Designing the selection cassette for the continuous, parallel, and iterative operation of genome engineering

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TOMINAGA Masahiro, Yo SHIMAMURA, Yoshiyuki SOWA, Ikuro KAWAGISHI, Kyoichi SAITO,and Daisuke UMENO

Department of Applied Chemistry and Biotechnology, Japan

For the direct editing of bacterial genome with single-base resolution, two-step recombination methods are widely used. Here, the target site is replaced with PCR-generated DNA cassette first, and the cassette is then replaced with exogenous genes. The DNA cassettes code for positive/negative markers in order to select both for the intermediate/final states. Here, we report the systematic effort to re-design the system to improve the robustness, throughput, and speed of this method to realize the seamless and automation-liable platform for genome engineering. In search for the robust negative genetic selection that can be operated by liquid handling, we found the nuclease kinase activity for non-natural nucleotide dP, originally developed for the directed evolution of genetic switches/circuits (Tashiro et al., Nuc. Acids Res., 39, e12 (2010)), provides robust and efficient selection for the recombinant cell only with liquid handling. Creating/testing the chimeric selectors, duplication of selector genes, tuning of the expression level of individual selector genes enabled us to eliminate the emergence of false-positive clones during the process. Having established the repeatable workflow where all the steps can be rapidly and seamlessly operated by liquid handling, we demonstrate the multi-step and in-parallel operation of genome modifications to generates a diverse set of isoprenoid-overproducing Escherichia coli strains.