dCas12a CRISPRi

Evaluation of dCas12a CRISPRi for downregulation of gene expression in baker's yeast
Conclusion:
CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for S. cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous β-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae. For analysis by flow cytometry, cultures were prepared from four colonies picked from transformation plates and inoculated in YEPD media supplemented with NTC (and G418 for dCas12a expressed from a plasmid) followed by incubation at 30°C, 550 rpm and 80% rh for two days to reach full saturation. Subsequently, cultures were diluted 20x in physiological salt and analysed with a BD FACSAria Fusion (BD). Detection of events was set such that 20.000 events were measured for single and double cells were excluded from the analysis. The signal of fluorescent proteins was detected with a bandpass filter set at 530/30 nm for eGFP, 450/50 nm for BFP and 610/20 nm for mCherry. The data was recorded using the BD FACSDiva 8.0.2 software to retrieve the geometric mean of the fluorescence distribution which was averaged for quadruplicates. Fluorescence obtained for eGFP, mCherry and BFP in arbitrary units was converted to molecules of equivalent fluorophores using Rainbow calibration beads with 8-peaks (BioLegend, London, UK) and the FlowCal Python package (39). Specifically, fluorescence of eGFP was expressed in Molecules of Equivalent FLuorescein (MEFL), mCherry in Molecules of Equivalent Phycoerythrin-TR (MEPTR) and BFP in Molecules of Equivalent BFP for BFP using values of calibration beads detected with channels ECD, FITC and BFP for mCherry, eGFP and BFP, respectively.