A Comparative Analysis of Various Cas Proteins in CRISPRi Gene Circuits

Cas proteins with deactivated cutting domains can be used as programmable components in genetic circuits in E. Coli. While dSpCas9 and dFnCas12a have proven functional in gene circuits, there exist Cas proteins from other species of bacteria that have yet to be explored and fully understood. A particularly new and mysterious Cas protein is CasX, which is completely distinct from both Cas9 and Cas12a. CasX is much smaller than both Cas9 and Cas12a; it could potentially be preferable for use in gene circuits. Cas9 and Cas12a proteins from varying species of bacteria recognize unique PAM sequences, and thus function slightly differently. To explore these differences, six versions of a CRISPRi inverter circuit that previously worked with FnCas12a in E. Coli were developed, each using the deactivated version of a different Cas protein (SaCas9, St1Cas9, TdCas9, NmCas9, AsCas12a, and CasX) programmed to target a cassette of tetA (tetracycline resistance) and sacB (sucrose sensitivity). The circuits are transformed into E. Coli, which are then selected for growth in tetracycline and sucrose. Additionally, another version of each circuit targeting GFP is tested, to confirm the results of the multiplexed tetA-sacB selection serially and to compare the effects of targeting different genes.