TY - JOUR
T1 - CRISPR-addressable yeast strains with applications in human G protein– coupled receptor profiling and synthetic biology
AU - Rowe, Jacob B.
AU - Taghon, Geoffrey J.
AU - Kapolka, Nicholas J.
AU - Morgan, William M.
AU - Isom, Daniel G.
N1 - Funding Information:
Funding and additional information—This work was supported by NIGMS, National Institutes of Health Grant R35GM119518 and by the Common Fund for Illuminating the Druggable Genome through National Institutes of Health Grant R03TR002908 (to D. G. I.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 Rowe et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020/6/12
Y1 - 2020/6/12
N2 - 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.
AB - 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.
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U2 - 10.1074/JBC.RA120.013066
DO - 10.1074/JBC.RA120.013066
M3 - Article
C2 - 32358068
AN - SCOPUS:85086497450
VL - 295
SP - 8262
EP - 8271
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 24
ER -