Figure 7.

Dependence on the Presence of a PAM at Proximity of the Desired Editing Site in the Native Type I-F CRISPR-Cas-Mediated Single Nucleotide Substitution (A Fragment in the mexB Gene as an Example)

Success of single nucleotide substitution (N to A) was examined in the PAM and PAM proximal region in a selected region in mexB. Editing plasmids containing a repair donor (U and D) for site specific point mutation were introduced into PA154197. For each of these mutagenesis, five randomly selected transformants with high luminescence intensity were subjected to colony PCR and DNA sequencing using primers Seq-F/Seq-R. Sequencing results demonstrated that mutations only in the PAM and PAM proximal 2 nucleotides can be efficiently achieved.