Genome stability is essential for engineering cell-based devices and reporter systems. With the advent of CRISPR technology, it is now possible to build such systems by installing the necessary genetic parts directly into an organism's genome. Here, we used this approach to build a set of 10 versatile yeast-based reporter strains for studying human G protein-coupled receptors (GPCRs), the largest class of membrane receptors in humans. These reporter strains contain the necessary genetically encoded parts for studying human GPCR signaling in yeast, as well as four CRISPR-addressable expression cassettes, i.e. landing pads, installed at known safe-harbor sites in the yeast genome. We showcase the utility of these strains in two applications. First, we demonstrate that increasing GPCR expression by incrementally increasing GPCR gene copy number potentiates Gα coupling of the pharmacologically dark receptor GPR68. Second, we used two CRISPR-addressable landing pads for autocrine activation of a GPCR (the somatostatin receptor SSTR5) with its peptide agonist SRIF-14. The utility of these reporter strains can be extended far beyond these select examples to include applications such as nanobody development, mutational analysis, drug discovery, and studies of GPCR chaperoning. Additionally, we present a BY4741 yeast strain created for broad applications in the yeast and synthetic biology communities that contains only the four CRISPR-addressable landing pads. The general utility of these yeast strains provides an inexpensive, scalable, and easy means of installing and expressing genes directly from the yeast genome to build genome-barcoded sensors, reporter systems, and cell-based factories.
Keywords: CRISPR/Cas; DNA payloads; G protein–coupled receptor (GPCR); Saccharomyces cerevisiae; bioengineering; genome barcoding; landing pad; pathway engineering; safe harbor locus; synthetic biology.
© 2020 Rowe et al.