Journal: bioRxiv
Article Title: Programmable domestication of thermophilic bacteria through removal of non-canonical defense systems
doi: 10.64898/2026.03.21.713436
Figure Lengend Snippet: Plasmid artificial modification–assisted conjugative engineering (PACE). Methylation-matched plasmids produced in engineered E. coli donor strains are transferred to Geobacillus recipients via pRK24-mediated conjugation. While host-specific methylation enables restriction evasion, intracellular defense systems limit plasmid establishment in wild strains. b, Architecture of the native Type II-C CRISPR–Cas system (GeoCas9EF) adapted for genome editing. A 21-bp spacer sgRNA cassette targets genomic loci adjacent to a protospacer-adjacent motif (PAM). c, Sequence logo of the preferred PAM recognized by GeoCas9EF in G. stearothermophilus EF60045, indicating a consensus 5’-NNNNCAAA- 3’ motif. d, CRISPR-mediated genome editing strategy. A conjugative plasmid carrying GeoCas9EF, sgRNA, and homologous repair arms enables genome modification via Cas9-induced cleavage and homologous recombination. Single-crossover (SCO) intermediates are resolved into double-crossover (DCO) mutants or false positives. Promotor optimization for Cas9 expression in strain SJEF4-2 is shown. e, PCR validation of representative gene deletions generated by CRISPR editing in SJEF4-2 and EF60045, targeting pyrimidine biosynthesis loci (e.g., pyrR and pyrFE ). Expected fragment sizes are indicated. f, Transformation efficiencies following sequential removal of endogenous defense systems. In EF60045 and SJEF4-2, deletion of native plasmids and non-canonical defense modules, including Wadjet II, BREX, CBASS, Gabija, Abi, and SspBCDE, dramatically increased electroporation efficiency (CFU µg ¹ DNA) and conjugation efficiency (transconjugants per recipient), revealing these systems as dominant barriers to genetic domestication.
Article Snippet: The broad-host-range conjugative plasmid pRK24 (Addgene plasmid # 51950, courtesy of Farren Isaacs) was first introduced into E. coli MC variants and selected on tetracycline (10 μg/mL).
Techniques: Plasmid Preparation, Modification, Methylation, Produced, Conjugation Assay, CRISPR, Sequencing, Homologous Recombination, Expressing, Biomarker Discovery, Generated, Transformation Assay, Electroporation
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