Lambert Lab CRISPR-Cas12a Xseq Last 14bp Mismatch
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. The crRNA contains one 14bp randomized region starting at the 5th position of the 20bp recognition sequence. 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
Last14