CRISPR-associated type V proteins as a tool for controlling mRNA stability in S. cerevisiae synthetic gene circuits.

Type V-A CRISPR-(d)Cas system has been used in multiplex genome editing and transcription regulation in both eukaryotes and prokaryotes. However, mRNA degradation through the endonuclease activity of Cas12a has never been studied. In this work, we present an efficient and powerful tool to induce mRNA degradation in the yeast Saccharomyces cerevisiae via the catalytic activity of (d)Cas12a on pre-crRNA structure. Our results point out that dFnCas12a, (d)LbCas12a, denAsCas12a and two variants (which carry either NLSs or NESs) perform significant mRNA degradation upon insertion of pre-crRNA fragments into the 5'- or 3' UTR of the target mRNA. The tool worked well with two more Cas12 proteins-(d)MbCas12a and Casϕ2-whereas failed by using type VI LwaCas13a, which further highlights the great potential of type V-A Cas proteins in yeast. We applied our tool to the construction of Boolean NOT, NAND, and IMPLY gates, whose logic operations are fully based on the control of the degradation of the mRNA encoding for a reporter protein. Compared to other methods for the regulation of mRNA stability in yeast synthetic gene circuits (such as RNAi and riboswitches/ribozymes), our system is far easier to engineer and ensure very high performance.