Figure 3.

Schematic diagram of the procedure of performing genetic modifications using the CRISPR/Cas9 system in C. acetobutylicum. Day 1: Transformation of the strain CAS2 with a plasmid containing gRNA and homology arms. Day 2: Transformants were grown to an optical density (OD)1.0 in CGM containing glucose and then spread on a CGM plate containing xylose and relevant antibiotic. Day 4: Colonies were screened by PCR, grown until OD 1.0 in CGM containing glucose, and then spread on plates containing 5-fluorouracil (5-FU). Day 6: Colonies on 5-FU were replicated plated on plates with and without antibiotic. Day 7: Antibiotic sensitive colonies were then screened to confirm the correct genotype. The strain was then ready to be characterized and/or ready for another round of genetic modification.