Fig. 1.

Transconjugant recovery and phenotype and sequencing evaluation of mutants from selected targets. (A) Transconjugants of L. interrogans serovar Canicola strain LAD-1 were recovered following conjugation with E. coli harboring either the empty plasmid pMaOriNHEJ.Inducible: Cas9 or plasmids containing sgRNA cassettes targeting LPS-related genes. Recovered colonies were cultured in liquid medium and subsequently analyzed by immunoblotting using anti-Canicola polyclonal antiserum (B). Selected clones were also subjected to PCR amplification of the target loci, followed by sequencing (C). The wild-type sequence from L. interrogans strain Fiocruz L1-130 is shown, with the protospacer region highlighted in blue and the PAM (5′-NGG-3′) in pink.