Cell-free expression and NGS-powered substrate profiling reveal a thermostable metagenomic meganuclease from an extreme environment with novel specificity.

A thermostable (>55°C) intron-encoded LAGLIDADG meganuclease, I-MG11, was discovered that recognizes a GC-rich 17-bp sequence and generates 4-nt palindromic 3'-overhangs.

• Why it matters: Sequence-homology mining of metagenomic databases often finds only close variants of known enzymes, leaving less-homologous meganucleases—especially from extreme environments—underexplored.
• What they did: The team combined wider metagenomic capture using metadata-aware homology with cell-free expression and NGS-based substrate profiling to assess cleavage patterns without gel assays.
• The result: Co-folding models of I-MG11 bound to DNA suggest specificity differences from other monomeric LAGLIDADG meganucleases (e.g., I-SceI) linked to InDels, enabling improved meganuclease identification and expanding genome-engineering options.

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