# Recombinant DNA technologies for multiplex genetic assays in human cells

> **NIH NIH R35** · CASE WESTERN RESERVE UNIVERSITY · 2021 · $402,500

## Abstract

PROJECT SUMMARY
 Advances in high throughput sequencing have already revealed millions of protein coding variants
within human exomes, and there are many millions of additional differences that likely exist but have not yet
been observed. Many of these variants likely play important roles influencing human health, but we lack the
clinical data required to associate each variant genotype with their corresponding phenotypes. This
disconnect is oftentimes referred to as the variant interpretation problem. Genetic experiments in model
systems play a critical role in uncovering the effects of protein coding variants, but traditional approaches
typically test variants one-by-one and will never address this glut of uncharacterized variants. Multiplex
genetic assays capable of simultaneously testing complex variant libraries have the required throughput, but
these approaches are still in their developmental infancy, and improvements are needed to increase the
capabilities, costs, efficiency, and usability of these techniques to successfully address this problem.
 Harnessing a palette of synthetic biology tools centered around the highly efficient Bxb1
bacteriophage DNA recombinase, I developed a user-friendly, highly customizable platform for expression of
complex variant libraries within individual cultured human cells. I previously paired this expression system
with a highly generalizable assay that identifies variants that are loss-of-function due to an reduced
intracellular steady-state abundance. I applied this assay to comprehensively study variants in four disease-
related proteins, and more collaborative projects are still in progress. Unfortunately, these methods alone will
not address the problem, and more orthogonal approaches are needed to tackle the millions of
uncharacterized disease-relevant variants that exist within people.
 The goal of this proposal is to build the next set of fundamental biotechnologies needed to enable
more high-throughput characterizations of protein variants. The individual directions described are each
highly generalizable and can be reapplied to study large swaths of the proteome with only slight
modification. Immediate directions include a functional complementation system to study essential genes, a
fluorescent transcriptional reporter system to study perturbations to intracellular signaling pathways, and a
barcoded ORFeome collection to identify genes that modulate phenotypes of interest when they are
overexpressed. A major purpose of these technologies is to facilitate adoption by other research groups,
especially those that are experts in other biological fields. These developments, along with the data we
generate in the process of demonstrating their utility, will directly address the variant interpretation problem
while also uncovering previously hidden biology underlying cancer-related molecular mechanisms critical to
cell function.

## Key facts

- **NIH application ID:** 10275903
- **Project number:** 1R35GM142886-01
- **Recipient organization:** CASE WESTERN RESERVE UNIVERSITY
- **Principal Investigator:** Kenneth A Matreyek
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $402,500
- **Award type:** 1
- **Project period:** 2021-08-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10275903

## Citation

> US National Institutes of Health, RePORTER application 10275903, Recombinant DNA technologies for multiplex genetic assays in human cells (1R35GM142886-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10275903. Licensed CC0.

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