title: Lambert Lab CRISPR-Cas12a Xseq Seed Region Crosstalk
design: 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 'seed' 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 ofsucrose. Cas12a binding is measured via selection with sucrose (SK), tetracycline (TK), or a kanamycin control (K).