PT - JOURNAL ARTICLE AU - Christopher D. Johnston AU - Sean Cotton AU - Susan R. Rittling AU - Jacqueline R. Starr AU - Gary Borisy AU - Floyd E. Dewhirst AU - Katherine P. Lemon TI - SyngenicDNA: stealth-based evasion of restriction-modification barriers during bacterial genetic engineering AID - 10.1101/387985 DP - 2018 Jan 01 TA - bioRxiv PG - 387985 4099 - http://biorxiv.org/content/early/2018/08/09/387985.short 4100 - http://biorxiv.org/content/early/2018/08/09/387985.full AB - Restriction-modification (RM) systems hinder the use of genetic approaches in the vast majority of bacteria. Here, we describe a systematic approach to adapt genetic tools for use in bacteria that are genetically intractable or poorly tractable owing to active RM defenses. In this process, we determine the genome and methylome of a bacterial strain and use this information to define the bacterium’s RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic tool in silico, synthesize an RM-silent ‘SyngenicDNA’ tool and propagate the tool as novel minicircle plasmids, termed SyMPL tools, before transformation. Using SyngenicDNA and SyMPL tools, we achieved a profound, >100,000- fold, improvement in the transformation of a clinically relevant USA300 strain of Staphylococcus aureus demonstrating the efficacy of these approaches for evading RM systems. The SyngenicDNA and SyMPL approaches are effective, flexible, and should be broadly applicable in microbial genetics. We expect these will facilitate a new era of microbial genetics free of the restraints of restriction-modification barriers.