CRISPR-associated type V proteins as a tool for controlling mRNA stability inS. cerevisiaesynthetic gene circuits

Abstract

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.

Laccase is a multitasking protein for synthetic gene circuits in the yeast Saccharomyces cerevisiae

“…Since this output signal is visible to the naked eye, the usage of laccase together with ABTS can speed up considerably the screening for strains containing a working synthetic gene circuit. We have already used TtLcc1 as a reporter to evaluate the efficiency of mRNA degradation caused by CRISPR type V proteins [ 17 ]. Here, we harnessed TtLcc1 to evaluate gene editing efficiency in a knockout circuit (see Fig.…”

“…PCR products and the cut-open vector were added into 15 μL Gibson isothermal mixture to obtain a final volume of 20 μL. These 20 μL were placed at 50 °C for 1 h. 5 μL were used to transform DH5α (Life Technology––18263–012) Escherichia coli cells (30 s heat shock at 42 °C) [ 15 ]. New plasmids were checked via Sanger-sequencing at Genewiz Inc., Suzhou (China).…”

“…SD plate containing 1 mM CuSO 4 (Innochem-Cat: 7758-98-7) and 0.5 mM ABTS (CAS number: 9003-99-0) used for laccase activity characterization were prepared as in Ref. [ 17 ]. Citrate pH 5.6 preparation: 12.514 g sodium citrate and 10.384 g citric acid were dissolved in 800 mL ddH 2 O, pH was adjusted with HCL and NaOH, and finally, the solution was added to 1000 mL ddH 2 0.…”

On the ever‐growing functional versatility of the CRISPR‐Cas13 system

“…In addition, it has been shown that some Cas9s are able to target RNA in a protospacer adjacent motif (PAM)‐independent way (Strutt et al., 2018 ; Zhang et al., 2022 ). Other endonucleases, such as Cas6f (class 1, type I; formerly known as Csy4) or Cas12a, have been used to cleave a specific mRNA and induce its degradation (Qi et al., 2012 ; Yu & Marchisio, 2023 ), although not in a programmable way (i.e. through genetic modification of the target).…”

Design and engineering of logic genetic-enzymatic gates based on the activity of the human CYP2C9 enzyme in permeabilized Saccharomyces cerevisiae cells

“…By leveraging our deep understanding of yeast biology and genetic resources, we have previously implemented a wide variety of biological circuits, especially biosensors, that were based on transcription/translation regulatory mechanisms in budding yeast [ 6 ]. The yeast S. cerevisiae represents a commonly used chassis for synthetic gene circuits because it is a unicellular eukaryotic organism whose genome is well annotated and relatively easy to manipulate by using homologous recombination [ 7 ].…”