CRISPR-enabled deaminase base editing has become a powerful tool for precisely editing nucleotides on the chromosome. In this study DNA helicases, such as Escherichia coli DnaB, were fused to activation-induced cytidine deaminase (AID) to form enzyme complexes which randomly introduces edited bases throughout the chromosome. DnaB-AID was found to increase 2.5 × 10³ fold relative to the mutagenesis frequency of wildtype. 97.9% of these edits were observed on the leading strand during DNA replication suggesting deamination to be highly coordinated with DNA replication. Using DnaB-AID, a 371.4% increase in β-carotene production was obtained following four rounds of editing. In Saccharomyces cerevisiae Helicase-AID was constructed by fusing AID to one of the subunits of eukaryotic helicase Mcm2-7 complex, MCM5. Using MCM5-AID, the average editing efficiency of five strains was 2.1 ± 0.4 × 10³ fold higher than the native genomic mutation rate. MCM5-AID was able to improve β-carotene production of S. cerevisiae 4742crt by 75.4% following eight rounds of editing. The S. cerevisiae MCM5-AID technique is the first biological tool for generating and accumulating single base mutations in eukaryotic chromosomes. Since the helicase complex is highly conservative in all eukaryotes, Helicase-AID could be adapted for various applications and research in all eukaryotic cells.

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Journal Article | Research Support, Non-U.S. Gov't

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Wang J, Zhao D, Li J, Hu M, Xin X, Price MA, Li Q, Liu L, Li S, Rosser SJ, ... Show all

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