Lambert Lab CRISPR-Cas12a Xseq Variable PAM Site
A compact synthetic dCas12a inverter containing randomized bases is used to probe the binding strength of Cas12a to its DNA target. All components are contained within a plasmid, and the experiment is conducted in E. coli. Here, the target is a constitutive promoter containing a 7bp randomized region, which corresponds to the Cas12a PAM recognition region. The crRNA sequence is fixed. The constitutive promoter drives the production of a tetA/sacB cassette, which is selected for in the presence of tetracycline and selected against in the presence of sucrose. Cas12a binding is measured via selection with sucrose (SK) or a kanamycin control (K).
SUB5844072
6PAM