FIG 5.

CRISPR/Cas9^D10A-mediated editing of the M. capsulatus sMMO hydroxylase mmoX chromosomal locus. (A) Nonsense codon (boldface) and nucleotide (underlined) substitutions for generation of the mmoX p.C151X (mmoX^TAA) mutation with the Cas9 PAM site shown (italicized). wt, wild type. (B) Representative agarose gel showing HpaI-digested mmoX PCR products from the wild type or a positive mmoX^TAA transconjugant. (C) Representative sequencing chromatogram of a Cas9^D10A-edited mmoX^TAA locus. (D) Wild-type and mmoX^TAA biomass on NMS agar with or without 5 µM CuSO₄. The activity of sMMO was assessed by naphthalene and o-dianisidine colorimetric assay. Positive sMMO activity is indicated by development of red coloration.