# Development of potent and predictable Cas9 gene activation tools through high-throughput screening

> **NIH NIH R01** · BRIGHAM AND WOMEN'S HOSPITAL · 2022 · $413,490

## Abstract

Project Summary
The ability to manipulate the expression of genes in cells and organisms is foundational to the
study of genetics. The CRISPR-Cas9 genome editing toolkit has revolutionized our ability to
modify the genome and epigenome precisely. While CRISPR tools have been optimized to allow
for robust, tunable, and predictable repression and inactivation of gene expression, approaches
to induce gene expression (CRISPR activation, or CRISPRa) are less robust and reproducible.
Through a combination of innovative high-throughput screens and cutting-edge computational
modeling, we will develop a cohort of simple, robust, tunable, and predictable CRISPR-based
tools to increase the expression of any mouse or human gene.
We have developed a high-throughput sequencing-based assay system, Self-sustaining Peptide
Activator Reporter-seq (SPARq), which enables quantitative screening of thousands of candidate
gene activating peptides and combinations thereof to monitor and optimize their gene activation
strength. In Aim 1, we will iteratively employ SPARq to systematically evaluate and optimize
multiple features of gene activating peptides, including activation peptide identity, combination,
linker, and CRISPRa method. We will perform SPARq screens in distinct cell types and with
distinct promoter architectures to identify tools that work consistently, designing a set of CRISPRa
tools that are significantly more potent and consistent than the current state-of-the-art.
In Aim 2, we will improve the predictability and consequently the utility of CRISPRa through a
novel high-throughput reporter assay and a computational effort to model the features associated
with CRISPRa potency. We have designed an approach, CRISPR Outcome and Phenotype
screening, that combines a sensitive reporter assay with a native genomic phenotypic
measurement to profile the activity of a CRISPRa tool at thousands of target sites. Using data
collected through this pipeline, we will develop an algorithm that takes as input one of the
CRISPRa tools developed in Aim 1, a cell type, gene, and CRISPR guide RNA and outputs an
accurate estimate of the expression of that gene following CRISPRa treatment. We will validate
the accuracy of this algorithm to enable tunable gene activation over an extensive dynamic range
in human HepG2 and K562 cells, providing it to the genetics community as a webtool.
Altogether, the efforts described in this proposal will pioneer a next generation toolkit to enable
more robust gain-of-function genetic manipulation and screening.

## Key facts

- **NIH application ID:** 10440841
- **Project number:** 1R01GM143249-01A1
- **Recipient organization:** BRIGHAM AND WOMEN'S HOSPITAL
- **Principal Investigator:** Richard I Sherwood
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $413,490
- **Award type:** 1
- **Project period:** 2022-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10440841

## Citation

> US National Institutes of Health, RePORTER application 10440841, Development of potent and predictable Cas9 gene activation tools through high-throughput screening (1R01GM143249-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10440841. Licensed CC0.

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