Fig. 2.

Evaluation of heptosyltransferase II (LIC11312) mutants. (A) Selected transconjugant colonies obtained via the inducible CRISPR/Cas9-NHEJ strategy were analyzed by immunoblotting using anti-Canicola polyclonal antiserum. These, along with additional colonies (n = 15), were subjected to PCR amplification of the LIC11312 locus, followed by Sanger sequencing which identified in-frame mutations (B). The protospacer region is highlighted, with the adjacent motif 5′-NGG-3′ indicated in pink, and each mutation is shown. Transconjugants recovered after conjugation with E. coli carrying the pMaOriPE plasmid (CRISPR-Prime editing), either alone or with a PEgRNA cassette targeting LIC11312 for a two-nucleotide deletion, were sequenced immediately after colony isolation (C) and again following growth in liquid medium supplemented with spectinomycin (D).