Fig. 4.
Crispr-cas12/cpf1-mediated genome-editing of submissive marker encoded by sulfur house-keeping genes MET25, MET6 and MET2. (A) Methionine biosynthetic pathway is encoded by a number of reduction steps to incorporate sulfide into the carbon backbone of homoserine. Sulfate was sequentially reduced to sulfide, blocking the sulfide incorporation pathway will result in the accumulation of sulfide (S2−) intracellularly. (B) Agar plate screening of MET25 indel mutations. Colonies with black sector indicates the successful mutation of MET25. (C) Re-streaking of the sectored colonies onto MLA plate to isolate genetically pure MET25 mutants. (D) A Taichi or “Yin-Yang” art is created by plating the wild type P01g strain and the MET25 mutant strains on the MLA plate. (E) On-target genome-editing efficiency for MET25, MET2 and MET6 with different crRNA configurations and modifications. N = 3, the reported data represents mean ± sd.