Schematic representation of the SyngenicDNA approach.

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Schematic representation of the SyngenicDNA approach. Schematic representation of the SyngenicDNA approach. (A) Identification of RM system target motifs by SMRTseq. Methylome analysis of polymerase kinetics during sequencing permits detection of methylated sites at single-nucleotide resolution across the genome, revealing the exact motifs targeted by innate RM systems (indicated by colored nucleotides; N is any nucleotide) (kinetic trace image adapted from www.pacb.com). (B) Assembly in silico of a genetic tool with a desired functionality, followed by screening for the presence of RM target sequences and sequence adaptation, using SNPs or synonymous codon substitutions in coding regions, to create an RM-silent template which is synthetized de novo to assemble a SyngenicDNA tool. (C) Artificial transformation of the bacterium of interest target bacterium. Inappropriately methylated target motifs of the original genetic tool are recognized as nonself-DNA and degraded by RM systems. In contrast, the SyngenicDNA variant retains the form and functionality of the genetic tool, but is uniquely designed at the nucleotide level to evade the RM systems and can operate as desired within the target bacterial host. Christopher D. Johnston et al. PNAS 2019;116:23:11454-11459 ©2019 by National Academy of Sciences