# Arrayed single-cell readout of pooled genetic perturbation libraries

> **NIH NIH R01** · BROAD INSTITUTE, INC. · 2020 · $1,112,161

## Abstract

Summary
Identifying gene function and impact on disease biology are overarching aims of life science research in the
post-genomic era. Functional genomics also underpins our ability to understand the meaning of genetic
variation in human populations. However, crucial gaps remain in the functional genomics tool set that will
slow our progress in applying genomics to unravel disease biology. Currently, efficient pooled methods
for genome-wide screening require either selection of cells based on growth advantage or physical purification
(eg by FACS). Many disease processes are characterized by complex cellular phenotypes including defects in
cell or organelle morphology, subcellular localization of molecular components, or cell motility. Other key
phenotypes of interest may involve transient states (eg mitosis), cell-cell interactions, or require dynamic
assays in live cells (eg, optical recording of electrophysiological activity of cardiac or neural cells). Image-
based, high-content screens using overexpression and RNA interference have uncovered novel genes
involved in complex phenotypes, including mitosis, synaptogenesis, and embryogenesis. However, such
microplate-based screens of clonal cell populations are not regularly conducted at the genomic scale due to
the expense, labor and automation expertise required. Although “living cell array” screens have reduced some
logistical hurdles, they still require individually synthesizing and arraying each gene perturbation reagent.
Other possible approaches for pooled screening such as single-cell transcriptomics cannot access the range of
complex and dynamic disease-associated phenotypes needed.
Here we propose to develop a new genomic perturbation and screening concept that combines major
advantages of pooled perturbation with imaging assays for single-cell arrayed readout of complex
phenotypes. Specifically, we will screen pooled genomic perturbations (with barcoded CRISPR-Cas9 single-
guide RNAs) using microscopy to read out phenotypes AND to identify perturbed genes at the single-cell level.
Perturbed genes will be identified by sgRNA-associated expressed barcodes in situ by read RNA fluorescence
in situ hybridization (FISH) or in situ sequencing (IS). This approach is highly scalable because the limiting cost
is microscope time for imaging. We image millions of cells per day on a standard research microscope and up
to 100 million cells per day on a high-throughput screening microscope. This means that genome-wide screens
can be routinely conducted on a general-purpose microscope in small labs. Even more ambitious screens
such as all pairwise combinations of 1000 genes with each pair represented across hundreds of cells ( 108
single-cell assays in toto) could be carried out routinely in a dedicated facility.

## Key facts

- **NIH application ID:** 9960539
- **Project number:** 5R01HG009283-04
- **Recipient organization:** BROAD INSTITUTE, INC.
- **Principal Investigator:** Paul Clark Blainey
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $1,112,161
- **Award type:** 5
- **Project period:** 2017-09-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9960539

## Citation

> US National Institutes of Health, RePORTER application 9960539, Arrayed single-cell readout of pooled genetic perturbation libraries (5R01HG009283-04). Retrieved via AI Analytics 2026-08-11 from https://api.ai-analytics.org/grant/nih/9960539. Licensed CC0.

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