RT Journal Article SR Electronic T1 Strategies to Identify and Edit Improvements in Synthetic Genome Segments Episomally JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.03.18.484905 DO 10.1101/2022.03.18.484905 A1 Alexandra Rudolph A1 Akos Nyerges A1 Anush Chiappino-Pepe A1 Matthieu Landon A1 Maximilien Baas-Thomas A1 George Church YR 2022 UL http://biorxiv.org/content/early/2022/03/19/2022.03.18.484905.abstract AB Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments on low copy number vectors. However, DNA synthesis and amplification have the potential to impose mutations on the contained DNA segments that can in turn reduce or prevent viability of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to ‘recoding’ an Escherichia coli strain to swap out seven codons to synonymous alternatives genome-wide. Ten 44,402 to 47,179 bp de novo synthesized BAC-contained DNA segments from the recoded strain were unable to complement deletion of the corresponding 33 to 61 wild type genes using a single antibiotic resistance marker. Next-Generation Sequencing was used to identify 1-7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the BAC-contained recoded segment when both the recoded and wild type copies of the mutated genes had to exist by necessity during the repair process. This strategy could be adapted to mutation identification and repair for other large-scale genome engineering projects, or for incorporation of small genetic engineering sites for quick protocol adjustments.Competing Interest StatementG.C. has founded the following companies: 64-x, EnEvolv, GRO Biosciences, ReadCoor, and Nabla.bio. A full list of his financial interests can be found at https://arep.med.harvard.edu/gmc/tech.html.