的表面活性素和核黄素滴度分别提高了42%和15倍。总体而言，MultiduBE 提供了一种方便有效的方法来进行多重原位诱变。