To our knowledge, this is the first time that a CRISPRi metabolite challenge assay has been applied both to test the growth of cells with CRISPRi system and to indirectly assay the binding of an anti-CRISPR protein to the pdCas9-sgRNA complex. The basis of this assay could be applied to numerous systems, especially cell culture where the phenotypes or survival of cells can be easily enumerated at different time points. In particular, we envision using this assay to interrogate the application of a CRISPR/anti-CRISPR switch to the treatment of metabolic diseases, as complex biochemical pathways can be easily halted or redirected by targeting certain enzymes.
iGEM Toronto has also created and demonstrated the utility of our light boxes by measuring the kinetics of our LacILOV-mCherry construct. In the future, these light boxes could be utilized by other teams to carefully regulate the intensity and duration of light pulses for other light-regulated synthetic biology constructs, in addition to reducing ambient light. To this end, we have created a guide to using these light boxes for CRISPR-Cas9 prokaryotic systems like our own, alongside a putative design for mammalian systems. Please see our guide page for more details.