Lambert Lab CRISPR-Cas12a Xseq Trunk Region Crosstalk (Overflow)
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 6bp randomized region, which corresponds to the Cas12a first 6 bp 'trunk' region. The crRNA also has a 6bp randomized region in the corresponding location. 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), tetracycline (TK), or a kanamycin control (K).
SUB5844072
Trunk