Direct, large-scale perturbation and mapping of protein function in human cells

The National Institutes of Health Office of the Director awarded the Trustees of Boston University $1,144,500 on September 4, 2026, under the Trans-NIH Research Support program (CFDA 93.310) to develop nanobody-based probes that enable precise, multiplexed, and genetically-encoded perturbations to protein conformations and states in human cells.

The work will generate a novel class of probes to realize large-scale, unbiased protein interaction mapping through pooled screening methodologies, addressing the absence of combinatorial proteome perturbation techniques comparable to CRISPR-based genetic screening. The technology leverages synthetic biology and continuous evolution to create protein-based tools that allow millions of simultaneous perturbations to signaling states without losing probe-phenotype linkage. The research will initially apply this platform to G-protein coupled receptors (GPCRs), a superfamily of cell membrane receptors involved in human physiology and disease, to generate interaction maps and understand how interconnected GPCR networks regulate cellular and disease phenotypes.

Performance occurs in Boston, Massachusetts. The period of performance extends from the award date through August 31, 2031. This is a Project Grant, a form of assistance award supporting hypothesis-driven research. The award is one of multiple Trans-NIH Research Support initiatives offered through the NIH Office of the Director for research projects selected through competitive merit review.

Abstract

The advent of crispr-based methodologies has transformed our ability to map the complex relationship between genotypes and phenotypes at scale, by enabling multiplexed and combinatorial genetic perturbations of the human genome. As powerful and informative as they have been, these methods are restricted to genetic perturbations and the genetic mapping of cellular phenotypes. However, many cellular phenotypes are driven by changes in molecular activities (e.g. proteins transitioning between multiple conformational or phosphorylation states) and not just the presence or absence of a gene product. To date, no method exists to combinatorially perturb the proteome with such scale and precision, and map how resulting changes to protein states drive critical cellular and disease phenotypes. What we need is an equivalent of pooled genetic perturbations for protein conformations, to create an equivalent of gene interaction maps for proteins. With support from the pioneer award, we will develop a technology to realize this unaddressed grand challenge in biology. Leveraging our expertise in synthetic biology and continuous evolution, we will generate a novel class of nanobody-based probes that enable precise, multiplexed, and genetically-encoded perturbations to protein conformations/states. These probes will empower a new generation of "crispr-like" pooled screens to generate large-scale, unbiased protein interaction maps. We will initially apply this technology to g-protein coupled receptors (gpcrs), a superfamily of cell membrane receptors involved in many aspects of human physiology and disease. Our protein-based probes will allow millions of simultaneous perturbations to the signaling states of gpcrs to be pooled without losing probe-phenotype linkage (much like grnas in crispr). Our technology will allow US to tackle the mystery of how interconnected gpcr networks regulate immune cell function, such as those involved in cancer immunology and therapeutics, and provide the first holistic descriptions of the gpcr-OME operating within human cells. This work, which takes our research into an exciting new direction, will provide a powerful new lens for basic biology and disease, and fosters a new therapeutic paradigm, in which we depart from drugging individual gpcr targets, and treat diseases by modulating sets of receptors with precision nanobodies. Finally, this work establishes a blueprint for using functional continuous evolution systems to generate protein modulators for many other classes of proteins to map the landscape of how individual molecular activities coordinate to produce cellular and disease phenotypes.