Bean, B. D. | bioRxiv Science

bioRxiv2021

The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 toolkit with three components: (i) a set of optimized S. pyogenes Cas9-guide RNA (gRNA) expression vectors with five selectable markers and the option to either pre-clone or co-transform the gRNAs; (ii) vectors for the one-step construction of integration cassettes expressing an untagged or GFP/RFP/HA-tagged gene of interest at one of three levels, supporting localization and overexpression studies; and (iii) integration cassettes containing moderately expressed GFP- or RFP-tagged compartmental markers for colocalization experiments. These components allow rapid, high efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. To demonstrate the ease of use, we applied our complete set of compartmental markers to co-label all target subcellular compartments with GFP and RFP. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle to simplify yeast research.

synthetic biology↗

bioRxiv2021

Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols

The yeast Saccharomyces cerevisiae is a powerful tool for studying G protein-coupled receptors (GPCRs) as they can be functionally coupled to its pheromone response pathway. However, some exogenous GPCRs, including the mu opioid receptor, are non-functional in yeast, which may be due to the presence of the fungal sterol ergosterol instead of the animal sterol cholesterol. We engineered yeast to produce cholesterol and introduced the human mu opioid receptor, creating an opioid biosensor capable of detecting the peptide DAMGO at an EC50 of 62 nM and the opiate morphine at an EC50 of 882 nM. Furthermore, introducing mu, delta, and kappa opioid receptors from diverse vertebrates consistently yielded active opioid biosensors that both recapitulated expected agonist binding profiles with EC50s as low as 2.5 nM and were inhibited by the antagonist naltrexone. Additionally, clinically relevant human mu opioid receptor alleles, or variants with terminal mutations, resulted in biosensors that largely displayed the expected changes in activity. We also tested mu opioid receptor-based biosensors with systematically adjusted biosynthetic intermediates of cholesterol, enabling us to relate sterol profiles with biosensor sensitivity. Finally, cholesterol-producing and sterol intermediate biosensor backgrounds were applied to other human GPCRs, resulting in SSTR5, 5-HTR4, FPR1 and NPY1R signaling with varying degrees of cholesterol dependence. Our sterol-optimized platform will be a valuable tool in generating human GPCR-based biosensors, aiding in ongoing receptor deorphanization efforts, and providing a framework for high-throughput screening of receptors and effectors.

synthetic biology↗